http://2010.igem.org/wiki/index.php?title=Special:Contributions/Gumilton&feed=atom&limit=50&target=Gumilton&year=&month=2010.igem.org - User contributions [en]2024-03-29T02:03:26ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Tsinghua/team/contactTeam:Tsinghua/team/contact2010-11-26T01:47:51Z<p>Gumilton: </p>
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<h1>Contact</h1><br />
<div class="content_block"><p><B>You are warmly welcomed to visit and find more information on our related websites.</B></p><br />
<p>You can drop in at<br />
<a href="http://www.facebook.com/pages/Tsinghua-iGEM/145908955419937" title="Tsinghua iGEM Team 2010 Facebook" target=_blank><img src="https://static.igem.org/mediawiki/2010/c/c8/Facebook-32x32.png"></a></p><br />
<p>Meanwhile RENREN is more popular in China.<br />
<a href="http://www.renren.com/tsinghuaigem" title="Tsinghua iGEM Team 2010 Renren Web" target=_blank><img src="https://static.igem.org/mediawiki/2010/6/69/Xiaonei.gif" width="32" height="32"></a><br />
</p><p><br />
Also you can also follow us on<br />
<a href="http://twitter.com/Tsinghua_iGEM" title="Tsinghua iGEM Team 2010 Twitter" target=_blank><img src="https://static.igem.org/mediawiki/2010/7/7e/Twitter-32x32.png" ></a><br />
</p><p><br />
For videos, check <a href="http://www.youtube.com/user/TsinghuaiGEM" title="Tsinghua iGEM Team 2010 YouTube" target=_blank><img src="https://static.igem.org/mediawiki/2010/3/38/Youtube-32x32.png"></a><br />
</p><p><br />
To contact us further, visit LinkedIn. <a href="http://cn.linkedin.com/pub/tsinghua-igem-thu/23/317/573" title="Tsinghua iGEM Team 2010 LinkedIn" target=_blank><img src="https://static.igem.org/mediawiki/2010/2/22/Linkedin-32x32.png" ></a><br />
</p><p><br />
To know more about our School of Life Science website, just visit the <a href="http://life.tsinghua.edu.cn/english" target=_blank>official website of School of Life Science, Tsinghua.</a></p><br />
</div><br />
<br />
<a name="en"></a><br />
<h1>New Enrollment</h1><br />
<div class="content_block"><br />
<p><br />
Tsinghua iGEM 2011 is on the way to constitute. Please come back and see more details updated.<br />
</p><p><br />
You're welcome to download the application form from this link.<br />
<a href="https://static.igem.org/mediawiki/2010/4/4e/TsinghuaiGEMTeam2011ApplicationForm.pdf" title="Tsinghua iGEM Team 2011 Application Form" target=_blank>Tsinghua iGEM Team 2011 Application Form</a><br />
</p><br />
<p><br />
All the applicants must fill out the form and send it to Tsinghua iGEM by email: igemthu@gmail.com without exception!<br />
</p><br />
<p><br />
Please proceed under the instruction in the form and IMPORTANT MENTION: the deadline is to be before 11:59 pm Dec 4th<br />
, 2010 (UTC+8:00, Beijing, Hong Kong Time). Manage your time well!<br />
</p><p><br />
All Best<br />
</p><p>Tsinghua iGEM 2011<br />
</p><br />
</div><br />
<br />
</div></div><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/team/contactTeam:Tsinghua/team/contact2010-11-25T17:10:50Z<p>Gumilton: </p>
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<h1>Contact</h1><br />
<div class="content_block"><p><B>You are warmly welcomed to visit and find more information on our related websites.</B></p><br />
<p>You can drop in at<br />
<a href="http://www.facebook.com/pages/Tsinghua-iGEM/145908955419937" title="Tsinghua iGEM Team 2010 Facebook" target=_blank><img src="https://static.igem.org/mediawiki/2010/c/c8/Facebook-32x32.png"></a></p><br />
<p>Meanwhile RENREN is more popular in China.<br />
<a href="http://www.renren.com/tsinghuaigem" title="Tsinghua iGEM Team 2010 Renren Web" target=_blank><img src="https://static.igem.org/mediawiki/2010/6/69/Xiaonei.gif" width="32" height="32"></a><br />
</p><p><br />
Also you can also follow us on<br />
<a href="http://twitter.com/Tsinghua_iGEM" title="Tsinghua iGEM Team 2010 Twitter" target=_blank><img src="https://static.igem.org/mediawiki/2010/7/7e/Twitter-32x32.png" ></a><br />
</p><p><br />
For videos, check <a href="http://www.youtube.com/user/TsinghuaiGEM" title="Tsinghua iGEM Team 2010 YouTube" target=_blank><img src="https://static.igem.org/mediawiki/2010/3/38/Youtube-32x32.png"></a><br />
</p><p><br />
To contact us further, visit LinkedIn. <a href="http://cn.linkedin.com/pub/tsinghua-igem-thu/23/317/573" title="Tsinghua iGEM Team 2010 LinkedIn" target=_blank><img src="https://static.igem.org/mediawiki/2010/2/22/Linkedin-32x32.png" ></a><br />
</p><p><br />
To know more about our School of Life Science website, just visit the <a href="http://life.tsinghua.edu.cn/english" target=_blank>official website of School of Life Science, Tsinghua.</a></p><br />
</div><br />
<br />
<a name="en"></a><br />
<h1>New Enrollment</h1><br />
<div class="content_block"><br />
<br />
Tsinghua iGEM 2011 is on the way to constitute. Please come back and see more details updated.<br />
</br><br />
You're welcome to download the application form from this link.<br />
<a href="https://static.igem.org/mediawiki/2010/4/4e/TsinghuaiGEMTeam2011ApplicationForm.pdf" title="Tsinghua iGEM Team 2011 Application Form" target=_blank>Tsinghua iGEM Team 2011 Application Form</a><br />
</div><br />
<br />
</div></div><br />
</html></div>Gumiltonhttp://2010.igem.org/File:TsinghuaiGEMTeam2011ApplicationForm.pdfFile:TsinghuaiGEMTeam2011ApplicationForm.pdf2010-11-25T17:09:18Z<p>Gumilton: </p>
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<div></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/team/contactTeam:Tsinghua/team/contact2010-11-16T21:54:49Z<p>Gumilton: </p>
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<h1>Contact</h1><br />
<div class="content_block"><p><B>You are warmly welcomed to visit and find more information on our related websites.</B></p><br />
<p>You can drop in at<br />
<a href="http://www.facebook.com/pages/Tsinghua-iGEM/145908955419937" title="Tsinghua iGEM Team 2010 Facebook" target=_blank><img src="https://static.igem.org/mediawiki/2010/c/c8/Facebook-32x32.png"></a></p><br />
<p>Meanwhile RENREN is more popular in China.<br />
<a href="http://www.renren.com/tsinghuaigem" title="Tsinghua iGEM Team 2010 Renren Web" target=_blank><img src="https://static.igem.org/mediawiki/2010/6/69/Xiaonei.gif" width="32" height="32"></a><br />
</p><p><br />
Also you can also follow us on<br />
<a href="http://twitter.com/Tsinghua_iGEM" title="Tsinghua iGEM Team 2010 Twitter" target=_blank><img src="https://static.igem.org/mediawiki/2010/7/7e/Twitter-32x32.png" ></a><br />
</p><p><br />
For videos, check <a href="http://www.youtube.com/user/TsinghuaiGEM" title="Tsinghua iGEM Team 2010 YouTube" target=_blank><img src="https://static.igem.org/mediawiki/2010/3/38/Youtube-32x32.png"></a><br />
</p><p><br />
To contact us further, visit LinkedIn. <a href="http://cn.linkedin.com/pub/tsinghua-igem-thu/23/317/573" title="Tsinghua iGEM Team 2010 LinkedIn" target=_blank><img src="https://static.igem.org/mediawiki/2010/2/22/Linkedin-32x32.png" ></a><br />
</p><p><br />
To know more about our School of Life Science website, just visit the <a href="http://life.tsinghua.edu.cn/english" target=_blank>official website of School of Life Science, Tsinghua.</a></p><br />
</div><br />
<br />
<a name="en"></a><br />
<h1>New Enrollment</h1><br />
<div class="content_block"><br />
<br />
Tsinghua iGEM 2011 is on the way to constitute. Please come back and see more details updated.<br />
</br><br />
You're welcome to download the application form from this link.<br />
</div><br />
<br />
</div></div><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/team/contactTeam:Tsinghua/team/contact2010-11-16T21:53:42Z<p>Gumilton: </p>
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<div id="main_content"><br />
<a name="cont"></a><br />
<h1>Contact</h1><br />
<div class="content_block"><p><B>You are warmly welcomed to visit and find more information on our related websites.</B></p><br />
<p>You can drop in at<br />
<a href="http://www.facebook.com/pages/Tsinghua-iGEM/145908955419937" title="Tsinghua iGEM Team 2010 Facebook" target=_blank><img src="https://static.igem.org/mediawiki/2010/c/c8/Facebook-32x32.png"></a></p><br />
<p>Meanwhile RENREN is more popular in China.<br />
<a href="http://www.renren.com/tsinghuaigem" title="Tsinghua iGEM Team 2010 Renren Web" target=_blank><img src="https://static.igem.org/mediawiki/2010/6/69/Xiaonei.gif" width="32" height="32"></a><br />
</p><p><br />
Also you can also follow us on<br />
<a href="http://twitter.com/Tsinghua_iGEM" title="Tsinghua iGEM Team 2010 Twitter" target=_blank><img src="https://static.igem.org/mediawiki/2010/7/7e/Twitter-32x32.png" ></a><br />
</p><p><br />
For videos, check <a href="http://www.youtube.com/user/TsinghuaiGEM" title="Tsinghua iGEM Team 2010 YouTube" target=_blank><img src="https://static.igem.org/mediawiki/2010/3/38/Youtube-32x32.png"></a><br />
</p><p><br />
To contact us further, visit LinkedIn. <a href="http://cn.linkedin.com/pub/tsinghua-igem-thu/23/317/573" title="Tsinghua iGEM Team 2010 LinkedIn" target=_blank><img src="https://static.igem.org/mediawiki/2010/2/22/Linkedin-32x32.png" ></a><br />
</p><p><br />
To know more about our School of Life Science website, just visit the <a href="http://life.tsinghua.edu.cn/english" target=_blank>official website of School of Life Science, Tsinghua.</a></p><br />
</div><br />
<br />
<a name="en"></a><br />
<h1>New Enrollment</h1><br />
<div class="content_block"><br />
<br />
Tsinghua iGEM 2011 is on the way to constitute. Please come back and see more details updated.<br />
</div><br />
<br />
</div></div><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/HumanPracticeTeam:Tsinghua/HumanPractice2010-11-16T07:33:21Z<p>Gumilton: /* BioEthics in Tsinghua iGEM */</p>
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=Ethics=<br />
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<br />
BioEthics regarding the Biological Ethics, discussing about biological issues, have been debated since ancient times. Bioethical issues gained widespread attention in recent years and are often extremely morally contentious. Considerable societal conflict exists regarding fundamental clinical issues such as: defining the beginning and end of life; appropriate standards of care; and broader environmental issues with potential social and health impacts such as growth of genetically-modified organisms and their use in the food supply.<br />
<br />
<br />
==BioEthics in Tsinghua iGEM==<br />
<br />
<br />
Here in Tsinghua iGEM 2010 Team, we are now focusing on some parts of BioEthics, such as animal handling requirement, care for animal welfare, appropriate standards of care, broader environmental issues with potential social and health impacts, impact of our products to be used in the manufacture and how to spread our ideas to make more people get this knowledge.<br />
<br />
Thus we, Tsinghua iGEM 2010, pay our attention specially to BioEthics and keep our promise by two ways. One is to teach every member about keeping healthy, safe and being environmentally friendly while conducting experiments. The other one is to try visit and talk with more people, discussing about our projects, the current biological issues and how to promote the environment where we are living as well as let more people know about and start to be interested in Synthetic Biology and iGEM.<br />
<br />
<html><br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> </html><br />
<br />
<br />
<html></div><div class="content_block"><br />
<a name="saf"></a></html><br />
<br />
='''Safety'''=<br />
According to the WHO biosafety is the prevention of unintentional exposure to pathogens and toxins, or their accidental release, whereas biosecurity is the prevention of loss, theft, misuse, diversion or intentional release of pathogens and toxins. (From https://2010.igem.org/Safety)<br />
<br />
We are always paying special care to the safety of our teammates and the people around. Thus we signed a document to keep in rules about the Safety in Laboratory, not only to protect ourselves but also help to keep a clean and healthy environment.<br />
<br />
The model animals we used are harmless engineering strains of Escherichia coli. They are widely used in all kinds of laboratories around the world. We answered the questions about safety on the Safety Page from iGEM(https://2010.igem.org/Safety) as following:<br />
<br />
<br />
<br />
'''Q1: Would any of your project ideas raise safety issues in terms of:<br />
<br />
researcher safety,<br />
<br />
public safety, or<br />
<br />
environmental safety?'''<br />
<br />
A: Our project is about how to use E Coli to simulate the production of Specific Antibodies. No serious safety problem can be caused by the harmless engineering strains of ''Escherichia coli'' and we kept our promise and rules which ensure the safety during the whole process.<br />
<br />
'''Q2: Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'''<br />
<br />
A: No any potential safety issues.<br />
<br />
'''Q3:Is there a local biosafety group, committee, or review board at your institution?'''<br />
<br />
If yes, what does your local biosafety group think about your project?<br />
<br />
A: Yes, it is. The safety check is operated frequently. This is organised and operated by the ''Laboratories and Facilities'' in Tsinghua University. During our project, they operated one of the safety checks in one year and they evaluated our laboratory as well as our project. Our lab construction is keeping in line with the policies in Tsinghua University and because our project is about how to use E Coli to simulate the production of Specific Antibodies and the models we use are ''Escherichia coli'' so they evaluated our project is in a safe category and meanwhile they spoke highly on our Tsinghua iGEM 2010 Safety Manual for "It helps to keep safety in everyone's mind and do really good job.".<br />
<br />
'''Q4:Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?'''<br />
<br />
A: The most invigorative way to inspire the teams to make up ways to deal with and improve the BioSafety is to establish a special prize in BioSafety, so that many teams could try their best to deal with the issues in BioSafety, like how to take experiments safely, how to build safe BioBricks and mostly how maintain the safe environment during our projects.<br />
<br />
Also to recognize that importance from the bottom of one's heart is the most effective approach for dealing with some issues. So if more education, announcement, lectures, courses and many other kinds of teaching methods can be carried out to draw attention on the safety, to let people keep this in mind, the Safety Issues could no longer be an issue but a common sense.<br />
<br />
<br/><br />
<br />
All the staffs and students operating the infrastructures, devices and anything related with experiments are required to wear the gloves all the time while working. To keep these in rules and in case of any accidents we carried out a document to keep in line with.<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/8/8f/Tsinghua_iGEM_2010_Safety_Manual.pdf" target=blank>Tsinghua iGEM 2010 Safety Manual</a></html><br />
<br />
Besides this there is also a document in Tsinghua University emphasizing on the common Laboratory Sfety. Here are the <html><a href="https://static.igem.org/mediawiki/2010/6/69/THUSAFETY.pdf" target=blank>Laboratory Safety Policy of Tsinghua University in Chinese Version</a><br />
and <a href="https://static.igem.org/mediawiki/2010/0/05/THULBM.pdf" target=blank>The Laboratory Biosafety Manual</a> released by WHO (World Health Organization) in which Tsinghua University keep align.<br />
<br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> <br />
<br />
<br />
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<a name="act"></a><br />
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<br />
='''Activities'''=<br />
<html><a name="shanghai"></a><div class="content_block"></html><br />
<br />
==Shanghai Meetup==<br />
<html><br />
More Info at <a href="https://2010.igem.org/Events/China_meetup" target=_blank>2010 iGEM China meetup</a><br><br><br />
<br />
<br />
This year, on August 5th, teams from universities in China, <br />
<a href="https://2010.igem.org/Team:SJTU-BioX-Shanghai" target=_blank>SJTU-BioX-Shanghai</a>,<br />
<a href="https://2010.igem.org/Team:Tsinghua" target=_blank>Tsinghua</a>,<br />
<a href="https://2010.igem.org/Team:Peking" target=_blank>Peking</a>, <br />
<a href="https://2010.igem.org/Team:ECUST-Shanghai" target=_blank>ECUST-Shanghai</a>, <br />
<a href="https://2010.igem.org/Team:USTC" target=_blank>USTC</a>, <br />
<a href="https://2010.igem.org/Team:USTC_Software" target=_blank>USTC_Software</a>, <br />
<a href="https://2010.igem.org/Team:ZJU-China" target=_blank>ZJU-China</a><br />
and Sun Yat-sen University all gathered in Shanghai and had a nice day at the iGEM 2010 China Meetup.<br />
<br />
<br><br />
<br><br />
<br />
<br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> <br />
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<br />
<a name="tsinghua"></a></html><br />
<br />
==Tsinghua University Lecture Day==<br />
[[Image:THULecturePost.jpg|400px]]<br />
<br />
On October 16th, we gave a lecture to the public in Tsinghua University. More than sixty Students of different majors and different grades all joined our lecture and participated in our heat discussion about bioethics and experimental safety. <br />
<br />
First, Teng Li, the leader of our team, introduced the basic concept and central principles of synthetic biology. We are rather glad that a small part of participants already have knowledge of this subject and have paid some special attention to this field, not only students majoring in life science, but also those from other departments. Then we focused on the introduction of iGEM competition. The origin of the competition, the development history and some well-done projects, were all included in this section. The standard parts were particularly pointed out in our lecture, for it’s the central spirit of this competition. Most audiences were greatly fascinated by synthetic biology and iGEM competition, which really encouraged us.<br />
<br />
After the introduction, another student of our team, Yunxiao Zhang, gave a presentation of our project to all the participants. He generally described the aim of our project, the experimental design and the results we have got as well. Questions and very good advices were proposed, which we really appreciate. Thank you very much!<br />
<br />
Then we iGEM team members and all the other participants had a heat discussion, mainly emphasize on the public health and safety of products from synthetic biology. Opinions are divided on this issue. Some people think that food or medicine yielded by bacteria is safe or usable if they can pass the series of tests. Others are against this idea for we cannot well control the metabolism products of bacteria, more complicated than we think. On the other hand, we find the fact that diverse education backgrounds are somehow relative to the opinions held by them. Most people who have little knowledge of biology and microorganism are just reluctant to use products from synthetic biology, due to the uncomfortable feeling about bacteria. However, students from school of life science pay more attention to the biological function and the clinical assay result of the products. Another interesting issue we discuss about is the name of the bacteria we use. Because the Chinese name of E.coli usually leaves bad impression on the public, ordinary people may prefer products from Lactobacillus than those from E.coli. The name may be more important and impressive than function to some degree in public eyes. That’s a rather interesting phenomenon.<br />
<br />
<br />
<html><br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> </html><br />
<br />
<br />
<html><a name="macquarie"></a></html><br />
<br />
==Survey and Discussion==<br />
<br />
'''Questionnaire'''<br/><br />
<br />
1. Have you ever heard about “synthetic biology”?<br />
<br />
A. Yes B. No<br />
<br />
2. Do you know something about “synthetic biology”?<br />
<br />
A. Pay special attention to it<br />
<br />
B. Only know a little about it <br />
<br />
C. Know nothing about it<br />
<br />
3. Are you willing to use any products in the field of synthetic biology? Such as food or medicine produced by E.coli?<br />
<br />
A. Yes<br />
<br />
B. Case by case<br />
<br />
C. No<br />
<br />
4. Should such reverse modification of organism be forbidden? <br />
<br />
A. Yes B. No<br />
<br />
5. Do you agree that synthetic life is somehow threatening?<br />
<br />
A. Yes B. Sometimes C. No<br />
<br />
6. Have you heard about “Bioethics”?<br />
<br />
A. Yes B. No<br />
<br />
7. Should the use of animals especially mammals in experiment be forbidden?<br />
<br />
A. Yes <br />
<br />
B. Case by case<br />
<br />
C. No<br />
<br />
8. Are you concerned with lab safety, such as the use of toxic reagents and potentially threatening apparatus? <br />
<br />
A. Yes B. No<br />
<br />
9. Have you ever received some relative training about experimental safety?<br />
<br />
A. Yes B. No<br />
<br />
10. Do you think it is potentially threatening to produce antibody with modified bacteria?<br />
<br />
A. Yes, because we cannot guarantee that it is safe.<br />
<br />
B. No, because we can solve these clinical problems with the development of medical research.<br />
<br />
11. Do you think it goes against principles of bioethics to produce antibody with modified bacteria?<br />
<br />
A. Yes B. No<br />
<br />
12. Do you think it will cause social disorder if bacteria for research spread out?<br />
<br />
A. Yes B. No<br />
<br />
<br />
<html><br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> </html><br />
<br />
==A Visit to Macquarie_iGEM==<br />
<br />
Tsinghua and Macquarie iGEM Teams have an impressive relationship both in the cooperation of projects as well as in Human Practice.<br />
<br />
[[Image:TSINGHUA&MAC1.JPG|500px]]<br />
<br />
<br />
On 27/9/2010, GU Xiang, one of the team students in Tsinghua iGEM paid a visit to the Macquarie iGEM. GU Xiang, Yagiz Aksoy, the leader of Macquarie iGEM Team, and Hilal Varinli met together on the beautiful campus of the University of Macquarie, in Sydney. They firstly visited the Life Science Building in Macquarie, "It is amazing" said by GU Xiang. Then they three dropped in the Biology Museum in that building. Later GU Xiang together with Yagiz and Hilal did some of their experiments on that day. The research devices and environment in the labs of Macquarie are absolutely at world leading-class level. They started with biosafety and ethics topics related to the iGEM. They both recognized the biosafety to be the first important issue in the Biological Research and Education. Then they exchanged some ideas about how to improve and draw everyone's attention to this issue. The strict biosafety regulations in Macquarie are good examples for many institutes to learn about.<br />
<br />
[[Image:THUMAC.JPG|500px]]<br />
<br />
After that, they introduced and talked about each other's projects. Through that they found the project of Macquarie iGEM can be <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3"><font face="Comic Sans MS" size="3">a potential screening method</font></a></html> for the project of Tsinghua iGEM. This was the climax of that day when they found this potential cooperation between these two teams and they followed further discussion immediately. As the Macquarie team was to build up a system which can reflect the change by showing different lights. Thus this could be useful in the screening for the antibodies. They finally agreed on this proposition and decided to find more details to see how to make this come true if they both had enough time. They took the first step of their collaboration together by running that day’s experiments with GU Xiang at Macquarie iGEM Labs.<br />
<br />
Also hearing that Macquarie iGEM is a newly built team, GU Xiang generously gave his ideas and experience in iGEM to Yagiz and Hilal, promoting them to build better wiki to let more people know about their ideas. Finally they three expressed freely about the issues in Biology and Life Science as well as the Chinese Culture, especially the Kungfu. They had a wonderful day!<br />
<br />
The leader and all the teammates from Tsinghua iGEM were pleased with this visit and cooperation, they said they were willing to cooperate with Macquarie iGEM to build up and finish a BioSystem together, especially in such a contemporary society where cooperation is extremely of significance.<br />
<br />
We hope that every team in iGEM could find this opportunity to cooperate with an other team to build up a system other than just by only one team. In this cooperation we Tsinghua iGEM got one new way to screen our antibodies which we never heard about and Macquarie iGEM supplied their system with another crucial application and even more applications based on this idea. Thus if all the teams in iGEM can cooperate with at least another one, or even more teams, we may can build up a wonderful Genetically Engineered Machine, definitely it is an international one, thus iGEM!!!<br />
<br />
<html><br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> </html><br />
<br />
<br />
<br />
<html><br />
</div><br />
</div></div><br />
</body><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/HumanPracticeTeam:Tsinghua/HumanPractice2010-11-16T07:28:55Z<p>Gumilton: /* BioEthics in Tsinghua iGEM */</p>
<hr />
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=Ethics=<br />
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<br />
BioEthics regarding the Biological Ethics, discussing about biological issues, have been debated since ancient times. Bioethical issues gained widespread attention in recent years and are often extremely morally contentious. Considerable societal conflict exists regarding fundamental clinical issues such as: defining the beginning and end of life; appropriate standards of care; and broader environmental issues with potential social and health impacts such as growth of genetically-modified organisms and their use in the food supply.<br />
<br />
<br />
==BioEthics in Tsinghua iGEM==<br />
<br />
<br />
Here in Tsinghua iGEM 2010 Team, we are now focusing on some parts of BioEthics, such as animal handling requirement, care for animal welfare, appropriate standards of care, broader environmental issues with potential social and health impacts, impact of our products to be used in the manufacture and how to spread our ideas to make more people get this knowledge.<br />
<br />
Thus we, Tsinghua iGEM 2010, pay our attention specially to BioEthics and keep our promise by two ways. One is to teach every member about keeping healthy, safe and being environmentally friendly while conducting experiments. The other one is to try visit and talk with more people, discussing about our projects, the current biological issues and how to promote the environment where we are living as well as let more people know about and start to be interested in Synthetic Biology and iGEM.。<br />
<br />
<html><br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> </html><br />
<br />
<br />
<html></div><div class="content_block"><br />
<a name="saf"></a></html><br />
<br />
='''Safety'''=<br />
According to the WHO biosafety is the prevention of unintentional exposure to pathogens and toxins, or their accidental release, whereas biosecurity is the prevention of loss, theft, misuse, diversion or intentional release of pathogens and toxins. (From https://2010.igem.org/Safety)<br />
<br />
We are always paying special care to the safety of our teammates and the people around. Thus we signed a document to keep in rules about the Safety in Laboratory, not only to protect ourselves but also help to keep a clean and healthy environment.<br />
<br />
The model animals we used are harmless engineering strains of Escherichia coli. They are widely used in all kinds of laboratories around the world. We answered the questions about safety on the Safety Page from iGEM(https://2010.igem.org/Safety) as following:<br />
<br />
<br />
<br />
'''Q1: Would any of your project ideas raise safety issues in terms of:<br />
<br />
researcher safety,<br />
<br />
public safety, or<br />
<br />
environmental safety?'''<br />
<br />
A: Our project is about how to use E Coli to simulate the production of Specific Antibodies. No serious safety problem can be caused by the harmless engineering strains of ''Escherichia coli'' and we kept our promise and rules which ensure the safety during the whole process.<br />
<br />
'''Q2: Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'''<br />
<br />
A: No any potential safety issues.<br />
<br />
'''Q3:Is there a local biosafety group, committee, or review board at your institution?'''<br />
<br />
If yes, what does your local biosafety group think about your project?<br />
<br />
A: Yes, it is. The safety check is operated frequently. This is organised and operated by the ''Laboratories and Facilities'' in Tsinghua University. During our project, they operated one of the safety checks in one year and they evaluated our laboratory as well as our project. Our lab construction is keeping in line with the policies in Tsinghua University and because our project is about how to use E Coli to simulate the production of Specific Antibodies and the models we use are ''Escherichia coli'' so they evaluated our project is in a safe category and meanwhile they spoke highly on our Tsinghua iGEM 2010 Safety Manual for "It helps to keep safety in everyone's mind and do really good job.".<br />
<br />
'''Q4:Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?'''<br />
<br />
A: The most invigorative way to inspire the teams to make up ways to deal with and improve the BioSafety is to establish a special prize in BioSafety, so that many teams could try their best to deal with the issues in BioSafety, like how to take experiments safely, how to build safe BioBricks and mostly how maintain the safe environment during our projects.<br />
<br />
Also to recognize that importance from the bottom of one's heart is the most effective approach for dealing with some issues. So if more education, announcement, lectures, courses and many other kinds of teaching methods can be carried out to draw attention on the safety, to let people keep this in mind, the Safety Issues could no longer be an issue but a common sense.<br />
<br />
<br/><br />
<br />
All the staffs and students operating the infrastructures, devices and anything related with experiments are required to wear the gloves all the time while working. To keep these in rules and in case of any accidents we carried out a document to keep in line with.<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/8/8f/Tsinghua_iGEM_2010_Safety_Manual.pdf" target=blank>Tsinghua iGEM 2010 Safety Manual</a></html><br />
<br />
Besides this there is also a document in Tsinghua University emphasizing on the common Laboratory Sfety. Here are the <html><a href="https://static.igem.org/mediawiki/2010/6/69/THUSAFETY.pdf" target=blank>Laboratory Safety Policy of Tsinghua University in Chinese Version</a><br />
and <a href="https://static.igem.org/mediawiki/2010/0/05/THULBM.pdf" target=blank>The Laboratory Biosafety Manual</a> released by WHO (World Health Organization) in which Tsinghua University keep align.<br />
<br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> <br />
<br />
<br />
</div><br />
<br />
<a name="act"></a><br />
</html><br />
<br />
='''Activities'''=<br />
<html><a name="shanghai"></a><div class="content_block"></html><br />
<br />
==Shanghai Meetup==<br />
<html><br />
More Info at <a href="https://2010.igem.org/Events/China_meetup" target=_blank>2010 iGEM China meetup</a><br><br><br />
<br />
<br />
This year, on August 5th, teams from universities in China, <br />
<a href="https://2010.igem.org/Team:SJTU-BioX-Shanghai" target=_blank>SJTU-BioX-Shanghai</a>,<br />
<a href="https://2010.igem.org/Team:Tsinghua" target=_blank>Tsinghua</a>,<br />
<a href="https://2010.igem.org/Team:Peking" target=_blank>Peking</a>, <br />
<a href="https://2010.igem.org/Team:ECUST-Shanghai" target=_blank>ECUST-Shanghai</a>, <br />
<a href="https://2010.igem.org/Team:USTC" target=_blank>USTC</a>, <br />
<a href="https://2010.igem.org/Team:USTC_Software" target=_blank>USTC_Software</a>, <br />
<a href="https://2010.igem.org/Team:ZJU-China" target=_blank>ZJU-China</a><br />
and Sun Yat-sen University all gathered in Shanghai and had a nice day at the iGEM 2010 China Meetup.<br />
<br />
<br><br />
<br><br />
<br />
<br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> <br />
<br />
<br />
<a name="tsinghua"></a></html><br />
<br />
==Tsinghua University Lecture Day==<br />
[[Image:THULecturePost.jpg|400px]]<br />
<br />
On October 16th, we gave a lecture to the public in Tsinghua University. More than sixty Students of different majors and different grades all joined our lecture and participated in our heat discussion about bioethics and experimental safety. <br />
<br />
First, Teng Li, the leader of our team, introduced the basic concept and central principles of synthetic biology. We are rather glad that a small part of participants already have knowledge of this subject and have paid some special attention to this field, not only students majoring in life science, but also those from other departments. Then we focused on the introduction of iGEM competition. The origin of the competition, the development history and some well-done projects, were all included in this section. The standard parts were particularly pointed out in our lecture, for it’s the central spirit of this competition. Most audiences were greatly fascinated by synthetic biology and iGEM competition, which really encouraged us.<br />
<br />
After the introduction, another student of our team, Yunxiao Zhang, gave a presentation of our project to all the participants. He generally described the aim of our project, the experimental design and the results we have got as well. Questions and very good advices were proposed, which we really appreciate. Thank you very much!<br />
<br />
Then we iGEM team members and all the other participants had a heat discussion, mainly emphasize on the public health and safety of products from synthetic biology. Opinions are divided on this issue. Some people think that food or medicine yielded by bacteria is safe or usable if they can pass the series of tests. Others are against this idea for we cannot well control the metabolism products of bacteria, more complicated than we think. On the other hand, we find the fact that diverse education backgrounds are somehow relative to the opinions held by them. Most people who have little knowledge of biology and microorganism are just reluctant to use products from synthetic biology, due to the uncomfortable feeling about bacteria. However, students from school of life science pay more attention to the biological function and the clinical assay result of the products. Another interesting issue we discuss about is the name of the bacteria we use. Because the Chinese name of E.coli usually leaves bad impression on the public, ordinary people may prefer products from Lactobacillus than those from E.coli. The name may be more important and impressive than function to some degree in public eyes. That’s a rather interesting phenomenon.<br />
<br />
<br />
<html><br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> </html><br />
<br />
<br />
<html><a name="macquarie"></a></html><br />
<br />
==Survey and Discussion==<br />
<br />
'''Questionnaire'''<br/><br />
<br />
1. Have you ever heard about “synthetic biology”?<br />
<br />
A. Yes B. No<br />
<br />
2. Do you know something about “synthetic biology”?<br />
<br />
A. Pay special attention to it<br />
<br />
B. Only know a little about it <br />
<br />
C. Know nothing about it<br />
<br />
3. Are you willing to use any products in the field of synthetic biology? Such as food or medicine produced by E.coli?<br />
<br />
A. Yes<br />
<br />
B. Case by case<br />
<br />
C. No<br />
<br />
4. Should such reverse modification of organism be forbidden? <br />
<br />
A. Yes B. No<br />
<br />
5. Do you agree that synthetic life is somehow threatening?<br />
<br />
A. Yes B. Sometimes C. No<br />
<br />
6. Have you heard about “Bioethics”?<br />
<br />
A. Yes B. No<br />
<br />
7. Should the use of animals especially mammals in experiment be forbidden?<br />
<br />
A. Yes <br />
<br />
B. Case by case<br />
<br />
C. No<br />
<br />
8. Are you concerned with lab safety, such as the use of toxic reagents and potentially threatening apparatus? <br />
<br />
A. Yes B. No<br />
<br />
9. Have you ever received some relative training about experimental safety?<br />
<br />
A. Yes B. No<br />
<br />
10. Do you think it is potentially threatening to produce antibody with modified bacteria?<br />
<br />
A. Yes, because we cannot guarantee that it is safe.<br />
<br />
B. No, because we can solve these clinical problems with the development of medical research.<br />
<br />
11. Do you think it goes against principles of bioethics to produce antibody with modified bacteria?<br />
<br />
A. Yes B. No<br />
<br />
12. Do you think it will cause social disorder if bacteria for research spread out?<br />
<br />
A. Yes B. No<br />
<br />
<br />
<html><br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> </html><br />
<br />
==A Visit to Macquarie_iGEM==<br />
<br />
Tsinghua and Macquarie iGEM Teams have an impressive relationship both in the cooperation of projects as well as in Human Practice.<br />
<br />
[[Image:TSINGHUA&MAC1.JPG|500px]]<br />
<br />
<br />
On 27/9/2010, GU Xiang, one of the team students in Tsinghua iGEM paid a visit to the Macquarie iGEM. GU Xiang, Yagiz Aksoy, the leader of Macquarie iGEM Team, and Hilal Varinli met together on the beautiful campus of the University of Macquarie, in Sydney. They firstly visited the Life Science Building in Macquarie, "It is amazing" said by GU Xiang. Then they three dropped in the Biology Museum in that building. Later GU Xiang together with Yagiz and Hilal did some of their experiments on that day. The research devices and environment in the labs of Macquarie are absolutely at world leading-class level. They started with biosafety and ethics topics related to the iGEM. They both recognized the biosafety to be the first important issue in the Biological Research and Education. Then they exchanged some ideas about how to improve and draw everyone's attention to this issue. The strict biosafety regulations in Macquarie are good examples for many institutes to learn about.<br />
<br />
[[Image:THUMAC.JPG|500px]]<br />
<br />
After that, they introduced and talked about each other's projects. Through that they found the project of Macquarie iGEM can be <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3"><font face="Comic Sans MS" size="3">a potential screening method</font></a></html> for the project of Tsinghua iGEM. This was the climax of that day when they found this potential cooperation between these two teams and they followed further discussion immediately. As the Macquarie team was to build up a system which can reflect the change by showing different lights. Thus this could be useful in the screening for the antibodies. They finally agreed on this proposition and decided to find more details to see how to make this come true if they both had enough time. They took the first step of their collaboration together by running that day’s experiments with GU Xiang at Macquarie iGEM Labs.<br />
<br />
Also hearing that Macquarie iGEM is a newly built team, GU Xiang generously gave his ideas and experience in iGEM to Yagiz and Hilal, promoting them to build better wiki to let more people know about their ideas. Finally they three expressed freely about the issues in Biology and Life Science as well as the Chinese Culture, especially the Kungfu. They had a wonderful day!<br />
<br />
The leader and all the teammates from Tsinghua iGEM were pleased with this visit and cooperation, they said they were willing to cooperate with Macquarie iGEM to build up and finish a BioSystem together, especially in such a contemporary society where cooperation is extremely of significance.<br />
<br />
We hope that every team in iGEM could find this opportunity to cooperate with an other team to build up a system other than just by only one team. In this cooperation we Tsinghua iGEM got one new way to screen our antibodies which we never heard about and Macquarie iGEM supplied their system with another crucial application and even more applications based on this idea. Thus if all the teams in iGEM can cooperate with at least another one, or even more teams, we may can build up a wonderful Genetically Engineered Machine, definitely it is an international one, thus iGEM!!!<br />
<br />
<html><br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> </html><br />
<br />
<br />
<br />
<html><br />
</div><br />
</div></div><br />
</body><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/HumanPracticeTeam:Tsinghua/HumanPractice2010-10-28T03:57:30Z<p>Gumilton: /* A Visit to Macquarie_iGEM */</p>
<hr />
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<br />
=Ethics=<br />
<html><div class="content_block"></html><br />
<br />
BioEthics regarding the Biological Ethics, discussing about biological issues, have been debated since ancient times. Bioethical issues gained widespread attention in recent years and are often extremely morally contentious. Considerable societal conflict exists regarding fundamental clinical issues such as: defining the beginning and end of life; appropriate standards of care; and broader environmental issues with potential social and health impacts such as growth of genetically-modified organisms and their use in the food supply.<br />
<br />
<br />
==BioEthics in Tsinghua iGEM==<br />
<br />
<br />
Here in Tsinghua iGEM 2010 Team, we are now focusing on some parts of BioEthics, such as animal handling requirement, care for animal welfare, appropriate standards of care, broader environmental issues with potential social and health impacts, impact of our products to be used in the manufacture and how to spread our ideas to make more people get this knowledge.<br />
<br />
Thus we, Tsinghua iGEM 2010, pay our attention specially to BioEthics and keep our promise by two ways. One is to teach every member about keeping healthy, safe and being environmentally friendly while conducting experiments. The other one is to try visit and talk with more people, discussing about our projects, the current biological issues and how to promote the environment where we are living as well as let more people know about and start to be interested in Synthetic Biology and iGEM.<br />
<br />
<html><br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> </html><br />
<br />
<br />
<html></div><div class="content_block"><br />
<a name="saf"></a></html><br />
<br />
='''Safety'''=<br />
According to the WHO biosafety is the prevention of unintentional exposure to pathogens and toxins, or their accidental release, whereas biosecurity is the prevention of loss, theft, misuse, diversion or intentional release of pathogens and toxins. (From https://2010.igem.org/Safety)<br />
<br />
We are always paying special care to the safety of our teammates and the people around. Thus we signed a document to keep in rules about the Safety in Laboratory, not only to protect ourselves but also help to keep a clean and healthy environment.<br />
<br />
The model animals we used are harmless engineering strains of Escherichia coli. They are widely used in all kinds of laboratories around the world. We answered the questions about safety on the Safety Page from iGEM(https://2010.igem.org/Safety) as following:<br />
<br />
<br />
<br />
'''Q1: Would any of your project ideas raise safety issues in terms of:<br />
<br />
researcher safety,<br />
<br />
public safety, or<br />
<br />
environmental safety?'''<br />
<br />
A: Our project is about how to use E Coli to simulate the production of Specific Antibodies. No serious safety problem can be caused by the harmless engineering strains of ''Escherichia coli'' and we kept our promise and rules which ensure the safety during the whole process.<br />
<br />
'''Q2: Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'''<br />
<br />
A: No any potential safety issues.<br />
<br />
'''Q3:Is there a local biosafety group, committee, or review board at your institution?'''<br />
<br />
If yes, what does your local biosafety group think about your project?<br />
<br />
A: Yes, it is. The safety check is operated frequently. This is organised and operated by the ''Laboratories and Facilities'' in Tsinghua University. During our project, they operated one of the safety checks in one year and they evaluated our laboratory as well as our project. Our lab construction is keeping in line with the policies in Tsinghua University and because our project is about how to use E Coli to simulate the production of Specific Antibodies and the models we use are ''Escherichia coli'' so they evaluated our project is in a safe category and meanwhile they spoke highly on our Tsinghua iGEM 2010 Safety Manual for "It helps to keep safety in everyone's mind and do really good job.".<br />
<br />
'''Q4:Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?'''<br />
<br />
A: The most invigorative way to inspire the teams to make up ways to deal with and improve the BioSafety is to establish a special prize in BioSafety, so that many teams could try their best to deal with the issues in BioSafety, like how to take experiments safely, how to build safe BioBricks and mostly how maintain the safe environment during our projects.<br />
<br />
Also to recognize that importance from the bottom of one's heart is the most effective approach for dealing with some issues. So if more education, announcement, lectures, courses and many other kinds of teaching methods can be carried out to draw attention on the safety, to let people keep this in mind, the Safety Issues could no longer be an issue but a common sense.<br />
<br />
<br/><br />
<br />
All the staffs and students operating the infrastructures, devices and anything related with experiments are required to wear the gloves all the time while working. To keep these in rules and in case of any accidents we carried out a document to keep in line with.<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/8/8f/Tsinghua_iGEM_2010_Safety_Manual.pdf" target=blank>Tsinghua iGEM 2010 Safety Manual</a></html><br />
<br />
Besides this there is also a document in Tsinghua University emphasizing on the common Laboratory Sfety. Here are the <html><a href="https://static.igem.org/mediawiki/2010/6/69/THUSAFETY.pdf" target=blank>Laboratory Safety Policy of Tsinghua University in Chinese Version</a><br />
and <a href="https://static.igem.org/mediawiki/2010/0/05/THULBM.pdf" target=blank>The Laboratory Biosafety Manual</a> released by WHO (World Health Organization) in which Tsinghua University keep align.<br />
<br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> <br />
<br />
<br />
</div><br />
<br />
<a name="act"></a><br />
</html><br />
<br />
='''Activities'''=<br />
<html><a name="shanghai"></a><div class="content_block"></html><br />
<br />
==Shanghai Meetup==<br />
<html><br />
More Info at <a href="https://2010.igem.org/Events/China_meetup" target=_blank>2010 iGEM China meetup</a><br><br><br />
<br />
<br />
This year, on August 5th, teams from universities in China, <br />
<a href="https://2010.igem.org/Team:SJTU-BioX-Shanghai" target=_blank>SJTU-BioX-Shanghai</a>,<br />
<a href="https://2010.igem.org/Team:Tsinghua" target=_blank>Tsinghua</a>,<br />
<a href="https://2010.igem.org/Team:Peking" target=_blank>Peking</a>, <br />
<a href="https://2010.igem.org/Team:ECUST-Shanghai" target=_blank>ECUST-Shanghai</a>, <br />
<a href="https://2010.igem.org/Team:USTC" target=_blank>USTC</a>, <br />
<a href="https://2010.igem.org/Team:USTC_Software" target=_blank>USTC_Software</a>, <br />
<a href="https://2010.igem.org/Team:ZJU-China" target=_blank>ZJU-China</a><br />
and Sun Yat-sen University all gathered in Shanghai and had a nice day at the iGEM 2010 China Meetup.<br />
<br />
<br><br />
<br><br />
<br />
<br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> <br />
<br />
<br />
<a name="tsinghua"></a></html><br />
<br />
==Tsinghua University Lecture Day==<br />
[[Image:THULecturePost.jpg|400px]]<br />
<br />
On October 16th, we gave a lecture to the public in Tsinghua University. More than sixty Students of different majors and different grades all joined our lecture and participated in our heat discussion about bioethics and experimental safety. <br />
<br />
First, Teng Li, the leader of our team, introduced the basic concept and central principles of synthetic biology. We are rather glad that a small part of participants already have knowledge of this subject and have paid some special attention to this field, not only students majoring in life science, but also those from other departments. Then we focused on the introduction of iGEM competition. The origin of the competition, the development history and some well-done projects, were all included in this section. The standard parts were particularly pointed out in our lecture, for it’s the central spirit of this competition. Most audiences were greatly fascinated by synthetic biology and iGEM competition, which really encouraged us.<br />
<br />
After the introduction, another student of our team, Yunxiao Zhang, gave a presentation of our project to all the participants. He generally described the aim of our project, the experimental design and the results we have got as well. Questions and very good advices were proposed, which we really appreciate. Thank you very much!<br />
<br />
Then we iGEM team members and all the other participants had a heat discussion, mainly emphasize on the public health and safety of products from synthetic biology. Opinions are divided on this issue. Some people think that food or medicine yielded by bacteria is safe or usable if they can pass the series of tests. Others are against this idea for we cannot well control the metabolism products of bacteria, more complicated than we think. On the other hand, we find the fact that diverse education backgrounds are somehow relative to the opinions held by them. Most people who have little knowledge of biology and microorganism are just reluctant to use products from synthetic biology, due to the uncomfortable feeling about bacteria. However, students from school of life science pay more attention to the biological function and the clinical assay result of the products. Another interesting issue we discuss about is the name of the bacteria we use. Because the Chinese name of E.coli usually leaves bad impression on the public, ordinary people may prefer products from Lactobacillus than those from E.coli. The name may be more important and impressive than function to some degree in public eyes. That’s a rather interesting phenomenon.<br />
<br />
<br />
<html><br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> </html><br />
<br />
<br />
<html><a name="macquarie"></a></html><br />
<br />
==Survey and Discussion==<br />
<br />
'''Questionnaire'''<br/><br />
<br />
1. Have you ever heard about “synthetic biology”?<br />
<br />
A. Yes B. No<br />
<br />
2. Do you know something about “synthetic biology”?<br />
<br />
A. Pay special attention to it<br />
<br />
B. Only know a little about it <br />
<br />
C. Know nothing about it<br />
<br />
3. Are you willing to use any products in the field of synthetic biology? Such as food or medicine produced by E.coli?<br />
<br />
A. Yes<br />
<br />
B. Case by case<br />
<br />
C. No<br />
<br />
4. Should such reverse modification of organism be forbidden? <br />
<br />
A. Yes B. No<br />
<br />
5. Do you agree that synthetic life is somehow threatening?<br />
<br />
A. Yes B. Sometimes C. No<br />
<br />
6. Have you heard about “Bioethics”?<br />
<br />
A. Yes B. No<br />
<br />
7. Should the use of animals especially mammals in experiment be forbidden?<br />
<br />
A. Yes <br />
<br />
B. Case by case<br />
<br />
C. No<br />
<br />
8. Are you concerned with lab safety, such as the use of toxic reagents and potentially threatening apparatus? <br />
<br />
A. Yes B. No<br />
<br />
9. Have you ever received some relative training about experimental safety?<br />
<br />
A. Yes B. No<br />
<br />
10. Do you think it is potentially threatening to produce antibody with modified bacteria?<br />
<br />
A. Yes, because we cannot guarantee that it is safe.<br />
<br />
B. No, because we can solve these clinical problems with the development of medical research.<br />
<br />
11. Do you think it goes against principles of bioethics to produce antibody with modified bacteria?<br />
<br />
A. Yes B. No<br />
<br />
12. Do you think it will cause social disorder if bacteria for research spread out?<br />
<br />
A. Yes B. No<br />
<br />
<br />
<html><br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> </html><br />
<br />
==A Visit to Macquarie_iGEM==<br />
<br />
Tsinghua and Macquarie iGEM Teams have an impressive relationship both in the cooperation of projects as well as in Human Practice.<br />
<br />
[[Image:TSINGHUA&MAC1.JPG|500px]]<br />
<br />
<br />
On 27/9/2010, GU Xiang, one of the team students in Tsinghua iGEM paid a visit to the Macquarie iGEM. GU Xiang, Yagiz Aksoy, the leader of Macquarie iGEM Team, and Hilal Varinli met together on the beautiful campus of the University of Macquarie, in Sydney. They firstly visited the Life Science Building in Macquarie, "It is amazing" said by GU Xiang. Then they three dropped in the Biology Museum in that building. Later GU Xiang together with Yagiz and Hilal did some of their experiments on that day. The research devices and environment in the labs of Macquarie are absolutely at world leading-class level. They started with biosafety and ethics topics related to the iGEM. They both recognized the biosafety to be the first important issue in the Biological Research and Education. Then they exchanged some ideas about how to improve and draw everyone's attention to this issue. The strict biosafety regulations in Macquarie are good examples for many institutes to learn about.<br />
<br />
[[Image:THUMAC.JPG|500px]]<br />
<br />
After that, they introduced and talked about each other's projects. Through that they found the project of Macquarie iGEM can be <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3"><font face="Comic Sans MS" size="3">a potential screening method</font></a></html> for the project of Tsinghua iGEM. This was the climax of that day when they found this potential cooperation between these two teams and they followed further discussion immediately. As the Macquarie team was to build up a system which can reflect the change by showing different lights. Thus this could be useful in the screening for the antibodies. They finally agreed on this proposition and decided to find more details to see how to make this come true if they both had enough time. They took the first step of their collaboration together by running that day’s experiments with GU Xiang at Macquarie iGEM Labs.<br />
<br />
Also hearing that Macquarie iGEM is a newly built team, GU Xiang generously gave his ideas and experience in iGEM to Yagiz and Hilal, promoting them to build better wiki to let more people know about their ideas. Finally they three expressed freely about the issues in Biology and Life Science as well as the Chinese Culture, especially the Kungfu. They had a wonderful day!<br />
<br />
The leader and all the teammates from Tsinghua iGEM were pleased with this visit and cooperation, they said they were willing to cooperate with Macquarie iGEM to build up and finish a BioSystem together, especially in such a contemporary society where cooperation is extremely of significance.<br />
<br />
We hope that every team in iGEM could find this opportunity to cooperate with an other team to build up a system other than just by only one team. In this cooperation we Tsinghua iGEM got one new way to screen our antibodies which we never heard about and Macquarie iGEM supplied their system with another crucial application and even more applications based on this idea. Thus if all the teams in iGEM can cooperate with at least another one, or even more teams, we may can build up a wonderful Genetically Engineered Machine, definitely it is an international one, thus iGEM!!!<br />
<br />
<html><br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> </html><br />
<br />
<br />
<br />
<html><br />
</div><br />
</div></div><br />
</body><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/HumanPracticeTeam:Tsinghua/HumanPractice2010-10-28T03:54:37Z<p>Gumilton: /* Survey and Discussion */</p>
<hr />
<div>__NOTOC__<br />
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<a name="Ethics"></a><br />
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=Ethics=<br />
<html><div class="content_block"></html><br />
<br />
BioEthics regarding the Biological Ethics, discussing about biological issues, have been debated since ancient times. Bioethical issues gained widespread attention in recent years and are often extremely morally contentious. Considerable societal conflict exists regarding fundamental clinical issues such as: defining the beginning and end of life; appropriate standards of care; and broader environmental issues with potential social and health impacts such as growth of genetically-modified organisms and their use in the food supply.<br />
<br />
<br />
==BioEthics in Tsinghua iGEM==<br />
<br />
<br />
Here in Tsinghua iGEM 2010 Team, we are now focusing on some parts of BioEthics, such as animal handling requirement, care for animal welfare, appropriate standards of care, broader environmental issues with potential social and health impacts, impact of our products to be used in the manufacture and how to spread our ideas to make more people get this knowledge.<br />
<br />
Thus we, Tsinghua iGEM 2010, pay our attention specially to BioEthics and keep our promise by two ways. One is to teach every member about keeping healthy, safe and being environmentally friendly while conducting experiments. The other one is to try visit and talk with more people, discussing about our projects, the current biological issues and how to promote the environment where we are living as well as let more people know about and start to be interested in Synthetic Biology and iGEM.<br />
<br />
<html><br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> </html><br />
<br />
<br />
<html></div><div class="content_block"><br />
<a name="saf"></a></html><br />
<br />
='''Safety'''=<br />
According to the WHO biosafety is the prevention of unintentional exposure to pathogens and toxins, or their accidental release, whereas biosecurity is the prevention of loss, theft, misuse, diversion or intentional release of pathogens and toxins. (From https://2010.igem.org/Safety)<br />
<br />
We are always paying special care to the safety of our teammates and the people around. Thus we signed a document to keep in rules about the Safety in Laboratory, not only to protect ourselves but also help to keep a clean and healthy environment.<br />
<br />
The model animals we used are harmless engineering strains of Escherichia coli. They are widely used in all kinds of laboratories around the world. We answered the questions about safety on the Safety Page from iGEM(https://2010.igem.org/Safety) as following:<br />
<br />
<br />
<br />
'''Q1: Would any of your project ideas raise safety issues in terms of:<br />
<br />
researcher safety,<br />
<br />
public safety, or<br />
<br />
environmental safety?'''<br />
<br />
A: Our project is about how to use E Coli to simulate the production of Specific Antibodies. No serious safety problem can be caused by the harmless engineering strains of ''Escherichia coli'' and we kept our promise and rules which ensure the safety during the whole process.<br />
<br />
'''Q2: Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'''<br />
<br />
A: No any potential safety issues.<br />
<br />
'''Q3:Is there a local biosafety group, committee, or review board at your institution?'''<br />
<br />
If yes, what does your local biosafety group think about your project?<br />
<br />
A: Yes, it is. The safety check is operated frequently. This is organised and operated by the ''Laboratories and Facilities'' in Tsinghua University. During our project, they operated one of the safety checks in one year and they evaluated our laboratory as well as our project. Our lab construction is keeping in line with the policies in Tsinghua University and because our project is about how to use E Coli to simulate the production of Specific Antibodies and the models we use are ''Escherichia coli'' so they evaluated our project is in a safe category and meanwhile they spoke highly on our Tsinghua iGEM 2010 Safety Manual for "It helps to keep safety in everyone's mind and do really good job.".<br />
<br />
'''Q4:Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?'''<br />
<br />
A: The most invigorative way to inspire the teams to make up ways to deal with and improve the BioSafety is to establish a special prize in BioSafety, so that many teams could try their best to deal with the issues in BioSafety, like how to take experiments safely, how to build safe BioBricks and mostly how maintain the safe environment during our projects.<br />
<br />
Also to recognize that importance from the bottom of one's heart is the most effective approach for dealing with some issues. So if more education, announcement, lectures, courses and many other kinds of teaching methods can be carried out to draw attention on the safety, to let people keep this in mind, the Safety Issues could no longer be an issue but a common sense.<br />
<br />
<br/><br />
<br />
All the staffs and students operating the infrastructures, devices and anything related with experiments are required to wear the gloves all the time while working. To keep these in rules and in case of any accidents we carried out a document to keep in line with.<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/8/8f/Tsinghua_iGEM_2010_Safety_Manual.pdf" target=blank>Tsinghua iGEM 2010 Safety Manual</a></html><br />
<br />
Besides this there is also a document in Tsinghua University emphasizing on the common Laboratory Sfety. Here are the <html><a href="https://static.igem.org/mediawiki/2010/6/69/THUSAFETY.pdf" target=blank>Laboratory Safety Policy of Tsinghua University in Chinese Version</a><br />
and <a href="https://static.igem.org/mediawiki/2010/0/05/THULBM.pdf" target=blank>The Laboratory Biosafety Manual</a> released by WHO (World Health Organization) in which Tsinghua University keep align.<br />
<br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> <br />
<br />
<br />
</div><br />
<br />
<a name="act"></a><br />
</html><br />
<br />
='''Activities'''=<br />
<html><a name="shanghai"></a><div class="content_block"></html><br />
<br />
==Shanghai Meetup==<br />
<html><br />
More Info at <a href="https://2010.igem.org/Events/China_meetup" target=_blank>2010 iGEM China meetup</a><br><br><br />
<br />
<br />
This year, on August 5th, teams from universities in China, <br />
<a href="https://2010.igem.org/Team:SJTU-BioX-Shanghai" target=_blank>SJTU-BioX-Shanghai</a>,<br />
<a href="https://2010.igem.org/Team:Tsinghua" target=_blank>Tsinghua</a>,<br />
<a href="https://2010.igem.org/Team:Peking" target=_blank>Peking</a>, <br />
<a href="https://2010.igem.org/Team:ECUST-Shanghai" target=_blank>ECUST-Shanghai</a>, <br />
<a href="https://2010.igem.org/Team:USTC" target=_blank>USTC</a>, <br />
<a href="https://2010.igem.org/Team:USTC_Software" target=_blank>USTC_Software</a>, <br />
<a href="https://2010.igem.org/Team:ZJU-China" target=_blank>ZJU-China</a><br />
and Sun Yat-sen University all gathered in Shanghai and had a nice day at the iGEM 2010 China Meetup.<br />
<br />
<br><br />
<br><br />
<br />
<br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> <br />
<br />
<br />
<a name="tsinghua"></a></html><br />
<br />
==Tsinghua University Lecture Day==<br />
[[Image:THULecturePost.jpg|400px]]<br />
<br />
On October 16th, we gave a lecture to the public in Tsinghua University. More than sixty Students of different majors and different grades all joined our lecture and participated in our heat discussion about bioethics and experimental safety. <br />
<br />
First, Teng Li, the leader of our team, introduced the basic concept and central principles of synthetic biology. We are rather glad that a small part of participants already have knowledge of this subject and have paid some special attention to this field, not only students majoring in life science, but also those from other departments. Then we focused on the introduction of iGEM competition. The origin of the competition, the development history and some well-done projects, were all included in this section. The standard parts were particularly pointed out in our lecture, for it’s the central spirit of this competition. Most audiences were greatly fascinated by synthetic biology and iGEM competition, which really encouraged us.<br />
<br />
After the introduction, another student of our team, Yunxiao Zhang, gave a presentation of our project to all the participants. He generally described the aim of our project, the experimental design and the results we have got as well. Questions and very good advices were proposed, which we really appreciate. Thank you very much!<br />
<br />
Then we iGEM team members and all the other participants had a heat discussion, mainly emphasize on the public health and safety of products from synthetic biology. Opinions are divided on this issue. Some people think that food or medicine yielded by bacteria is safe or usable if they can pass the series of tests. Others are against this idea for we cannot well control the metabolism products of bacteria, more complicated than we think. On the other hand, we find the fact that diverse education backgrounds are somehow relative to the opinions held by them. Most people who have little knowledge of biology and microorganism are just reluctant to use products from synthetic biology, due to the uncomfortable feeling about bacteria. However, students from school of life science pay more attention to the biological function and the clinical assay result of the products. Another interesting issue we discuss about is the name of the bacteria we use. Because the Chinese name of E.coli usually leaves bad impression on the public, ordinary people may prefer products from Lactobacillus than those from E.coli. The name may be more important and impressive than function to some degree in public eyes. That’s a rather interesting phenomenon.<br />
<br />
<br />
<html><br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> </html><br />
<br />
<br />
<html><a name="macquarie"></a></html><br />
<br />
==Survey and Discussion==<br />
<br />
'''Questionnaire'''<br/><br />
<br />
1. Have you ever heard about “synthetic biology”?<br />
<br />
A. Yes B. No<br />
<br />
2. Do you know something about “synthetic biology”?<br />
<br />
A. Pay special attention to it<br />
<br />
B. Only know a little about it <br />
<br />
C. Know nothing about it<br />
<br />
3. Are you willing to use any products in the field of synthetic biology? Such as food or medicine produced by E.coli?<br />
<br />
A. Yes<br />
<br />
B. Case by case<br />
<br />
C. No<br />
<br />
4. Should such reverse modification of organism be forbidden? <br />
<br />
A. Yes B. No<br />
<br />
5. Do you agree that synthetic life is somehow threatening?<br />
<br />
A. Yes B. Sometimes C. No<br />
<br />
6. Have you heard about “Bioethics”?<br />
<br />
A. Yes B. No<br />
<br />
7. Should the use of animals especially mammals in experiment be forbidden?<br />
<br />
A. Yes <br />
<br />
B. Case by case<br />
<br />
C. No<br />
<br />
8. Are you concerned with lab safety, such as the use of toxic reagents and potentially threatening apparatus? <br />
<br />
A. Yes B. No<br />
<br />
9. Have you ever received some relative training about experimental safety?<br />
<br />
A. Yes B. No<br />
<br />
10. Do you think it is potentially threatening to produce antibody with modified bacteria?<br />
<br />
A. Yes, because we cannot guarantee that it is safe.<br />
<br />
B. No, because we can solve these clinical problems with the development of medical research.<br />
<br />
11. Do you think it goes against principles of bioethics to produce antibody with modified bacteria?<br />
<br />
A. Yes B. No<br />
<br />
12. Do you think it will cause social disorder if bacteria for research spread out?<br />
<br />
A. Yes B. No<br />
<br />
<br />
<html><br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> </html><br />
<br />
==A Visit to Macquarie_iGEM==<br />
<br />
Tsinghua and Macquarie iGEM Teams have an impressive relationship both in the cooperation of projects as well as in Human Practice.<br />
<br />
[[Image:TSINGHUA&MAC1.JPG|500px]]<br />
<br />
<br />
On 27/9/2010, GU Xiang, one of the team students in Tsinghua iGEM paid a visit to the Macquarie iGEM. GU Xiang, Yagiz Aksoy, the leader of Macquarie iGEM Team, and Hilal Varinli met together on the beautiful campus of the University of Macquarie, in Sydney. They firstly visited the Life Science Building in Macquarie, "It is amazing" said by GU Xiang. Then they three dropped in the Biology Museum in that building. Later GU Xiang together with Yagiz and Hilal did some of their experiments on that day. The research devices and environment in the labs of Macquarie are absolutely at world leading-class level. They started with biosafety and ethics topics related to the iGEM. They both recognized the biosafety to be the first important issue in the Biological Research and Education. Then they exchanged some ideas about how to improve and draw everyone's attention to this issue. The strict biosafety regulations in Macquarie are good examples for many institutes to learn about.<br />
<br />
[[Image:THUMAC.JPG|500px]]<br />
<br />
After that, they introduced and talked about each other's projects. Through that they found the project of Macquarie iGEM can be a potential screening method for the project of Tsinghua iGEM. This was the climax of that day when they found this potential cooperation between these two teams and they followed further discussion immediately. As the Macquarie team was to build up a system which can reflect the change by showing different lights. Thus this could be useful in the screening for the antibodies. They finally agreed on this proposition and decided to find more details to see how to make this come true if they both had enough time. They took the first step of their collaboration together by running that day’s experiments with GU Xiang at Macquarie iGEM Labs.<br />
<br />
Also hearing that Macquarie iGEM is a newly built team, GU Xiang generously gave his ideas and experience in iGEM to Yagiz and Hilal, promoting them to build better wiki to let more people know about their ideas. Finally they three expressed freely about the issues in Biology and Life Science as well as the Chinese Culture, especially the Kungfu. They had a wonderful day!<br />
<br />
The leader and all the teammates from Tsinghua iGEM were pleased with this visit and cooperation, they said they were willing to cooperate with Macquarie iGEM to build up and finish a BioSystem together, especially in such a contemporary society where cooperation is extremely of significance.<br />
<br />
We hope that every team in iGEM could find this opportunity to cooperate with an other team to build up a system other than just by only one team. In this cooperation we Tsinghua iGEM got one new way to screen our antibodies which we never heard about and Macquarie iGEM supplied their system with another crucial application and even more applications based on this idea. Thus if all the teams in iGEM can cooperate with at least another one, or even more teams, we may can build up a wonderful Genetically Engineered Machine, definitely it is an international one, thus iGEM!!!<br />
<br />
<html><br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> </html><br />
<br />
<br />
<br />
<html><br />
</div><br />
</div></div><br />
</body><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/HumanPracticeTeam:Tsinghua/HumanPractice2010-10-27T18:14:01Z<p>Gumilton: /* Safety */</p>
<hr />
<div>__NOTOC__<br />
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<br />
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<br />
=Ethics=<br />
<html><div class="content_block"></html><br />
<br />
BioEthics regarding the Biological Ethics, discussing about biological issues, have been debated since ancient times. Bioethical issues gained widespread attention in recent years and are often extremely morally contentious. Considerable societal conflict exists regarding fundamental clinical issues such as: defining the beginning and end of life; appropriate standards of care; and broader environmental issues with potential social and health impacts such as growth of genetically-modified organisms and their use in the food supply.<br />
<br />
<br />
==BioEthics in Tsinghua iGEM==<br />
<br />
<br />
Here in Tsinghua iGEM 2010 Team, we are now focusing on some parts of BioEthics, such as animal handling requirement, care for animal welfare, appropriate standards of care, broader environmental issues with potential social and health impacts, impact of our products to be used in the manufacture and how to spread our ideas to make more people get this knowledge.<br />
<br />
Thus we, Tsinghua iGEM 2010, pay our attention specially to BioEthics and keep our promise by two ways. One is to teach every member about keeping healthy, safe and being environmentally friendly while conducting experiments. The other one is to try visit and talk with more people, discussing about our projects, the current biological issues and how to promote the environment where we are living as well as let more people know about and start to be interested in Synthetic Biology and iGEM.<br />
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='''Safety'''=<br />
According to the WHO biosafety is the prevention of unintentional exposure to pathogens and toxins, or their accidental release, whereas biosecurity is the prevention of loss, theft, misuse, diversion or intentional release of pathogens and toxins. (From https://2010.igem.org/Safety)<br />
<br />
We are always paying special care to the safety of our teammates and the people around. Thus we signed a document to keep in rules about the Safety in Laboratory, not only to protect ourselves but also help to keep a clean and healthy environment.<br />
<br />
The model animals we used are harmless engineering strains of Escherichia coli. They are widely used in all kinds of laboratories around the world. We answered the questions about safety on the Safety Page from iGEM(https://2010.igem.org/Safety) as following:<br />
<br />
<br />
<br />
'''Q1: Would any of your project ideas raise safety issues in terms of:<br />
<br />
researcher safety,<br />
<br />
public safety, or<br />
<br />
environmental safety?'''<br />
<br />
A: Our project is about how to use E Coli to simulate the production of Specific Antibodies. No serious safety problem can be caused by the harmless engineering strains of ''Escherichia coli'' and we kept our promise and rules which ensure the safety during the whole process.<br />
<br />
'''Q2: Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'''<br />
<br />
A: No any potential safety issues.<br />
<br />
'''Q3:Is there a local biosafety group, committee, or review board at your institution?'''<br />
<br />
If yes, what does your local biosafety group think about your project?<br />
<br />
A: Yes, it is. The safety check is operated frequently. This is organised and operated by the ''Laboratories and Facilities'' in Tsinghua University. During our project, they operated one of the safety checks in one year and they evaluated our laboratory as well as our project. Our lab construction is keeping in line with the policies in Tsinghua University and because our project is about how to use E Coli to simulate the production of Specific Antibodies and the models we use are ''Escherichia coli'' so they evaluated our project is in a safe category and meanwhile they spoke highly on our Tsinghua iGEM 2010 Safety Manual for "It helps to keep safety in everyone's mind and do really good job.".<br />
<br />
'''Q4:Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?'''<br />
<br />
A: The most invigorative way to inspire the teams to make up ways to deal with and improve the BioSafety is to establish a special prize in BioSafety, so that many teams could try their best to deal with the issues in BioSafety, like how to take experiments safely, how to build safe BioBricks and mostly how maintain the safe environment during our projects.<br />
<br />
Also to recognize that importance from the bottom of one's heart is the most effective approach for dealing with some issues. So if more education, announcement, lectures, courses and many other kinds of teaching methods can be carried out to draw attention on the safety, to let people keep this in mind, the Safety Issues could no longer be an issue but a common sense.<br />
<br />
<br/><br />
<br />
All the staffs and students operating the infrastructures, devices and anything related with experiments are required to wear the gloves all the time while working. To keep these in rules and in case of any accidents we carried out a document to keep in line with.<br />
<br />
<html><a href="https://static.igem.org/mediawiki/2010/8/8f/Tsinghua_iGEM_2010_Safety_Manual.pdf" target=blank>Tsinghua iGEM 2010 Safety Manual</a></html><br />
<br />
Besides this there is also a document in Tsinghua University emphasizing on the common Laboratory Sfety. Here are the <html><a href="https://static.igem.org/mediawiki/2010/6/69/THUSAFETY.pdf" target=blank>Laboratory Safety Policy of Tsinghua University in Chinese Version</a><br />
and <a href="https://static.igem.org/mediawiki/2010/0/05/THULBM.pdf" target=blank>The Laboratory Biosafety Manual</a> released by WHO (World Health Organization) in which Tsinghua University keep align.<br />
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='''Activities'''=<br />
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==Shanghai Meetup==<br />
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More Info at <a href="https://2010.igem.org/Events/China_meetup" target=_blank>2010 iGEM China meetup</a><br><br><br />
<br />
<br />
This year, on August 5th, teams from universities in China, <br />
<a href="https://2010.igem.org/Team:SJTU-BioX-Shanghai" target=_blank>SJTU-BioX-Shanghai</a>,<br />
<a href="https://2010.igem.org/Team:Tsinghua" target=_blank>Tsinghua</a>,<br />
<a href="https://2010.igem.org/Team:Peking" target=_blank>Peking</a>, <br />
<a href="https://2010.igem.org/Team:ECUST-Shanghai" target=_blank>ECUST-Shanghai</a>, <br />
<a href="https://2010.igem.org/Team:USTC" target=_blank>USTC</a>, <br />
<a href="https://2010.igem.org/Team:USTC_Software" target=_blank>USTC_Software</a>, <br />
<a href="https://2010.igem.org/Team:ZJU-China" target=_blank>ZJU-China</a><br />
and Sun Yat-sen University all gathered in Shanghai and had a nice day at the iGEM 2010 China Meetup.<br />
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<br><br />
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==Tsinghua University Lecture Day==<br />
[[Image:THULecturePost.jpg|400px]]<br />
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==Survey and Discussion==<br />
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==A Visit to Macquarie_iGEM==<br />
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Tsinghua and Macquarie iGEM Teams have an impressive relationship both in the cooperation of projects as well as in Human Practice.<br />
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[[Image:TSINGHUA&MAC1.JPG|500px]]<br />
<br />
<br />
On 27/9/2010, GU Xiang, one of the team students in Tsinghua iGEM paid a visit to the Macquarie iGEM. GU Xiang, Yagiz Aksoy, the leader of Macquarie iGEM Team, and Hilal Varinli met together on the beautiful campus of the University of Macquarie, in Sydney. They firstly visited the Life Science Building in Macquarie, "It is amazing" said by GU Xiang. Then they three dropped in the Biology Museum in that building. Later GU Xiang together with Yagiz and Hilal did some of their experiments on that day. The research devices and environment in the labs of Macquarie are absolutely at world leading-class level. They started with biosafety and ethics topics related to the iGEM. They both recognized the biosafety to be the first important issue in the Biological Research and Education. Then they exchanged some ideas about how to improve and draw everyone's attention to this issue. The strict biosafety regulations in Macquarie are good examples for many institutes to learn about.<br />
<br />
[[Image:THUMAC.JPG|500px]]<br />
<br />
After that, they introduced and talked about each other's projects. Through that they found the project of Macquarie iGEM can be a potential screening method for the project of Tsinghua iGEM. This was the climax of that day when they found this potential cooperation between these two teams and they followed further discussion immediately. As the Macquarie team was to build up a system which can reflect the change by showing different lights. Thus this could be useful in the screening for the antibodies. They finally agreed on this proposition and decided to find more details to see how to make this come true if they both had enough time. They took the first step of their collaboration together by running that day’s experiments with GU Xiang at Macquarie iGEM Labs.<br />
<br />
Also hearing that Macquarie iGEM is a newly built team, GU Xiang generously gave his ideas and experience in iGEM to Yagiz and Hilal, promoting them to build better wiki to let more people know about their ideas. Finally they three expressed freely about the issues in Biology and Life Science as well as the Chinese Culture, especially the Kungfu. They had a wonderful day!<br />
<br />
The leader and all the teammates from Tsinghua iGEM were pleased with this visit and cooperation, they said they were willing to cooperate with Macquarie iGEM to build up and finish a BioSystem together, especially in such a contemporary society where cooperation is extremely of significance.<br />
<br />
We hope that every team in iGEM could find this opportunity to cooperate with an other team to build up a system other than just by only one team. In this cooperation we Tsinghua iGEM got one new way to screen our antibodies which we never heard about and Macquarie iGEM supplied their system with another crucial application and even more applications based on this idea. Thus if all the teams in iGEM can cooperate with at least another one, or even more teams, we may can build up a wonderful Genetically Engineered Machine, definitely it is an international one, thus iGEM!!!<br />
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</html></div>Gumiltonhttp://2010.igem.org/File:Tsinghua_iGEM_2010_Safety_Manual.pdfFile:Tsinghua iGEM 2010 Safety Manual.pdf2010-10-27T18:13:23Z<p>Gumilton: </p>
<hr />
<div></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/experiments/dryTeam:Tsinghua/experiments/dry2010-10-27T17:40:59Z<p>Gumilton: </p>
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<div>__NOTOC__<br />
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<h1>Dry Experiments Records</h1><br />
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<p>Our Experiments were carried out by the following nine groups which are somewhat independent from each other. Here come the Groups and Their Tasks.</p><br />
<p><br />
Group 1A:Landing pad (LP) construction and Insertion with Tranditional Att Recomination method(Promoter Inside)<br/><br />
</p><p><br />
Group 1B:Landing pad (LP) construction and Insertion with Innovational Att Recomination method(Promoter Outside)<br/><br />
</p><p><br />
Group 1C:Landing pad (LP) construction and Insertion: Inducement and Recombination<br />
</p><p><br />
<br />
Group 2A:Donor Plasmid Construction: Two Fragments Construction (Three Fragments Ligation)<br />
</p><p><br />
<br />
Group 2B:Donor Plasmid Construction: Traditional Four Fragments Construction<br />
</p><p><br />
<br />
Group 2C:Donor Plasmid Construction: Four Fragments Construction with Dra III<br />
</p><p><br />
<br />
Group 2D:Donor Plasmid Construction: Four Fragments Construction with Novel Methods<br />
</p><p><br />
<br />
Group 3:Helper Plasmid (HP) Insertion, Donor Plasmid Insertion, Recombination Induction & Removal of HP<br />
</p><p><br />
<br />
Group 4:Screening Strategy<br />
</p><p><br />
</div></div><br />
<a name="prt"></a><br />
<br />
<div class="content_block"><br />
<h1>Protocols</h1><br />
<br />
<p><br />
<br />
Here are our Experimental Protocols in PDF version. You're advised to view in <a href="http://www.adobe.com/acom/" target=blank>Acrobat</a> or <a href="http://get.adobe.com/reader/" target=blank>Adobe Reader</a> which you can download free from the<a href="http://www.adobe.com" target=blank> Home Website of Adobe® Company</a>, for other viewers we can not guarantee the right format of viewing. Those files are encrypted to keep copyrights, if you want to print them out of copy to your documents, to share with others our ideas and methods, you are welcome to ask us for the unencrypted files definitely for free~<br />
</p><br />
<br />
<h3>Molecular Cloning</h3><br />
<br />
<p><a href="https://static.igem.org/mediawiki/2010/0/0a/THUProtocol_1-1_Isolatio_of_plasmid_DNA.pdf" target=blank>THUProtocol_1-1_Isolatio_of_plasmid_DNA</a><br><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/d/d8/THUProtocol_1-2_DNA_Gel_Extraction.pdf" target=blank>THUProtocol_1-2_DNA_Gel_Extraction</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/5/59/THUProtocol_1-3_Restriction_Enzyme_Digestion.pdf" target=blank>THUProtocol_1-3_Restriction_Enzyme_Digestion</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/b/b7/THUProtocol_1-4_Ligation_of_DNA_Fragments.pdf" target=blank>THUProtocol_1-4_Ligation_of_DNA_Fragments</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c3/THUProtocol_1-5_PCR.pdf" target=blank>THUProtocol_1-5_PCR</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c0/THUProtocol_1-6_Preparation_of_Competent_Cell_for_Electro_Transformation.pdf" target=blank>THUProtocol_1-6_Preparation_of_Competent_Cell_for_Electro_Transformation</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/7/77/THUProtocol_1-7_Chemical_Transformation_of_Recombinant_DNA.pdf" target=blank>THUProtocol_1-7_Chemical_Transformation_of_Recombinant_DNA</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/4/40/THUProtocol_1-8_Electro_Transformation_of_Recombinant_DNA.pdf" target=blank>THUProtocol_1-8_Electro_Transformation_of_Recombinant_DNA</a><br />
</p><p><br />
<h3>Protein Isolation and Identification</h3><br />
<p><br />
<a href="https://static.igem.org/mediawiki/2010/f/f0/THUProtocol_2-1_Protein_Isolation_for_Prokaryotes.pdf" target=blank>THUProtocol_2-1_Protein_Isolation_for_Prokaryotes</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/0/08/THUProtocol_2-2_Protein_Identification_SDS-PAGE.pdf" target=blank>THUProtocol_2-2_Protein_Identification_SDS-PAGE</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c3/THUProtocol_1-5_PCR.pdf" target=blank>THUProtocol_1-5_PCR</a><br />
</p><br />
<br />
</div><br />
<br />
</div><br />
</div></div><br />
</body><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/experimentsTeam:Tsinghua/experiments2010-10-27T17:40:01Z<p>Gumilton: </p>
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<h1>Experiment Records</h1><br />
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<p>Our Experiments were carried out by the following nine groups which are somewhat independent from each other. Here come the Groups and Their Tasks.</p><br />
<p><br />
Group 1A:Landing pad (LP) construction and Insertion with Tranditional Att Recomination method(Promoter Inside)<br/><br />
</p><p><br />
Group 1B:Landing pad (LP) construction and Insertion with Innovational Att Recomination method(Promoter Outside)<br/><br />
</p><p><br />
Group 1C:Landing pad (LP) construction and Insertion: Inducement and Recombination<br />
</p><p><br />
<br />
Group 2A:Donor Plasmid Construction: Two Fragments Construction (Three Fragments Ligation)<br />
</p><p><br />
<br />
Group 2B:Donor Plasmid Construction: Traditional Four Fragments Construction<br />
</p><p><br />
<br />
Group 2C:Donor Plasmid Construction: Four Fragments Construction with Dra III<br />
</p><p><br />
<br />
Group 2D:Donor Plasmid Construction: Four Fragments Construction with Novel Methods<br />
</p><p><br />
<br />
Group 3:Helper Plasmid (HP) Insertion, Donor Plasmid Insertion, Recombination Induction & Removal of HP<br />
</p><p><br />
<br />
Group 4:Screening Strategy<br />
</p><p><br />
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<a name="prt"></a><br />
<br />
<div class="content_block"><br />
<h1>Protocols</h1><br />
<br />
<p><br />
<br />
Here are our Experimental Protocols in PDF version. You're advised to view in <a href="http://www.adobe.com/acom/" target=blank>Acrobat</a> or <a href="http://get.adobe.com/reader/" target=blank>Adobe Reader</a> which you can download free from the<a href="http://www.adobe.com" target=blank> Home Website of Adobe® Company</a>, for other viewers we can not guarantee the right format of viewing. Those files are encrypted to keep copyrights, if you want to print them out of copy to your documents, to share with others our ideas and methods, you are welcome to ask us for the unencrypted files definitely for free~<br />
</p><br />
<br />
<h3>Molecular Cloning</h3><br />
<br />
<p><a href="https://static.igem.org/mediawiki/2010/0/0a/THUProtocol_1-1_Isolatio_of_plasmid_DNA.pdf" target=blank>THUProtocol_1-1_Isolatio_of_plasmid_DNA</a><br><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/d/d8/THUProtocol_1-2_DNA_Gel_Extraction.pdf" target=blank>THUProtocol_1-2_DNA_Gel_Extraction</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/5/59/THUProtocol_1-3_Restriction_Enzyme_Digestion.pdf" target=blank>THUProtocol_1-3_Restriction_Enzyme_Digestion</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/b/b7/THUProtocol_1-4_Ligation_of_DNA_Fragments.pdf" target=blank>THUProtocol_1-4_Ligation_of_DNA_Fragments</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c3/THUProtocol_1-5_PCR.pdf" target=blank>THUProtocol_1-5_PCR</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c0/THUProtocol_1-6_Preparation_of_Competent_Cell_for_Electro_Transformation.pdf" target=blank>THUProtocol_1-6_Preparation_of_Competent_Cell_for_Electro_Transformation</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/7/77/THUProtocol_1-7_Chemical_Transformation_of_Recombinant_DNA.pdf" target=blank>THUProtocol_1-7_Chemical_Transformation_of_Recombinant_DNA</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/4/40/THUProtocol_1-8_Electro_Transformation_of_Recombinant_DNA.pdf" target=blank>THUProtocol_1-8_Electro_Transformation_of_Recombinant_DNA</a><br />
</p><p><br />
<h3>Protein Isolation and Identification</h3><br />
<p><br />
<a href="https://static.igem.org/mediawiki/2010/f/f0/THUProtocol_2-1_Protein_Isolation_for_Prokaryotes.pdf" target=blank>THUProtocol_2-1_Protein_Isolation_for_Prokaryotes</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/0/08/THUProtocol_2-2_Protein_Identification_SDS-PAGE.pdf" target=blank>THUProtocol_2-2_Protein_Identification_SDS-PAGE</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c3/THUProtocol_1-5_PCR.pdf" target=blank>THUProtocol_1-5_PCR</a><br />
</p><br />
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</body><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/experiments/resultsTeam:Tsinghua/experiments/results2010-10-27T17:39:17Z<p>Gumilton: </p>
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<h1>Results</h1><br />
<div class="content_block"><br />
<h2>Landing Pad Construction</h2><br />
<p><br />
<img src="http://www.htys.org/results/image002.jpg" />&nbsp;&nbsp;<img src="http://www.htys.org/results/image003.jpg" /></p><p><br />
Left:The landing pad (LP) vector backbone and tetracycline gene with landing pad on its side, with size of 1k and 1.4k<br /><br />
Right:extraction of LP vector, about 2.4k.<br />
</p><br />
<h2>Landing Pad Insertion</h2><br />
<p><img src="http://www.htys.org/results/image004.jpg" /> </p><br />
<p>Colony PCR: Upper primer is in the E.coli genome, 500bp before att site, while the downer primer is in the landing pad. The positive result comes out only when the recombination occurs, shown as the figure. The last lane acts as a negative control, using the untransformed E.coli<br />
</p><br />
<h2>Helper Plasmid Insertion</h2><br />
<p><img src="http://www.htys.org/results/image005.jpg" /> </p> <br />
<p>We transformed the helper plasmid (HP) into E.coli, and extracted the vector, about 6k.</p><br />
<br />
<h2>Donor Plasmid(2) Construction</h2><br />
<p><img src="http://www.htys.org/results/image006.jpg" /> &nbsp;&nbsp;<br />
<img src="http://www.htys.org/results/image007.jpg" /> </p><br />
<p>Here we use a multiple ligation method to construct our donor plasmid (containing two genes)</p> <br />
<br />
<p>Left:the size of every part (vector backbone, chloromycetin , and kanamycin): 1k,1.6k,1.1k<br /><br />
Right:We transformed the ligation product and extracted the DP,and first superhelix is about 2.3k<br />
</p><br />
<h2>Donor Plasmid(4) Construction</h2><br />
<p><img src="http://www.htys.org/results/image008.jpg" />&nbsp;&nbsp;<br />
<img src="http://www.htys.org/results/image009.jpg" /> </p><br />
<p>PCR: eGFP, mCherry, Kan and Chlr four genes separately from PIB-eGFP, PBS34, PKD13 and PKD3 plasmids.</p><br />
<br />
<p><img src="http://www.htys.org/results/image010.jpg" /> </p><br />
<p>PCR to check pUC19+eGFP and pUC19+ChlrPCR. All 12 samples are positive.</p><br />
<br />
<p><img src="http://www.htys.org/results/image011.jpg" /> </p><br />
<p>Double digest PE1 and mCherry with SalI and BamHI, double digest PC1</p><br />
<br />
<p><img src="http://www.htys.org/results/image012.jpg" /> </p> <br />
<p>Using sequencing primers to PCR PEM2+Kan. Lane 1 and 5 are positive.</p><br />
<br />
<p><img src="http://www.htys.org/results/image013.jpg" /> </p><br />
<p>Use sequencing primers to PCR PEMK1+Chlr. Lane 3-5 are positive.</p><br />
<br />
<h2>DP Insertion and Recombination Induction</h2><br />
<h3>Single Gene Recombination (Kan)</h3><br />
<p><img src="http://www.htys.org/results/image014.jpg" /> </p> <br />
<p>PCR checking:Upper primer is in the E.coli genome, 500bp before att site, while the downer primer is in the landing pad. The positive result comes out only when the recombination occurs, shown as the figure. The left six lane are belong to a group of Negative Control. Kanamycin is about 700bp, while the recombination rate is 100%</p><br />
<br />
<h3>Two Genes Recombination (Kan and Chlr)</h3><br />
<br />
<p><img src="http://www.htys.org/results/image015.jpg" /><img src="http://www.htys.org/results/image016.jpg" /> </p><br />
<p>PCR checking: method as before. Close to the marker, we choose an untransformed E.coli as the negative control. The following eight lanes are randomly picked experimental group, half of which show as we expected. These colonies are picked from the plate with Kan resistance.<br />
</p><br />
<p><img src="http://www.htys.org/results/image017.jpg" /><img src="http://www.htys.org/results/image018.jpg" /> </p><br />
<p>PCR checking: method as before. Close to the marker, we choose an untransformed E.coli as the negative control. The following eight lanes are randomly picked experimental group, three of which show as we expected. These colonies are picked from the plate with Chlr resistance.<br />
</p><br />
<br />
<p><img src="http://www.htys.org/results/image019.jpg" />&nbsp;&nbsp;<img src="http://www.htys.org/results/image020.jpg" /> </p><br />
<p>Here we picked colonies from Kan resistance plate and Chlr resistance plate in the same time. Left figure shows the Kan positive results in the 1,2,4 lanes, while right figure shows the Chlr positive results in the 3,4,7,8 lanes.<br />
</p><br />
<br />
<h2>Calculation of Recombination Rate</h2><br />
<p>Recombination rate=<br />
(Positive Colony Numbers in Kan Plate + Positive Colony Numbers in Chlr Plate)/ <br />
(Positive Colony Numbers in Kan Plate + Positive Colony Numbers in Chlr Plate + Colony Numbers in Tet Plate)<br />
</p><p><br />
We have counted the total number of colonies in the Kan plate, the number is 213, and its effective rate is 0.75;<br />
The total number of colonies in the Chlr plate, the number is 602, and its effective rate is 0.68;<br />
Colonies form Kan and Chlr are coated in Tet resistance plate, the number is 451 and 417.<br />
</p><p><br />
So our Recombination Rate= (213*0.75+602*0.68) / (213*0.75+602*0.68+451+417) = 39.6%<br />
</p><br />
<h2>CBD presentation system</h2><br />
<p><img src="http://www.htys.org/results/image021.jpg" /> </p><br />
<p>PCR check: we ligated CBD coding gene and OmpA coding gene to the targeted vector, then transformed to E.coli and extracted the vector. All these four vectors show the positive results, about 1k.</p><br />
<br />
<p><img src="http://www.htys.org/results/image022.jpg" /> </p><br />
<p>SDS-PAGE Check: CBD-OmpA band is circled in the figure.</p><br />
</div></div><br />
<br />
</div></div><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/experiments/dryTeam:Tsinghua/experiments/dry2010-10-27T17:38:18Z<p>Gumilton: </p>
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<p>Our Experiments were carried out by the following nine groups which are somewhat independent from each other. Here come the Groups and Their Tasks.</p><br />
<p><br />
Group 1A:Landing pad (LP) construction and Insertion with Tranditional Att Recomination method(Promoter Inside)<br/><br />
</p><p><br />
Group 1B:Landing pad (LP) construction and Insertion with Innovational Att Recomination method(Promoter Outside)<br/><br />
</p><p><br />
Group 1C:Landing pad (LP) construction and Insertion: Inducement and Recombination<br />
</p><p><br />
<br />
Group 2A:Donor Plasmid Construction: Two Fragments Construction (Three Fragments Ligation)<br />
</p><p><br />
<br />
Group 2B:Donor Plasmid Construction: Traditional Four Fragments Construction<br />
</p><p><br />
<br />
Group 2C:Donor Plasmid Construction: Four Fragments Construction with Dra III<br />
</p><p><br />
<br />
Group 2D:Donor Plasmid Construction: Four Fragments Construction with Novel Methods<br />
</p><p><br />
<br />
Group 3:Helper Plasmid (HP) Insertion, Donor Plasmid Insertion, Recombination Induction & Removal of HP<br />
</p><p><br />
<br />
Group 4:Screening Strategy<br />
</p><p><br />
</div></div><br />
<a name="prt"></a><br />
<br />
<div class="content_block"><br />
<h1>Protocols</h1><br />
<br />
<p><br />
<br />
Here are our Experimental Protocols in PDF version. You're advised to view in <a href="http://www.adobe.com/acom/" target=blank>Acrobat</a> or <a href="http://get.adobe.com/reader/" target=blank>Adobe Reader</a> which you can download free from the<a href="http://www.adobe.com" target=blank> Home Website of Adobe® Company</a>, for other viewers we can not guarantee the right format of viewing. Those files are encrypted to keep copyrights, if you want to print them out of copy to your documents, to share with others our ideas and methods, you are welcome to ask us for the unencrypted files definitely for free~<br />
</p><br />
<br />
<h3>Molecular Cloning</h3><br />
<br />
<p><a href="https://static.igem.org/mediawiki/2010/0/0a/THUProtocol_1-1_Isolatio_of_plasmid_DNA.pdf" target=blank>THUProtocol_1-1_Isolatio_of_plasmid_DNA</a><br><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/d/d8/THUProtocol_1-2_DNA_Gel_Extraction.pdf" target=blank>THUProtocol_1-2_DNA_Gel_Extraction</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/5/59/THUProtocol_1-3_Restriction_Enzyme_Digestion.pdf" target=blank>THUProtocol_1-3_Restriction_Enzyme_Digestion</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/b/b7/THUProtocol_1-4_Ligation_of_DNA_Fragments.pdf" target=blank>THUProtocol_1-4_Ligation_of_DNA_Fragments</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c3/THUProtocol_1-5_PCR.pdf" target=blank>THUProtocol_1-5_PCR</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c0/THUProtocol_1-6_Preparation_of_Competent_Cell_for_Electro_Transformation.pdf" target=blank>THUProtocol_1-6_Preparation_of_Competent_Cell_for_Electro_Transformation</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/7/77/THUProtocol_1-7_Chemical_Transformation_of_Recombinant_DNA.pdf" target=blank>THUProtocol_1-7_Chemical_Transformation_of_Recombinant_DNA</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/4/40/THUProtocol_1-8_Electro_Transformation_of_Recombinant_DNA.pdf" target=blank>THUProtocol_1-8_Electro_Transformation_of_Recombinant_DNA</a><br />
</p><p><br />
<h3>Protein Isolation and Identification</h3><br />
<p><br />
<a href="https://static.igem.org/mediawiki/2010/f/f0/THUProtocol_2-1_Protein_Isolation_for_Prokaryotes.pdf" target=blank>THUProtocol_2-1_Protein_Isolation_for_Prokaryotes</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/0/08/THUProtocol_2-2_Protein_Identification_SDS-PAGE.pdf" target=blank>THUProtocol_2-2_Protein_Identification_SDS-PAGE</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c3/THUProtocol_1-5_PCR.pdf" target=blank>THUProtocol_1-5_PCR</a><br />
</p><br />
<br />
</div><br />
<br />
</div><br />
</div></div><br />
</body><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/experiments/dryTeam:Tsinghua/experiments/dry2010-10-27T17:37:02Z<p>Gumilton: </p>
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<p>Our Experiments were carried out by the following nine groups which are somewhat independent from each other. Here come the Groups and Their Tasks.</p><br />
<p><br />
Group 1A:Landing pad (LP) construction and Insertion with Tranditional Att Recomination method(Promoter Inside)<br/><br />
</p><p><br />
Group 1B:Landing pad (LP) construction and Insertion with Innovational Att Recomination method(Promoter Outside)<br/><br />
</p><p><br />
Group 1C:Landing pad (LP) construction and Insertion: Inducement and Recombination<br />
</p><p><br />
<br />
Group 2A:Donor Plasmid Construction: Two Fragments Construction (Three Fragments Ligation)<br />
</p><p><br />
<br />
Group 2B:Donor Plasmid Construction: Traditional Four Fragments Construction<br />
</p><p><br />
<br />
Group 2C:Donor Plasmid Construction: Four Fragments Construction with Dra III<br />
</p><p><br />
<br />
Group 2D:Donor Plasmid Construction: Four Fragments Construction with Novel Methods<br />
</p><p><br />
<br />
Group 3:Helper Plasmid (HP) Insertion, Donor Plasmid Insertion, Recombination Induction & Removal of HP<br />
</p><p><br />
<br />
Group 4:Screening Strategy<br />
</p><p><br />
</div></div><br />
<a name="prt"></a><br />
<br />
<div class="content_block"><br />
<h1>Protocols</h1><br />
<br />
<p><br />
<br />
Here are our Experimental Protocols in PDF version. You're advised to view in <a href="http://www.adobe.com/acom/" target=blank>Acrobat</a> or <a href="http://get.adobe.com/reader/" target=blank>Adobe Reader</a> which you can download free from the<a href="http://www.adobe.com" target=blank> Home Website of Adobe® Company</a>, for other viewers we can not guarantee the right format of viewing. Those files are encrypted to keep copyrights, if you want to print them out of copy to your documents, to share with others our ideas and methods, you are welcome to ask us for the unencrypted files definitely for free~<br />
</p><br />
<br />
<h3>Molecular Cloning</h3><br />
<br />
<p><a href="https://static.igem.org/mediawiki/2010/0/0a/THUProtocol_1-1_Isolatio_of_plasmid_DNA.pdf" target=blank>THUProtocol_1-1_Isolatio_of_plasmid_DNA</a><br><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/d/d8/THUProtocol_1-2_DNA_Gel_Extraction.pdf" target=blank>THUProtocol_1-2_DNA_Gel_Extraction</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/5/59/THUProtocol_1-3_Restriction_Enzyme_Digestion.pdf" target=blank>THUProtocol_1-3_Restriction_Enzyme_Digestion</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/b/b7/THUProtocol_1-4_Ligation_of_DNA_Fragments.pdf" target=blank>THUProtocol_1-4_Ligation_of_DNA_Fragments</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c3/THUProtocol_1-5_PCR.pdf" target=blank>THUProtocol_1-5_PCR</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c0/THUProtocol_1-6_Preparation_of_Competent_Cell_for_Electro_Transformation.pdf" target=blank>THUProtocol_1-6_Preparation_of_Competent_Cell_for_Electro_Transformation</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/7/77/THUProtocol_1-7_Chemical_Transformation_of_Recombinant_DNA.pdf" target=blank>THUProtocol_1-7_Chemical_Transformation_of_Recombinant_DNA</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/4/40/THUProtocol_1-8_Electro_Transformation_of_Recombinant_DNA.pdf" target=blank>THUProtocol_1-8_Electro_Transformation_of_Recombinant_DNA</a><br />
</p><p><br />
<h3>Protein Isolation and Identification</h3><br />
<p><br />
<a href="https://static.igem.org/mediawiki/2010/f/f0/THUProtocol_2-1_Protein_Isolation_for_Prokaryotes.pdf" target=blank>THUProtocol_2-1_Protein_Isolation_for_Prokaryotes</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/0/08/THUProtocol_2-2_Protein_Identification_SDS-PAGE.pdf" target=blank>THUProtocol_2-2_Protein_Identification_SDS-PAGE</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c3/THUProtocol_1-5_PCR.pdf" target=blank>THUProtocol_1-5_PCR</a><br />
</p><br />
<br />
</div><br />
<br />
</div><br />
</div></div><br />
</body><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/experiments/dryTeam:Tsinghua/experiments/dry2010-10-27T17:36:11Z<p>Gumilton: </p>
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|{{#calendar: year=2010 | month=07 | title=Team:Tsinghua/Notebook}}<br />
|{{#calendar: year=2010 | month=08 | title=Team:Tsinghua/Notebook}}<br />
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|<br />
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<html><br />
<p>Our Experiments were carried out by the following nine groups which are somewhat independent from each other. Here come the Groups and Their Tasks.</p><br />
<p><br />
Group 1A:Landing pad (LP) construction and Insertion with Tranditional Att Recomination method(Promoter Inside)<br/><br />
</p><p><br />
Group 1B:Landing pad (LP) construction and Insertion with Innovational Att Recomination method(Promoter Outside)<br/><br />
</p><p><br />
Group 1C:Landing pad (LP) construction and Insertion: Inducement and Recombination<br />
</p><p><br />
<br />
Group 2A:Donor Plasmid Construction: Two Fragments Construction (Three Fragments Ligation)<br />
</p><p><br />
<br />
Group 2B:Donor Plasmid Construction: Traditional Four Fragments Construction<br />
</p><p><br />
<br />
Group 2C:Donor Plasmid Construction: Four Fragments Construction with Dra III<br />
</p><p><br />
<br />
Group 2D:Donor Plasmid Construction: Four Fragments Construction with Novel Methods<br />
</p><p><br />
<br />
Group 3:Helper Plasmid (HP) Insertion, Donor Plasmid Insertion, Recombination Induction & Removal of HP<br />
</p><p><br />
<br />
Group 4:Screening Strategy<br />
</p><p><br />
</div></div><br />
<a name="prt"></a><br />
<br />
<div class="content_block"><br />
<h1>Protocols</h1><br />
<br />
<p><br />
<br />
Here are our Experimental Protocols in PDF version. You're advised to view in <a href="http://www.adobe.com/acom/" target=blank>Acrobat</a> or <a href="http://get.adobe.com/reader/" target=blank>Adobe Reader</a> which you can download free from the<a href="http://www.adobe.com" target=blank> Home Website of Adobe® Company</a>, for other viewers we can not guarantee the right format of viewing. Those files are encrypted to keep copyrights, if you want to print them out of copy to your documents, to share with others our ideas and methods, you are welcome to ask us for the unencrypted files definitely for free~<br />
</p><br />
<br />
<h3>Molecular Cloning</h3><br />
<br />
<p><a href="https://static.igem.org/mediawiki/2010/0/0a/THUProtocol_1-1_Isolatio_of_plasmid_DNA.pdf" target=blank>THUProtocol_1-1_Isolatio_of_plasmid_DNA</a><br><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/d/d8/THUProtocol_1-2_DNA_Gel_Extraction.pdf" target=blank>THUProtocol_1-2_DNA_Gel_Extraction</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/5/59/THUProtocol_1-3_Restriction_Enzyme_Digestion.pdf" target=blank>THUProtocol_1-3_Restriction_Enzyme_Digestion</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/b/b7/THUProtocol_1-4_Ligation_of_DNA_Fragments.pdf" target=blank>THUProtocol_1-4_Ligation_of_DNA_Fragments</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c3/THUProtocol_1-5_PCR.pdf" target=blank>THUProtocol_1-5_PCR</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c0/THUProtocol_1-6_Preparation_of_Competent_Cell_for_Electro_Transformation.pdf" target=blank>THUProtocol_1-6_Preparation_of_Competent_Cell_for_Electro_Transformation</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/7/77/THUProtocol_1-7_Chemical_Transformation_of_Recombinant_DNA.pdf" target=blank>THUProtocol_1-7_Chemical_Transformation_of_Recombinant_DNA</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/4/40/THUProtocol_1-8_Electro_Transformation_of_Recombinant_DNA.pdf" target=blank>THUProtocol_1-8_Electro_Transformation_of_Recombinant_DNA</a><br />
</p><p><br />
<h3>Protein Isolation and Identification</h3><br />
<p><br />
<a href="https://static.igem.org/mediawiki/2010/f/f0/THUProtocol_2-1_Protein_Isolation_for_Prokaryotes.pdf" target=blank>THUProtocol_2-1_Protein_Isolation_for_Prokaryotes</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/0/08/THUProtocol_2-2_Protein_Identification_SDS-PAGE.pdf" target=blank>THUProtocol_2-2_Protein_Identification_SDS-PAGE</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c3/THUProtocol_1-5_PCR.pdf" target=blank>THUProtocol_1-5_PCR</a><br />
</p><br />
<br />
</div><br />
<br />
</div><br />
</div></div><br />
</body><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/experiments/dryTeam:Tsinghua/experiments/dry2010-10-27T17:35:47Z<p>Gumilton: </p>
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<h1>Experiment Records</h1><br />
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<p>Our Experiments were carried out by the following nine groups which are somewhat independent from each other. Here come the Groups and Their Tasks.</p><br />
<p><br />
Group 1A:Landing pad (LP) construction and Insertion with Tranditional Att Recomination method(Promoter Inside)<br/><br />
</p><p><br />
Group 1B:Landing pad (LP) construction and Insertion with Innovational Att Recomination method(Promoter Outside)<br/><br />
</p><p><br />
Group 1C:Landing pad (LP) construction and Insertion: Inducement and Recombination<br />
</p><p><br />
<br />
Group 2A:Donor Plasmid Construction: Two Fragments Construction (Three Fragments Ligation)<br />
</p><p><br />
<br />
Group 2B:Donor Plasmid Construction: Traditional Four Fragments Construction<br />
</p><p><br />
<br />
Group 2C:Donor Plasmid Construction: Four Fragments Construction with Dra III<br />
</p><p><br />
<br />
Group 2D:Donor Plasmid Construction: Four Fragments Construction with Novel Methods<br />
</p><p><br />
<br />
Group 3:Helper Plasmid (HP) Insertion, Donor Plasmid Insertion, Recombination Induction & Removal of HP<br />
</p><p><br />
<br />
Group 4:Screening Strategy<br />
</p><p><br />
</div></div><br />
<a name="prt"></a><br />
<br />
<div class="content_block"><br />
<h1>Protocols</h1><br />
<br />
<p><br />
<br />
Here are our Experimental Protocols in PDF version. You're advised to view in <a href="http://www.adobe.com/acom/" target=blank>Acrobat</a> or <a href="http://get.adobe.com/reader/" target=blank>Adobe Reader</a> which you can download free from the<a href="http://www.adobe.com" target=blank> Home Website of Adobe® Company</a>, for other viewers we can not guarantee the right format of viewing. Those files are encrypted to keep copyrights, if you want to print them out of copy to your documents, to share with others our ideas and methods, you are welcome to ask us for the unencrypted files definitely for free~<br />
</p><br />
<br />
<h3>Molecular Cloning</h3><br />
<br />
<p><a href="https://static.igem.org/mediawiki/2010/0/0a/THUProtocol_1-1_Isolatio_of_plasmid_DNA.pdf" target=blank>THUProtocol_1-1_Isolatio_of_plasmid_DNA</a><br><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/d/d8/THUProtocol_1-2_DNA_Gel_Extraction.pdf" target=blank>THUProtocol_1-2_DNA_Gel_Extraction</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/5/59/THUProtocol_1-3_Restriction_Enzyme_Digestion.pdf" target=blank>THUProtocol_1-3_Restriction_Enzyme_Digestion</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/b/b7/THUProtocol_1-4_Ligation_of_DNA_Fragments.pdf" target=blank>THUProtocol_1-4_Ligation_of_DNA_Fragments</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c3/THUProtocol_1-5_PCR.pdf" target=blank>THUProtocol_1-5_PCR</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c0/THUProtocol_1-6_Preparation_of_Competent_Cell_for_Electro_Transformation.pdf" target=blank>THUProtocol_1-6_Preparation_of_Competent_Cell_for_Electro_Transformation</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/7/77/THUProtocol_1-7_Chemical_Transformation_of_Recombinant_DNA.pdf" target=blank>THUProtocol_1-7_Chemical_Transformation_of_Recombinant_DNA</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/4/40/THUProtocol_1-8_Electro_Transformation_of_Recombinant_DNA.pdf" target=blank>THUProtocol_1-8_Electro_Transformation_of_Recombinant_DNA</a><br />
</p><p><br />
<h3>Protein Isolation and Identification</h3><br />
<p><br />
<a href="https://static.igem.org/mediawiki/2010/f/f0/THUProtocol_2-1_Protein_Isolation_for_Prokaryotes.pdf" target=blank>THUProtocol_2-1_Protein_Isolation_for_Prokaryotes</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/0/08/THUProtocol_2-2_Protein_Identification_SDS-PAGE.pdf" target=blank>THUProtocol_2-2_Protein_Identification_SDS-PAGE</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c3/THUProtocol_1-5_PCR.pdf" target=blank>THUProtocol_1-5_PCR</a><br />
</p><br />
<br />
</div><br />
<br />
</div><br />
</div></div><br />
</body><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/experiments/dryTeam:Tsinghua/experiments/dry2010-10-27T17:34:49Z<p>Gumilton: </p>
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<h1>Experiment Records</h1><br />
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|<br />
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<p>Our Experiments were carried out by the following nine groups which are somewhat independent from each other. Here come the Groups and Their Tasks.</p><br />
<p><br />
Group 1A:Landing pad (LP) construction and Insertion with Tranditional Att Recomination method(Promoter Inside)<br/><br />
</p><p><br />
Group 1B:Landing pad (LP) construction and Insertion with Innovational Att Recomination method(Promoter Outside)<br/><br />
</p><p><br />
Group 1C:Landing pad (LP) construction and Insertion: Inducement and Recombination<br />
</p><p><br />
<br />
Group 2A:Donor Plasmid Construction: Two Fragments Construction (Three Fragments Ligation)<br />
</p><p><br />
<br />
Group 2B:Donor Plasmid Construction: Traditional Four Fragments Construction<br />
</p><p><br />
<br />
Group 2C:Donor Plasmid Construction: Four Fragments Construction with Dra III<br />
</p><p><br />
<br />
Group 2D:Donor Plasmid Construction: Four Fragments Construction with Novel Methods<br />
</p><p><br />
<br />
Group 3:Helper Plasmid (HP) Insertion, Donor Plasmid Insertion, Recombination Induction & Removal of HP<br />
</p><p><br />
<br />
Group 4:Screening Strategy<br />
</p><p><br />
</div></div><br />
<a name="prt"></a><br />
<br />
<div class="content_block"><br />
<h1>Protocols</h1><br />
<br />
<p><br />
<br />
Here are our Experimental Protocols in PDF version. You're advised to view in <a href="http://www.adobe.com/acom/" target=blank>Acrobat</a> or <a href="http://get.adobe.com/reader/" target=blank>Adobe Reader</a> which you can download free from the<a href="http://www.adobe.com" target=blank> Home Website of Adobe® Company</a>, for other viewers we can not guarantee the right format of viewing. Those files are encrypted to keep copyrights, if you want to print them out of copy to your documents, to share with others our ideas and methods, you are welcome to ask us for the unencrypted files definitely for free~<br />
</p><br />
<br />
<h3>Molecular Cloning</h3><br />
<br />
<p><a href="https://static.igem.org/mediawiki/2010/0/0a/THUProtocol_1-1_Isolatio_of_plasmid_DNA.pdf" target=blank>THUProtocol_1-1_Isolatio_of_plasmid_DNA</a><br><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/d/d8/THUProtocol_1-2_DNA_Gel_Extraction.pdf" target=blank>THUProtocol_1-2_DNA_Gel_Extraction</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/5/59/THUProtocol_1-3_Restriction_Enzyme_Digestion.pdf" target=blank>THUProtocol_1-3_Restriction_Enzyme_Digestion</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/b/b7/THUProtocol_1-4_Ligation_of_DNA_Fragments.pdf" target=blank>THUProtocol_1-4_Ligation_of_DNA_Fragments</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c3/THUProtocol_1-5_PCR.pdf" target=blank>THUProtocol_1-5_PCR</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c0/THUProtocol_1-6_Preparation_of_Competent_Cell_for_Electro_Transformation.pdf" target=blank>THUProtocol_1-6_Preparation_of_Competent_Cell_for_Electro_Transformation</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/7/77/THUProtocol_1-7_Chemical_Transformation_of_Recombinant_DNA.pdf" target=blank>THUProtocol_1-7_Chemical_Transformation_of_Recombinant_DNA</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/4/40/THUProtocol_1-8_Electro_Transformation_of_Recombinant_DNA.pdf" target=blank>THUProtocol_1-8_Electro_Transformation_of_Recombinant_DNA</a><br />
</p><p><br />
<h3>Protein Isolation and Identification</h3><br />
<p><br />
<a href="https://static.igem.org/mediawiki/2010/f/f0/THUProtocol_2-1_Protein_Isolation_for_Prokaryotes.pdf" target=blank>THUProtocol_2-1_Protein_Isolation_for_Prokaryotes</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/0/08/THUProtocol_2-2_Protein_Identification_SDS-PAGE.pdf" target=blank>THUProtocol_2-2_Protein_Identification_SDS-PAGE</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c3/THUProtocol_1-5_PCR.pdf" target=blank>THUProtocol_1-5_PCR</a><br />
</p><br />
<br />
</div><br />
<br />
</div><br />
</div></div><br />
</body><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/experiments/dryTeam:Tsinghua/experiments/dry2010-10-27T17:32:00Z<p>Gumilton: New page: __NOTOC__ {{:Team:Tsinghua/header}}<html> <body id="bd_experiment"> <div id="content_box"> </html> {{:Team:Tsinghua/leftbar}} <html> <script> navlist= new Array("Records", "Protocols", "...</p>
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<h1>Experiment Records</h1><br />
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|{{#calendar: year=2010 | month=09 | title=Team:Tsinghua/Notebook}}<br />
|{{#calendar: year=2010 | month=10 | title=Team:Tsinghua/Notebook}}<br />
|<br />
|}<br />
<html><br />
<p>Our Experiments were carried out by the following nine groups which are somewhat independent from each other. Here come the Groups and Their Tasks.</p><br />
<p><br />
Group 1A:Landing pad (LP) construction and Insertion with Tranditional Att Recomination method(Promoter Inside)<br/><br />
</p><p><br />
Group 1B:Landing pad (LP) construction and Insertion with Innovational Att Recomination method(Promoter Outside)<br/><br />
</p><p><br />
Group 1C:Landing pad (LP) construction and Insertion: Inducement and Recombination<br />
</p><p><br />
<br />
Group 2A:Donor Plasmid Construction: Two Fragments Construction (Three Fragments Ligation)<br />
</p><p><br />
<br />
Group 2B:Donor Plasmid Construction: Traditional Four Fragments Construction<br />
</p><p><br />
<br />
Group 2C:Donor Plasmid Construction: Four Fragments Construction with Dra III<br />
</p><p><br />
<br />
Group 2D:Donor Plasmid Construction: Four Fragments Construction with Novel Methods<br />
</p><p><br />
<br />
Group 3:Helper Plasmid (HP) Insertion, Donor Plasmid Insertion, Recombination Induction & Removal of HP<br />
</p><p><br />
<br />
Group 4:Screening Strategy<br />
</p><p><br />
</div></div><br />
<a name="prt"></a><br />
<br />
<div class="content_block"><br />
<h1>Protocols</h1><br />
<br />
<p><br />
<br />
Here are our Experimental Protocols in PDF version. You're advised to view in <a href="http://www.adobe.com/acom/" target=blank>Acrobat</a> or <a href="http://get.adobe.com/reader/" target=blank>Adobe Reader</a> which you can download free from the<a href="http://www.adobe.com" target=blank> Home Website of Adobe® Company</a>, for other viewers we can not guarantee the right format of viewing. Those files are encrypted to keep copyrights, if you want to print them out of copy to your documents, to share with others our ideas and methods, you are welcome to ask us for the unencrypted files definitely for free~<br />
</p><br />
<br />
<h3>Molecular Cloning</h3><br />
<br />
<p><a href="https://static.igem.org/mediawiki/2010/0/0a/THUProtocol_1-1_Isolatio_of_plasmid_DNA.pdf" target=blank>THUProtocol_1-1_Isolatio_of_plasmid_DNA</a><br><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/d/d8/THUProtocol_1-2_DNA_Gel_Extraction.pdf" target=blank>THUProtocol_1-2_DNA_Gel_Extraction</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/5/59/THUProtocol_1-3_Restriction_Enzyme_Digestion.pdf" target=blank>THUProtocol_1-3_Restriction_Enzyme_Digestion</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/b/b7/THUProtocol_1-4_Ligation_of_DNA_Fragments.pdf" target=blank>THUProtocol_1-4_Ligation_of_DNA_Fragments</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c3/THUProtocol_1-5_PCR.pdf" target=blank>THUProtocol_1-5_PCR</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c0/THUProtocol_1-6_Preparation_of_Competent_Cell_for_Electro_Transformation.pdf" target=blank>THUProtocol_1-6_Preparation_of_Competent_Cell_for_Electro_Transformation</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/7/77/THUProtocol_1-7_Chemical_Transformation_of_Recombinant_DNA.pdf" target=blank>THUProtocol_1-7_Chemical_Transformation_of_Recombinant_DNA</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/4/40/THUProtocol_1-8_Electro_Transformation_of_Recombinant_DNA.pdf" target=blank>THUProtocol_1-8_Electro_Transformation_of_Recombinant_DNA</a><br />
</p><p><br />
<h3>Protein Isolation and Identification</h3><br />
<p><br />
<a href="https://static.igem.org/mediawiki/2010/f/f0/THUProtocol_2-1_Protein_Isolation_for_Prokaryotes.pdf" target=blank>THUProtocol_2-1_Protein_Isolation_for_Prokaryotes</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/0/08/THUProtocol_2-2_Protein_Identification_SDS-PAGE.pdf" target=blank>THUProtocol_2-2_Protein_Identification_SDS-PAGE</a><br />
</p><p><br />
<a href="https://static.igem.org/mediawiki/2010/c/c3/THUProtocol_1-5_PCR.pdf" target=blank>THUProtocol_1-5_PCR</a><br />
</p><br />
<br />
</div><br />
<br />
</div><br />
</div></div><br />
</body><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/HumanPracticeTeam:Tsinghua/HumanPractice2010-10-27T17:26:09Z<p>Gumilton: /* A Visit to Macquarie_iGEM */</p>
<hr />
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=Ethics=<br />
<html><div class="content_block"></html><br />
<br />
BioEthics regarding the Biological Ethics, discussing about biological issues, have been debated since ancient times. Bioethical issues gained widespread attention in recent years and are often extremely morally contentious. Considerable societal conflict exists regarding fundamental clinical issues such as: defining the beginning and end of life; appropriate standards of care; and broader environmental issues with potential social and health impacts such as growth of genetically-modified organisms and their use in the food supply.<br />
<br />
<br />
==BioEthics in Tsinghua iGEM==<br />
<br />
<br />
Here in Tsinghua iGEM 2010 Team, we are now focusing on some parts of BioEthics, such as animal handling requirement, care for animal welfare, appropriate standards of care, broader environmental issues with potential social and health impacts, impact of our products to be used in the manufacture and how to spread our ideas to make more people get this knowledge.<br />
<br />
Thus we, Tsinghua iGEM 2010, pay our attention specially to BioEthics and keep our promise by two ways. One is to teach every member about keeping healthy, safe and being environmentally friendly while conducting experiments. The other one is to try visit and talk with more people, discussing about our projects, the current biological issues and how to promote the environment where we are living as well as let more people know about and start to be interested in Synthetic Biology and iGEM.<br />
<br />
<html><br><br>&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a> </html><br />
<br />
<br />
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<br />
='''Safety'''=<br />
According to the WHO biosafety is the prevention of unintentional exposure to pathogens and toxins, or their accidental release, whereas biosecurity is the prevention of loss, theft, misuse, diversion or intentional release of pathogens and toxins. (From https://2010.igem.org/Safety)<br />
<br />
We are always paying special care to the safety of our teammates and the people around. Thus we signed a document to keep in rules about the Safety in Laboratory, not only to protect ourselves but also help to keep a clean and healthy environment.<br />
<br />
The model animals we used are harmless engineering strains of Escherichia coli. They are widely used in all kinds of laboratories around the world. We answered the questions about safety on the Safety Page from iGEM(https://2010.igem.org/Safety) as following:<br />
<br />
<br />
<br />
'''Q1: Would any of your project ideas raise safety issues in terms of:<br />
<br />
researcher safety,<br />
<br />
public safety, or<br />
<br />
environmental safety?'''<br />
<br />
A: Our project is about how to use E Coli to simulate the production of Specific Antibodies. No serious safety problem can be caused by the harmless engineering strains of ''Escherichia coli'' and we kept our promise and rules which ensure the safety during the whole process.<br />
<br />
'''Q2: Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'''<br />
<br />
A: No any potential safety issues.<br />
<br />
'''Q3:Is there a local biosafety group, committee, or review board at your institution?'''<br />
<br />
If yes, what does your local biosafety group think about your project?<br />
<br />
A: Yes, it is. The safety check is operated frequently. This is organised and operated by the ''Laboratories and Facilities'' in Tsinghua University. During our project, they operated one of the safety checks in one year and they evaluated our laboratory as well as our project. Our lab construction is keeping in line with the policies in Tsinghua University and because our project is about how to use E Coli to simulate the production of Specific Antibodies and the models we use are ''Escherichia coli'' so they evaluated our project is in a safe category and meanwhile they spoke highly on our Tsinghua iGEM 2010 Safety Manual for "It helps to keep safety in everyone's mind and do really good job.".<br />
<br />
'''Q4:Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?'''<br />
<br />
A: The most invigorative way to inspire the teams to make up ways to deal with and improve the BioSafety is to establish a special prize in BioSafety, so that many teams could try their best to deal with the issues in BioSafety, like how to take experiments safely, how to build safe BioBricks and mostly how maintain the safe environment during our projects.<br />
<br />
Also to recognize that importance from the bottom of one's heart is the most effective approach for dealing with some issues. So if more education, announcement, lectures, courses and many other kinds of teaching methods can be carried out to draw attention on the safety, to let people keep this in mind, the Safety Issues could no longer be an issue but a common sense.<br />
<br />
<br/><br />
<br />
All the staffs and students operating the infrastructures, devices and anything related with experiments are required to wear the gloves all the time while working. To keep these in rules and in case of any accidents we carried out a document to keep in line with.<br />
<br />
<html><a href="https://static.igem.org/mediawiki/igem.org/8/8f/Tsinghua_iGEM_2010_Safety_Manual.pdf" target=blank>Tsinghua iGEM 2010 Safety Manual</a></html><br />
<br />
Besides this there is also a document in Tsinghua University emphasizing on the common Laboratory Sfety. Here are the <html><a href="https://static.igem.org/mediawiki/2010/6/69/THUSAFETY.pdf" target=blank>Laboratory Safety Policy of Tsinghua University in Chinese Version</a><br />
and <a href="https://static.igem.org/mediawiki/2010/0/05/THULBM.pdf" target=blank>The Laboratory Biosafety Manual</a> released by WHO (World Health Organization) in which Tsinghua University keep align.<br />
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==Shanghai Meetup==<br />
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More Info at <a href="https://2010.igem.org/Events/China_meetup" target=_blank>2010 iGEM China meetup</a><br><br><br />
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This year, on August 5th, teams from universities in China, <br />
<a href="https://2010.igem.org/Team:SJTU-BioX-Shanghai" target=_blank>SJTU-BioX-Shanghai</a>,<br />
<a href="https://2010.igem.org/Team:Tsinghua" target=_blank>Tsinghua</a>,<br />
<a href="https://2010.igem.org/Team:Peking" target=_blank>Peking</a>, <br />
<a href="https://2010.igem.org/Team:ECUST-Shanghai" target=_blank>ECUST-Shanghai</a>, <br />
<a href="https://2010.igem.org/Team:USTC" target=_blank>USTC</a>, <br />
<a href="https://2010.igem.org/Team:USTC_Software" target=_blank>USTC_Software</a>, <br />
<a href="https://2010.igem.org/Team:ZJU-China" target=_blank>ZJU-China</a><br />
and Sun Yat-sen University all gathered in Shanghai and had a nice day at the iGEM 2010 China Meetup.<br />
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==Tsinghua University Lecture Day==<br />
[[Image:THULecturePost.jpg|400px]]<br />
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==Survey and Discussion==<br />
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==A Visit to Macquarie_iGEM==<br />
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Tsinghua and Macquarie iGEM Teams have an impressive relationship both in the cooperation of projects as well as in Human Practice.<br />
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[[Image:TSINGHUA&MAC1.JPG|500px]]<br />
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On 27/9/2010, GU Xiang, one of the team students in Tsinghua iGEM paid a visit to the Macquarie iGEM. GU Xiang, Yagiz Aksoy, the leader of Macquarie iGEM Team, and Hilal Varinli met together on the beautiful campus of the University of Macquarie, in Sydney. They firstly visited the Life Science Building in Macquarie, "It is amazing" said by GU Xiang. Then they three dropped in the Biology Museum in that building. Later GU Xiang together with Yagiz and Hilal did some of their experiments on that day. The research devices and environment in the labs of Macquarie are absolutely at world leading-class level. They started with biosafety and ethics topics related to the iGEM. They both recognized the biosafety to be the first important issue in the Biological Research and Education. Then they exchanged some ideas about how to improve and draw everyone's attention to this issue. The strict biosafety regulations in Macquarie are good examples for many institutes to learn about.<br />
<br />
[[Image:THUMAC.JPG|500px]]<br />
<br />
After that, they introduced and talked about each other's projects. Through that they found the project of Macquarie iGEM can be a potential screening method for the project of Tsinghua iGEM. This was the climax of that day when they found this potential cooperation between these two teams and they followed further discussion immediately. As the Macquarie team was to build up a system which can reflect the change by showing different lights. Thus this could be useful in the screening for the antibodies. They finally agreed on this proposition and decided to find more details to see how to make this come true if they both had enough time. They took the first step of their collaboration together by running that day’s experiments with GU Xiang at Macquarie iGEM Labs.<br />
<br />
Also hearing that Macquarie iGEM is a newly built team, GU Xiang generously gave his ideas and experience in iGEM to Yagiz and Hilal, promoting them to build better wiki to let more people know about their ideas. Finally they three expressed freely about the issues in Biology and Life Science as well as the Chinese Culture, especially the Kungfu. They had a wonderful day!<br />
<br />
The leader and all the teammates from Tsinghua iGEM were pleased with this visit and cooperation, they said they were willing to cooperate with Macquarie iGEM to build up and finish a BioSystem together, especially in such a contemporary society where cooperation is extremely of significance.<br />
<br />
We hope that every team in iGEM could find this opportunity to cooperate with an other team to build up a system other than just by only one team. In this cooperation we Tsinghua iGEM got one new way to screen our antibodies which we never heard about and Macquarie iGEM supplied their system with another crucial application and even more applications based on this idea. Thus if all the teams in iGEM can cooperate with at least another one, or even more teams, we may can build up a wonderful Genetically Engineered Machine, definitely it is an international one, thus iGEM!!!<br />
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==Module II==<br />
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===Strategy 1 '''Bacterial Based Microarray'''===<br />
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[[Image:TSModule2m.PNG|500px]]<br />
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<br/><br />
====Outline of screening desired antibody by bacterial based microarray====<br />
Microarray technique is a kind of pretty mature biomedical technology applied to diverse areas from pharmacology research to clinical use, such as genetics diagnose. Due to the advantage that large amount of various substances can be screened in microarray, microarray can be used for selection of desired antibodies with specific affinity in our project. However, current existing microarray technology rely on specific DNA or protein attached substrate and our project lean on bacteria expressing specific surface protein used for screening for desired antibody. Therefore, traditional protocols and materials used in microarray will be adjusted in our project. '''We believe that our adjustment will improve the efficiency of antibody screening and help our project achieve its ultimate goal.'''<br />
<br />
<br/><br />
<br />
====Specific description of the details of the experiments====<br />
In this part, we want to transform two batches of E coli with vectors carrying specific genes expressing various kinds of antibody achieved by the recombination methods in our project and specific antigen or some other protein used to select antibody from the aforementioned mix of antibodies. By fusion antibody and antigen gene with the genes of membrane integral displaying protein called OmpA, we can manage to display our protein to the surface of the bacteria. Therefore, the selection will process through interaction between antibody and antigen at the surface of the bacteria. Then, by linking the gene of display protein to the gene of one kind of protein called cellulose binding domain which can bind to cellulose, we are able to anchor the bacteria expressing CBD to the surface of the specific microarray coated with cellulose substrate. The whole process is illustrated in the following figure.<br />
[[Image:THUProjectFigure9.jpg|550px]]<br />
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<br />
====Information concerning the specific proteins====<br />
=====Cellulose binding domain=====<br />
[[Image:THUProjectFigure10.jpg|230px|left]]<br />
<br />
The protein used in this part comes from CipC gene found in Clostridium cellulolyticum. CipC gene encodes a protein called cohesion which participate in degradation of cellulose for the benefits of the bacteria Clostridium cellulolyticum. The protein encoded in this gene contains a specific domain called cellulose binding domain which bind to cellulose with pretty high binding affinity and thus facilitate the degradation of the cellulose. The structure of CBD has been resolved shown in the figure below. Our purpose is to utilize the ability of CBD to bind to cellulose and express CBD in E coli and thus force it to bind to microarray coated with cellulose substrate.<br />
<br />
<br><br />
<br />
=====Displaying protein OmpA=====<br />
At present, two kinds of membrane protein are known, that is, alpha helix protein from cytoplasmic membrane and beta-barrel protein in bacteria outer-membrane, exemplified by porin protein. There are two membrane layers in gram-negative bacteria, separately called inner membrane and outer membrane. OmpA is located in outer membrane and belongs to beta-barrel category. This protein is responsible for normal physiological functions in gene regulation in E coli. Because OmpA is located in outer membrane, intensive investigation has been conducted in OmpA because OmpA plays an important role in signal transduction and much work has focused on the mechanism how OmpA was transported from cytosol out of the membrane. Because of the clarified function of the protein, some bioengineers managed to utilize its function to display protein to achieve certain purposes. Therefore, we try to take advantage of the properties of OmpA to display protein, that is, to display CBD, antibody and antigen. The structure of OmpA has been determined, illustrated in the following figure.<br />
[[Image:THUProjectFigure11.jpg|300px]][[Image:THUProjectFigure12.jpg|300px]]<br />
<br />
Previous work has demonstrated that 46-159 amino acid is adequate to anchor OmpA to membrane and from the topology figure, we can find out that the fragment of AA 46-159 comprise the transmembrane part of the protein, as the red box indicates. Besides, to ensure the transport of OmpA from cytosol to cell membrane, we need to add a signal peptide to the N terminal of OmpA. Previous work found that one kind of lipoprotein in E coli contains a signal peptide consisting of nine amino acids, which ensures the successful transport of the protein outside the membrane. Mining through the parts provided by iGEM, we found one part which provides exactly the same sequence we wants. This part contains N terminal signal peptide and the sequence AA 46-159 and the linker region downstream of OmpA which ensures the folding of the attached domain, such as CBD. The engineered OmpA is illustrated as following.<br />
<br />
[[Image:THUProjectFigure13.jpg|500px]]<br />
<br/><br />
=====The selection of antibodies=====<br />
Due to commercialization of antibody, we have no access to the cDNA of these antibodies. Therefore, we managed to synthesize the cDNA.<br />
<br/><br />
=====Design of microarray=====<br />
Microarray is a solid substrate based 2 dimensinoal array, which can be utilized to detect large sum of bio-substance. After decade of developing, various kinds of microarrays have been invented, such as DNA microarray, protein microarray, tissue microarray and so on. Different microarrays can be used to detect different components based on different substrates. Our project belongs to cellular microarray, aiming to select different kinds of antibodies based on the interaction between different proteins displayed on bacteria membrane.<br />
This part of our project aims to attach CBD-expressing bacteria to the surface of microarray coated with cellulose. Weizmannn Institute has developed zephyrin-based microarray, in which the microarray plate is dotted with cellulose. Thus, this innovation provides potential for application in our project.<br />
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<html><a name="m2s2"></a></html><br />
===Strategy 2 '''ToxR-based Transmembrane Signaling Pathway Method'''===<br />
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<br />
[[Image:TSModule2s.PNG|600px]]<br />
<br />
====Propose of This Step====<br />
In order to select a specific antibody from a large number of antibodies, we studied the formation of mammalian antibodies. In the process of antibody production in mammals, mediated Annexin antibodies-mediated antibody activation play an important role. '''This section hopes to use a membrane receptor of E. coli to simulate this process, to achieve the purpose of screening.'''<br />
<br/><br />
====Principle and Background====<br />
To achieve this, a ToxR-based two-hybrid system was introduced into E.coli cells. The Vibrio cholerae transcriptional regulator ToxR is anchored in the cytoplasmic membrane by a single transmembrane segment, its C-terminal domain facing the periplasm. Most of its N-terminal cytoplasmic domain shares sequence similarity with the winged helix–turn–helix (wHTH) motif of OmpR-like transcriptional regulators. The ToxR protein of Vibrio cholerae regulates the expression of several virulence factors that play important roles in the pathogenesis of cholera.<br />
<br />
ToxR stimulates transcription from the cholera toxin gene promoter ctx by direct binding to DNA element ITITTGAT present in different isolates of Vcholerae in three to eight tandemly repeated copies upstream of the ctxAB structural genes.<br />
<br />
Transcription activation is thought to be initiated by environmental stimuli which cause the periplasmic ToxR domain to form a homodimer. This, in turn, tethers together the two cytoplasmic ToxR domains, which can now bind to the control region of the ctx promoter. A second membraneassociated protein, ToxS (Mr 19 000), is required for maximal activation of the ctx promoter; most plausibly it stabilizes the ToxR dimer by direct contact.<br />
<br />
[[Image:THUProjectFigure14.jpg|600px]]<br />
<br />
'''Figure Shows the Original structure of ToxR system in Vibrio cholerae'''<br />
<br />
Fusion protein was made between the inner- and trans-membrane parts of ToxR and of the recombinant antibody.<br />
<br><br><br><br />
[[Image:THUProjectFigure15.jpg|80px|left]]<br />
<br />
When the specific antigens recognized by antibodies, antibodies with the same antigen recognize site will be close to each other through the antigen,inner-membrane part of “Antibody-ToxR” Protein dimerize, ctx promoter active, Resistance gene starts to be transcribed.<br />
<br><br><br><br><br />
[[Image:THUProjectFigure16.jpg|600px]]<br />
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Use “Anti-Histag Antibody” and “Protein with His-tag” as “antibody” and “antigen” to verify the effect of this system.<br />
<br />
Antibody: ToxR + Anti-Histag Antibody scFv Light Chain<br />
<br />
Plus: ToxR + Anti-Histag Antibody scFv Heavy Chain<br />
<br />
Antigen: His tag + Protein A + Histag<br />
<br />
===Protocol===<br />
<br />
We need to achieve the construction of the selection vector under the control of ctx promoter, then induce to the bacteria.<br />
Measure the expression of the two genes and measure the distribution in membrane.<br />
Use the exogenous protein carried His-tag as the antigen.<br />
Treat with antibiotics.<br />
Compare the expression level between the experiment group and the control, then define the effect of our system indirectly.<br />
<br />
Reference:A ToxR-based two-hybrid system for the detection of periplasmic and cytoplasmic protein–protein interactions in Escherichia coli: minimal requirements for specific DNA binding and transcriptional activation<br />
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===Strategy 3===<br />
Cooperation with <html><a href="https://2010.igem.org/Team:Macquarie_Australia" target=blank><font face="Comic Sans MS" size="4">Macquarie Australia</font></a><br />
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<h1>Conclusion</h1><br />
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==Comparison between natural antibody production and E. Coli system==<br />
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===VDJ Recombination vs Lading Pad Recombination===<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is the mechanism by which immunoglobulin (Ig) and T cell receptors (TCR) are generated in the immune system. Through V(D)J recombination, Variable, Diverse, and Joining gene segments are randomly combined, thus encoding various kinds of immunoglobins and T cell receptors to recognize potential antigens. <br />
During the process of B cell maturation, gene segments are rearranged and thus transcribe mRNA that can produce diverse types of antibody for recognition of billions of potential epitopes. The rearrangement brings three gene segments (termed V, D, and J) in close proximity, which are then joined together head to tail, the process of which is known as V(D)J recombination based on the V,D,J gene segments involved. Multiple copies of the V, D, and J genes in the human genome are shuffled in the process, and a specific antibody will be generate among millions of other possible combinations. Because gene are joined together permanently, one mature B cell will produce only one specific antibody.<br />
<br />
Our system is aimed to mimic this recombination process, using E.coli as the carrier.<br />
Through previous research in immunology field, each kind of gene segments (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and recombination occurs when VDJ recombinase recognize the specific signal sequence and mediate the recombination process. In our project, we design a sequence, which carries separated parts of one complete gene and integrate synthesized DNA fragment into the genome of E coli via landing pad method. Then we use I-Sel enzyme to cut the gene segments out of E coli genome, into which gene segments are inserted and choose RecA enzyme to recognize the previously designed signal sequence flanking our target sequence and recombine the gene segments, just like what recombinase accomplish in B cells. The whole process is called landing pad recombination.<br />
<br />
===Somatic hyper-mutation vs Junctional mutation===<br />
<br />
Somatic hypermutation involves a programmed process of mutation affecting the variable regions of immunoglobulin genes. During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is at least 105-106 fold greater than the normal rate of mutation across the genome. Variation is mainly in the form of single base substitutions, with insertions and deletions being less common. These mutations occur mostly at “hotspots” in the DNA, known as hypervariable regions. These regions correspond to the complementarity determining regions; the sites involved in antigen recognition on the immunoglobulin. This directed hypermutation allows for the selection of B cells that express immunoglobulin receptors possessing an enhanced ability to recognize and bind a specific foreign antigen.<br />
<br />
In addition, the cross ligation, namely Landing pad recombination, has higher opportunity to mutate in the link sequence. The introduction of 4 junctional shuffler 64 will meet the need of the rest 107 diversity, perfectly undertaking the work in the antibody diversity construction that hyper-mutation does in the highly variable region. In this way, we have finished a building library, waiting for the specialized antigen to select.<br />
<br />
===Antigen-specific Selection vs CBD-Based Microarray and ToxR-Based Transmembrane pathway method===<br />
In mammalian immune system, billions of antibodies will be generated. Then how can the immune system find out specific antibody that indiscriminately recognize the specific antigen that comes in. Generally speaking, due to the fact that one mature B cell only generates one specific immunoglobin, once the membrane integral immunoglobin bind to the specific antigen, a series of intracellular response, such as gene activation and cell differetiation will be triggered in B cell. Then, B cells expressing the specific antibodies that recognize the specific antigen will proliferate and generate enormous amount of antibodies that help eliminate the specific antigen.<br />
<br />
Simply put, antibody selection is accomplished through the specific interaction between the membrane immunoglobin and the specific antigen. Therefore, in our project, we also plan to mimic this process through interaction between the antibodies generated through landing pad recombination and the antigen we target. CBD-based Microarray and ToxR-based Transmembrane pathway are the two methods by which we achieve this goal.<br />
CBD domain, which interacts with cellulose, potentially provides us way to locate our E.coli to the microarray coated with cellulose. OmpA (outer membrane protein A), displays the antibody and antigen out to the membrane surface. Then by applying the antibodies generated through landing pad recombination to the microarray, antibodies that bind to the antigen will be retained and thus selected out of the pool of antibodies.<br />
<br />
Alternatively, the ToxR-based transmembrane pathway method shows a different strategy for us to seek the right folded antibody. Once the antigen comes, ToxR outer part will form dimer shape and the downstream signal occur, supplying the approach of reporters, such as GFP, to examine and pick out what we want.<br />
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<h1>Background</h1><br />
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<img src="/wiki/images/d/d8/Usa.jpg" /><br />
</html>Antibodies are a kind of magic substance.<br />
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Antibody-related research topics can be divided into two directions:<br />
<br />
Firstly, in the natural science area, studying the antibody production mechanism in mammals is central. The core issue is how B-cells can produce numerous antibodies using limited gene fragments in the<br />
chromosome. Consequently, when required, producing plenty of proper antibodies to respond to the changing external environment.<br />
<br />
Secondly, in the engineering field, the main goal lies in developing an antibody production technology. The core question here is how to develop an economical and effective method to manufacture various highly<br />
specific antibodies.<br />
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We believe that the two are essentially uniform.<br />
<br />
<br />
antibody generation. Thanks to the simple and easy-to-industrialize nature of prokaryotic systems, such an antibody generation system, once established, will facilitate the cheap and efficient production<br />
of antibodies.<br />
<br />
Production of antibodies in the mammalian immune system involves two steps:<br />
<br />
-Random production of a large numbers of antibodies<br />
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-Selection of a specific antibody matching the antigen.<br />
<br />
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Therefore, our Antibody Generation System would be composed of two devices:<br />
<br />
<html><h3>Module I: Generation of antibody library.</h3></html><br />
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Using an efficient bacterial in-vivo recombination method, we simulate the complex restructuring process during the development of immune B-cells with the simple recombination during the proliferation of the<br />
Escherichia coli bacteria, with the purpose of using a single strain of bacteria to generate an antibody library. <br />
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In our project we also use mathematical modeling as a tool, to prove the feasibility of this system in theory. /to prove the theoretical feasibility of this model.<br />
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<html><h3>Module II: Selection of specific antibodies</h3></html><br />
<br />
For antibody screening, the immune system has evolved a smart way to pick out the antibodies it needs. However, we need a simple way to industrialize the selection of antibodies independent of the eukaryotic<br />
system. Our antibody generation system will try to combine the advantages of both the mammalian immune system and the prokaryotic reproduction system.Several strategies have been developed to achieve<br />
these goal:<br />
<br />
1. Using a combination of proteins on the E.coli membrane to simulate the recognition of antigen and antibody, and use the signaling mechanism of prokaryotic cells to transfer the correct<br />
recognition to the downstream signal.<br />
<br />
2. Using the extracellular presenting system of the Prokaryotes to change the antibodies screening to the simple "Filter" screening.<br />
<br />
3. Using the interaction and combination system of the Prokaryotes to change the antibodies screening to the simple antibiotic resistence screening.<br />
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<img src="/wiki/images/f/fc/China.gif" /></html>抗体是一类神奇的物质。<br />
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与抗体有关的科学问题主要分为两个方向:<br />
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一是在自然科学领域,研究哺乳动物体内抗体的产生机制及其作用机理,其核心问题是,免疫系统如何利用有限的基因片段产生理论上无限多种抗体,并在特定情况下大量产生适当抗体,从而应对不断变化的外界环境。<br />
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二是在工程学领域,研究抗体生产技术,其核心问题是:能否发展一套迅速、便宜生产各种高特异性抗体的系统。<br />
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而本质上,这两个方向却是统一的。<br />
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根据合成生物学的思想,清华大学iGEM10项目希望以原核生物为基础,构建一个完全模拟免疫系统'''抗体生成过程的人造系统''',通过该系统,研究抗体形成的有关问题;而由于原核生物本身的特点,这个系统同时具有了抗体工业化生产所必需的高效、便宜的特性。实现了科学与工程的完美统一!<br />
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免疫系统的抗体生成过程包括两个步骤:<br />
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-随机产生大量多样的抗体<br />
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-对某种特定抗体进行筛选<br />
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因此,我们的系统由两部分组成:<br />
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<html><h3>模块 I: 抗体库的构建</h3></html><br />
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使用一种高效的大肠杆菌体内重组(in-vivo recombination)方法,使细菌在增殖过程中的重组来模拟免疫B细胞发育过程中复杂的重组过程,从而达到使用单一菌株简单构建抗体库的目的。<br />
同时,利用数学建模工具在理论上印证此系统能产生的抗体数目,可达到与免疫系统相当的数量级。<br />
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<html><h3>模块 II: 特异性抗体的筛选</h3></html><br />
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在抗体筛选中,免疫系统所使用的方法显然要简单、高效的多。而脱离开复杂生物系统的工业生产中的筛选方法,则更易于操作。在我们的系统中,这两个筛选策略的优势得以结合。几种基于不同思路的方法被开发出来,以实现这些目的。目前主要包括:<br />
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1、 应用原核生物跨膜信号系统模拟免疫系统膜联抗体与抗原结合后所启动的应答机制,从而将抗体筛选转化成下游简单的营养型筛选。<br />
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2、应用原核生物胞外呈递系统将抗体筛选转化成简单的“过滤”筛选。<br />
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3、应用原核生物结合系统将抗体筛选转化成抗性筛选。<br />
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</html>Antikörper sind eine Art magische Substanz<br />
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Forschung in Antikörpern kann in zwei Gebiete eingeteilt werden:<br />
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Erstens, im naturwissenschaftlichen Bereich, ist das Studium der Produktionsmechanismen von Antikörpern in Säugetieren zentral. Der Kernpunkt hier ist die Frage wie B-Zellen unzählige Antikörper aus einer begrenzten Menge Genfragmente im Chromosom herstellen könne. Infolgedessen können sie mit eine Vielzahl korrekter Antikörper auf Änderungen in ihrer Umwelt reagieren.<br />
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Zweitens, im Ingenieurbereich, ist es das Ziel eine Technologie zu entwickeln zur Antikörperproduktion. Die zentrale Frage hier ist wie man eine kostengünstige und effektive Methode zur Produktion von hoch spezifischen Antikörpern entwickeln kann. <br />
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Wir glauben das beides im wesentlichen uniform ist.<br />
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Inspiriert vom Konzept der synthetischen Biologie ist das Tsinghua iGEM 2010 Projekt auf die Entwicklung eines Systems zur künstlichen Herstellung von Antikörpern (Artificial Antibody Generation System) mit Hilfe von prokaryotischen Zellen, die einen neuen Weg zur Lösung des Problems anbieten, fokussiert. Dank der simplen und leicht zu industrialisierenden Natur des prokaryotischem Systems wird ein solches antikörperproduzierendes System, sobald es etabliert ist, die kostengünstige und effiziente Produktion von Antikörpern möglich mache, <br />
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Die Produktion von Antikörpern im Immunsystem von Säugetieren besteht aus zwei Schritte:<br />
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-Die Produktion einer grossen Anzahl zufälliger Antikörper <br />
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-Die Selektion der gewünschten Antikörper die zum entsprechendem Antigen passen<br />
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Aus diesem Grund besteht unser Antikörper produzierendes System aus zwei Komponenten: <br />
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<html><h3>Modul I: Erstellung einer Antikörper-Bibliothek</h3></html><br />
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Mit Hilfe einer effizienten bakteriellen in-vivo Rekombinationsmethode simulieren wir den komplizierten Umstrukturierungsprozess, der während er Entwicklung von B-Zellen im Immunsystem stattfindet, mit der simplen Rekombination von Bakterien, Escherichia coli, die während der Vermehrung statt findet. Um dieses Ziel zu erreichen wir ein einziger Bakterienstamm zur Herstellung der Antikörper-Bibliothek verwendet. <br />
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In unserem Projekt verwenden wir auch mathematische Modellierung als ein Werkzeug um das System theoretisch zu beweisen. <br />
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<html><h3>Modul II: Selektion eines spezifischenAntikörpers</h3></html><br />
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In der Antikörperselektion hat das Immunsystem einen intelligenten Weg entwickelt um die Antikörper die es braucht auszuwählen. Allerdings brauchen wir eine einfachen Weise um die Selektion von Antikörpern, unabhängig vom eukaryotischen System, zu industrialisieren. Unser Antikörperherstellungssystem wird versuchen die Vorteile vom Immunsystem der Säugetieren mit denen des Reproduktionssystem der Prokaryoten zu vereinen. Mehrere Strategien sind entwickelt worden um dieses Ziel zu erreichen:<br />
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1. Durch den Gebrauch einer Kombination von Proteinen auf der E. Coli Membran wird der Erkennungsmechanismus zwischen Antigen und Antikörper simuliert und der Signalisierungsmechanismus innerhalb der prokaryotischen Zellen wird gebraucht um die korrekte Erkennung zum nachgeschaltetem Signal weiterzugeben。<br />
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2. Durch den Gebrauch des extrazellulären Präsentiersystems der Prokaryoten, wird das Antikörper-Screening zum einfachen "Filter"-Screening.<br />
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3. Durch den Gebrauch des Interaktions- und Kombinationssystems der Prokaryoten wird das Antikörper-Screening zum simplen Antikörper-Resistenz-Screening.<br />
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</html>Anticuerpo es una especie de sustancia mágica.<br />
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Los temas de investigación relacionados con el anticuerpo se puede dividir en dos áreas:<br />
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En primer lugar, en el área de las ciencias naturales, estudiando el mecanismo de producción de anticuerpos en los mamíferos. El tema central es cómo las células B producen numerosos anticuerpos utilizando limitados fragmentos del gen en el cromosoma. En consecuencia, en cierta ocasión, producen gran cantidad de anticuerpos adecuados para responder a los cambios del medio ambiente.<br />
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En segundo lugar, en el campo de la ingeniería, desarrollando una tecnología de producción de anticuerpos. El tema central es cómo podemos desarrollar un método económico y eficaz para la fabricación de diversos específicos anticuerpos.<br />
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Creemos que los dos son esencialmente uniformes.<br />
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Inspirado en el concepto de la biología sintética, Tsinghua iGEM 2010, el proyecto se centra en el desarrollo Sistema de Generación artificial de un anticuerpo dentro de las células procariotas para proporcionar una nueva forma para resolver los problemas sobre la generación de anticuerpos. Gracias a la simpleza y facilidad para ser industrializado el sistema procariótico, como un sistema de generación de anticuerpos, una vez establecido, facilitará la producción económica y eficiente de los anticuerpos.<br />
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La producción de anticuerpos en el sistema inmune de los mamíferos consta de dos etapas:<br />
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-Producir un gran número de anticuerpos al azar<br />
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-Seleccionar de los específicos de acuerdo con el antígeno.<br />
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Por lo tanto, nuestro Sistema de Generación de anticuerpos sería también compuesto de dos dispositivos:<br />
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<html><h3>Módulo I: La generación de la colección de anticuerpos</h3></html><br />
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Utilizando un método de recombinación eficiente bacteriana in vivo, se simula el complejo proceso de reestructuración durante el desarrollo de las células B inmunes con la recombinación simple durante la proliferación de bacterias Escherichia coli, para lograr el propósito de utilizar una sola cepa de bacterias para generar una colección de anticuerpos.<br />
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Mientras tanto, este proyecto utiliza las herramientas de modelación matemática para demostrar la viabilidad de este sistema en la teoría.<br />
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<html><h3>Módulo II: Selección de anticuerpos específicos</h3></html><br />
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En la detección de anticuerpos, el sistema inmune se desarrolla de una manera inteligente para seleccionar el anticuerpo que necesita. Sin embargo, se necesita una manera simple de industrializar la selección de anticuerpos independiente del sistema de eucariotas. Nuestro sistema de generación de anticuerpos tratará de combinar las ventajas de ambos sistemas: sistema inmune de mamíferos y el sistema de reproducción de procariotas. Varias estrategias se desarrollan para alcanzar tal objetivo:<br />
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1. Usar la combinación de proteínas en la membrana de E. coli para simular el reconocimiento entre el antígeno y el anticuerpo, y utilizar el mecanismo de señalización dentro de las células procariotas para transferir el reconocimiento correcto en la señal de corriente.<br />
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2. Utilizar el sistema extracelular que presenta los procariotas para cambiar la detección de anticuerpos a la simple "filtro" de selección.<br />
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3. Utilizar el sistema de interacción y combinación de los procariotas para cambiar los anticuerpos de detección para la proyección simple de resistencia a los antibióticos.<br />
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</html>Антитело—волшебное вещество.<br />
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А научная проблема ,которая связывается с антителом ,делится два направления:<br />
Во-первых, по естественной науке , изучают продукцию и механизм антитела в млекопитающем. И на этой стороне, вопрос сути обсуждения --- как В-клетка может использовать лимитированные фрагменты генов выработать многочисленные антитела в хромосомах.Следовательно,в требуемом моменте, можно выроботать множество надлежащего антитела, которое может адаптироваться заменяющую внешнюю среду. <br />
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Во-вторых, по инженерной области , изучают технику , вырабатывающую антителою. А вопрос сути обсуждения ---можно или нет развить и экономный действующий метод , который изготовит разные своеоразные антитела. И мы верим , что по существу это две стороны единообразны . Благодаря синтетической биологии , проект Университета Цинхуа (iGEM10) хочет использовать прокариоты установить искусственные системы, которые совсем имтируют процесс генерации антитела иммунной системы. Благодаря этой системе , нам можно изучать волросы о генерировании антитела . А так как у прокариотовесть свои особенности, в то же время у этой системы эффективные и дешёвые особенности , которые нужны промышленному производстве антител.<br />
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Процесс генерации антитело иммунной системы существует две ступени : <br />
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---Произвольно выдаётся множество разных антител.<br />
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---Выбирают некоторые своеобразные антитела. <br />
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Для этого, наша система составится из двух частей :<br />
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'''МодульⅠ:выработка сбор антител'''<br />
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По эффективному метод , который использует рекомбинацию кишечной палочки. Потом бактерии рекомбинируют ,когда бактерии пролиферируют , и нам можно дальше имитировать перестройку иммунных В-клеток в своём развитии. Потому и мы сделаем с целью выработать сбор антител единственным видом бактерии.<br />
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В то же время , в нашем проекте , по моделированию математики , докажем осуществимость этого проекта.<br />
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'''МодульⅡ: Выбор специфических антител'''<br />
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Для скрининга антител, способ выделить антитела иммунной системы Более простой и более эффективной . Тем не менее, мы должны простой способ индустриализации выбор антител зависит от эукариотических системы. Наша система антител поколения будет пытаться совместить преимущества обоих млекопитающих иммунной системы и прокариотических системы воспроизводства. Некоторые стратегии были разработаны для достижения этих целей:<br />
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1. Используя сочетание белков на мембраны E.coli для имитации признание антигена и антитела, и использовать механизм сигнализации прокариотических клетках для передачи правильного распознавания на выходе сигнал.<br />
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2. Использование внеклеточных представления системы Прокариоты изменить антител отбора для простых "Фильтр" скрининга.<br />
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3. Использование взаимодействие и комбинация системы Прокариоты изменить антител отбора для простых антибиотиков скрининга сопротивления.<br />
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</html>抗体とは、不思議な物質である。抗体に関する科学問題には二つの種類がある。<br />
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一つは自然科学の領域で、哺乳動物の体の中の抗体の発生体制およびその原理である。その中核の問題はどのようにして、限られた遺伝子の一部を利用し、無限のさまざまな抗体を作るのか、そして、変わりつつある外界の環境を応対するため、どうやって特定な場合にたくさんの適当な抗体を発生させるのか、ということである。もう一つは工程学の領域で、抗体を作成する技術を研究することである。その中核の問題は、さまざまな高特異抗体をすばやく、安く作成できるかどうかということである。本質的に言えば、この二つの種類は一緒である。合成生物学の思想により、原核生物を基づき、清華大学iGEM10プロジェクトは免疫システム抗体の発生のプロセスを全て模擬する人口的なシステムを構成しようとしている。このシステムを通じて、抗体発生に関する問題を研究できるし、抗体工業化生産には必要な効率的と安い特性も持っていて、科学と工程の結びつきを実現した!<br />
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免疫システムの抗体発生のプロセスには二つの段取りがある:<br />
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ランダムに多種多様な抗体を作成する;<br />
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ある特定抗体を選択する。<br />
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したがって、われわれのシステムが二つの部分からなっている:<br />
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<html><h3>モジュール1:抗体庫の構成</h3></html><br />
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効果がよい大腸菌体内リーコンビネーションを使い、増殖する際に細菌を再編させ、免疫B細胞の発育中の複雑なプロセスをまね、それで単一菌株を使い抗体庫を構成する目的を達する。それと同時に、数学の建模道具で論理的にこのシステムでできる抗体の数を裏付ける。<br />
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<html><h3>モジュール2:特異性抗体の選別</h3></html><br />
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抗体の選別の際、免疫システムで使われる方法のほうが明らかにやさしく効果がよい。また、複雑な生物システムの工業生産脱の選別方法がなおさら操作しやすい。私たちのシステムには、これら二つの選別策略が結ばれた。異なる思想に基づくいくつかの方法が開発され、実現された。今の段階では、次のようなものがる。<br />
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1.原核生物跨膜信号システム模擬免疫システム模联抗体が抗体と結ばれた後起動された応対機制を利用し、抗体を簡単な栄養形選別に転換される;<br />
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2.原核生物胞外呈递システムを利用し、抗体を簡単な濾過選別に転換させる。<br />
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3.原核生物結合システムを利用し、抗体を抗性選別に転換させる。<br />
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</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/project/conTeam:Tsinghua/project/con2010-10-27T16:42:52Z<p>Gumilton: </p>
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<h1>Outline</h1><br />
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In order to realize the production of antibodies in prokaryotic cells, we firstly investigate the whole process of antibody production in mammalian immune system and try to simulate the whole process via the methods and concepts of synthetic biology. Finally, we successfully constructed the desired system. The comparison between the key steps involved in antibody generation and corresponding synthetic methods are shown in the chart below.<br />
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<table border="2" bordercolor="maroon" bgcolor="silver"><br />
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<th colspan=3>Antibody Production</th><br />
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<th colspan=2>E Coli. Production System</th></tr><br />
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<td rowspan=4 width=70px><a href="#VDJ_Recombination_vs_Lading_Pad_Recombination">VDJ Recombination</a></td><br />
<td rowspan=3 width=70px>Preparation</td><br />
<td width=70px>RSS Sequence</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">Landing Pad Insertion</a></td><br />
<td rowspan=4 width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1">Module I: Recombination System</a></td><br />
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<td>VDJ Recombinase</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">Helper Plasmid Insertion</a></td><br />
</tr><br />
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<td>VDJ Library</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">Donor Plasmid Construction</a></td><br />
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<tr><br />
<td>Recombination</td><br />
<td colspan=2><img src="https://static.igem.org/mediawiki/2010/d/d6/THUarrow2.png" width=180></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">DP Insertion and Recombination Induction</a></td><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="#Somatic_hyper-mutation_vs_Junctional_mutation">Somatic Hypermutation</a></td><br />
<td rowspan=2 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
<!---------<td>Junctional Diversity</td>----------------------><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">CBD-based Microarray</a></td><br />
<td rowspan=4><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2">Module II: Selection System</a></td><br />
</tr><br />
<tr><br />
<td rowspan=3><a href="#Antigen-specific_Selection_vs_CBD-Based_Microarray_and_ToxR-Based_Transmembrane_pathway_method">Antigen Selection</a></td><br />
<td rowspan=3 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">ToxR-based Transmembrane Pathway</a></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">Cooperation With Macquarie_Australia</a></td><br />
</tr><br />
</tbody><br />
</table><br />
</html><br />
<br/><br />
Generally speaking, antibody production in our project can be divided into two modules.<br />
<br />
<html><br />
<body bgcolor="#336699" text="#ffffff" link="#60a179"><br />
<br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1"><font face="Comic Sans MS" size="4">Module I: Generation of antibody library</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2"><font face="Comic Sans MS" size="4">Module II: Selection of specific antibodies</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
</html><br />
<br />
==Project Design==<br />
<br />
<br />
<br />
===Module 1===<br />
<br />
'''Antibodies Library Diversity & Randomicity'''<br />
[[Image:TSModule1.PNG|650px]]<br />
<br />
<br />
The first module is carried out by a novel method called E coli in vivo recombination system. The theoretical and experimental basis comes from the work of Thomas E. Kuhlman and Edward C. Cox. We managed to modify their system, wishing to utilize it for simulation of antibody recombination.<br />
<br />
Firstly, we try to insert a DNA segment called”Landing Pad(LP)” into the specific sites of E coli via Att recombination. Landing pad sequence consists of the following parts(from 5’ to 3’ in the sequence):<br />
<br />
1)25bp-long random sequence<br />
<br />
2)15bp-long recognition sequence of restriction enzyme I-scel<br />
<br />
3) antibiotic resistance gene used for antibody selection<br />
<br />
4) 15bp-long recognition sequence of restriction enzyme I-scel(corresponding to 2)<br />
<br />
5) 25bp-long random sequence (corresponding to 1)<br />
<br />
After integrating DNA segment of Landing pad into the genome of E coli, we completed the genetic engineering of the genome of E coli. All the five parts of landing pad are designed for subsequent recombination. <br />
<br />
Besides, in order to achieve different recombination goals, we designed several landing pads of different sequences.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion"><font face="Comic Sans MS" size=3>'Landing Pad Construction and Insertion'</font></a></html> to learn about the details of Landing Pad design. <br />
<br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#att"><font face="Comic Sans MS" size=3>'link'</font></a></html> to learn more about ATT recombination.<br />
<br />
<br />
After the integration of landing pad, we have to insert another DNA fragment called Helper Plasmid(HP), which provides necessary tools for in vivo recombination. <br />
<br />
In this plasmid(Helper Plasmid), there are two genes used for recombination. <br />
<br />
The first one encodes restriction enzyme I-scel after induction of L-arabinose. I-scel is able to recognize the 15bp-length sequence flanking a target DNA fragment and cut out the fragment, therefore providing DNA segments for recombination mediated by other components.<br />
<br />
The second one encodes the enzyme responsible for recombination called Lamda-Red. With the presence of IPTG, Lamda-Red will recombine DNA fragments flanked by the same DNA sequence recognized by Lamda-Red.<br />
<br />
In addition, Helper Plasmid contains a specific temperature-sensitive replicon, which is used to control the replication via temperature changes. We can remove the replicon when necessary.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi"><font face="Comic Sans MS" size=3>'HP insertion'</font></a></html> to learn more about Helper Plasmid.<br />
<br />
<br />
Up till now, we successfully engineered a ‘recepient strain’. Then we have to transform the ‘recepient strain’ with the Donor Plasmid(DP) containing recombination fragments. Donor Plasmid contains several Insertion Fragments and each Insertion Fragment contains following five parts:<br />
<br />
1) one 15bp-long restriction enzyme I-scel recognition site<br />
<br />
2) one 25bp-long random sequence<br />
<br />
3) one fragment for insertion and recombination<br />
<br />
4) one 25bp-long random sequence<br />
<br />
5) one 15bp-long restriction enzyme I-scel recognition site(in correspondence with 1)<br />
<br />
(Note: the order of the five parts differ from that of landing pad)<br />
<br />
As we previously mentioned, Donor plasmid contains several Insertion Fragments. Based on the flanking landing pad sequence, we can ascribe Insertion Fragments to the same group as long as the flanking landing pad sequences of those Insertion Fragments are the same.<br />
<br />
In order to achieve different goals, we design different Donor Plasmids, different Donor Plasmids contain different number of Insertion Fragments, which belong to different groups.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc"><font face="Comic Sans MS" size=3>‘DP construction’</font></a></html> to learn about the details of Donor Plasmid and Insertion Fragments and the strategies employed in our design. <br />
<br />
<br />
As we all know, antibody diversity mainly lean on the number of the fragments used for recombination. Therefore, in order to mimic the antibody generation process, we need to construct Donor Plasmid which contains large sum of Insertion Fragments. Theoretically, we can construct Donor Plasmid of this kind. However, due to the time limit of the competition, we didn’t try to construct DP containing many Insertion Fragments.<br />
<br />
In spite of this, in order to meet the needs, we designed two methods to construct plasmids on a large scale and attempted the usage in parts of our Donor Plasmids.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Strategies"><font face="Comic Sans MS" size=3>'DP construction and Strategies'</font></a></html> for the methods of rapid plasmid construction. <br />
<br />
<br />
Besides, we have applied for two BBFRFC, which are BBFRFC61 and BBFRFC 62 respectively.<br />
<br />
Then, after transformation of the prepared ‘recepient strain’ with Donor Plasmid and the induction of corresponding agents, I-scel enzyme cuts out DNA fragments from DP and LP, generating numerous Insertion Fragments. Besides, antibiotic resistance gene integrated into E coli genome is also cut out, providing recombination sites for Insertion Fragments.<br />
<br />
Provided with Insertion Fragments mentioned above and Lamda-Red expressed by Donor Plasmid, Insertion Fragments recombine with the corresponding landing pad sequence in the genome and thus get inserted into the correct site of the genome. Based on our design of Landing pad, we can ensure that only one Insertion Fragment from one group is randomly inserted into the genome, therefore mimicking the VDJ recombination process.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri"><font face="Comic Sans MS" size=3>‘DP Insertion and Recombination Induction’</font></a></html> to learn more about this part. <br />
<br />
<br />
Engineered E coli cells which have undergone successful recombination can be selected out of the pool through specific screening methods. After tests on different strains, our project achieve a recombination rate above 50% based on our current techniques.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/experiments#res"><font face="Comic Sans MS" size=3>‘Result’</font></a></html> to learn more about the identification of recombination rate. <br />
<br />
<br/><br/><br />
<br />
<br />
<br />
<br />
===Module 2===<br />
<br />
The goal of the second module in our project is aimed at selection of specific antibodies. Due to the maturity of existing antibody screening technique, our work mainly focuses on imitation of mammalian antibody selection, thus providing possible alternatives for existing methods.<br />
<br />
The outline of our ideas is to find mechanisms similar to in vivo antibody selection and take in,to account the industry production costs, controllability and reliability, thus developing new methods for antibody selection.<br />
In fact, in mammalian system, antibody selection is achieved through the activation of B lymphocytes and thus the rapid proliferation of the B lymphocytes that express the specific antibodies that bind to antigen. Simply put, the whole process relies on the interaction between antigen epitopes and membrane integral immunoglobins.<br />
<br />
To mimic the activation mediated by membrane integral immunoglobins, we develop ToxR-mediated transmembrane activation signal system. In this system, the interaction between antibodies and antigen triggered downstream expression of reporter genes, thus providing signals for selection.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2"><font face="Comic Sans MS" size=3>‘ToxR-based Transmembrane Signaling Pathway Method’</font></a></html> for detailed description of this method.<br />
<br />
<br />
In addition, we develop a technique called ‘Bacterial based Microarray’ for selection purpose. In this method, we combine membrane display technique and high throughput microarray technique, that is, a bacterial based microarray method.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1"><font face="Comic Sans MS" size=3>’Bacterial based microarray’</font></a></html> for details.<br />
<br />
<br />
In order to find methods for antibody selection, we cooperate with other iGEM teams. For example, we talked with Macquarie_Australia iGEM team about the project. The techniques involved in their project might be useful for our selection methods. Therefore, members from both teams worked together to research on this problem.<br />
<br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3"><font face="Comic Sans MS" size=3>'link'</font></a></html> to learn more about our cooperation.<br />
<br />
<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens. <br />
Our system is aiming at imitating this recombination process, using E.coli as the gene carrier.<br />
It is known to all that regional genes (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and the recombination occurs when VDJ recombinase are expressed. We choose RecA enzyme to induce the recombination, while use I-Sel enzyme to cut the genome, just mimicking the process happened in B cells.<br />
<br/><br />
<br/><br />
<br />
==Comparison between natural antibody production and E. Coli system==<br />
<br/><br />
===VDJ Recombination vs Lading Pad Recombination===<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is the mechanism by which immunoglobulin (Ig) and T cell receptors (TCR) are generated in the immune system. Through V(D)J recombination, Variable, Diverse, and Joining gene segments are randomly combined, thus encoding various kinds of immunoglobins and T cell receptors to recognize potential antigens. <br />
During the process of B cell maturation, gene segments are rearranged and thus transcribe mRNA that can produce diverse types of antibody for recognition of billions of potential epitopes. The rearrangement brings three gene segments (termed V, D, and J) in close proximity, which are then joined together head to tail, the process of which is known as V(D)J recombination based on the V,D,J gene segments involved. Multiple copies of the V, D, and J genes in the human genome are shuffled in the process, and a specific antibody will be generate among millions of other possible combinations. Because gene are joined together permanently, one mature B cell will produce only one specific antibody.<br />
<br />
Our system is aimed to mimic this recombination process, using E.coli as the carrier.<br />
Through previous research in immunology field, each kind of gene segments (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and recombination occurs when VDJ recombinase recognize the specific signal sequence and mediate the recombination process. In our project, we design a sequence, which carries separated parts of one complete gene and integrate synthesized DNA fragment into the genome of E coli via landing pad method. Then we use I-Sel enzyme to cut the gene segments out of E coli genome, into which gene segments are inserted and choose RecA enzyme to recognize the previously designed signal sequence flanking our target sequence and recombine the gene segments, just like what recombinase accomplish in B cells. The whole process is called landing pad recombination.<br />
<br />
===Somatic hyper-mutation vs Junctional mutation===<br />
<br />
Somatic hypermutation involves a programmed process of mutation affecting the variable regions of immunoglobulin genes. During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is at least 105-106 fold greater than the normal rate of mutation across the genome. Variation is mainly in the form of single base substitutions, with insertions and deletions being less common. These mutations occur mostly at “hotspots” in the DNA, known as hypervariable regions. These regions correspond to the complementarity determining regions; the sites involved in antigen recognition on the immunoglobulin. This directed hypermutation allows for the selection of B cells that express immunoglobulin receptors possessing an enhanced ability to recognize and bind a specific foreign antigen.<br />
<br />
In addition, the cross ligation, namely Landing pad recombination, has higher opportunity to mutate in the link sequence. The introduction of 4 junctional shuffler 64 will meet the need of the rest 107 diversity, perfectly undertaking the work in the antibody diversity construction that hyper-mutation does in the highly variable region. In this way, we have finished a building library, waiting for the specialized antigen to select.<br />
<br />
===Antigen-specific Selection vs CBD-Based Microarray and ToxR-Based Transmembrane pathway method===<br />
In mammalian immune system, billions of antibodies will be generated. Then how can the immune system find out specific antibody that indiscriminately recognize the specific antigen that comes in. Generally speaking, due to the fact that one mature B cell only generates one specific immunoglobin, once the membrane integral immunoglobin bind to the specific antigen, a series of intracellular response, such as gene activation and cell differetiation will be triggered in B cell. Then, B cells expressing the specific antibodies that recognize the specific antigen will proliferate and generate enormous amount of antibodies that help eliminate the specific antigen.<br />
<br />
Simply put, antibody selection is accomplished through the specific interaction between the membrane immunoglobin and the specific antigen. Therefore, in our project, we also plan to mimic this process through interaction between the antibodies generated through landing pad recombination and the antigen we target. CBD-based Microarray and ToxR-based Transmembrane pathway are the two methods by which we achieve this goal.<br />
CBD domain, which interacts with cellulose, potentially provides us way to locate our E.coli to the microarray coated with cellulose. OmpA (outer membrane protein A), displays the antibody and antigen out to the membrane surface. Then by applying the antibodies generated through landing pad recombination to the microarray, antibodies that bind to the antigen will be retained and thus selected out of the pool of antibodies.<br />
<br />
Alternatively, the ToxR-based transmembrane pathway method shows a different strategy for us to seek the right folded antibody. Once the antigen comes, ToxR outer part will form dimer shape and the downstream signal occur, supplying the approach of reporters, such as GFP, to examine and pick out what we want.<br />
<br />
<html><br />
&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a><br />
</body><br />
</div><br />
<br />
</div><br />
</div></div></body><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/project/outlineTeam:Tsinghua/project/outline2010-10-27T16:42:08Z<p>Gumilton: </p>
<hr />
<div>__NOTOC__<br />
{{:Team:Tsinghua/header}}<br />
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<div id="content_box"> <br />
</html><br />
{{:Team:Tsinghua/leftbar}}<br />
<html><br />
<script><br />
navlist=new Array("Background", "Project Outline", "Module I", "Module II", "Conclusion", "Future");<br />
linkl = new Array("/Team:Tsinghua/project", "#outline", "/Team:Tsinghua/project/outline/m1", "/Team:Tsinghua/project/outline/m2", "con", "future");<br />
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writenav(navlist, linkl, 1, subs);<br />
</script><br />
<div id="main_content"><a name="outline"></a><br />
<h1>Outline</h1><br />
<br />
<div class="content_block"><br />
<br />
<br/><br />
In order to realize the production of antibodies in prokaryotic cells, we firstly investigate the whole process of antibody production in mammalian immune system and try to simulate the whole process via the methods and concepts of synthetic biology. Finally, we successfully constructed the desired system. The comparison between the key steps involved in antibody generation and corresponding synthetic methods are shown in the chart below.<br />
<br/><br />
<br />
<table border="2" bordercolor="maroon" bgcolor="silver"><br />
<tbody><br />
<tr><br />
<th colspan=3>Antibody Production</th><br />
<th width=50px></th><br />
<th colspan=2>E Coli. Production System</th></tr><br />
<tr><br />
<td rowspan=4 width=70px><a href="#VDJ_Recombination_vs_Lading_Pad_Recombination">VDJ Recombination</a></td><br />
<td rowspan=3 width=70px>Preparation</td><br />
<td width=70px>RSS Sequence</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">Landing Pad Insertion</a></td><br />
<td rowspan=4 width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1">Module I: Recombination System</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Recombinase</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">Helper Plasmid Insertion</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Library</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">Donor Plasmid Construction</a></td><br />
</tr><br />
<tr><br />
<td>Recombination</td><br />
<td colspan=2><img src="https://static.igem.org/mediawiki/2010/d/d6/THUarrow2.png" width=180></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">DP Insertion and Recombination Induction</a></td><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="#Somatic_hyper-mutation_vs_Junctional_mutation">Somatic Hypermutation</a></td><br />
<td rowspan=2 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
<!---------<td>Junctional Diversity</td>----------------------><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">CBD-based Microarray</a></td><br />
<td rowspan=4><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2">Module II: Selection System</a></td><br />
</tr><br />
<tr><br />
<td rowspan=3><a href="#Antigen-specific_Selection_vs_CBD-Based_Microarray_and_ToxR-Based_Transmembrane_pathway_method">Antigen Selection</a></td><br />
<td rowspan=3 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">ToxR-based Transmembrane Pathway</a></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">Cooperation With Macquarie_Australia</a></td><br />
</tr><br />
</tbody><br />
</table><br />
</html><br />
<br/><br />
Generally speaking, antibody production in our project can be divided into two modules.<br />
<br />
<html><br />
<body bgcolor="#336699" text="#ffffff" link="#60a179"><br />
<br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1"><font face="Comic Sans MS" size="4">Module I: Generation of antibody library</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2"><font face="Comic Sans MS" size="4">Module II: Selection of specific antibodies</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
</html><br />
<br />
==Project Design==<br />
<br />
<br />
<br />
===Module 1===<br />
<br />
'''Antibodies Library Diversity & Randomicity'''<br />
[[Image:TSModule1.PNG|650px]]<br />
<br />
<br />
The first module is carried out by a novel method called E coli in vivo recombination system. The theoretical and experimental basis comes from the work of Thomas E. Kuhlman and Edward C. Cox. We managed to modify their system, wishing to utilize it for simulation of antibody recombination.<br />
<br />
Firstly, we try to insert a DNA segment called”Landing Pad(LP)” into the specific sites of E coli via Att recombination, the manner in which lambda phage integrates its DNA into E Coli genome. Landing pad sequence consists of the following parts(from 5’ to 3’ in the sequence):<br />
<br />
1)25bp-long random sequence<br />
<br />
2)15bp-long recognition sequence of restriction enzyme I-scel<br />
<br />
3) antibiotic resistance gene used for antibody selection<br />
<br />
4) 15bp-long recognition sequence of restriction enzyme I-scel(corresponding to 2)<br />
<br />
5) 25bp-long random sequence (corresponding to 1)<br />
<br />
After integrating DNA segment of Landing pad into the genome of E coli, we completed the genetic engineering of the genome of E coli. All the five parts of landing pad are designed for subsequent recombination. <br />
<br />
Besides, in order to achieve different recombination goals, we designed several landing pads of different sequences.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion"><font face="Comic Sans MS" size=3>'Landing Pad Construction and Insertion'</font></a></html> to learn about the details of Landing Pad design. <br />
<br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#att"><font face="Comic Sans MS" size=3>'link'</font></a></html> to learn more about ATT recombination.<br />
<br />
<br />
After the integration of landing pad, we have to insert another DNA fragment called Helper Plasmid(HP), which provides necessary tools for in vivo recombination. <br />
<br />
In this plasmid(Helper Plasmid), there are two genes used for recombination. <br />
<br />
The first one encodes restriction enzyme I-scel after induction of L-arabinose. I-scel is able to recognize the 15bp-length sequence flanking a target DNA fragment and cut out the fragment, therefore providing DNA segments for recombination mediated by other components.<br />
<br />
The second one encodes the enzyme responsible for recombination called Lamda-Red. With the presence of IPTG, Lamda-Red will recombine DNA fragments flanked by the same DNA sequence recognized by Lamda-Red.<br />
<br />
In addition, Helper Plasmid contains a specific temperature-sensitive replicon, which is used to control the replication via temperature changes. We can remove the replicon when necessary.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi"><font face="Comic Sans MS" size=3>'HP insertion'</font></a></html> to learn more about Helper Plasmid.<br />
<br />
<br />
Up till now, we successfully engineered a ‘recepient strain’. Then we have to transform the ‘recepient strain’ with the Donor Plasmid(DP) containing recombination fragments. Donor Plasmid contains several Insertion Fragments and each Insertion Fragment contains following five parts:<br />
<br />
1) one 15bp-long restriction enzyme I-scel recognition site<br />
<br />
2) one 25bp-long random sequence<br />
<br />
3) one fragment for insertion and recombination<br />
<br />
4) one 25bp-long random sequence<br />
<br />
5) one 15bp-long restriction enzyme I-scel recognition site(in correspondence with 1)<br />
<br />
(Note: the order of the five parts differ from that of landing pad)<br />
<br />
As we previously mentioned, Donor plasmid contains several Insertion Fragments. Based on the flanking landing pad sequence, we can ascribe Insertion Fragments to the same group as long as the flanking landing pad sequences of those Insertion Fragments are the same.<br />
<br />
In order to achieve different goals, we design different Donor Plasmids, different Donor Plasmids contain different number of Insertion Fragments, which belong to different groups.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc"><font face="Comic Sans MS" size=3>‘DP construction’</font></a></html> to learn about the details of Donor Plasmid and Insertion Fragments and the strategies employed in our design. <br />
<br />
<br />
As we all know, antibody diversity mainly lean on the number of the fragments used for recombination. Therefore, in order to mimic the antibody generation process, we need to construct Donor Plasmid which contains large sum of Insertion Fragments. Theoretically, we can construct Donor Plasmid of this kind. However, due to the time limit of the competition, we didn’t try to construct DP containing many Insertion Fragments.<br />
<br />
In spite of this, in order to meet the needs, we designed two methods to construct plasmids on a large scale and attempted the usage in parts of our Donor Plasmids.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Strategies"><font face="Comic Sans MS" size=3>'DP construction and Strategies'</font></a></html> for the methods of rapid plasmid construction. <br />
<br />
<br />
Besides, we have applied for two BBFRFC, which are BBFRFC61 and BBFRFC 62 respectively.<br />
<br />
Then, after transformation of the prepared ‘recepient strain’ with Donor Plasmid and the induction of corresponding agents, I-scel enzyme cuts out DNA fragments from DP and LP, generating numerous Insertion Fragments. Besides, antibiotic resistance gene integrated into E coli genome is also cut out, providing recombination sites for Insertion Fragments.<br />
<br />
Provided with Insertion Fragments mentioned above and Lamda-Red expressed by Donor Plasmid, Insertion Fragments recombine with the corresponding landing pad sequence in the genome and thus get inserted into the correct site of the genome. Based on our design of Landing pad, we can ensure that only one Insertion Fragment from one group is randomly inserted into the genome, therefore mimicking the VDJ recombination process.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri"><font face="Comic Sans MS" size=3>‘DP Insertion and Recombination Induction’</font></a></html> to learn more about this part. <br />
<br />
<br />
Engineered E coli cells which have undergone successful recombination can be selected out of the pool through specific screening methods. After tests on different strains, our project achieve a recombination rate above 50% based on our current techniques.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/experiments#res"><font face="Comic Sans MS" size=3>‘Result’</font></a></html> to learn more about the identification of recombination rate. <br />
<br />
<br/><br/><br />
<br />
===Module 2===<br />
<br />
The goal of the second module in our project is aimed at selection of specific antibodies. Due to the maturity of existing antibody screening technique, our work mainly focuses on imitation of mammalian antibody selection, thus providing possible alternatives for existing methods.<br />
<br />
The outline of our ideas is to find mechanisms similar to in vivo antibody selection and take in,to account the industry production costs, controllability and reliability, thus developing new methods for antibody selection.<br />
In fact, in mammalian system, antibody selection is achieved through the activation of B lymphocytes and thus the rapid proliferation of the B lymphocytes that express the specific antibodies that bind to antigen. Simply put, the whole process relies on the interaction between antigen epitopes and membrane integral immunoglobins.<br />
<br />
To mimic the activation mediated by membrane integral immunoglobins, we develop ToxR-mediated transmembrane activation signal system. In this system, the interaction between antibodies and antigen triggered downstream expression of reporter genes, thus providing signals for selection.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2"><font face="Comic Sans MS" size=3>‘ToxR-based Transmembrane Signaling Pathway Method’</font></a></html> for detailed description of this method.<br />
<br />
<br />
In addition, we develop a technique called ‘Bacterial based Microarray’ for selection purpose. In this method, we combine membrane display technique and high throughput microarray technique, that is, a bacterial based microarray method.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1"><font face="Comic Sans MS" size=3>’Bacterial based microarray’</font></a></html> for details.<br />
<br />
<br />
In order to find methods for antibody selection, we cooperate with other iGEM teams. For example, we talked with Macquarie_Australia iGEM team about the project. The techniques involved in their project might be useful for our selection methods. Therefore, members from both teams worked together to research on this problem.<br />
<br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3"><font face="Comic Sans MS" size=3>'link'</font></a></html> to learn more about our cooperation.<br />
<br />
<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens. <br />
Our system is aiming at imitating this recombination process, using E.coli as the gene carrier.<br />
It is known to all that regional genes (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and the recombination occurs when VDJ recombinase are expressed. We choose RecA enzyme to induce the recombination, while use I-Sel enzyme to cut the genome, just mimicking the process happened in B cells.<br />
<br/><br />
<br/><br />
<br />
==Comparison between natural antibody production and E. Coli system==<br />
<br/><br />
===VDJ Recombination vs Lading Pad Recombination===<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is the mechanism by which immunoglobulin (Ig) and T cell receptors (TCR) are generated in the immune system. Through V(D)J recombination, Variable, Diverse, and Joining gene segments are randomly combined, thus encoding various kinds of immunoglobins and T cell receptors to recognize potential antigens. <br />
During the process of B cell maturation, gene segments are rearranged and thus transcribe mRNA that can produce diverse types of antibody for recognition of billions of potential epitopes. The rearrangement brings three gene segments (termed V, D, and J) in close proximity, which are then joined together head to tail, the process of which is known as V(D)J recombination based on the V,D,J gene segments involved. Multiple copies of the V, D, and J genes in the human genome are shuffled in the process, and a specific antibody will be generate among millions of other possible combinations. Because gene are joined together permanently, one mature B cell will produce only one specific antibody.<br />
<br />
Our system is aimed to mimic this recombination process, using E.coli as the carrier.<br />
Through previous research in immunology field, each kind of gene segments (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and recombination occurs when VDJ recombinase recognize the specific signal sequence and mediate the recombination process. In our project, we design a sequence, which carries separated parts of one complete gene and integrate synthesized DNA fragment into the genome of E coli via landing pad method. Then we use I-Sel enzyme to cut the gene segments out of E coli genome, into which gene segments are inserted and choose RecA enzyme to recognize the previously designed signal sequence flanking our target sequence and recombine the gene segments, just like what recombinase accomplish in B cells. The whole process is called landing pad recombination.<br />
<br />
===Somatic hyper-mutation vs Junctional mutation===<br />
<br />
Somatic hypermutation involves a programmed process of mutation affecting the variable regions of immunoglobulin genes. During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is at least 105-106 fold greater than the normal rate of mutation across the genome. Variation is mainly in the form of single base substitutions, with insertions and deletions being less common. These mutations occur mostly at “hotspots” in the DNA, known as hypervariable regions. These regions correspond to the complementarity determining regions; the sites involved in antigen recognition on the immunoglobulin. This directed hypermutation allows for the selection of B cells that express immunoglobulin receptors possessing an enhanced ability to recognize and bind a specific foreign antigen.<br />
<br />
In addition, the cross ligation, namely Landing pad recombination, has higher opportunity to mutate in the link sequence. The introduction of 4 junctional shuffler 64 will meet the need of the rest 107 diversity, perfectly undertaking the work in the antibody diversity construction that hyper-mutation does in the highly variable region. In this way, we have finished a building library, waiting for the specialized antigen to select.<br />
<br />
===Antigen-specific Selection vs CBD-Based Microarray and ToxR-Based Transmembrane pathway method===<br />
In mammalian immune system, billions of antibodies will be generated. Then how can the immune system find out specific antibody that indiscriminately recognize the specific antigen that comes in. Generally speaking, due to the fact that one mature B cell only generates one specific immunoglobin, once the membrane integral immunoglobin bind to the specific antigen, a series of intracellular response, such as gene activation and cell differetiation will be triggered in B cell. Then, B cells expressing the specific antibodies that recognize the specific antigen will proliferate and generate enormous amount of antibodies that help eliminate the specific antigen.<br />
<br />
Simply put, antibody selection is accomplished through the specific interaction between the membrane immunoglobin and the specific antigen. Therefore, in our project, we also plan to mimic this process through interaction between the antibodies generated through landing pad recombination and the antigen we target. CBD-based Microarray and ToxR-based Transmembrane pathway are the two methods by which we achieve this goal.<br />
CBD domain, which interacts with cellulose, potentially provides us way to locate our E.coli to the microarray coated with cellulose. OmpA (outer membrane protein A), displays the antibody and antigen out to the membrane surface. Then by applying the antibodies generated through landing pad recombination to the microarray, antibodies that bind to the antigen will be retained and thus selected out of the pool of antibodies.<br />
<br />
Alternatively, the ToxR-based transmembrane pathway method shows a different strategy for us to seek the right folded antibody. Once the antigen comes, ToxR outer part will form dimer shape and the downstream signal occur, supplying the approach of reporters, such as GFP, to examine and pick out what we want.<br />
<br />
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&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a><br />
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</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/project/futureTeam:Tsinghua/project/future2010-10-27T16:41:11Z<p>Gumilton: </p>
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<div>__NOTOC__<br />
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<h1>Future Work</h1><br />
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==Construction of recombination library==<br />
<br />
We just demonstrated the feasibility of construction of library of recombination segments like the one in humans. In future work, we hope to finish this part and make real antibody libraries.<br />
<br />
In human beings, our antibody comprises of two parts, the light chain and the heavy chain. During formation, the heavy chain undergoes recombination of V, D and J segments while the light one between V and J. The recombination of these gene segments gives rise to diversity to the scale of <math>10^5</math> to <math>10^6</math>.<br />
<br />
In order to construct a real antibody library, we need to ligate several dozen segments into a vector. The protocol for molecular manipulation is clear but we may need a larger vector, such as the BACs to harbor all the segments.<br />
<br />
<br />
<br />
==Test of antibody function==<br />
<br />
Once the antibody library is accomplished, the major focus will be whether such antibodies can have normal function. We will settle such issues with antibody expression and subsequent assays. Immunohistochemistry or immunofluorescence offers the opportunity to compare the specificity of such antibodies while equilibrium dialysis can measure its affinity.<br />
<br />
===Specificity===<br />
<br />
Immunohistochemistry or immunofluorescence stains certain proteins in the tissue marked with the antibody, which is a perfect check for antibody specificity. With the serum antibody as control, specificity can be checked by simply measuring the background and cross-reaction. This technique offers an intuitive way for checking specificity.<br />
<br />
Western blotting is a more precise method. Western blotting of cell lysates can reveal cross-reaction of the antibody, which will appear as extra bands on the film.<br />
<br />
<br />
<br />
===Affinity===<br />
<br />
Affinity can be checked with equilibrium dialysis, in which the antibody for a small hapten molecule is kept in the dialysis membrane with the hapten in the solution outside. When the system reaches its equilibrium, the concentration of the antigen reveals the equilibrium constant of antibody binding, which indicates the affinity quantitatively.<br />
<br />
<br />
[[Image:Dialysis.png]]<br />
<br />
==Other applications==<br />
<br />
The recombination method is useful in other aspects.<br />
<br />
Nowadays, microbe based environmental monitoring is fast and real-time. However, certain phenomenon might be instant but reveal the hint for potential environmental hazards. In this case, our system might be quite useful, for recombination records the instant event permanently in the genome of the bacteria, just like a log. Thus, detection will discover the appearance of the hazards even if it's transient. For instance, the increase in phosphate concentration in some districts might be transient but will cause algae bloom in lakes downstream. With this recombination apparatus, the bacteria which have encountered such abnormal phosphate concentration can undergo recombination and record this event permanently. Culture and analysis will reveal such harmful events.<br />
<br />
Besides, since the signal can be recorded firmly with recombination, it can be amplified during proliferation of the bacteria. This is crucial for detecting some weak yet important changes.<br />
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</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/project/conTeam:Tsinghua/project/con2010-10-27T16:40:15Z<p>Gumilton: </p>
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<div id="main_content"><a name="outline"></a><br />
<h1>Outline</h1><br />
<br />
<div class="content_block"><br />
<br />
<br/><br />
In order to realize the production of antibodies in prokaryotic cells, we firstly investigate the whole process of antibody production in mammalian immune system and try to simulate the whole process via the methods and concepts of synthetic biology. Finally, we successfully constructed the desired system. The comparison between the key steps involved in antibody generation and corresponding synthetic methods are shown in the chart below.<br />
<br/><br />
<br />
<table border="2" bordercolor="maroon" bgcolor="silver"><br />
<tbody><br />
<tr><br />
<th colspan=3>Antibody Production</th><br />
<th width=50px></th><br />
<th colspan=2>E Coli. Production System</th></tr><br />
<tr><br />
<td rowspan=4 width=70px><a href="#VDJ_Recombination_vs_Lading_Pad_Recombination">VDJ Recombination</a></td><br />
<td rowspan=3 width=70px>Preparation</td><br />
<td width=70px>RSS Sequence</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">Landing Pad Insertion</a></td><br />
<td rowspan=4 width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1">Module I: Recombination System</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Recombinase</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">Helper Plasmid Insertion</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Library</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">Donor Plasmid Construction</a></td><br />
</tr><br />
<tr><br />
<td>Recombination</td><br />
<td colspan=2><img src="https://static.igem.org/mediawiki/2010/d/d6/THUarrow2.png" width=180></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">DP Insertion and Recombination Induction</a></td><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="#Somatic_hyper-mutation_vs_Junctional_mutation">Somatic Hypermutation</a></td><br />
<td rowspan=2 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
<!---------<td>Junctional Diversity</td>----------------------><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">CBD-based Microarray</a></td><br />
<td rowspan=4><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2">Module II: Selection System</a></td><br />
</tr><br />
<tr><br />
<td rowspan=3><a href="#Antigen-specific_Selection_vs_CBD-Based_Microarray_and_ToxR-Based_Transmembrane_pathway_method">Antigen Selection</a></td><br />
<td rowspan=3 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">ToxR-based Transmembrane Pathway</a></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">Cooperation With Macquarie_Australia</a></td><br />
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</table><br />
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<br/><br />
Generally speaking, antibody production in our project can be divided into two modules.<br />
<br />
<html><br />
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<br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1"><font face="Comic Sans MS" size="4">Module I: Generation of antibody library</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
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<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2"><font face="Comic Sans MS" size="4">Module II: Selection of specific antibodies</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
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<br />
==Project Design==<br />
<br />
<br />
<br />
===Module 1===<br />
<br />
'''Antibodies Library Diversity & Randomicity'''<br />
[[Image:TSModule1.PNG|650px]]<br />
<br />
<br />
The first module is carried out by a novel method called E coli in vivo recombination system. The theoretical and experimental basis comes from the work of Thomas E. Kuhlman and Edward C. Cox. We managed to modify their system, wishing to utilize it for simulation of antibody recombination.<br />
<br />
Firstly, we try to insert a DNA segment called”Landing Pad(LP)” into the specific sites of E coli via Att recombination. Landing pad sequence consists of the following parts(from 5’ to 3’ in the sequence):<br />
<br />
1)25bp-long random sequence<br />
<br />
2)15bp-long recognition sequence of restriction enzyme I-scel<br />
<br />
3) antibiotic resistance gene used for antibody selection<br />
<br />
4) 15bp-long recognition sequence of restriction enzyme I-scel(corresponding to 2)<br />
<br />
5) 25bp-long random sequence (corresponding to 1)<br />
<br />
After integrating DNA segment of Landing pad into the genome of E coli, we completed the genetic engineering of the genome of E coli. All the five parts of landing pad are designed for subsequent recombination. <br />
<br />
Besides, in order to achieve different recombination goals, we designed several landing pads of different sequences.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion"><font face="Comic Sans MS" size=3>'Landing Pad Construction and Insertion'</font></a></html> to learn about the details of Landing Pad design. <br />
<br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#att"><font face="Comic Sans MS" size=3>'link'</font></a></html> to learn more about ATT recombination.<br />
<br />
<br />
After the integration of landing pad, we have to insert another DNA fragment called Helper Plasmid(HP), which provides necessary tools for in vivo recombination. <br />
<br />
In this plasmid(Helper Plasmid), there are two genes used for recombination. <br />
<br />
The first one encodes restriction enzyme I-scel after induction of L-arabinose. I-scel is able to recognize the 15bp-length sequence flanking a target DNA fragment and cut out the fragment, therefore providing DNA segments for recombination mediated by other components.<br />
<br />
The second one encodes the enzyme responsible for recombination called Lamda-Red. With the presence of IPTG, Lamda-Red will recombine DNA fragments flanked by the same DNA sequence recognized by Lamda-Red.<br />
<br />
In addition, Helper Plasmid contains a specific temperature-sensitive replicon, which is used to control the replication via temperature changes. We can remove the replicon when necessary.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi"><font face="Comic Sans MS" size=3>'HP insertion'</font></a></html> to learn more about Helper Plasmid.<br />
<br />
<br />
Up till now, we successfully engineered a ‘recepient strain’. Then we have to transform the ‘recepient strain’ with the Donor Plasmid(DP) containing recombination fragments. Donor Plasmid contains several Insertion Fragments and each Insertion Fragment contains following five parts:<br />
<br />
1) one 15bp-long restriction enzyme I-scel recognition site<br />
<br />
2) one 25bp-long random sequence<br />
<br />
3) one fragment for insertion and recombination<br />
<br />
4) one 25bp-long random sequence<br />
<br />
5) one 15bp-long restriction enzyme I-scel recognition site(in correspondence with 1)<br />
<br />
(Note: the order of the five parts differ from that of landing pad)<br />
<br />
As we previously mentioned, Donor plasmid contains several Insertion Fragments. Based on the flanking landing pad sequence, we can ascribe Insertion Fragments to the same group as long as the flanking landing pad sequences of those Insertion Fragments are the same.<br />
<br />
In order to achieve different goals, we design different Donor Plasmids, different Donor Plasmids contain different number of Insertion Fragments, which belong to different groups.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc"><font face="Comic Sans MS" size=3>‘DP construction’</font></a></html> to learn about the details of Donor Plasmid and Insertion Fragments and the strategies employed in our design. <br />
<br />
<br />
As we all know, antibody diversity mainly lean on the number of the fragments used for recombination. Therefore, in order to mimic the antibody generation process, we need to construct Donor Plasmid which contains large sum of Insertion Fragments. Theoretically, we can construct Donor Plasmid of this kind. However, due to the time limit of the competition, we didn’t try to construct DP containing many Insertion Fragments.<br />
<br />
In spite of this, in order to meet the needs, we designed two methods to construct plasmids on a large scale and attempted the usage in parts of our Donor Plasmids.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Strategies"><font face="Comic Sans MS" size=3>'DP construction and Strategies'</font></a></html> for the methods of rapid plasmid construction. <br />
<br />
<br />
Besides, we have applied for two BBFRFC, which are BBFRFC61 and BBFRFC 62 respectively.<br />
<br />
Then, after transformation of the prepared ‘recepient strain’ with Donor Plasmid and the induction of corresponding agents, I-scel enzyme cuts out DNA fragments from DP and LP, generating numerous Insertion Fragments. Besides, antibiotic resistance gene integrated into E coli genome is also cut out, providing recombination sites for Insertion Fragments.<br />
<br />
Provided with Insertion Fragments mentioned above and Lamda-Red expressed by Donor Plasmid, Insertion Fragments recombine with the corresponding landing pad sequence in the genome and thus get inserted into the correct site of the genome. Based on our design of Landing pad, we can ensure that only one Insertion Fragment from one group is randomly inserted into the genome, therefore mimicking the VDJ recombination process.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri"><font face="Comic Sans MS" size=3>‘DP Insertion and Recombination Induction’</font></a></html> to learn more about this part. <br />
<br />
<br />
Engineered E coli cells which have undergone successful recombination can be selected out of the pool through specific screening methods. After tests on different strains, our project achieve a recombination rate above 50% based on our current techniques.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/experiments#res"><font face="Comic Sans MS" size=3>‘Result’</font></a></html> to learn more about the identification of recombination rate. <br />
<br />
<br/><br/><br />
<br />
<br />
<br />
<br />
===Module 2===<br />
<br />
The goal of the second module in our project is aimed at selection of specific antibodies. Due to the maturity of existing antibody screening technique, our work mainly focuses on imitation of mammalian antibody selection, thus providing possible alternatives for existing methods.<br />
<br />
The outline of our ideas is to find mechanisms similar to in vivo antibody selection and take in,to account the industry production costs, controllability and reliability, thus developing new methods for antibody selection.<br />
In fact, in mammalian system, antibody selection is achieved through the activation of B lymphocytes and thus the rapid proliferation of the B lymphocytes that express the specific antibodies that bind to antigen. Simply put, the whole process relies on the interaction between antigen epitopes and membrane integral immunoglobins.<br />
<br />
To mimic the activation mediated by membrane integral immunoglobins, we develop ToxR-mediated transmembrane activation signal system. In this system, the interaction between antibodies and antigen triggered downstream expression of reporter genes, thus providing signals for selection.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2"><font face="Comic Sans MS" size=3>‘ToxR-based Transmembrane Signaling Pathway Method’</font></a></html> for detailed description of this method.<br />
<br />
<br />
In addition, we develop a technique called ‘Bacterial based Microarray’ for selection purpose. In this method, we combine membrane display technique and high throughput microarray technique, that is, a bacterial based microarray method.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1"><font face="Comic Sans MS" size=3>’Bacterial based microarray’</font></a></html> for details.<br />
<br />
<br />
In order to find methods for antibody selection, we cooperate with other iGEM teams. For example, we talked with Macquarie_Australia iGEM team about the project. The techniques involved in their project might be useful for our selection methods. Therefore, members from both teams worked together to research on this problem.<br />
<br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3"><font face="Comic Sans MS" size=3>'link'</font></a></html> to learn more about our cooperation.<br />
<br />
<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens. <br />
Our system is aiming at imitating this recombination process, using E.coli as the gene carrier.<br />
It is known to all that regional genes (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and the recombination occurs when VDJ recombinase are expressed. We choose RecA enzyme to induce the recombination, while use I-Sel enzyme to cut the genome, just mimicking the process happened in B cells.<br />
<br/><br />
<br/><br />
<br />
==Comparison between natural antibody production and E. Coli system==<br />
<br/><br />
===VDJ Recombination vs Lading Pad Recombination===<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is the mechanism by which immunoglobulin (Ig) and T cell receptors (TCR) are generated in the immune system. Through V(D)J recombination, Variable, Diverse, and Joining gene segments are randomly combined, thus encoding various kinds of immunoglobins and T cell receptors to recognize potential antigens. <br />
During the process of B cell maturation, gene segments are rearranged and thus transcribe mRNA that can produce diverse types of antibody for recognition of billions of potential epitopes. The rearrangement brings three gene segments (termed V, D, and J) in close proximity, which are then joined together head to tail, the process of which is known as V(D)J recombination based on the V,D,J gene segments involved. Multiple copies of the V, D, and J genes in the human genome are shuffled in the process, and a specific antibody will be generate among millions of other possible combinations. Because gene are joined together permanently, one mature B cell will produce only one specific antibody.<br />
<br />
Our system is aimed to mimic this recombination process, using E.coli as the carrier.<br />
Through previous research in immunology field, each kind of gene segments (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and recombination occurs when VDJ recombinase recognize the specific signal sequence and mediate the recombination process. In our project, we design a sequence, which carries separated parts of one complete gene and integrate synthesized DNA fragment into the genome of E coli via landing pad method. Then we use I-Sel enzyme to cut the gene segments out of E coli genome, into which gene segments are inserted and choose RecA enzyme to recognize the previously designed signal sequence flanking our target sequence and recombine the gene segments, just like what recombinase accomplish in B cells. The whole process is called landing pad recombination.<br />
<br />
===Somatic hyper-mutation vs Junctional mutation===<br />
<br />
Somatic hypermutation involves a programmed process of mutation affecting the variable regions of immunoglobulin genes. During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is at least 105-106 fold greater than the normal rate of mutation across the genome. Variation is mainly in the form of single base substitutions, with insertions and deletions being less common. These mutations occur mostly at “hotspots” in the DNA, known as hypervariable regions. These regions correspond to the complementarity determining regions; the sites involved in antigen recognition on the immunoglobulin. This directed hypermutation allows for the selection of B cells that express immunoglobulin receptors possessing an enhanced ability to recognize and bind a specific foreign antigen.<br />
<br />
In addition, the cross ligation, namely Landing pad recombination, has higher opportunity to mutate in the link sequence. The introduction of 4 junctional shuffler 64 will meet the need of the rest 107 diversity, perfectly undertaking the work in the antibody diversity construction that hyper-mutation does in the highly variable region. In this way, we have finished a building library, waiting for the specialized antigen to select.<br />
<br />
===Antigen-specific Selection vs CBD-Based Microarray and ToxR-Based Transmembrane pathway method===<br />
In mammalian immune system, billions of antibodies will be generated. Then how can the immune system find out specific antibody that indiscriminately recognize the specific antigen that comes in. Generally speaking, due to the fact that one mature B cell only generates one specific immunoglobin, once the membrane integral immunoglobin bind to the specific antigen, a series of intracellular response, such as gene activation and cell differetiation will be triggered in B cell. Then, B cells expressing the specific antibodies that recognize the specific antigen will proliferate and generate enormous amount of antibodies that help eliminate the specific antigen.<br />
<br />
Simply put, antibody selection is accomplished through the specific interaction between the membrane immunoglobin and the specific antigen. Therefore, in our project, we also plan to mimic this process through interaction between the antibodies generated through landing pad recombination and the antigen we target. CBD-based Microarray and ToxR-based Transmembrane pathway are the two methods by which we achieve this goal.<br />
CBD domain, which interacts with cellulose, potentially provides us way to locate our E.coli to the microarray coated with cellulose. OmpA (outer membrane protein A), displays the antibody and antigen out to the membrane surface. Then by applying the antibodies generated through landing pad recombination to the microarray, antibodies that bind to the antigen will be retained and thus selected out of the pool of antibodies.<br />
<br />
Alternatively, the ToxR-based transmembrane pathway method shows a different strategy for us to seek the right folded antibody. Once the antigen comes, ToxR outer part will form dimer shape and the downstream signal occur, supplying the approach of reporters, such as GFP, to examine and pick out what we want.<br />
<br />
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<h1>Outline</h1><br />
<br />
<div class="content_block"><br />
<br />
<br/><br />
In order to realize the production of antibodies in prokaryotic cells, we firstly investigate the whole process of antibody production in mammalian immune system and try to simulate the whole process via the methods and concepts of synthetic biology. Finally, we successfully constructed the desired system. The comparison between the key steps involved in antibody generation and corresponding synthetic methods are shown in the chart below.<br />
<br/><br />
<br />
<table border="2" bordercolor="maroon" bgcolor="silver"><br />
<tbody><br />
<tr><br />
<th colspan=3>Antibody Production</th><br />
<th width=50px></th><br />
<th colspan=2>E Coli. Production System</th></tr><br />
<tr><br />
<td rowspan=4 width=70px><a href="#VDJ_Recombination_vs_Lading_Pad_Recombination">VDJ Recombination</a></td><br />
<td rowspan=3 width=70px>Preparation</td><br />
<td width=70px>RSS Sequence</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">Landing Pad Insertion</a></td><br />
<td rowspan=4 width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1">Module I: Recombination System</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Recombinase</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">Helper Plasmid Insertion</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Library</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">Donor Plasmid Construction</a></td><br />
</tr><br />
<tr><br />
<td>Recombination</td><br />
<td colspan=2><img src="https://static.igem.org/mediawiki/2010/d/d6/THUarrow2.png" width=180></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">DP Insertion and Recombination Induction</a></td><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="#Somatic_hyper-mutation_vs_Junctional_mutation">Somatic Hypermutation</a></td><br />
<td rowspan=2 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
<!---------<td>Junctional Diversity</td>----------------------><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">CBD-based Microarray</a></td><br />
<td rowspan=4><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2">Module II: Selection System</a></td><br />
</tr><br />
<tr><br />
<td rowspan=3><a href="#Antigen-specific_Selection_vs_CBD-Based_Microarray_and_ToxR-Based_Transmembrane_pathway_method">Antigen Selection</a></td><br />
<td rowspan=3 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">ToxR-based Transmembrane Pathway</a></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">Cooperation With Macquarie_Australia</a></td><br />
</tr><br />
</tbody><br />
</table><br />
</html><br />
<br/><br />
Generally speaking, antibody production in our project can be divided into two modules.<br />
<br />
<html><br />
<body bgcolor="#336699" text="#ffffff" link="#60a179"><br />
<br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1"><font face="Comic Sans MS" size="4">Module I: Generation of antibody library</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2"><font face="Comic Sans MS" size="4">Module II: Selection of specific antibodies</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
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<br />
==Project Design==<br />
<br />
<br />
<br />
===Module 1===<br />
<br />
'''Antibodies Library Diversity & Randomicity'''<br />
[[Image:TSModule1.PNG|650px]]<br />
<br />
<br />
The first module is carried out by a novel method called E coli in vivo recombination system. The theoretical and experimental basis comes from the work of Thomas E. Kuhlman and Edward C. Cox. We managed to modify their system, wishing to utilize it for simulation of antibody recombination.<br />
<br />
Firstly, we try to insert a DNA segment called”Landing Pad(LP)” into the specific sites of E coli via Att recombination. Landing pad sequence consists of the following parts(from 5’ to 3’ in the sequence):<br />
<br />
1)25bp-long random sequence<br />
<br />
2)15bp-long recognition sequence of restriction enzyme I-scel<br />
<br />
3) antibiotic resistance gene used for antibody selection<br />
<br />
4) 15bp-long recognition sequence of restriction enzyme I-scel(corresponding to 2)<br />
<br />
5) 25bp-long random sequence (corresponding to 1)<br />
<br />
After integrating DNA segment of Landing pad into the genome of E coli, we completed the genetic engineering of the genome of E coli. All the five parts of landing pad are designed for subsequent recombination. <br />
<br />
Besides, in order to achieve different recombination goals, we designed several landing pads of different sequences.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion"><font face="Comic Sans MS" size=3>'Landing Pad Construction and Insertion'</font></a></html> to learn about the details of Landing Pad design. <br />
<br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#att"><font face="Comic Sans MS" size=3>'link'</font></a></html> to learn more about ATT recombination.<br />
<br />
<br />
After the integration of landing pad, we have to insert another DNA fragment called Helper Plasmid(HP), which provides necessary tools for in vivo recombination. <br />
<br />
In this plasmid(Helper Plasmid), there are two genes used for recombination. <br />
<br />
The first one encodes restriction enzyme I-scel after induction of L-arabinose. I-scel is able to recognize the 15bp-length sequence flanking a target DNA fragment and cut out the fragment, therefore providing DNA segments for recombination mediated by other components.<br />
<br />
The second one encodes the enzyme responsible for recombination called Lamda-Red. With the presence of IPTG, Lamda-Red will recombine DNA fragments flanked by the same DNA sequence recognized by Lamda-Red.<br />
<br />
In addition, Helper Plasmid contains a specific temperature-sensitive replicon, which is used to control the replication via temperature changes. We can remove the replicon when necessary.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi"><font face="Comic Sans MS" size=3>'HP insertion'</font></a></html> to learn more about Helper Plasmid.<br />
<br />
<br />
Up till now, we successfully engineered a ‘recepient strain’. Then we have to transform the ‘recepient strain’ with the Donor Plasmid(DP) containing recombination fragments. Donor Plasmid contains several Insertion Fragments and each Insertion Fragment contains following five parts:<br />
<br />
1) one 15bp-long restriction enzyme I-scel recognition site<br />
<br />
2) one 25bp-long random sequence<br />
<br />
3) one fragment for insertion and recombination<br />
<br />
4) one 25bp-long random sequence<br />
<br />
5) one 15bp-long restriction enzyme I-scel recognition site(in correspondence with 1)<br />
<br />
(Note: the order of the five parts differ from that of landing pad)<br />
<br />
As we previously mentioned, Donor plasmid contains several Insertion Fragments. Based on the flanking landing pad sequence, we can ascribe Insertion Fragments to the same group as long as the flanking landing pad sequences of those Insertion Fragments are the same.<br />
<br />
In order to achieve different goals, we design different Donor Plasmids, different Donor Plasmids contain different number of Insertion Fragments, which belong to different groups.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc"><font face="Comic Sans MS" size=3>‘DP construction’</font></a></html> to learn about the details of Donor Plasmid and Insertion Fragments and the strategies employed in our design. <br />
<br />
<br />
As we all know, antibody diversity mainly lean on the number of the fragments used for recombination. Therefore, in order to mimic the antibody generation process, we need to construct Donor Plasmid which contains large sum of Insertion Fragments. Theoretically, we can construct Donor Plasmid of this kind. However, due to the time limit of the competition, we didn’t try to construct DP containing many Insertion Fragments.<br />
<br />
In spite of this, in order to meet the needs, we designed two methods to construct plasmids on a large scale and attempted the usage in parts of our Donor Plasmids.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Strategies"><font face="Comic Sans MS" size=3>'DP construction and Strategies'</font></a></html> for the methods of rapid plasmid construction. <br />
<br />
<br />
Besides, we have applied for two BBFRFC, which are BBFRFC61 and BBFRFC 62 respectively.<br />
<br />
Then, after transformation of the prepared ‘recepient strain’ with Donor Plasmid and the induction of corresponding agents, I-scel enzyme cuts out DNA fragments from DP and LP, generating numerous Insertion Fragments. Besides, antibiotic resistance gene integrated into E coli genome is also cut out, providing recombination sites for Insertion Fragments.<br />
<br />
Provided with Insertion Fragments mentioned above and Lamda-Red expressed by Donor Plasmid, Insertion Fragments recombine with the corresponding landing pad sequence in the genome and thus get inserted into the correct site of the genome. Based on our design of Landing pad, we can ensure that only one Insertion Fragment from one group is randomly inserted into the genome, therefore mimicking the VDJ recombination process.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri"><font face="Comic Sans MS" size=3>‘DP Insertion and Recombination Induction’</font></a></html> to learn more about this part. <br />
<br />
<br />
Engineered E coli cells which have undergone successful recombination can be selected out of the pool through specific screening methods. After tests on different strains, our project achieve a recombination rate above 50% based on our current techniques.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/experiments#res"><font face="Comic Sans MS" size=3>‘Result’</font></a></html> to learn more about the identification of recombination rate. <br />
<br />
<br/><br/><br />
<br />
<br />
<br />
<br />
===Module 2===<br />
<br />
The goal of the second module in our project is aimed at selection of specific antibodies. Due to the maturity of existing antibody screening technique, our work mainly focuses on imitation of mammalian antibody selection, thus providing possible alternatives for existing methods.<br />
<br />
The outline of our ideas is to find mechanisms similar to in vivo antibody selection and take in,to account the industry production costs, controllability and reliability, thus developing new methods for antibody selection.<br />
In fact, in mammalian system, antibody selection is achieved through the activation of B lymphocytes and thus the rapid proliferation of the B lymphocytes that express the specific antibodies that bind to antigen. Simply put, the whole process relies on the interaction between antigen epitopes and membrane integral immunoglobins.<br />
<br />
To mimic the activation mediated by membrane integral immunoglobins, we develop ToxR-mediated transmembrane activation signal system. In this system, the interaction between antibodies and antigen triggered downstream expression of reporter genes, thus providing signals for selection.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2"><font face="Comic Sans MS" size=3>‘ToxR-based Transmembrane Signaling Pathway Method’</font></a></html> for detailed description of this method.<br />
<br />
<br />
In addition, we develop a technique called ‘Bacterial based Microarray’ for selection purpose. In this method, we combine membrane display technique and high throughput microarray technique, that is, a bacterial based microarray method.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1"><font face="Comic Sans MS" size=3>’Bacterial based microarray’</font></a></html> for details.<br />
<br />
<br />
In order to find methods for antibody selection, we cooperate with other iGEM teams. For example, we talked with Macquarie_Australia iGEM team about the project. The techniques involved in their project might be useful for our selection methods. Therefore, members from both teams worked together to research on this problem.<br />
<br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3"><font face="Comic Sans MS" size=3>'link'</font></a></html> to learn more about our cooperation.<br />
<br />
<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens. <br />
Our system is aiming at imitating this recombination process, using E.coli as the gene carrier.<br />
It is known to all that regional genes (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and the recombination occurs when VDJ recombinase are expressed. We choose RecA enzyme to induce the recombination, while use I-Sel enzyme to cut the genome, just mimicking the process happened in B cells.<br />
<br/><br />
<br/><br />
<br />
==Comparison between natural antibody production and E. Coli system==<br />
<br/><br />
===VDJ Recombination vs Lading Pad Recombination===<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is the mechanism by which immunoglobulin (Ig) and T cell receptors (TCR) are generated in the immune system. Through V(D)J recombination, Variable, Diverse, and Joining gene segments are randomly combined, thus encoding various kinds of immunoglobins and T cell receptors to recognize potential antigens. <br />
During the process of B cell maturation, gene segments are rearranged and thus transcribe mRNA that can produce diverse types of antibody for recognition of billions of potential epitopes. The rearrangement brings three gene segments (termed V, D, and J) in close proximity, which are then joined together head to tail, the process of which is known as V(D)J recombination based on the V,D,J gene segments involved. Multiple copies of the V, D, and J genes in the human genome are shuffled in the process, and a specific antibody will be generate among millions of other possible combinations. Because gene are joined together permanently, one mature B cell will produce only one specific antibody.<br />
<br />
Our system is aimed to mimic this recombination process, using E.coli as the carrier.<br />
Through previous research in immunology field, each kind of gene segments (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and recombination occurs when VDJ recombinase recognize the specific signal sequence and mediate the recombination process. In our project, we design a sequence, which carries separated parts of one complete gene and integrate synthesized DNA fragment into the genome of E coli via landing pad method. Then we use I-Sel enzyme to cut the gene segments out of E coli genome, into which gene segments are inserted and choose RecA enzyme to recognize the previously designed signal sequence flanking our target sequence and recombine the gene segments, just like what recombinase accomplish in B cells. The whole process is called landing pad recombination.<br />
<br />
===Somatic hyper-mutation vs Junctional mutation===<br />
<br />
Somatic hypermutation involves a programmed process of mutation affecting the variable regions of immunoglobulin genes. During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is at least 105-106 fold greater than the normal rate of mutation across the genome. Variation is mainly in the form of single base substitutions, with insertions and deletions being less common. These mutations occur mostly at “hotspots” in the DNA, known as hypervariable regions. These regions correspond to the complementarity determining regions; the sites involved in antigen recognition on the immunoglobulin. This directed hypermutation allows for the selection of B cells that express immunoglobulin receptors possessing an enhanced ability to recognize and bind a specific foreign antigen.<br />
<br />
In addition, the cross ligation, namely Landing pad recombination, has higher opportunity to mutate in the link sequence. The introduction of 4 junctional shuffler 64 will meet the need of the rest 107 diversity, perfectly undertaking the work in the antibody diversity construction that hyper-mutation does in the highly variable region. In this way, we have finished a building library, waiting for the specialized antigen to select.<br />
<br />
===Antigen-specific Selection vs CBD-Based Microarray and ToxR-Based Transmembrane pathway method===<br />
In mammalian immune system, billions of antibodies will be generated. Then how can the immune system find out specific antibody that indiscriminately recognize the specific antigen that comes in. Generally speaking, due to the fact that one mature B cell only generates one specific immunoglobin, once the membrane integral immunoglobin bind to the specific antigen, a series of intracellular response, such as gene activation and cell differetiation will be triggered in B cell. Then, B cells expressing the specific antibodies that recognize the specific antigen will proliferate and generate enormous amount of antibodies that help eliminate the specific antigen.<br />
<br />
Simply put, antibody selection is accomplished through the specific interaction between the membrane immunoglobin and the specific antigen. Therefore, in our project, we also plan to mimic this process through interaction between the antibodies generated through landing pad recombination and the antigen we target. CBD-based Microarray and ToxR-based Transmembrane pathway are the two methods by which we achieve this goal.<br />
CBD domain, which interacts with cellulose, potentially provides us way to locate our E.coli to the microarray coated with cellulose. OmpA (outer membrane protein A), displays the antibody and antigen out to the membrane surface. Then by applying the antibodies generated through landing pad recombination to the microarray, antibodies that bind to the antigen will be retained and thus selected out of the pool of antibodies.<br />
<br />
Alternatively, the ToxR-based transmembrane pathway method shows a different strategy for us to seek the right folded antibody. Once the antigen comes, ToxR outer part will form dimer shape and the downstream signal occur, supplying the approach of reporters, such as GFP, to examine and pick out what we want.<br />
<br />
<html><br />
&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a><br />
</body><br />
</div><br />
<br />
</div><br />
</div></div></body><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/project/outlineTeam:Tsinghua/project/outline2010-10-27T16:23:11Z<p>Gumilton: /* Module 2 */</p>
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<h1>Outline</h1><br />
<br />
<div class="content_block"><br />
<br />
<br/><br />
In order to realize the production of antibodies in prokaryotic cells, we firstly investigate the whole process of antibody production in mammalian immune system and try to simulate the whole process via the methods and concepts of synthetic biology. Finally, we successfully constructed the desired system. The comparison between the key steps involved in antibody generation and corresponding synthetic methods are shown in the chart below.<br />
<br/><br />
<br />
<table border="2" bordercolor="maroon" bgcolor="silver"><br />
<tbody><br />
<tr><br />
<th colspan=3>Antibody Production</th><br />
<th width=50px></th><br />
<th colspan=2>E Coli. Production System</th></tr><br />
<tr><br />
<td rowspan=4 width=70px><a href="#VDJ_Recombination_vs_Lading_Pad_Recombination">VDJ Recombination</a></td><br />
<td rowspan=3 width=70px>Preparation</td><br />
<td width=70px>RSS Sequence</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">Landing Pad Insertion</a></td><br />
<td rowspan=4 width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1">Module I: Recombination System</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Recombinase</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">Helper Plasmid Insertion</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Library</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">Donor Plasmid Construction</a></td><br />
</tr><br />
<tr><br />
<td>Recombination</td><br />
<td colspan=2><img src="https://static.igem.org/mediawiki/2010/d/d6/THUarrow2.png" width=180></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">DP Insertion and Recombination Induction</a></td><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="#Somatic_hyper-mutation_vs_Junctional_mutation">Somatic Hypermutation</a></td><br />
<td rowspan=2 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
<!---------<td>Junctional Diversity</td>----------------------><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">CBD-based Microarray</a></td><br />
<td rowspan=4><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2">Module II: Selection System</a></td><br />
</tr><br />
<tr><br />
<td rowspan=3><a href="#Antigen-specific_Selection_vs_CBD-Based_Microarray_and_ToxR-Based_Transmembrane_pathway_method">Antigen Selection</a></td><br />
<td rowspan=3 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">ToxR-based Transmembrane Pathway</a></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">Cooperation With Macquarie_Australia</a></td><br />
</tr><br />
</tbody><br />
</table><br />
</html><br />
<br/><br />
Generally speaking, antibody production in our project can be divided into two modules.<br />
<br />
<html><br />
<body bgcolor="#336699" text="#ffffff" link="#60a179"><br />
<br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1"><font face="Comic Sans MS" size="4">Module I: Generation of antibody library</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2"><font face="Comic Sans MS" size="4">Module II: Selection of specific antibodies</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
</html><br />
<br />
==Project Design==<br />
<br />
===Module 1===<br />
<br />
'''Antibodies Library Diversity & Randomicity'''<br />
[[Image:TSModule1.PNG|650px]]<br />
<br />
<br />
The first module is carried out by a novel method called E coli in vivo recombination system. The theoretical and experimental basis comes from the work of Thomas E. Kuhlman and Edward C. Cox. We managed to modify their system, wishing to utilize it for simulation of antibody recombination.<br />
<br />
Firstly, we try to insert a DNA segment called”Landing Pad(LP)” into the specific sites of E coli via Att recombination. Landing pad sequence consists of the following parts(from 5’ to 3’ in the sequence):<br />
<br />
1)25bp-long random sequence<br />
<br />
2)15bp-long recognition sequence of restriction enzyme I-scel<br />
<br />
3) antibiotic resistance gene used for antibody selection<br />
<br />
4) 15bp-long recognition sequence of restriction enzyme I-scel(corresponding to 2)<br />
<br />
5) 25bp-long random sequence (corresponding to 1)<br />
<br />
After integrating DNA segment of Landing pad into the genome of E coli, we completed the genetic engineering of the genome of E coli. All the five parts of landing pad are designed for subsequent recombination. <br />
<br />
Besides, in order to achieve different recombination goals, we designed several landing pads of different sequences.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion"><font face="Comic Sans MS" size=3>'Landing Pad Construction and Insertion'</font></a></html> to learn about the details of Landing Pad design. <br />
<br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#att"><font face="Comic Sans MS" size=3>'link'</font></a></html> to learn more about ATT recombination.<br />
<br />
<br />
After the integration of landing pad, we have to insert another DNA fragment called Helper Plasmid(HP), which provides necessary tools for in vivo recombination. <br />
<br />
In this plasmid(Helper Plasmid), there are two genes used for recombination. <br />
<br />
The first one encodes restriction enzyme I-scel after induction of L-arabinose. I-scel is able to recognize the 15bp-length sequence flanking a target DNA fragment and cut out the fragment, therefore providing DNA segments for recombination mediated by other components.<br />
<br />
The second one encodes the enzyme responsible for recombination called Lamda-Red. With the presence of IPTG, Lamda-Red will recombine DNA fragments flanked by the same DNA sequence recognized by Lamda-Red.<br />
<br />
In addition, Helper Plasmid contains a specific temperature-sensitive replicon, which is used to control the replication via temperature changes. We can remove the replicon when necessary.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi"><font face="Comic Sans MS" size=3>'HP insertion'</font></a></html> to learn more about Helper Plasmid.<br />
<br />
<br />
Up till now, we successfully engineered a ‘recepient strain’. Then we have to transform the ‘recepient strain’ with the Donor Plasmid(DP) containing recombination fragments. Donor Plasmid contains several Insertion Fragments and each Insertion Fragment contains following five parts:<br />
<br />
1) one 15bp-long restriction enzyme I-scel recognition site<br />
<br />
2) one 25bp-long random sequence<br />
<br />
3) one fragment for insertion and recombination<br />
<br />
4) one 25bp-long random sequence<br />
<br />
5) one 15bp-long restriction enzyme I-scel recognition site(in correspondence with 1)<br />
<br />
(Note: the order of the five parts differ from that of landing pad)<br />
<br />
As we previously mentioned, Donor plasmid contains several Insertion Fragments. Based on the flanking landing pad sequence, we can ascribe Insertion Fragments to the same group as long as the flanking landing pad sequences of those Insertion Fragments are the same.<br />
<br />
In order to achieve different goals, we design different Donor Plasmids, different Donor Plasmids contain different number of Insertion Fragments, which belong to different groups.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc"><font face="Comic Sans MS" size=3>‘DP construction’</font></a></html> to learn about the details of Donor Plasmid and Insertion Fragments and the strategies employed in our design. <br />
<br />
<br />
As we all know, antibody diversity mainly lean on the number of the fragments used for recombination. Therefore, in order to mimic the antibody generation process, we need to construct Donor Plasmid which contains large sum of Insertion Fragments. Theoretically, we can construct Donor Plasmid of this kind. However, due to the time limit of the competition, we didn’t try to construct DP containing many Insertion Fragments.<br />
<br />
In spite of this, in order to meet the needs, we designed two methods to construct plasmids on a large scale and attempted the usage in parts of our Donor Plasmids.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Strategies"><font face="Comic Sans MS" size=3>'DP construction and Strategies'</font></a></html> for the methods of rapid plasmid construction. <br />
<br />
<br />
Besides, we have applied for two BBFRFC, which are BBFRFC61 and BBFRFC 62 respectively.<br />
<br />
Then, after transformation of the prepared ‘recepient strain’ with Donor Plasmid and the induction of corresponding agents, I-scel enzyme cuts out DNA fragments from DP and LP, generating numerous Insertion Fragments. Besides, antibiotic resistance gene integrated into E coli genome is also cut out, providing recombination sites for Insertion Fragments.<br />
<br />
Provided with Insertion Fragments mentioned above and Lamda-Red expressed by Donor Plasmid, Insertion Fragments recombine with the corresponding landing pad sequence in the genome and thus get inserted into the correct site of the genome. Based on our design of Landing pad, we can ensure that only one Insertion Fragment from one group is randomly inserted into the genome, therefore mimicking the VDJ recombination process.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri"><font face="Comic Sans MS" size=3>‘DP Insertion and Recombination Induction’</font></a></html> to learn more about this part. <br />
<br />
<br />
Engineered E coli cells which have undergone successful recombination can be selected out of the pool through specific screening methods. After tests on different strains, our project achieve a recombination rate above 50% based on our current techniques.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/experiments#res"><font face="Comic Sans MS" size=3>‘Result’</font></a></html> to learn more about the identification of recombination rate. <br />
<br />
<br/><br/><br />
===Module 2===<br />
<br />
The goal of the second module in our project is aimed at selection of specific antibodies. Due to the maturity of existing antibody screening technique, our work mainly focuses on imitation of mammalian antibody selection, thus providing possible alternatives for existing methods.<br />
<br />
The outline of our ideas is to find mechanisms similar to in vivo antibody selection and take in,to account the industry production costs, controllability and reliability, thus developing new methods for antibody selection.<br />
In fact, in mammalian system, antibody selection is achieved through the activation of B lymphocytes and thus the rapid proliferation of the B lymphocytes that express the specific antibodies that bind to antigen. Simply put, the whole process relies on the interaction between antigen epitopes and membrane integral immunoglobins.<br />
<br />
To mimic the activation mediated by membrane integral immunoglobins, we develop ToxR-mediated transmembrane activation signal system. In this system, the interaction between antibodies and antigen triggered downstream expression of reporter genes, thus providing signals for selection.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2"><font face="Comic Sans MS" size=3>‘ToxR-based Transmembrane Signaling Pathway Method’</font></a></html> for detailed description of this method.<br />
<br />
<br />
In addition, we develop a technique called ‘Bacterial based Microarray’ for selection purpose. In this method, we combine membrane display technique and high throughput microarray technique, that is, a bacterial based microarray method.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1"><font face="Comic Sans MS" size=3>’Bacterial based microarray’</font></a></html> for details.<br />
<br />
<br />
In order to find methods for antibody selection, we cooperate with other iGEM teams. For example, we talked with Macquarie_Australia iGEM team about the project. The techniques involved in their project might be useful for our selection methods. Therefore, members from both teams worked together to research on this problem.<br />
<br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3"><font face="Comic Sans MS" size=3>'link'</font></a></html> to learn more about our cooperation.<br />
<br />
<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens. <br />
Our system is aiming at imitating this recombination process, using E.coli as the gene carrier.<br />
It is known to all that regional genes (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and the recombination occurs when VDJ recombinase are expressed. We choose RecA enzyme to induce the recombination, while use I-Sel enzyme to cut the genome, just mimicking the process happened in B cells.<br />
<br/><br />
<br/><br />
<br />
==Comparison between natural antibody production and E. Coli system==<br />
<br/><br />
===VDJ Recombination vs Lading Pad Recombination===<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is the mechanism by which immunoglobulin (Ig) and T cell receptors (TCR) are generated in the immune system. Through V(D)J recombination, Variable, Diverse, and Joining gene segments are randomly combined, thus encoding various kinds of immunoglobins and T cell receptors to recognize potential antigens. <br />
During the process of B cell maturation, gene segments are rearranged and thus transcribe mRNA that can produce diverse types of antibody for recognition of billions of potential epitopes. The rearrangement brings three gene segments (termed V, D, and J) in close proximity, which are then joined together head to tail, the process of which is known as V(D)J recombination based on the V,D,J gene segments involved. Multiple copies of the V, D, and J genes in the human genome are shuffled in the process, and a specific antibody will be generate among millions of other possible combinations. Because gene are joined together permanently, one mature B cell will produce only one specific antibody.<br />
<br />
Our system is aimed to mimic this recombination process, using E.coli as the carrier.<br />
Through previous research in immunology field, each kind of gene segments (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and recombination occurs when VDJ recombinase recognize the specific signal sequence and mediate the recombination process. In our project, we design a sequence, which carries separated parts of one complete gene and integrate synthesized DNA fragment into the genome of E coli via landing pad method. Then we use I-Sel enzyme to cut the gene segments out of E coli genome, into which gene segments are inserted and choose RecA enzyme to recognize the previously designed signal sequence flanking our target sequence and recombine the gene segments, just like what recombinase accomplish in B cells. The whole process is called landing pad recombination.<br />
<br />
===Somatic hyper-mutation vs Junctional mutation===<br />
<br />
Somatic hypermutation involves a programmed process of mutation affecting the variable regions of immunoglobulin genes. During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is at least 105-106 fold greater than the normal rate of mutation across the genome. Variation is mainly in the form of single base substitutions, with insertions and deletions being less common. These mutations occur mostly at “hotspots” in the DNA, known as hypervariable regions. These regions correspond to the complementarity determining regions; the sites involved in antigen recognition on the immunoglobulin. This directed hypermutation allows for the selection of B cells that express immunoglobulin receptors possessing an enhanced ability to recognize and bind a specific foreign antigen.<br />
<br />
In addition, the cross ligation, namely Landing pad recombination, has higher opportunity to mutate in the link sequence. The introduction of 4 junctional shuffler 64 will meet the need of the rest 107 diversity, perfectly undertaking the work in the antibody diversity construction that hyper-mutation does in the highly variable region. In this way, we have finished a building library, waiting for the specialized antigen to select.<br />
<br />
===Antigen-specific Selection vs CBD-Based Microarray and ToxR-Based Transmembrane pathway method===<br />
In mammalian immune system, billions of antibodies will be generated. Then how can the immune system find out specific antibody that indiscriminately recognize the specific antigen that comes in. Generally speaking, due to the fact that one mature B cell only generates one specific immunoglobin, once the membrane integral immunoglobin bind to the specific antigen, a series of intracellular response, such as gene activation and cell differetiation will be triggered in B cell. Then, B cells expressing the specific antibodies that recognize the specific antigen will proliferate and generate enormous amount of antibodies that help eliminate the specific antigen.<br />
<br />
Simply put, antibody selection is accomplished through the specific interaction between the membrane immunoglobin and the specific antigen. Therefore, in our project, we also plan to mimic this process through interaction between the antibodies generated through landing pad recombination and the antigen we target. CBD-based Microarray and ToxR-based Transmembrane pathway are the two methods by which we achieve this goal.<br />
CBD domain, which interacts with cellulose, potentially provides us way to locate our E.coli to the microarray coated with cellulose. OmpA (outer membrane protein A), displays the antibody and antigen out to the membrane surface. Then by applying the antibodies generated through landing pad recombination to the microarray, antibodies that bind to the antigen will be retained and thus selected out of the pool of antibodies.<br />
<br />
Alternatively, the ToxR-based transmembrane pathway method shows a different strategy for us to seek the right folded antibody. Once the antigen comes, ToxR outer part will form dimer shape and the downstream signal occur, supplying the approach of reporters, such as GFP, to examine and pick out what we want.<br />
<br />
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</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/project/outlineTeam:Tsinghua/project/outline2010-10-27T16:22:16Z<p>Gumilton: /* Project Design */</p>
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<div id="main_content"><a name="outline"></a><br />
<h1>Outline</h1><br />
<br />
<div class="content_block"><br />
<br />
<br/><br />
In order to realize the production of antibodies in prokaryotic cells, we firstly investigate the whole process of antibody production in mammalian immune system and try to simulate the whole process via the methods and concepts of synthetic biology. Finally, we successfully constructed the desired system. The comparison between the key steps involved in antibody generation and corresponding synthetic methods are shown in the chart below.<br />
<br/><br />
<br />
<table border="2" bordercolor="maroon" bgcolor="silver"><br />
<tbody><br />
<tr><br />
<th colspan=3>Antibody Production</th><br />
<th width=50px></th><br />
<th colspan=2>E Coli. Production System</th></tr><br />
<tr><br />
<td rowspan=4 width=70px><a href="#VDJ_Recombination_vs_Lading_Pad_Recombination">VDJ Recombination</a></td><br />
<td rowspan=3 width=70px>Preparation</td><br />
<td width=70px>RSS Sequence</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">Landing Pad Insertion</a></td><br />
<td rowspan=4 width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1">Module I: Recombination System</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Recombinase</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">Helper Plasmid Insertion</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Library</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">Donor Plasmid Construction</a></td><br />
</tr><br />
<tr><br />
<td>Recombination</td><br />
<td colspan=2><img src="https://static.igem.org/mediawiki/2010/d/d6/THUarrow2.png" width=180></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">DP Insertion and Recombination Induction</a></td><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="#Somatic_hyper-mutation_vs_Junctional_mutation">Somatic Hypermutation</a></td><br />
<td rowspan=2 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
<!---------<td>Junctional Diversity</td>----------------------><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">CBD-based Microarray</a></td><br />
<td rowspan=4><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2">Module II: Selection System</a></td><br />
</tr><br />
<tr><br />
<td rowspan=3><a href="#Antigen-specific_Selection_vs_CBD-Based_Microarray_and_ToxR-Based_Transmembrane_pathway_method">Antigen Selection</a></td><br />
<td rowspan=3 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">ToxR-based Transmembrane Pathway</a></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">Cooperation With Macquarie_Australia</a></td><br />
</tr><br />
</tbody><br />
</table><br />
</html><br />
<br/><br />
Generally speaking, antibody production in our project can be divided into two modules.<br />
<br />
<html><br />
<body bgcolor="#336699" text="#ffffff" link="#60a179"><br />
<br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1"><font face="Comic Sans MS" size="4">Module I: Generation of antibody library</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2"><font face="Comic Sans MS" size="4">Module II: Selection of specific antibodies</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
</html><br />
<br />
==Project Design==<br />
<br />
===Module 1===<br />
<br />
'''Antibodies Library Diversity & Randomicity'''<br />
[[Image:TSModule1.PNG|650px]]<br />
<br />
<br />
The first module is carried out by a novel method called E coli in vivo recombination system. The theoretical and experimental basis comes from the work of Thomas E. Kuhlman and Edward C. Cox. We managed to modify their system, wishing to utilize it for simulation of antibody recombination.<br />
<br />
Firstly, we try to insert a DNA segment called”Landing Pad(LP)” into the specific sites of E coli via Att recombination. Landing pad sequence consists of the following parts(from 5’ to 3’ in the sequence):<br />
<br />
1)25bp-long random sequence<br />
<br />
2)15bp-long recognition sequence of restriction enzyme I-scel<br />
<br />
3) antibiotic resistance gene used for antibody selection<br />
<br />
4) 15bp-long recognition sequence of restriction enzyme I-scel(corresponding to 2)<br />
<br />
5) 25bp-long random sequence (corresponding to 1)<br />
<br />
After integrating DNA segment of Landing pad into the genome of E coli, we completed the genetic engineering of the genome of E coli. All the five parts of landing pad are designed for subsequent recombination. <br />
<br />
Besides, in order to achieve different recombination goals, we designed several landing pads of different sequences.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion"><font face="Comic Sans MS" size=3>'Landing Pad Construction and Insertion'</font></a></html> to learn about the details of Landing Pad design. <br />
<br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#att"><font face="Comic Sans MS" size=3>'link'</font></a></html> to learn more about ATT recombination.<br />
<br />
<br />
After the integration of landing pad, we have to insert another DNA fragment called Helper Plasmid(HP), which provides necessary tools for in vivo recombination. <br />
<br />
In this plasmid(Helper Plasmid), there are two genes used for recombination. <br />
<br />
The first one encodes restriction enzyme I-scel after induction of L-arabinose. I-scel is able to recognize the 15bp-length sequence flanking a target DNA fragment and cut out the fragment, therefore providing DNA segments for recombination mediated by other components.<br />
<br />
The second one encodes the enzyme responsible for recombination called Lamda-Red. With the presence of IPTG, Lamda-Red will recombine DNA fragments flanked by the same DNA sequence recognized by Lamda-Red.<br />
<br />
In addition, Helper Plasmid contains a specific temperature-sensitive replicon, which is used to control the replication via temperature changes. We can remove the replicon when necessary.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi"><font face="Comic Sans MS" size=3>'HP insertion'</font></a></html> to learn more about Helper Plasmid.<br />
<br />
<br />
Up till now, we successfully engineered a ‘recepient strain’. Then we have to transform the ‘recepient strain’ with the Donor Plasmid(DP) containing recombination fragments. Donor Plasmid contains several Insertion Fragments and each Insertion Fragment contains following five parts:<br />
<br />
1) one 15bp-long restriction enzyme I-scel recognition site<br />
<br />
2) one 25bp-long random sequence<br />
<br />
3) one fragment for insertion and recombination<br />
<br />
4) one 25bp-long random sequence<br />
<br />
5) one 15bp-long restriction enzyme I-scel recognition site(in correspondence with 1)<br />
<br />
(Note: the order of the five parts differ from that of landing pad)<br />
<br />
As we previously mentioned, Donor plasmid contains several Insertion Fragments. Based on the flanking landing pad sequence, we can ascribe Insertion Fragments to the same group as long as the flanking landing pad sequences of those Insertion Fragments are the same.<br />
<br />
In order to achieve different goals, we design different Donor Plasmids, different Donor Plasmids contain different number of Insertion Fragments, which belong to different groups.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc"><font face="Comic Sans MS" size=3>‘DP construction’</font></a></html> to learn about the details of Donor Plasmid and Insertion Fragments and the strategies employed in our design. <br />
<br />
<br />
As we all know, antibody diversity mainly lean on the number of the fragments used for recombination. Therefore, in order to mimic the antibody generation process, we need to construct Donor Plasmid which contains large sum of Insertion Fragments. Theoretically, we can construct Donor Plasmid of this kind. However, due to the time limit of the competition, we didn’t try to construct DP containing many Insertion Fragments.<br />
<br />
In spite of this, in order to meet the needs, we designed two methods to construct plasmids on a large scale and attempted the usage in parts of our Donor Plasmids.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Strategies"><font face="Comic Sans MS" size=3>'DP construction and Strategies'</font></a></html> for the methods of rapid plasmid construction. <br />
<br />
<br />
Besides, we have applied for two BBFRFC, which are BBFRFC61 and BBFRFC 62 respectively.<br />
<br />
Then, after transformation of the prepared ‘recepient strain’ with Donor Plasmid and the induction of corresponding agents, I-scel enzyme cuts out DNA fragments from DP and LP, generating numerous Insertion Fragments. Besides, antibiotic resistance gene integrated into E coli genome is also cut out, providing recombination sites for Insertion Fragments.<br />
<br />
Provided with Insertion Fragments mentioned above and Lamda-Red expressed by Donor Plasmid, Insertion Fragments recombine with the corresponding landing pad sequence in the genome and thus get inserted into the correct site of the genome. Based on our design of Landing pad, we can ensure that only one Insertion Fragment from one group is randomly inserted into the genome, therefore mimicking the VDJ recombination process.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri"><font face="Comic Sans MS" size=3>‘DP Insertion and Recombination Induction’</font></a></html> to learn more about this part. <br />
<br />
<br />
Engineered E coli cells which have undergone successful recombination can be selected out of the pool through specific screening methods. After tests on different strains, our project achieve a recombination rate above 50% based on our current techniques.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/experiments#res"><font face="Comic Sans MS" size=3>‘Result’</font></a></html> to learn more about the identification of recombination rate. <br />
<br />
===Module 2===<br />
<br />
The goal of the second module in our project is aimed at selection of specific antibodies. Due to the maturity of existing antibody screening technique, our work mainly focuses on imitation of mammalian antibody selection, thus providing possible alternatives for existing methods.<br />
<br />
The outline of our ideas is to find mechanisms similar to in vivo antibody selection and take in,to account the industry production costs, controllability and reliability, thus developing new methods for antibody selection.<br />
In fact, in mammalian system, antibody selection is achieved through the activation of B lymphocytes and thus the rapid proliferation of the B lymphocytes that express the specific antibodies that bind to antigen. Simply put, the whole process relies on the interaction between antigen epitopes and membrane integral immunoglobins.<br />
<br />
To mimic the activation mediated by membrane integral immunoglobins, we develop ToxR-mediated transmembrane activation signal system. In this system, the interaction between antibodies and antigen triggered downstream expression of reporter genes, thus providing signals for selection.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2"><font face="Comic Sans MS" size=3>‘ToxR-based Transmembrane Signaling Pathway Method’</font></a></html> for detailed description of this method.<br />
<br />
<br />
In addition, we develop a technique called ‘Bacterial based Microarray’ for selection purpose. In this method, we combine membrane display technique and high throughput microarray technique, that is, a bacterial based microarray method.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1"><font face="Comic Sans MS" size=3>’Bacterial based microarray’</font></a></html> for details.<br />
<br />
<br />
In order to find methods for antibody selection, we cooperate with other iGEM teams. For example, we talked with Macquarie_Australia iGEM team about the project. The techniques involved in their project might be useful for our selection methods. Therefore, members from both teams worked together to research on this problem.<br />
<br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3"><font face="Comic Sans MS" size=3>'link'</font></a></html> to learn more about our cooperation.<br />
<br />
<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens. <br />
Our system is aiming at imitating this recombination process, using E.coli as the gene carrier.<br />
It is known to all that regional genes (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and the recombination occurs when VDJ recombinase are expressed. We choose RecA enzyme to induce the recombination, while use I-Sel enzyme to cut the genome, just mimicking the process happened in B cells.<br />
<br/><br />
<br/><br />
<br />
==Comparison between natural antibody production and E. Coli system==<br />
<br/><br />
===VDJ Recombination vs Lading Pad Recombination===<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is the mechanism by which immunoglobulin (Ig) and T cell receptors (TCR) are generated in the immune system. Through V(D)J recombination, Variable, Diverse, and Joining gene segments are randomly combined, thus encoding various kinds of immunoglobins and T cell receptors to recognize potential antigens. <br />
During the process of B cell maturation, gene segments are rearranged and thus transcribe mRNA that can produce diverse types of antibody for recognition of billions of potential epitopes. The rearrangement brings three gene segments (termed V, D, and J) in close proximity, which are then joined together head to tail, the process of which is known as V(D)J recombination based on the V,D,J gene segments involved. Multiple copies of the V, D, and J genes in the human genome are shuffled in the process, and a specific antibody will be generate among millions of other possible combinations. Because gene are joined together permanently, one mature B cell will produce only one specific antibody.<br />
<br />
Our system is aimed to mimic this recombination process, using E.coli as the carrier.<br />
Through previous research in immunology field, each kind of gene segments (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and recombination occurs when VDJ recombinase recognize the specific signal sequence and mediate the recombination process. In our project, we design a sequence, which carries separated parts of one complete gene and integrate synthesized DNA fragment into the genome of E coli via landing pad method. Then we use I-Sel enzyme to cut the gene segments out of E coli genome, into which gene segments are inserted and choose RecA enzyme to recognize the previously designed signal sequence flanking our target sequence and recombine the gene segments, just like what recombinase accomplish in B cells. The whole process is called landing pad recombination.<br />
<br />
===Somatic hyper-mutation vs Junctional mutation===<br />
<br />
Somatic hypermutation involves a programmed process of mutation affecting the variable regions of immunoglobulin genes. During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is at least 105-106 fold greater than the normal rate of mutation across the genome. Variation is mainly in the form of single base substitutions, with insertions and deletions being less common. These mutations occur mostly at “hotspots” in the DNA, known as hypervariable regions. These regions correspond to the complementarity determining regions; the sites involved in antigen recognition on the immunoglobulin. This directed hypermutation allows for the selection of B cells that express immunoglobulin receptors possessing an enhanced ability to recognize and bind a specific foreign antigen.<br />
<br />
In addition, the cross ligation, namely Landing pad recombination, has higher opportunity to mutate in the link sequence. The introduction of 4 junctional shuffler 64 will meet the need of the rest 107 diversity, perfectly undertaking the work in the antibody diversity construction that hyper-mutation does in the highly variable region. In this way, we have finished a building library, waiting for the specialized antigen to select.<br />
<br />
===Antigen-specific Selection vs CBD-Based Microarray and ToxR-Based Transmembrane pathway method===<br />
In mammalian immune system, billions of antibodies will be generated. Then how can the immune system find out specific antibody that indiscriminately recognize the specific antigen that comes in. Generally speaking, due to the fact that one mature B cell only generates one specific immunoglobin, once the membrane integral immunoglobin bind to the specific antigen, a series of intracellular response, such as gene activation and cell differetiation will be triggered in B cell. Then, B cells expressing the specific antibodies that recognize the specific antigen will proliferate and generate enormous amount of antibodies that help eliminate the specific antigen.<br />
<br />
Simply put, antibody selection is accomplished through the specific interaction between the membrane immunoglobin and the specific antigen. Therefore, in our project, we also plan to mimic this process through interaction between the antibodies generated through landing pad recombination and the antigen we target. CBD-based Microarray and ToxR-based Transmembrane pathway are the two methods by which we achieve this goal.<br />
CBD domain, which interacts with cellulose, potentially provides us way to locate our E.coli to the microarray coated with cellulose. OmpA (outer membrane protein A), displays the antibody and antigen out to the membrane surface. Then by applying the antibodies generated through landing pad recombination to the microarray, antibodies that bind to the antigen will be retained and thus selected out of the pool of antibodies.<br />
<br />
Alternatively, the ToxR-based transmembrane pathway method shows a different strategy for us to seek the right folded antibody. Once the antigen comes, ToxR outer part will form dimer shape and the downstream signal occur, supplying the approach of reporters, such as GFP, to examine and pick out what we want.<br />
<br />
<html><br />
&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a><br />
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</div></div></body><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/project/outlineTeam:Tsinghua/project/outline2010-10-27T16:19:53Z<p>Gumilton: /* Project Design */</p>
<hr />
<div>__NOTOC__<br />
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<script><br />
navlist=new Array("Background", "Project Outline", "Module I", "Module II", "Future");<br />
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<div id="main_content"><a name="outline"></a><br />
<h1>Outline</h1><br />
<br />
<div class="content_block"><br />
<br />
<br/><br />
In order to realize the production of antibodies in prokaryotic cells, we firstly investigate the whole process of antibody production in mammalian immune system and try to simulate the whole process via the methods and concepts of synthetic biology. Finally, we successfully constructed the desired system. The comparison between the key steps involved in antibody generation and corresponding synthetic methods are shown in the chart below.<br />
<br/><br />
<br />
<table border="2" bordercolor="maroon" bgcolor="silver"><br />
<tbody><br />
<tr><br />
<th colspan=3>Antibody Production</th><br />
<th width=50px></th><br />
<th colspan=2>E Coli. Production System</th></tr><br />
<tr><br />
<td rowspan=4 width=70px><a href="#VDJ_Recombination_vs_Lading_Pad_Recombination">VDJ Recombination</a></td><br />
<td rowspan=3 width=70px>Preparation</td><br />
<td width=70px>RSS Sequence</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">Landing Pad Insertion</a></td><br />
<td rowspan=4 width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1">Module I: Recombination System</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Recombinase</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">Helper Plasmid Insertion</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Library</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">Donor Plasmid Construction</a></td><br />
</tr><br />
<tr><br />
<td>Recombination</td><br />
<td colspan=2><img src="https://static.igem.org/mediawiki/2010/d/d6/THUarrow2.png" width=180></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">DP Insertion and Recombination Induction</a></td><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="#Somatic_hyper-mutation_vs_Junctional_mutation">Somatic Hypermutation</a></td><br />
<td rowspan=2 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
<!---------<td>Junctional Diversity</td>----------------------><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">CBD-based Microarray</a></td><br />
<td rowspan=4><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2">Module II: Selection System</a></td><br />
</tr><br />
<tr><br />
<td rowspan=3><a href="#Antigen-specific_Selection_vs_CBD-Based_Microarray_and_ToxR-Based_Transmembrane_pathway_method">Antigen Selection</a></td><br />
<td rowspan=3 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">ToxR-based Transmembrane Pathway</a></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">Cooperation With Macquarie_Australia</a></td><br />
</tr><br />
</tbody><br />
</table><br />
</html><br />
<br/><br />
Generally speaking, antibody production in our project can be divided into two modules.<br />
<br />
<html><br />
<body bgcolor="#336699" text="#ffffff" link="#60a179"><br />
<br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1"><font face="Comic Sans MS" size="4">Module I: Generation of antibody library</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2"><font face="Comic Sans MS" size="4">Module II: Selection of specific antibodies</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
</html><br />
<br />
==Project Design==<br />
<br />
===Module 1===<br />
<br />
'''Antibodies Library Diversity & Randomicity'''<br />
[[Image:TSModule1.PNG|650px]]<br />
<br />
<br />
The first module is carried out by a novel method called E coli in vivo recombination system. The theoretical and experimental basis comes from the work of Thomas E. Kuhlman and Edward C. Cox. We managed to modify their system, wishing to utilize it for simulation of antibody recombination.<br />
<br />
Firstly, we try to insert a DNA segment called”Landing Pad(LP)” into the specific sites of E coli via Att recombination. Landing pad sequence consists of the following parts(from 5’ to 3’ in the sequence):<br />
<br />
1)25bp-long random sequence<br />
<br />
2)15bp-long recognition sequence of restriction enzyme I-scel<br />
<br />
3) antibiotic resistance gene used for antibody selection<br />
<br />
4) 15bp-long recognition sequence of restriction enzyme I-scel(corresponding to 2)<br />
<br />
5) 25bp-long random sequence (corresponding to 1)<br />
<br />
After integrating DNA segment of Landing pad into the genome of E coli, we completed the genetic engineering of the genome of E coli. All the five parts of landing pad are designed for subsequent recombination. <br />
<br />
Besides, in order to achieve different recombination goals, we designed several landing pads of different sequences.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion"><font face="Comic Sans MS" size=3>'Landing Pad Construction and Insertion'</font></a></html> to learn about the details of Landing Pad design. <br />
<br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#att"><font face="Comic Sans MS" size=3>'link'</font></a></html> to learn more about ATT recombination.<br />
<br />
<br />
After the integration of landing pad, we have to insert another DNA fragment called Helper Plasmid(HP), which provides necessary tools for in vivo recombination. <br />
<br />
In this plasmid(Helper Plasmid), there are two genes used for recombination. <br />
<br />
The first one encodes restriction enzyme I-scel after induction of L-arabinose. I-scel is able to recognize the 15bp-length sequence flanking a target DNA fragment and cut out the fragment, therefore providing DNA segments for recombination mediated by other components.<br />
<br />
The second one encodes the enzyme responsible for recombination called Lamda-Red. With the presence of IPTG, Lamda-Red will recombine DNA fragments flanked by the same DNA sequence recognized by Lamda-Red.<br />
<br />
In addition, Helper Plasmid contains a specific temperature-sensitive replicon, which is used to control the replication via temperature changes. We can remove the replicon when necessary.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi"><font face="Comic Sans MS" size=3>'HP insertion'</font></a></html> to learn more about Helper Plasmid.<br />
<br />
<br />
Up till now, we successfully engineered a ‘recepient strain’. Then we have to transform the ‘recepient strain’ with the Donor Plasmid(DP) containing recombination fragments. Donor Plasmid contains several Insertion Fragments and each Insertion Fragment contains following five parts:<br />
<br />
1) one 15bp-long restriction enzyme I-scel recognition site<br />
<br />
2) one 25bp-long random sequence<br />
<br />
3) one fragment for insertion and recombination<br />
<br />
4) one 25bp-long random sequence<br />
<br />
5) one 15bp-long restriction enzyme I-scel recognition site(in correspondence with 1)<br />
<br />
(Note: the order of the five parts differ from that of landing pad)<br />
<br />
As we previously mentioned, Donor plasmid contains several Insertion Fragments. Based on the flanking landing pad sequence, we can ascribe Insertion Fragments to the same group as long as the flanking landing pad sequences of those Insertion Fragments are the same.<br />
<br />
In order to achieve different goals, we design different Donor Plasmids, different Donor Plasmids contain different number of Insertion Fragments, which belong to different groups.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc"><font face="Comic Sans MS" size=3>‘DP construction’</font></a></html> to learn about the details of Donor Plasmid and Insertion Fragments and the strategies employed in our design. <br />
<br />
<br />
As we all know, antibody diversity mainly lean on the number of the fragments used for recombination. Therefore, in order to mimic the antibody generation process, we need to construct Donor Plasmid which contains large sum of Insertion Fragments. Theoretically, we can construct Donor Plasmid of this kind. However, due to the time limit of the competition, we didn’t try to construct DP containing many Insertion Fragments.<br />
<br />
In spite of this, in order to meet the needs, we designed two methods to construct plasmids on a large scale and attempted the usage in parts of our Donor Plasmids.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Strategies"><font face="Comic Sans MS" size=3>'DP construction and Strategies'</font></a></html> for the methods of rapid plasmid construction. <br />
<br />
<br />
Besides, we have applied for two BBFRFC, which are BBFRFC61 and BBFRFC 62 respectively.<br />
<br />
Then, after transformation of the prepared ‘recepient strain’ with Donor Plasmid and the induction of corresponding agents, I-scel enzyme cuts out DNA fragments from DP and LP, generating numerous Insertion Fragments. Besides, antibiotic resistance gene integrated into E coli genome is also cut out, providing recombination sites for Insertion Fragments.<br />
<br />
Provided with Insertion Fragments mentioned above and Lamda-Red expressed by Donor Plasmid, Insertion Fragments recombine with the corresponding landing pad sequence in the genome and thus get inserted into the correct site of the genome. Based on our design of Landing pad, we can ensure that only one Insertion Fragment from one group is randomly inserted into the genome, therefore mimicking the VDJ recombination process.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri"><font face="Comic Sans MS" size=3>‘DP Insertion and Recombination Induction’</font></a></html> to learn more about this part. <br />
<br />
<br />
Engineered E coli cells which have undergone successful recombination can be selected out of the pool through specific screening methods. After tests on different strains, our project achieve a recombination rate above 50% based on our current techniques.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/experiments#res"><font face="Comic Sans MS" size=3>‘Result’</font></a></html> to learn more about the identification of recombination rate. <br />
<br />
<br />
The goal of the second module in our project is aimed at selection of specific antibodies. Due to the maturity of existing antibody screening technique, our work mainly focuses on imitation of mammalian antibody selection, thus providing possible alternatives for existing methods.<br />
<br />
The outline of our ideas is to find mechanisms similar to in vivo antibody selection and take in,to account the industry production costs, controllability and reliability, thus developing new methods for antibody selection.<br />
In fact, in mammalian system, antibody selection is achieved through the activation of B lymphocytes and thus the rapid proliferation of the B lymphocytes that express the specific antibodies that bind to antigen. Simply put, the whole process relies on the interaction between antigen epitopes and membrane integral immunoglobins.<br />
<br />
To mimic the activation mediated by membrane integral immunoglobins, we develop ToxR-mediated transmembrane activation signal system. In this system, the interaction between antibodies and antigen triggered downstream expression of reporter genes, thus providing signals for selection.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2"><font face="Comic Sans MS" size=3>‘ToxR-based Transmembrane Signaling Pathway Method’</font></a></html> for detailed description of this method.<br />
<br />
<br />
In addition, we develop a technique called ‘Bacterial based Microarray’ for selection purpose. In this method, we combine membrane display technique and high throughput microarray technique, that is, a bacterial based microarray method.<br />
<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1"><font face="Comic Sans MS" size=3>’Bacterial based microarray’</font></a></html> for details.<br />
<br />
<br />
In order to find methods for antibody selection, we cooperate with other iGEM teams. For example, we talked with Macquarie_Australia iGEM team about the project. The techniques involved in their project might be useful for our selection methods. Therefore, members from both teams worked together to research on this problem.<br />
<br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3"><font face="Comic Sans MS" size=3>'link'</font></a></html> to learn more about our cooperation.<br />
<br />
<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens. <br />
Our system is aiming at imitating this recombination process, using E.coli as the gene carrier.<br />
It is known to all that regional genes (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and the recombination occurs when VDJ recombinase are expressed. We choose RecA enzyme to induce the recombination, while use I-Sel enzyme to cut the genome, just mimicking the process happened in B cells.<br />
<br/><br />
<br/><br />
<br />
==Comparison between natural antibody production and E. Coli system==<br />
<br/><br />
===VDJ Recombination vs Lading Pad Recombination===<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is the mechanism by which immunoglobulin (Ig) and T cell receptors (TCR) are generated in the immune system. Through V(D)J recombination, Variable, Diverse, and Joining gene segments are randomly combined, thus encoding various kinds of immunoglobins and T cell receptors to recognize potential antigens. <br />
During the process of B cell maturation, gene segments are rearranged and thus transcribe mRNA that can produce diverse types of antibody for recognition of billions of potential epitopes. The rearrangement brings three gene segments (termed V, D, and J) in close proximity, which are then joined together head to tail, the process of which is known as V(D)J recombination based on the V,D,J gene segments involved. Multiple copies of the V, D, and J genes in the human genome are shuffled in the process, and a specific antibody will be generate among millions of other possible combinations. Because gene are joined together permanently, one mature B cell will produce only one specific antibody.<br />
<br />
Our system is aimed to mimic this recombination process, using E.coli as the carrier.<br />
Through previous research in immunology field, each kind of gene segments (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and recombination occurs when VDJ recombinase recognize the specific signal sequence and mediate the recombination process. In our project, we design a sequence, which carries separated parts of one complete gene and integrate synthesized DNA fragment into the genome of E coli via landing pad method. Then we use I-Sel enzyme to cut the gene segments out of E coli genome, into which gene segments are inserted and choose RecA enzyme to recognize the previously designed signal sequence flanking our target sequence and recombine the gene segments, just like what recombinase accomplish in B cells. The whole process is called landing pad recombination.<br />
<br />
===Somatic hyper-mutation vs Junctional mutation===<br />
<br />
Somatic hypermutation involves a programmed process of mutation affecting the variable regions of immunoglobulin genes. During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is at least 105-106 fold greater than the normal rate of mutation across the genome. Variation is mainly in the form of single base substitutions, with insertions and deletions being less common. These mutations occur mostly at “hotspots” in the DNA, known as hypervariable regions. These regions correspond to the complementarity determining regions; the sites involved in antigen recognition on the immunoglobulin. This directed hypermutation allows for the selection of B cells that express immunoglobulin receptors possessing an enhanced ability to recognize and bind a specific foreign antigen.<br />
<br />
In addition, the cross ligation, namely Landing pad recombination, has higher opportunity to mutate in the link sequence. The introduction of 4 junctional shuffler 64 will meet the need of the rest 107 diversity, perfectly undertaking the work in the antibody diversity construction that hyper-mutation does in the highly variable region. In this way, we have finished a building library, waiting for the specialized antigen to select.<br />
<br />
===Antigen-specific Selection vs CBD-Based Microarray and ToxR-Based Transmembrane pathway method===<br />
In mammalian immune system, billions of antibodies will be generated. Then how can the immune system find out specific antibody that indiscriminately recognize the specific antigen that comes in. Generally speaking, due to the fact that one mature B cell only generates one specific immunoglobin, once the membrane integral immunoglobin bind to the specific antigen, a series of intracellular response, such as gene activation and cell differetiation will be triggered in B cell. Then, B cells expressing the specific antibodies that recognize the specific antigen will proliferate and generate enormous amount of antibodies that help eliminate the specific antigen.<br />
<br />
Simply put, antibody selection is accomplished through the specific interaction between the membrane immunoglobin and the specific antigen. Therefore, in our project, we also plan to mimic this process through interaction between the antibodies generated through landing pad recombination and the antigen we target. CBD-based Microarray and ToxR-based Transmembrane pathway are the two methods by which we achieve this goal.<br />
CBD domain, which interacts with cellulose, potentially provides us way to locate our E.coli to the microarray coated with cellulose. OmpA (outer membrane protein A), displays the antibody and antigen out to the membrane surface. Then by applying the antibodies generated through landing pad recombination to the microarray, antibodies that bind to the antigen will be retained and thus selected out of the pool of antibodies.<br />
<br />
Alternatively, the ToxR-based transmembrane pathway method shows a different strategy for us to seek the right folded antibody. Once the antigen comes, ToxR outer part will form dimer shape and the downstream signal occur, supplying the approach of reporters, such as GFP, to examine and pick out what we want.<br />
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==Module I==<br />
<br />
'''Antibodies Library Diversity & Randomicity'''<br />
[[Image:TSModule1.PNG|650px]]<br />
<br />
This is an overall flowchart about our Module I and in this figure you can find each step is symbolizing each one of the content below. You can find the details about how we carried out experiments and achieved our goals by scrolling down and reading through them. You're warmly welcome to have a discussion with us.<br />
<br/><br />
===Abstract===<br />
<br />
===Flow chart===<br />
[[Image:THUProjectFlowChart.JPG|600px]]<br />
<br />
<br />
===Landing Pad Construction and Insertion===<br />
<br><br />
<br />
'''Purpose of this step'''<br />
<br />
Landing pad insertion is the first step of our two-step recombination system. By doing this step we induced our “RSS Sequence” into E.coli.<br />
<br />
We insert a “landing pad” fragment which includes a promoter (placIQ1) and a tetracycline resistance gene (tetA) flanked by I-SceI recognition sites and 20-bp landing pad regions (LP1 and LP2) into Escherichia coli chromosome via att recombination. Then the helper plasmids encoding I-SceI endonuclease and λ-Red and donor plasmid encoding various antibiotic genes flanked by I-SceI recognition sites and same landing pad regions (LP1 and LP2) are transformed, which will be introduced in detail in next part. I-SceI expression is induced via the addition of L-arabinose. I-SceI recognition sites in the donor plasmid and chromosome are cleaved. Integration of the fragment in donor plasmid is facilitated by IPTG-induced λ-Red expression.<br />
<br />
This two-step recombination method allows for the insertion of very large fragments into a specific location in ''Escherichia coli'' chromosome and in any orientation. <br />
<br />
<br />
'''Construction of landing pad'''<br />
<br />
Landing pad consists of the following parts: a promoter (placIQ1), two landing pad regions, two I-SceI recognition sites and a tetracycline resistance gene (tetA).<br />
<br />
[[Image:THUProjectFigure1.PNG|650px]]<br />
<br />
Another construction strategy is shown as below:<br />
<br />
[[Image:THUProjectFigure1_1.JPG|650px]]<br />
<br />
'''The first construction is used as the four-gene donor plasmid method, while the latter is for the two-gene donor plasmid method.''' We take the advantage of the former construction in the final recombination, because the promoter will not be cut when the recombination happens, thus saving us lots of time. As the latter construction, we can easily add a promoter before the gene, in two-gene donor plasmid A(shown in the Donor Plasmid Construction Part). <br />
<br />
First we obtain two target DNAs – promoter and tetA with PCR. Then We use the overlap extension polymerase chain reaction (or OE-PCR) to link two fragments together. Primer 2 and primer 3 have homologous sequences, so one segment can anneal to the other in certain conditions, in other words, the two segments can overlap into one segment after extension. If necessary, operate PCR again with primer 1 and primer 4 to obtain more products. <br />
<br><br />
[[Image:THUOverlap.JPG|500px]]<br />
<br />
'''Landing pad insertion-- ATT RECOMBINATION'''<br />
<br />
Our approach is based on genome targeting systems that utilize plasmids carrying a conditional-replication origin and a phage attachment (attP) site. We refer to our plasmids as CRIM (conditionalreplication, integration, and modular) plasmids. CRIM plasmids can be integrated into or retrieved from their bacterial attachment (attB) site by supplying phage integrase (Int) without or with excisionase (Xis) in trans.<br />
<br />
We got a strain with plasmid pUK2 from LAB. Then we develop a E.coli strain contains the helper plasmid AH69. These two plasmids are shown below. In order to match other parts of our whole project, the modification that kan-exon should be replaced with tet-SDS was necessary. We got tet-SDS from another plasmid named pkts-cs. Then we use PCR to get the two fragments as PT (from pkts-cs) and V (from pUK2). We ligate PT and V after digestion and modification to form a new plasmid. This new plasmid was named as pUKIP and contains a promoter region, a tet-SDS region and phage attachment site (attP). As reported in the paper, there exist one bacterial attachment site (attB) of HK002 in TorT-TorS gene of the chromosomal DNAs of E.coli K12 strain. That is to say, the PT fragment will integrate into the cell genome after the plasmid was transferred in cells. <br />
<html><a name="att"></a></html><br />
[[Image:THUProjectFlowchart.jpg|700px]]<br />
<br />
<html><a name="hpi"></a></html><br />
<br />
===Helper Plasmid(HP) Insertion===<br />
'''CRIM plasmid integration'''<br />
<br />
Cells carrying a CRIM helper plasmid were grown in 20 ml of LB cultures with ampicillin at 30°C to an optical density of 600 nm of ca. 0.6 and then made electrocompetent. Following electroporation, cells were suspended in LB without ampicillin, incubated at 30°C for 30 min, at 42°C for 30 min and at 30°C for 30 min, and then spread onto selective agar (tet) and incubated at 37°C. Colonies were purified once nonselectively and then tested for antibiotic resistance for stable integration and loss of the helper plasmid and by PCR for copy number.<br />
<br />
'''CRIM plasmid excision'''<br />
<br />
Cells were transformed with the respective Xis/Int CRIM helper plasmid and then spread on ampicillin agar media at 30°C. Colonies were purified once or twice nonselectively on plates that were incubated for 1 h at 42°C and overnight at 37°C. They were then tested for antibiotic sensitivities and by PCR for loss of the integrated plasmid.<br />
<br><br />
<br />
===Helper Plasmid Insertion: “Applying Recombinase Coding Gene”===<br />
We transferred our helper plasmid, which contains RecA enzyme coding gene and I-Sel enzyme coding gene, into E.coli. These two enzymes will act exactly as what the recombinase does.<br />
<br />
<br />
<br />
<br />
<html><a name="dpc"></a></html><br />
===Donor Plasmid(DP) Construction===<br />
<br />
<B>Constructing Our “Antibody Coding Gene Library”</B><br />
<br />
<br />
<br />
<br />
'''Purpose of this step:'''<br />
<br />
The construction of DP provides us multiple genes, which vary from each other, forming the library for us to select.<br />
After inserting landing pad and helper plasmid to E.coli, we must construct a series of donor plasmids to demonstrate that this system can truly realize the recombinant process in E.coli, thus we can further use this module to simulate the recombination of antibody gene in mammalian B cells. We not only need to test the efficiency of recombination, but also ensure that genes we get from this recombinant process can be expressed correctly and have their original function. So we intend to construct four plasmids to test the system.<br />
<br />
<br />
'''Experiment design and expected results:'''<br />
<br />
'''[1] Donor plasmid A'''<br />
<br />
[[Image:THUPA.jpg|500px]]<br />
<br />
In donor plasmid A, we insert only one gene, kanamycin resistant gene (Kanr) to test our recombination. At the 5’ end of Kanr, we add I-scel recognizing sequence (which is represented by the white arrow) and recombination sequence 1 (which is shown in red). At the 3’ end, we add another recombination sequence (which is shown in blue) and also I-scel recognizing sequence.<br />
<br />
After construction, we will transform this donor plasmid to E.coli with landing pad and helper plasmid. After arabinose and IPTG inducing, the restriction enzyme I-scel will cut down Kanr (containing recombination sequences), which can recombine with the bacterial chromosome.<br />
<br/><br/><br />
'''[2] Donor plasmid B'''<br />
<br />
[[Image:THUProjectFigure2.jpg|500px]]<br />
<br />
In donor plasmid B, we insert two genes, kanamycin resistant gene (Kanr) and Chloromycetin resistant gene (Chlr). At the 5’ end of these two genes, we add I-scel recognizing sequence (which is represented by the white arrow) and recombination sequence 1 (which is shown in red). At the 3’ end, we add another recombination sequence (which is shown in blue) and also I-scel recognizing sequence.<br />
<br />
After construction, we will transform this donor plasmid to E.coli with landing pad and helper plasmid. After arabinose and IPTG inducing, the restriction enzyme I-scel will cut down these two genes (containing recombination sequences), which can recombine with the bacterial chromosome. In our expectation, either kanr or chlr will replace the landing pad, resulting in the bacteria resistance to either kanamycin or chloromycetin, but not both. This process will be random, so we can get as many colonies resistant to kanamycin as those resistant to chloromycetin.<br />
<br/><br/><br />
'''[3] Donor plasmid C'''<br />
<br />
[[Image:THUProjectFigure3.jpg|500px]]<br />
<br />
Donor plasmid C includes four genes, GFP, mCherry, Kanr, Chlr, respectively. The same with genes in donor plasmid A, we add recombination sequences and I-scel recognition sequences to the ends of each genes. The recombination sequences of GFP and mCherry are identical, and those of Kanr and Chlr are the same. Note that recombination sequence at 3’ end of GFP (mCherry) and that at 5’ end of Kanr (Chlr) are the same, so we can get a random recombination of two genes, one is a fluorescence gene and the other is resistant gene, creating 2X2=4 different results.<br />
<br/><br/><br />
'''[4] Donor plasmid D'''<br />
<br />
[[Image:THUProjectFigure4.JPG|500px]]<br />
<br />
For constructing donor plasmid D, we first cut the GFP and mCherry to 2 fragments respectively. Then we insert these four fragments into the plasmid in the order shown in the above picture. We expect that we can see either green or red fluorescence after transforming and inducing. Through this experiment, we can tell whether or not recombination sequence will affect the normal function of genes, further demonstrate that antibody producing by our system will be effective. On the other hand, we should note that the sequence length is another significant reason of antibody diversity, so the effect of recombination sequence on antibody will much small than that on fluorescence genes.<br />
<br/><br />
<br />
====Strategies====<br />
<br />
Traditionally, we can construct these four plasmids by inserting genes into the plasmid one by one. Take donor plasmid C as a typical example. We choose plasmid PUC19 as our vector for the four genes. After identifying the multiple clone sites of PUC19 and four genes carefully, we select out four sites, including HindIII, SalI, BamHI, KpnI and EcoRI. We first insert the gene eGFP, amplified by PCR primers with HindIII and SalI sites, into the vector digested by HindIII and SalI. Then we use this plasmid (PUC19+eGFP) as our next vector for gene mCherry with SalI and BamHI sites. Repeat these processes of enzyme digestion and ligation, we can eventually get donor plasmid C with four genes as we want.<br />
<br />
However, considering the huge number of antibody fragments, we try our best to seek other strategies to complete the ligation of multiple fragments, which can be done more quickly and efficiently.<br />
<br />
=====Principle=====<br />
<br />
The fragments include different landing pad regions and endoclease recognition site, and are grouped together by sharing the same landing pad region.<br />
<br />
=====Method===== <br />
<br />
In the demand of larger amount of fragments and constructed plasmids, we find two ways to realize such purpose, regarded as the key point of our whole project-guarantee the large variation of antibody.<br />
<br />
'''Method 1: Multi-Fragments ligation method based on restriction enzyme DraIII'''<br />
<br />
'''Concept:'''<br />
<br />
The recognition site of endonuclease DraIII is as follows: 5' CACNNNGTG 3', so we can design more than one recognition sequence added to multiple fragments and complete the excision in one procedure.<br />
<br />
'''Procedure:'''<br />
<br />
In our experiment, three different recognition sequences are designed and added to six ends of three fragments with every two of the ends sharing the identical sequence. These three fragments, one encoding Km resistant gene, another Cm resistant gene, and the third replication origin, then can be excised in one sample, and ligated to construct a plasmid.<br />
<br />
Both the Km and Cm resistant gene are flanked with the identical landing pad region, thus, expected to be recombined into the E. coli chromosome with the same efficiency.<br />
<br />
In the same way, we can excise and ligate three fragments or even more fragments. In the later experiment, we try to construct plasmid with four or five fragments.<br />
<br />
As introduced above, a key problem to produce antibodies based on our design is how to develop an efficient and quick method, which can be applied to construct antibody genes library. Therefore, we try to establish a special method, named “multiple fragments ligation in order”, the principle of which is, briefly, based on a very kind of restriction enzyme-DraIII. <br />
<br />
DraIII is somehow distinguished from other restriction enzymes for its specific cutting site, as shown below:<br />
<br />
CAC NNN▼GTG<br />
<br />
GTG▲NNN CAC<br />
<br />
N stands for any of the base A, T, C, G. It is obvious that multiple different short sequences can be cut by Dra III as long as the sequence starts with CAC and ends with GTG. Two fragments with complementary “NNN” can be combined together while those without noncomplementary “NNN” cannot. The following figure shows the principle and process:<br />
<br />
[[Image:THUProjectFigure5.JPG|650px]]<br />
<br />
[[Image:THUProjectFigure6.JPG|650px]]<br />
<br />
<br />
We carried out the series of experiments based on this document.<br />
[[Image:BBF RFC 61.pdf]]<br />
<br />
<br />
'''Method 2: Complementary ligation'''<br />
<br />
This method depends on sequence and ligation-independent cloning (SLIC), which allows the assembly of multiple DNA fragments in a single reaction using in vitro homologous recombination and single-strand annealing. SLIC mimics in vivo homologous recombination by relying on exonuclease-generated ssDNA overhangs in insert and vector fragments, and the assembly of these fragments by recombination in vitro. Homologous recombination in vivo depends upon a double-stranded break, generation of ssDNA by exonucleases, homology searching by recombinases, annealing of homologous stretches, and repair of overhangs and gaps by enzymes that include resolvases, nucleases and polymerases. It is possible to generate recombination intermediates in vitro and introduce these into cells to allow the cell endogenous repair machinery to finish the repair to generate recombinant DNA.<br />
<br />
The experiment generally includes the following steps:<br />
<br />
Generate the vector by cleavage with a restriction enzyme and generate the insert by PCR.<br />
<br />
Treat both the vector and the insert with T4 DNA polymerase in the absence of dNTPs to chew back 5’ strand to reveal ssDNA overhangs.<br />
<br />
Incubate vector and insert with RecA protein and ATP to promoter recombination.<br />
<br />
Transform the products into ''E.coli''.<br />
<br />
[[Image:THUMethod2.png]]<br />
<br />
<br />
We carried out the series of experiments based on this document.<br />
[[Image:BBF RFC 62.pdf]]<br />
<br />
===Removal of Helper Plasmid(HP)===<br />
Helper plasmid (HP) includes a temperature sensitive pSC101 replication origin, which maintains the plasmid at low copy number. This plasmid is thus easily removed by growth at 42℃ and screening against spectinomycin resistance. Of more concern is the donor plasmid, which is cured by I-SceI cleavage, and this process is very efficient, with only about 1% of cells retaining the donor plasmid.<br />
<br />
<html><a name="dpiri"></a></html><br />
===Donor Plasmid(DP) Insertion & Recombination Induction: The "Very VDJ Recombination"===<br />
'''Propose of this step:'''<br />
<br />
When our DP and HP are inserted, we have finished our preparing and begin to operate our system, just as the recombinase dose in B cells.<br />
<br />
After the reform of the E.coli genome and the construction of the donor plasmid, we need to test our module’s function. First we should insert the donor plasmid(DP) into the E.coli, and then induce the recombination by adding IPTG and arabinose. Arabinose active the I-Sel restriction enzyme, then cut DP and genome at the same site. After the digestion, we add IPTG to help the recombination, using the homologous sequence near the cohesive end.<br />
<br />
'''DP Insertion: '''<br />
[[Image:THUProjectFigure7.png|350px|right]]<br />
<br />
As we said before, we use a pre-altered template to amplify landing pad fragments using the landing pad regions as standardized priming sites. Here we used the conventional electroporation method to transform the DP into E.coli genome, then incubate the plate at 37°overnight.<br />
Electroporation is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. It is a dynamic phenomenon that depends on the local transmembrane voltage (we used 1 V) at each point on the cell membrane. If E.coli and DP are mixed together, the plasmids can be transferred into the cell after electroporation. This procedure is highly efficient than chemical transformation. (Partly from wikipedia)<br />
<br/><br/><br />
'''Recombination Induction:'''<br />
<br />
Individual colonies were inoculated into 5 ml of EZ-Rich Defined Medium (RDM; Teknova) +0.5% glycerol, 2mM IPTG, and 0.2% w/v L-arabinose. After growing at 37°C for 1 h in a shaking water bath, we transfer the medium to 30_C shaking water bath for 4 h, then 100 mg/ml spectinomycin was added. At first the I-Sel enzyme is expressed to cut the genome and DP at the same site. And this step is used to constitutive express the Rec A enzyme, thus initiating the recombination by the homologous region.<br />
<br />
The appropriate antibiotic for the given insertion fragment was then added (25 mg/ml kanamycin, 34 mg/ml chloramphenicol), and the cultures were grown overnight. The next day, 100 ml sample was plated on LB plates with the appropriate antibiotic and grown at 37°C. We test the sample by screening it on LB plates containing 100 mg/ml ampicillin or 10 mg/ml tetracycline to verify the loss of the landing pad and donor plasmid.<br />
<br />
[[Image:THUProjectFigure8.JPG|650px]]<br />
<br />
===Helper Plasmid (HP) Removal: “Inactivation of Recombinase”===<br />
<br />
Helper plasmid (HP) includes a temperature sensitive pSC101 replication origin, which maintains the plasmid at low copy number. This plasmid is thus easily removed by growth at 42℃ and screening against spectinomycin resistance. Of more concern is the donor plasmid, which is cured by I-SceI cleavage, and this process is very efficient, with only about 1% of cells retaining the donor plasmid.<br />
<br />
<br />
===Verification of Recombination efficiency===<br />
<br />
To examine the efficiency of the recombination system, we carry out the experiments as follow.<br />
<br />
Using the E. coli which are already been transformation with Landing Pad and Helper plasmid to construct competent cell for electro transformation. The detail can be found at Tsinghua iGEM 2010 Protocol 1-6:Preparation of Competent Cell for Electro Transformation. Transformation the Donor Plasmid and spread the bacteria onto the plate with propriety antibiotics.<br />
<br />
There are two ways to induce. One is induction directly, the other is induction while culture.<br />
<br />
Induction directly:<br />
1. Pick colony into TB (0.2% L-arabinose, 2mM IPTG) without any antibiotics. Culture at 37℃ for 1 hour.<br />
<br />
2. Add 100ug/ml spectinomycin, culture at 30℃ for 4 hours.<br />
<br />
3. Add addition antibiotics according to the insertion gene on the Donor plasmid. Culture overnight at 30℃.<br />
<br />
4. Dilute at propriety concentration and spread at plate with antibiotics to count.<br />
<br />
Induction while culture:<br />
<br />
1. Pick colony into multi-antibiotics (tet, spe, and other antibiotics according to the gene insertion in the Donor plasmid) LB. Culture at 30℃.<br />
<br />
2. Transfer the bacteria into LB with spe and one other antibiotic to select. Culture until OD value reaches 0.6 and then add 0.2% L-arabinose, 2mM IPTG. Culture at 30℃ for half an hour.<br />
<br />
3. Culture at 37℃ for 20 minutes.<br />
<br />
4. Culture at 30℃ overnight.<br />
<br />
5. Dilute at propriety concentration and spread at plate shown blow.<br />
<br />
(1) tet only: to test whether the Helper plasmid exist.<br />
<br />
(2) one of the antibiotic among the Donor plasmid only (depend on the Donor plasmid) for count.<br />
<br />
(3) tet + IPTG + arabinose + spectinomycin.<br />
<br />
(4) one of the antibiotic + IPTG + arabinose + spectinomycin.<br />
<br />
6. Pick colonies from plate, using well designed primers to detect. The principle of primer design is that the up-stream primer is on the genome next to the recombination site and the down-stream one is on the combination gene. Calculate the rate of recombination last.<br />
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==Module I==<br />
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'''Antibodies Library Diversity & Randomicity'''<br />
[[Image:TSModule1.PNG|650px]]<br />
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This is an overall flowchart about our Module I and in this figure you can find each step is symbolizing each one of the content below. You can find the details about how we carried out experiments and achieved our goals by scrolling down and reading through them. You're warmly welcome to have a discussion with us.<br />
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===Abstract===<br />
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===Flow chart===<br />
[[Image:THUProjectFlowChart.JPG|600px]]<br />
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===Landing Pad Construction and Insertion===<br />
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'''Purpose of this step'''<br />
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Landing pad insertion is the first step of our two-step recombination system. By doing this step we induced our “RSS Sequence” into E.coli.<br />
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We insert a “landing pad” fragment which includes a promoter (placIQ1) and a tetracycline resistance gene (tetA) flanked by I-SceI recognition sites and 20-bp landing pad regions (LP1 and LP2) into Escherichia coli chromosome via att recombination. Then the helper plasmids encoding I-SceI endonuclease and λ-Red and donor plasmid encoding various antibiotic genes flanked by I-SceI recognition sites and same landing pad regions (LP1 and LP2) are transformed, which will be introduced in detail in next part. I-SceI expression is induced via the addition of L-arabinose. I-SceI recognition sites in the donor plasmid and chromosome are cleaved. Integration of the fragment in donor plasmid is facilitated by IPTG-induced λ-Red expression.<br />
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This two-step recombination method allows for the insertion of very large fragments into a specific location in ''Escherichia coli'' chromosome and in any orientation. <br />
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'''Construction of landing pad'''<br />
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Landing pad consists of the following parts: a promoter (placIQ1), two landing pad regions, two I-SceI recognition sites and a tetracycline resistance gene (tetA).<br />
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[[Image:THUProjectFigure1.PNG|650px]]<br />
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Another construction strategy is shown as below:<br />
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[[Image:THUProjectFigure1_1.JPG|650px]]<br />
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'''The first construction is used as the four-gene donor plasmid method, while the latter is for the two-gene donor plasmid method.''' We take the advantage of the former construction in the final recombination, because the promoter will not be cut when the recombination happens, thus saving us lots of time. As the latter construction, we can easily add a promoter before the gene, in two-gene donor plasmid A(shown in the Donor Plasmid Construction Part). <br />
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First we obtain two target DNAs – promoter and tetA with PCR. Then We use the overlap extension polymerase chain reaction (or OE-PCR) to link two fragments together. Primer 2 and primer 3 have homologous sequences, so one segment can anneal to the other in certain conditions, in other words, the two segments can overlap into one segment after extension. If necessary, operate PCR again with primer 1 and primer 4 to obtain more products. <br />
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[[Image:THUOverlap.JPG|500px]]<br />
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'''Landing pad insertion-- ATT RECOMBINATION'''<br />
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Our approach is based on genome targeting systems that utilize plasmids carrying a conditional-replication origin and a phage attachment (attP) site. We refer to our plasmids as CRIM (conditionalreplication, integration, and modular) plasmids. CRIM plasmids can be integrated into or retrieved from their bacterial attachment (attB) site by supplying phage integrase (Int) without or with excisionase (Xis) in trans.<br />
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We got a strain with plasmid pUK2 from LAB. Then we develop a E.coli strain contains the helper plasmid AH69. These two plasmids are shown below. In order to match other parts of our whole project, the modification that kan-exon should be replaced with tet-SDS was necessary. We got tet-SDS from another plasmid named pkts-cs. Then we use PCR to get the two fragments as PT (from pkts-cs) and V (from pUK2). We ligate PT and V after digestion and modification to form a new plasmid. This new plasmid was named as pUKIP and contains a promoter region, a tet-SDS region and phage attachment site (attP). As reported in the paper, there exist one bacterial attachment site (attB) of HK002 in TorT-TorS gene of the chromosomal DNAs of E.coli K12 strain. That is to say, the PT fragment will integrate into the cell genome after the plasmid was transferred in cells. <br />
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[[Image:THUProjectFlowchart.jpg|700px]]<br />
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===Helper Plasmid(HP) Insertion===<br />
'''CRIM plasmid integration'''<br />
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Cells carrying a CRIM helper plasmid were grown in 20 ml of LB cultures with ampicillin at 30°C to an optical density of 600 nm of ca. 0.6 and then made electrocompetent. Following electroporation, cells were suspended in LB without ampicillin, incubated at 30°C for 30 min, at 42°C for 30 min and at 30°C for 30 min, and then spread onto selective agar (tet) and incubated at 37°C. Colonies were purified once nonselectively and then tested for antibiotic resistance for stable integration and loss of the helper plasmid and by PCR for copy number.<br />
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'''CRIM plasmid excision'''<br />
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Cells were transformed with the respective Xis/Int CRIM helper plasmid and then spread on ampicillin agar media at 30°C. Colonies were purified once or twice nonselectively on plates that were incubated for 1 h at 42°C and overnight at 37°C. They were then tested for antibiotic sensitivities and by PCR for loss of the integrated plasmid.<br />
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===Helper Plasmid Insertion: “Applying Recombinase Coding Gene”===<br />
We transferred our helper plasmid, which contains RecA enzyme coding gene and I-Sel enzyme coding gene, into E.coli. These two enzymes will act exactly as what the recombinase does.<br />
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===Donor Plasmid(DP) Construction===<br />
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<B>Constructing Our “Antibody Coding Gene Library”</B><br />
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'''Purpose of this step:'''<br />
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The construction of DP provides us multiple genes, which vary from each other, forming the library for us to select.<br />
After inserting landing pad and helper plasmid to E.coli, we must construct a series of donor plasmids to demonstrate that this system can truly realize the recombinant process in E.coli, thus we can further use this module to simulate the recombination of antibody gene in mammalian B cells. We not only need to test the efficiency of recombination, but also ensure that genes we get from this recombinant process can be expressed correctly and have their original function. So we intend to construct four plasmids to test the system.<br />
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'''Experiment design and expected results:'''<br />
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'''[1] Donor plasmid A'''<br />
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[[Image:THUPA.jpg|500px]]<br />
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In donor plasmid A, we insert only one gene, kanamycin resistant gene (Kanr) to test our recombination. At the 5’ end of Kanr, we add I-scel recognizing sequence (which is represented by the white arrow) and recombination sequence 1 (which is shown in red). At the 3’ end, we add another recombination sequence (which is shown in blue) and also I-scel recognizing sequence.<br />
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After construction, we will transform this donor plasmid to E.coli with landing pad and helper plasmid. After arabinose and IPTG inducing, the restriction enzyme I-scel will cut down Kanr (containing recombination sequences), which can recombine with the bacterial chromosome.<br />
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'''[2] Donor plasmid B'''<br />
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[[Image:THUProjectFigure2.jpg|500px]]<br />
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In donor plasmid B, we insert two genes, kanamycin resistant gene (Kanr) and Chloromycetin resistant gene (Chlr). At the 5’ end of these two genes, we add I-scel recognizing sequence (which is represented by the white arrow) and recombination sequence 1 (which is shown in red). At the 3’ end, we add another recombination sequence (which is shown in blue) and also I-scel recognizing sequence.<br />
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After construction, we will transform this donor plasmid to E.coli with landing pad and helper plasmid. After arabinose and IPTG inducing, the restriction enzyme I-scel will cut down these two genes (containing recombination sequences), which can recombine with the bacterial chromosome. In our expectation, either kanr or chlr will replace the landing pad, resulting in the bacteria resistance to either kanamycin or chloromycetin, but not both. This process will be random, so we can get as many colonies resistant to kanamycin as those resistant to chloromycetin.<br />
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'''[3] Donor plasmid C'''<br />
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[[Image:THUProjectFigure3.jpg|500px]]<br />
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Donor plasmid C includes four genes, GFP, mCherry, Kanr, Chlr, respectively. The same with genes in donor plasmid A, we add recombination sequences and I-scel recognition sequences to the ends of each genes. The recombination sequences of GFP and mCherry are identical, and those of Kanr and Chlr are the same. Note that recombination sequence at 3’ end of GFP (mCherry) and that at 5’ end of Kanr (Chlr) are the same, so we can get a random recombination of two genes, one is a fluorescence gene and the other is resistant gene, creating 2X2=4 different results.<br />
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'''[4] Donor plasmid D'''<br />
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[[Image:THUProjectFigure4.JPG|500px]]<br />
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For constructing donor plasmid D, we first cut the GFP and mCherry to 2 fragments respectively. Then we insert these four fragments into the plasmid in the order shown in the above picture. We expect that we can see either green or red fluorescence after transforming and inducing. Through this experiment, we can tell whether or not recombination sequence will affect the normal function of genes, further demonstrate that antibody producing by our system will be effective. On the other hand, we should note that the sequence length is another significant reason of antibody diversity, so the effect of recombination sequence on antibody will much small than that on fluorescence genes.<br />
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====Strategies====<br />
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Traditionally, we can construct these four plasmids by inserting genes into the plasmid one by one. Take donor plasmid C as a typical example. We choose plasmid PUC19 as our vector for the four genes. After identifying the multiple clone sites of PUC19 and four genes carefully, we select out four sites, including HindIII, SalI, BamHI, KpnI and EcoRI. We first insert the gene eGFP, amplified by PCR primers with HindIII and SalI sites, into the vector digested by HindIII and SalI. Then we use this plasmid (PUC19+eGFP) as our next vector for gene mCherry with SalI and BamHI sites. Repeat these processes of enzyme digestion and ligation, we can eventually get donor plasmid C with four genes as we want.<br />
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However, considering the huge number of antibody fragments, we try our best to seek other strategies to complete the ligation of multiple fragments, which can be done more quickly and efficiently.<br />
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=====Principle=====<br />
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The fragments include different landing pad regions and endoclease recognition site, and are grouped together by sharing the same landing pad region.<br />
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=====Method===== <br />
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In the demand of larger amount of fragments and constructed plasmids, we find two ways to realize such purpose, regarded as the key point of our whole project-guarantee the large variation of antibody.<br />
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'''Method 1: Multi-Fragments ligation method based on restriction enzyme DraIII'''<br />
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'''Concept:'''<br />
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The recognition site of endonuclease DraIII is as follows: 5' CACNNNGTG 3', so we can design more than one recognition sequence added to multiple fragments and complete the excision in one procedure.<br />
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'''Procedure:'''<br />
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In our experiment, three different recognition sequences are designed and added to six ends of three fragments with every two of the ends sharing the identical sequence. These three fragments, one encoding Km resistant gene, another Cm resistant gene, and the third replication origin, then can be excised in one sample, and ligated to construct a plasmid.<br />
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Both the Km and Cm resistant gene are flanked with the identical landing pad region, thus, expected to be recombined into the E. coli chromosome with the same efficiency.<br />
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In the same way, we can excise and ligate three fragments or even more fragments. In the later experiment, we try to construct plasmid with four or five fragments.<br />
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As introduced above, a key problem to produce antibodies based on our design is how to develop an efficient and quick method, which can be applied to construct antibody genes library. Therefore, we try to establish a special method, named “multiple fragments ligation in order”, the principle of which is, briefly, based on a very kind of restriction enzyme-DraIII. <br />
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DraIII is somehow distinguished from other restriction enzymes for its specific cutting site, as shown below:<br />
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CAC NNN▼GTG<br />
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GTG▲NNN CAC<br />
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N stands for any of the base A, T, C, G. It is obvious that multiple different short sequences can be cut by Dra III as long as the sequence starts with CAC and ends with GTG. Two fragments with complementary “NNN” can be combined together while those without noncomplementary “NNN” cannot. The following figure shows the principle and process:<br />
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[[Image:THUProjectFigure5.JPG|650px]]<br />
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[[Image:THUProjectFigure6.JPG|650px]]<br />
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'''Method 2: Complementary ligation'''<br />
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This method depends on sequence and ligation-independent cloning (SLIC), which allows the assembly of multiple DNA fragments in a single reaction using in vitro homologous recombination and single-strand annealing. SLIC mimics in vivo homologous recombination by relying on exonuclease-generated ssDNA overhangs in insert and vector fragments, and the assembly of these fragments by recombination in vitro. Homologous recombination in vivo depends upon a double-stranded break, generation of ssDNA by exonucleases, homology searching by recombinases, annealing of homologous stretches, and repair of overhangs and gaps by enzymes that include resolvases, nucleases and polymerases. It is possible to generate recombination intermediates in vitro and introduce these into cells to allow the cell endogenous repair machinery to finish the repair to generate recombinant DNA.<br />
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The experiment generally includes the following steps:<br />
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Generate the vector by cleavage with a restriction enzyme and generate the insert by PCR.<br />
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Treat both the vector and the insert with T4 DNA polymerase in the absence of dNTPs to chew back 5’ strand to reveal ssDNA overhangs.<br />
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Incubate vector and insert with RecA protein and ATP to promoter recombination.<br />
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Transform the products into ''E.coli''.<br />
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[[Image:THUMethod2.png]]<br />
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===Removal of Helper Plasmid(HP)===<br />
Helper plasmid (HP) includes a temperature sensitive pSC101 replication origin, which maintains the plasmid at low copy number. This plasmid is thus easily removed by growth at 42℃ and screening against spectinomycin resistance. Of more concern is the donor plasmid, which is cured by I-SceI cleavage, and this process is very efficient, with only about 1% of cells retaining the donor plasmid.<br />
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===Donor Plasmid(DP) Insertion & Recombination Induction: The "Very VDJ Recombination"===<br />
'''Propose of this step:'''<br />
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When our DP and HP are inserted, we have finished our preparing and begin to operate our system, just as the recombinase dose in B cells.<br />
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After the reform of the E.coli genome and the construction of the donor plasmid, we need to test our module’s function. First we should insert the donor plasmid(DP) into the E.coli, and then induce the recombination by adding IPTG and arabinose. Arabinose active the I-Sel restriction enzyme, then cut DP and genome at the same site. After the digestion, we add IPTG to help the recombination, using the homologous sequence near the cohesive end.<br />
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'''DP Insertion: '''<br />
[[Image:THUProjectFigure7.png|350px|right]]<br />
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As we said before, we use a pre-altered template to amplify landing pad fragments using the landing pad regions as standardized priming sites. Here we used the conventional electroporation method to transform the DP into E.coli genome, then incubate the plate at 37°overnight.<br />
Electroporation is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. It is a dynamic phenomenon that depends on the local transmembrane voltage (we used 1 V) at each point on the cell membrane. If E.coli and DP are mixed together, the plasmids can be transferred into the cell after electroporation. This procedure is highly efficient than chemical transformation. (Partly from wikipedia)<br />
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'''Recombination Induction:'''<br />
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Individual colonies were inoculated into 5 ml of EZ-Rich Defined Medium (RDM; Teknova) +0.5% glycerol, 2mM IPTG, and 0.2% w/v L-arabinose. After growing at 37°C for 1 h in a shaking water bath, we transfer the medium to 30_C shaking water bath for 4 h, then 100 mg/ml spectinomycin was added. At first the I-Sel enzyme is expressed to cut the genome and DP at the same site. And this step is used to constitutive express the Rec A enzyme, thus initiating the recombination by the homologous region.<br />
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The appropriate antibiotic for the given insertion fragment was then added (25 mg/ml kanamycin, 34 mg/ml chloramphenicol), and the cultures were grown overnight. The next day, 100 ml sample was plated on LB plates with the appropriate antibiotic and grown at 37°C. We test the sample by screening it on LB plates containing 100 mg/ml ampicillin or 10 mg/ml tetracycline to verify the loss of the landing pad and donor plasmid.<br />
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[[Image:THUProjectFigure8.JPG|650px]]<br />
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===Helper Plasmid (HP) Removal: “Inactivation of Recombinase”===<br />
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Helper plasmid (HP) includes a temperature sensitive pSC101 replication origin, which maintains the plasmid at low copy number. This plasmid is thus easily removed by growth at 42℃ and screening against spectinomycin resistance. Of more concern is the donor plasmid, which is cured by I-SceI cleavage, and this process is very efficient, with only about 1% of cells retaining the donor plasmid.<br />
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===Verification of Recombination efficiency===<br />
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To examine the efficiency of the recombination system, we carry out the experiments as follow.<br />
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Using the E. coli which are already been transformation with Landing Pad and Helper plasmid to construct competent cell for electro transformation. The detail can be found at Tsinghua iGEM 2010 Protocol 1-6:Preparation of Competent Cell for Electro Transformation. Transformation the Donor Plasmid and spread the bacteria onto the plate with propriety antibiotics.<br />
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There are two ways to induce. One is induction directly, the other is induction while culture.<br />
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Induction directly:<br />
1. Pick colony into TB (0.2% L-arabinose, 2mM IPTG) without any antibiotics. Culture at 37℃ for 1 hour.<br />
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2. Add 100ug/ml spectinomycin, culture at 30℃ for 4 hours.<br />
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3. Add addition antibiotics according to the insertion gene on the Donor plasmid. Culture overnight at 30℃.<br />
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4. Dilute at propriety concentration and spread at plate with antibiotics to count.<br />
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Induction while culture:<br />
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1. Pick colony into multi-antibiotics (tet, spe, and other antibiotics according to the gene insertion in the Donor plasmid) LB. Culture at 30℃.<br />
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2. Transfer the bacteria into LB with spe and one other antibiotic to select. Culture until OD value reaches 0.6 and then add 0.2% L-arabinose, 2mM IPTG. Culture at 30℃ for half an hour.<br />
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3. Culture at 37℃ for 20 minutes.<br />
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4. Culture at 30℃ overnight.<br />
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5. Dilute at propriety concentration and spread at plate shown blow.<br />
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(1) tet only: to test whether the Helper plasmid exist.<br />
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(2) one of the antibiotic among the Donor plasmid only (depend on the Donor plasmid) for count.<br />
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(3) tet + IPTG + arabinose + spectinomycin.<br />
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(4) one of the antibiotic + IPTG + arabinose + spectinomycin.<br />
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6. Pick colonies from plate, using well designed primers to detect. The principle of primer design is that the up-stream primer is on the genome next to the recombination site and the down-stream one is on the combination gene. Calculate the rate of recombination last.<br />
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</html></div>Gumiltonhttp://2010.igem.org/File:THUMethod2.pngFile:THUMethod2.png2010-10-27T16:04:17Z<p>Gumilton: </p>
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<div></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/HumanPracticeTeam:Tsinghua/HumanPractice2010-10-27T15:56:59Z<p>Gumilton: /* A Visit to Macquarie_iGEM */</p>
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=Ethics=<br />
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BioEthics regarding the Biological Ethics, discussing about biological issues, have been debated since ancient times. Bioethical issues gained widespread attention in recent years and are often extremely morally contentious. Considerable societal conflict exists regarding fundamental clinical issues such as: defining the beginning and end of life; appropriate standards of care; and broader environmental issues with potential social and health impacts such as growth of genetically-modified organisms and their use in the food supply.<br />
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==BioEthics in Tsinghua iGEM==<br />
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Here in Tsinghua iGEM 2010 Team, we are now focusing on some parts of BioEthics, such as animal handling requirement, care for animal welfare, appropriate standards of care, broader environmental issues with potential social and health impacts, impact of our products to be used in the manufacture and how to spread our ideas to make more people get this knowledge.<br />
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Thus we, Tsinghua iGEM 2010, pay our attention specially to BioEthics and keep our promise by two ways. One is to teach every member about keeping healthy, safe and being environmentally friendly while conducting experiments. The other one is to try visit and talk with more people, discussing about our projects, the current biological issues and how to promote the environment where we are living as well as let more people know about and start to be interested in Synthetic Biology and iGEM.<br />
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='''Safety'''=<br />
According to the WHO biosafety is the prevention of unintentional exposure to pathogens and toxins, or their accidental release, whereas biosecurity is the prevention of loss, theft, misuse, diversion or intentional release of pathogens and toxins. (From https://2010.igem.org/Safety)<br />
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We are always paying special care to the safety of our teammates and the people around. Thus we signed a document to keep in rules about the Safety in Laboratory, not only to protect ourselves but also help to keep a clean and healthy environment.<br />
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The model animals we used are harmless engineering strains of Escherichia coli. They are widely used in all kinds of laboratories around the world. We answered the questions about safety on the Safety Page from iGEM(https://2010.igem.org/Safety) as following:<br />
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'''Q1: Would any of your project ideas raise safety issues in terms of:<br />
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researcher safety,<br />
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public safety, or<br />
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environmental safety?'''<br />
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A: Our project is about how to use E Coli to simulate the production of Specific Antibodies. No serious safety problem can be caused by the harmless engineering strains of ''Escherichia coli'' and we kept our promise and rules which ensure the safety during the whole process.<br />
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'''Q2: Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'''<br />
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A: No any potential safety issues.<br />
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'''Q3:Is there a local biosafety group, committee, or review board at your institution?'''<br />
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If yes, what does your local biosafety group think about your project?<br />
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A: Yes, it is. The safety check is operated frequently. This is organised and operated by the ''Laboratories and Facilities'' in Tsinghua University. During our project, they operated one of the safety checks in one year and they evaluated our laboratory as well as our project. Our lab construction is keeping in line with the policies in Tsinghua University and because our project is about how to use E Coli to simulate the production of Specific Antibodies and the models we use are ''Escherichia coli'' so they evaluated our project is in a safe category and meanwhile they spoke highly on our Tsinghua iGEM 2010 Safety Manual for "It helps to keep safety in everyone's mind and do really good job.".<br />
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'''Q4:Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?'''<br />
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A: The most invigorative way to inspire the teams to make up ways to deal with and improve the BioSafety is to establish a special prize in BioSafety, so that many teams could try their best to deal with the issues in BioSafety, like how to take experiments safely, how to build safe BioBricks and mostly how maintain the safe environment during our projects.<br />
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Also to recognize that importance from the bottom of one's heart is the most effective approach for dealing with some issues. So if more education, announcement, lectures, courses and many other kinds of teaching methods can be carried out to draw attention on the safety, to let people keep this in mind, the Safety Issues could no longer be an issue but a common sense.<br />
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All the staffs and students operating the infrastructures, devices and anything related with experiments are required to wear the gloves all the time while working. To keep these in rules and in case of any accidents we carried out a document to keep in line with.<br />
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<html><a href="https://static.igem.org/mediawiki/igem.org/8/8f/Tsinghua_iGEM_2010_Safety_Manual.pdf" target=blank>Tsinghua iGEM 2010 Safety Manual</a></html><br />
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Besides this there is also a document in Tsinghua University emphasizing on the common Laboratory Sfety. Here are the <html><a href="https://static.igem.org/mediawiki/2010/6/69/THUSAFETY.pdf" target=blank>Laboratory Safety Policy of Tsinghua University in Chinese Version</a><br />
and <a href="https://static.igem.org/mediawiki/2010/0/05/THULBM.pdf" target=blank>The Laboratory Biosafety Manual</a> released by WHO (World Health Organization) in which Tsinghua University keep align.<br />
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='''Activities'''=<br />
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==Shanghai Meetup==<br />
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More Info at <a href="https://2010.igem.org/Events/China_meetup" target=_blank>2010 iGEM China meetup</a><br><br><br />
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This year, on August 5th, teams from universities in China, <br />
<a href="https://2010.igem.org/Team:SJTU-BioX-Shanghai" target=_blank>SJTU-BioX-Shanghai</a>,<br />
<a href="https://2010.igem.org/Team:Tsinghua" target=_blank>Tsinghua</a>,<br />
<a href="https://2010.igem.org/Team:Peking" target=_blank>Peking</a>, <br />
<a href="https://2010.igem.org/Team:ECUST-Shanghai" target=_blank>ECUST-Shanghai</a>, <br />
<a href="https://2010.igem.org/Team:USTC" target=_blank>USTC</a>, <br />
<a href="https://2010.igem.org/Team:USTC_Software" target=_blank>USTC_Software</a>, <br />
<a href="https://2010.igem.org/Team:ZJU-China" target=_blank>ZJU-China</a><br />
and Sun Yat-sen University all gathered in Shanghai and had a nice day at the iGEM 2010 China Meetup.<br />
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==Tsinghua University Lecture Day==<br />
[[Image:THULecturePost.jpg|400px]]<br />
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==Survey and Discussion==<br />
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==A Visit to Macquarie_iGEM==<br />
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Tsinghua and Macquarie iGEM Teams have an impressive relationship both in the cooperation of projects as well as in Human Practice.<br />
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[[Image:TSINGHUA&MAC1.JPG|500px]]<br />
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On 27/9/2010, GU Xiang, one of the team students in Tsinghua iGEM paid a visit to the Macquarie iGEM. GU Xiang, Yagiz Aksoy, the leader of Macquarie iGEM Team, and Hilal Varinli met together on the beautiful campus of the University of Macquarie, in Sydney. They firstly visited the Life Science Building in Macquarie, "It is amazing" said by GU Xiang. Then they three dropped in the Biology Museum in that building. Later GU Xiang together with Yagiz and Hilal did some of their experiments on that day. The research devices and environment in the labs of Macquarie are absolutely at world leading-class level.<br />
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[[Image:THUMAC.JPG|500px]]<br />
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After that, they introduced and talked about each other's projects. Through that they found the project of Macquarie iGEM can be a potential screening method for the project of Tsinghua iGEM. Also hearing that Macquarie iGEM is a newly built team, GU Xiang generously gave his ideas and experience in iGEM to Yagiz and Hilal, promoting them to build better wiki to let more people know about their ideas. Finally they three expressed freely about the issues in Biology and Life Science as well as the Chinese Culture, especially the Kungfu. They had a wonderful day!<br />
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</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/supportTeam:Tsinghua/support2010-10-27T15:12:45Z<p>Gumilton: /* Acknowledgement */</p>
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=Acknowledgement=<br />
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Firstly we want to thank and acknowledge our sponsors below. This project would not be possible without you!<br />
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<a href="http://www.tsinghua.edu.cn/eng/index.jsp" title="Tsinghua University" target=_blank>Tsinghua University<br/><img src="https://static.igem.org/mediawiki/2010/0/0e/THUCENCEL.jpg" width="500px"></a><br />
<br/><br/><br />
<a href="http://life.tsinghua.edu.cn/english/" title="School of Life Science, Tsinghua University" target=_blank>School of Life Science in Tsinghua University<br/><img src="https://static.igem.org/mediawiki/2010/a/a2/TsinghuaLIFE.gif" width="500px"></a><br />
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<a href="http://www.phys.tsinghua.edu.cn:8080/english/" title="Department of Physics, School of Sciences" target=_blank>Department of Physics, School of Sciences, Tsinghua University<br/><img src="https://static.igem.org/mediawiki/2010/e/e0/TsinghuaWULI.jpg" width="500px"></a><br />
<br/><br/><br />
<a href="http://ad.tsinghua.edu.cn/yxweb_en/index.jsp" title="Academy of Arts and Design, Tsinghua University" target=_blank>Academy of Arts and Design, Tsinghua University<br/><img src="https://static.igem.org/mediawiki/2010/4/4d/TsinghuaMEIYUAN.jpg" width="500px"></a><br />
<br/><br/><br />
<a href="http://www.tsinghua.edu.cn/docsn/wb/waiban-eng.htm" title="Office of International Cooperation and Exchange" target=_blank>Office of International Cooperation and Exchange<br/> Office for Affairs of Hong Kong,Macao and Taiwan<br/> Tsinghua University<br/><img src="https://static.igem.org/mediawiki/2010/2/22/THUOICE.png" width="500px"></a><br />
<br/><br/><br />
<a href="http://www.tsinghua.edu.cn/docsn/jwc/office/indexe.htm" title="Tsinghua University Academic Affair Office" target=_blank>Tsinghua University Academic Affair Office<br/><img src="https://static.igem.org/mediawiki/2010/f/f0/THUAAO.jpg" width="500px"></a><br />
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Center of Theoretical Biology<br />
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Stem Cell Research Center in Tsinghua University<br />
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We thank ''José Torres Díaz''<html><sup>1</sup></html> and ''Julio González''<html><sup>2</sup></html> for their efforts to help us translate our Project Background into Spanish.<br />
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We thank ''WANG Shiqi''<html><sup>3</sup></html> for Japanese translation amending and ''LIANG Zecai''<html><sup>3</sup></html> for support, ''ZHANG Ying''<html><sup>4</sup></html> for Russian translation, ''QI Weijie''<html><sup>5</sup></html> for Japanese translation, ''Monika''<html><sup>6</sup></html> for German translation.<br />
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1. From School Electrical & Electronic Engineering, University of Tarapacá. Arica, Chile.<br />
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2. From School of Industrial Engineering, University of Tarapacá. Arica, Chile.<br />
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3. From School of Life Science, Tsinghua University<br />
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4. From Dalian University of Foreign Languages, now in РГСУ, Российский Государственный Социальный Университет.<br />
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5. From Shanghai International Studies University<br />
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6. From Denmark and got her Bachelor Degree in Germany.<br />
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=Reference=<br />
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1. Stephan Frey, Martin Haslbeck, Otmar Hainzl and Johannes Buchner: Synthesis and characterization of a functional intact IgG in a prokaryotic cell-free expression system Biol. Chem., Vol. 389, pp. 37–45<br />
<br />
2. Thomas E. Kuhlman ,Edward C. Cox:Site-specific chromosomal integration of largesynthetic constructs. Nucleic Acids Research, 2009, 1–10.<br />
<br />
3. Alex Pautsch and Georg E. Schulz Structure of the outer membrane protein A transmembrane domain Nature Structural biology 1998,5,11, 1013-1017<br />
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4. JOSEPH A. FRANCISCO* Transport and anchoring of 8-lactamase to the external surface of Escherichia coli PNAS 1992 89, 2713-2717<br />
<br />
5. Keren Ofir, Yevgeny Berdichevsky Versatile protein microarray based on carbohydrate-binding modules Proteomics 2005, 5, 1806–1814<br />
<br />
6. Ming-xiong He, Hong Feng, Yi-zheng Zhang, Construction of a novel cell-surface display system for heterologous gene expression in Escherichia coli by using an outer membrane protein of Zymomonas mobilis as anchor motif, Biotechnol Lett (2008) 30:2111–2117<br />
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7. Yariv Mazor, Thomas Van Blarcom, Robert Mabry, Brent L Iverson & George Georgiou, Isolation of engineered, fulllength antibodies from libraries expressed in Escherichia coli, NATURE BIOTECHNOLOGY<br />
<br />
8. Yariv Mazor, Thomas Van Blarcom, Brent L Iverson & George Georgiou, E-clonal antibodies: selection of full-length IgG antibodies using bacterial periplasmic display, NATURE PROTOCOLS 1766 VOL.3<br />
<br />
9. Christian W. Cobaugh, Juan C. Almagro, Mark Pogson, Brent Iverson and George Georgiou, Synthetic Antibody Libraries Focused Towards Peptide Ligands, J. Mol. Biol. (2008) 378, 622–633<br />
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10. Micka¨el Desvaux, The cellulosome of Clostridium cellulolyticum Enzyme and Microbial, Technology 37 (2005) 373–385<br />
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11. SANDRINE PAGE`S ANNE BE´LAI¨CH , HENRI-PIERRE FIEROBE, CHANTAL TARDIF, CHRISTIAN GAUDIN,AND JEAN-PIERRE BE´LAI¨CH, Sequence Analysis of Scaffolding Protein CipC and ORFXp, a New Cohesin-Containing Protein in Clostridium cellulolyticum: Comparison of Various Cohesin Domains and Subcellular Localization of ORFXp, JOURNAL OF BACTERIOLOGY, Mar. 1999, p. 1801–1810<br />
<br />
12. Sachdev S Sidhu, Full-length antibodies on display, NATURE BIOTECHNOLOGY VOLUME 25 NUMBER 5 MAY 2007<br />
<br />
13. Sang Yup Lee, Hyun Uk Kim, Jin Hwan Park, Jong Myung Park and Tae Yong Kim, Metabolic engineering of microorganisms: general strategies and drug production, Drug Discovery Today _ Volume 14, Numbers 1/2<br />
<br />
14. Ario de Marco, Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli, Microbial Cell Factories 2009, 8:26<br />
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<!--- To Gu Xiang -----><br />
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</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/project/outline/m1Team:Tsinghua/project/outline/m12010-10-27T15:05:40Z<p>Gumilton: /* Helper Plasmid (HP) Removal: “Inactivation of Recombinase” */</p>
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==Module I==<br />
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'''Antibodies Library Diversity & Randomicity'''<br />
[[Image:TSModule1.PNG|650px]]<br />
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This is an overall flowchart about our Module I and in this figure you can find each step is symbolizing each one of the content below. You can find the details about how we carried out experiments and achieved our goals by scrolling down and reading through them. You're warmly welcome to have a discussion with us.<br />
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===Abstract===<br />
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===Flow chart===<br />
[[Image:THUProjectFlowChart.JPG|600px]]<br />
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===Landing Pad Construction and Insertion===<br />
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'''Purpose of this step'''<br />
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Landing pad insertion is the first step of our two-step recombination system. By doing this step we induced our “RSS Sequence” into E.coli.<br />
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We insert a “landing pad” fragment which includes a promoter (placIQ1) and a tetracycline resistance gene (tetA) flanked by I-SceI recognition sites and 20-bp landing pad regions (LP1 and LP2) into Escherichia coli chromosome via att recombination. Then the helper plasmids encoding I-SceI endonuclease and λ-Red and donor plasmid encoding various antibiotic genes flanked by I-SceI recognition sites and same landing pad regions (LP1 and LP2) are transformed, which will be introduced in detail in next part. I-SceI expression is induced via the addition of L-arabinose. I-SceI recognition sites in the donor plasmid and chromosome are cleaved. Integration of the fragment in donor plasmid is facilitated by IPTG-induced λ-Red expression.<br />
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This two-step recombination method allows for the insertion of very large fragments into a specific location in ''Escherichia coli'' chromosome and in any orientation. <br />
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'''Construction of landing pad'''<br />
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Landing pad consists of the following parts: a promoter (placIQ1), two landing pad regions, two I-SceI recognition sites and a tetracycline resistance gene (tetA).<br />
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[[Image:THUProjectFigure1.PNG|650px]]<br />
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Another construction strategy is shown as below:<br />
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[[Image:THUProjectFigure1_1.JPG|650px]]<br />
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'''The first construction is used as the four-gene donor plasmid method, while the latter is for the two-gene donor plasmid method.''' We take the advantage of the former construction in the final recombination, because the promoter will not be cut when the recombination happens, thus saving us lots of time. As the latter construction, we can easily add a promoter before the gene, in two-gene donor plasmid A(shown in the Donor Plasmid Construction Part). <br />
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First we obtain two target DNAs – promoter and tetA with PCR. Then We use the overlap extension polymerase chain reaction (or OE-PCR) to link two fragments together. Primer 2 and primer 3 have homologous sequences, so one segment can anneal to the other in certain conditions, in other words, the two segments can overlap into one segment after extension. If necessary, operate PCR again with primer 1 and primer 4 to obtain more products. <br />
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[[Image:THUOverlap.JPG|500px]]<br />
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'''Landing pad insertion-- ATT RECOMBINATION'''<br />
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Our approach is based on genome targeting systems that utilize plasmids carrying a conditional-replication origin and a phage attachment (attP) site. We refer to our plasmids as CRIM (conditionalreplication, integration, and modular) plasmids. CRIM plasmids can be integrated into or retrieved from their bacterial attachment (attB) site by supplying phage integrase (Int) without or with excisionase (Xis) in trans.<br />
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We got a strain with plasmid pUK2 from LAB. Then we develop a E.coli strain contains the helper plasmid AH69. These two plasmids are shown below. In order to match other parts of our whole project, the modification that kan-exon should be replaced with tet-SDS was necessary. We got tet-SDS from another plasmid named pkts-cs. Then we use PCR to get the two fragments as PT (from pkts-cs) and V (from pUK2). We ligate PT and V after digestion and modification to form a new plasmid. This new plasmid was named as pUKIP and contains a promoter region, a tet-SDS region and phage attachment site (attP). As reported in the paper, there exist one bacterial attachment site (attB) of HK002 in TorT-TorS gene of the chromosomal DNAs of E.coli K12 strain. That is to say, the PT fragment will integrate into the cell genome after the plasmid was transferred in cells. <br />
<html><a name="att"></a></html><br />
[[Image:THUProjectFlowchart.jpg|700px]]<br />
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===Helper Plasmid(HP) Insertion===<br />
'''CRIM plasmid integration'''<br />
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Cells carrying a CRIM helper plasmid were grown in 20 ml of LB cultures with ampicillin at 30°C to an optical density of 600 nm of ca. 0.6 and then made electrocompetent. Following electroporation, cells were suspended in LB without ampicillin, incubated at 30°C for 30 min, at 42°C for 30 min and at 30°C for 30 min, and then spread onto selective agar (tet) and incubated at 37°C. Colonies were purified once nonselectively and then tested for antibiotic resistance for stable integration and loss of the helper plasmid and by PCR for copy number.<br />
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'''CRIM plasmid excision'''<br />
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Cells were transformed with the respective Xis/Int CRIM helper plasmid and then spread on ampicillin agar media at 30°C. Colonies were purified once or twice nonselectively on plates that were incubated for 1 h at 42°C and overnight at 37°C. They were then tested for antibiotic sensitivities and by PCR for loss of the integrated plasmid.<br />
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===Helper Plasmid Insertion: “Applying Recombinase Coding Gene”===<br />
We transferred our helper plasmid, which contains RecA enzyme coding gene and I-Sel enzyme coding gene, into E.coli. These two enzymes will act exactly as what the recombinase does.<br />
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===Donor Plasmid(DP) Construction===<br />
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<B>Constructing Our “Antibody Coding Gene Library”</B><br />
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'''Purpose of this step:'''<br />
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The construction of DP provides us multiple genes, which vary from each other, forming the library for us to select.<br />
After inserting landing pad and helper plasmid to E.coli, we must construct a series of donor plasmids to demonstrate that this system can truly realize the recombinant process in E.coli, thus we can further use this module to simulate the recombination of antibody gene in mammalian B cells. We not only need to test the efficiency of recombination, but also ensure that genes we get from this recombinant process can be expressed correctly and have their original function. So we intend to construct four plasmids to test the system.<br />
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'''Experiment design and expected results:'''<br />
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'''[1] Donor plasmid A'''<br />
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[[Image:THUPA.jpg|500px]]<br />
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In donor plasmid A, we insert only one gene, kanamycin resistant gene (Kanr) to test our recombination. At the 5’ end of Kanr, we add I-scel recognizing sequence (which is represented by the white arrow) and recombination sequence 1 (which is shown in red). At the 3’ end, we add another recombination sequence (which is shown in blue) and also I-scel recognizing sequence.<br />
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After construction, we will transform this donor plasmid to E.coli with landing pad and helper plasmid. After arabinose and IPTG inducing, the restriction enzyme I-scel will cut down Kanr (containing recombination sequences), which can recombine with the bacterial chromosome.<br />
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'''[2] Donor plasmid B'''<br />
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[[Image:THUProjectFigure2.jpg|500px]]<br />
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In donor plasmid B, we insert two genes, kanamycin resistant gene (Kanr) and Chloromycetin resistant gene (Chlr). At the 5’ end of these two genes, we add I-scel recognizing sequence (which is represented by the white arrow) and recombination sequence 1 (which is shown in red). At the 3’ end, we add another recombination sequence (which is shown in blue) and also I-scel recognizing sequence.<br />
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After construction, we will transform this donor plasmid to E.coli with landing pad and helper plasmid. After arabinose and IPTG inducing, the restriction enzyme I-scel will cut down these two genes (containing recombination sequences), which can recombine with the bacterial chromosome. In our expectation, either kanr or chlr will replace the landing pad, resulting in the bacteria resistance to either kanamycin or chloromycetin, but not both. This process will be random, so we can get as many colonies resistant to kanamycin as those resistant to chloromycetin.<br />
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'''[3] Donor plasmid C'''<br />
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[[Image:THUProjectFigure3.jpg|500px]]<br />
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Donor plasmid C includes four genes, GFP, mCherry, Kanr, Chlr, respectively. The same with genes in donor plasmid A, we add recombination sequences and I-scel recognition sequences to the ends of each genes. The recombination sequences of GFP and mCherry are identical, and those of Kanr and Chlr are the same. Note that recombination sequence at 3’ end of GFP (mCherry) and that at 5’ end of Kanr (Chlr) are the same, so we can get a random recombination of two genes, one is a fluorescence gene and the other is resistant gene, creating 2X2=4 different results.<br />
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'''[4] Donor plasmid D'''<br />
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[[Image:THUProjectFigure4.JPG|500px]]<br />
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For constructing donor plasmid D, we first cut the GFP and mCherry to 2 fragments respectively. Then we insert these four fragments into the plasmid in the order shown in the above picture. We expect that we can see either green or red fluorescence after transforming and inducing. Through this experiment, we can tell whether or not recombination sequence will affect the normal function of genes, further demonstrate that antibody producing by our system will be effective. On the other hand, we should note that the sequence length is another significant reason of antibody diversity, so the effect of recombination sequence on antibody will much small than that on fluorescence genes.<br />
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====Strategies====<br />
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Traditionally, we can construct these four plasmids by inserting genes into the plasmid one by one. Take donor plasmid C as a typical example. We choose plasmid PUC19 as our vector for the four genes. After identifying the multiple clone sites of PUC19 and four genes carefully, we select out four sites, including HindIII, SalI, BamHI, KpnI and EcoRI. We first insert the gene eGFP, amplified by PCR primers with HindIII and SalI sites, into the vector digested by HindIII and SalI. Then we use this plasmid (PUC19+eGFP) as our next vector for gene mCherry with SalI and BamHI sites. Repeat these processes of enzyme digestion and ligation, we can eventually get donor plasmid C with four genes as we want.<br />
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However, considering the huge number of antibody fragments, we try our best to seek other strategies to complete the ligation of multiple fragments, which can be done more quickly and efficiently.<br />
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=====Principle=====<br />
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The fragments include different landing pad regions and endoclease recognition site, and are grouped together by sharing the same landing pad region.<br />
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=====Method===== <br />
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In the demand of larger amount of fragments and constructed plasmids, we find two ways to realize such purpose, regarded as the key point of our whole project-guarantee the large variation of antibody.<br />
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'''Method 1:'''<br />
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'''Concept:'''<br />
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The recognition site of endonuclease DraIII is as follows: 5' CACNNNGTG 3', so we can design more than one recognition sequence added to multiple fragments and complete the excision in one procedure.<br />
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'''Procedure:'''<br />
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In our experiment, three different recognition sequences are designed and added to six ends of three fragments with every two of the ends sharing the identical sequence. These three fragments, one encoding Km resistant gene, another Cm resistant gene, and the third replication origin, then can be excised in one sample, and ligated to construct a plasmid.<br />
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Both the Km and Cm resistant gene are flanked with the identical landing pad region, thus, expected to be recombined into the E. coli chromosome with the same efficiency.<br />
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In the same way, we can excise and ligate three fragments or even more fragments. In the later experiment, we try to construct plasmid with four or five fragments.<br />
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As introduced above, a key problem to produce antibodies based on our design is how to develop an efficient and quick method, which can be applied to construct antibody genes library. Therefore, we try to establish a special method, named “multiple fragments ligation in order”, the principle of which is, briefly, based on a very kind of restriction enzyme-DraIII. <br />
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DraIII is somehow distinguished from other restriction enzymes for its specific cutting site, as shown below:<br />
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CAC NNN▼GTG<br />
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GTG▲NNN CAC<br />
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N stands for any of the base A, T, C, G. It is obvious that multiple different short sequences can be cut by Dra III as long as the sequence starts with CAC and ends with GTG. Two fragments with complementary “NNN” can be combined together while those without noncomplementary “NNN” cannot. The following figure shows the principle and process:<br />
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[[Image:THUProjectFigure5.JPG|650px]]<br />
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[[Image:THUProjectFigure6.JPG|650px]]<br />
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===Removal of Helper Plasmid(HP)===<br />
Helper plasmid (HP) includes a temperature sensitive pSC101 replication origin, which maintains the plasmid at low copy number. This plasmid is thus easily removed by growth at 42℃ and screening against spectinomycin resistance. Of more concern is the donor plasmid, which is cured by I-SceI cleavage, and this process is very efficient, with only about 1% of cells retaining the donor plasmid.<br />
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<html><a name="dpiri"></a></html><br />
===Donor Plasmid(DP) Insertion & Recombination Induction: The "Very VDJ Recombination"===<br />
'''Propose of this step:'''<br />
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When our DP and HP are inserted, we have finished our preparing and begin to operate our system, just as the recombinase dose in B cells.<br />
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After the reform of the E.coli genome and the construction of the donor plasmid, we need to test our module’s function. First we should insert the donor plasmid(DP) into the E.coli, and then induce the recombination by adding IPTG and arabinose. Arabinose active the I-Sel restriction enzyme, then cut DP and genome at the same site. After the digestion, we add IPTG to help the recombination, using the homologous sequence near the cohesive end.<br />
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'''DP Insertion: '''<br />
[[Image:THUProjectFigure7.png|350px|right]]<br />
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As we said before, we use a pre-altered template to amplify landing pad fragments using the landing pad regions as standardized priming sites. Here we used the conventional electroporation method to transform the DP into E.coli genome, then incubate the plate at 37°overnight.<br />
Electroporation is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. It is a dynamic phenomenon that depends on the local transmembrane voltage (we used 1 V) at each point on the cell membrane. If E.coli and DP are mixed together, the plasmids can be transferred into the cell after electroporation. This procedure is highly efficient than chemical transformation. (Partly from wikipedia)<br />
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'''Recombination Induction:'''<br />
<br />
Individual colonies were inoculated into 5 ml of EZ-Rich Defined Medium (RDM; Teknova) +0.5% glycerol, 2mM IPTG, and 0.2% w/v L-arabinose. After growing at 37°C for 1 h in a shaking water bath, we transfer the medium to 30_C shaking water bath for 4 h, then 100 mg/ml spectinomycin was added. At first the I-Sel enzyme is expressed to cut the genome and DP at the same site. And this step is used to constitutive express the Rec A enzyme, thus initiating the recombination by the homologous region.<br />
<br />
The appropriate antibiotic for the given insertion fragment was then added (25 mg/ml kanamycin, 34 mg/ml chloramphenicol), and the cultures were grown overnight. The next day, 100 ml sample was plated on LB plates with the appropriate antibiotic and grown at 37°C. We test the sample by screening it on LB plates containing 100 mg/ml ampicillin or 10 mg/ml tetracycline to verify the loss of the landing pad and donor plasmid.<br />
<br />
[[Image:THUProjectFigure8.JPG|650px]]<br />
<br />
===Helper Plasmid (HP) Removal: “Inactivation of Recombinase”===<br />
<br />
Helper plasmid (HP) includes a temperature sensitive pSC101 replication origin, which maintains the plasmid at low copy number. This plasmid is thus easily removed by growth at 42℃ and screening against spectinomycin resistance. Of more concern is the donor plasmid, which is cured by I-SceI cleavage, and this process is very efficient, with only about 1% of cells retaining the donor plasmid.<br />
<br />
<br />
===Verification of Recombination efficiency===<br />
<br />
To examine the efficiency of the recombination system, we carry out the experiments as follow.<br />
<br />
Using the E. coli which are already been transformation with Landing Pad and Helper plasmid to construct competent cell for electro transformation. The detail can be found at Tsinghua iGEM 2010 Protocol 1-6:Preparation of Competent Cell for Electro Transformation. Transformation the Donor Plasmid and spread the bacteria onto the plate with propriety antibiotics.<br />
<br />
There are two ways to induce. One is induction directly, the other is induction while culture.<br />
<br />
Induction directly:<br />
1. Pick colony into TB (0.2% L-arabinose, 2mM IPTG) without any antibiotics. Culture at 37℃ for 1 hour.<br />
<br />
2. Add 100ug/ml spectinomycin, culture at 30℃ for 4 hours.<br />
<br />
3. Add addition antibiotics according to the insertion gene on the Donor plasmid. Culture overnight at 30℃.<br />
<br />
4. Dilute at propriety concentration and spread at plate with antibiotics to count.<br />
<br />
Induction while culture:<br />
<br />
1. Pick colony into multi-antibiotics (tet, spe, and other antibiotics according to the gene insertion in the Donor plasmid) LB. Culture at 30℃.<br />
<br />
2. Transfer the bacteria into LB with spe and one other antibiotic to select. Culture until OD value reaches 0.6 and then add 0.2% L-arabinose, 2mM IPTG. Culture at 30℃ for half an hour.<br />
<br />
3. Culture at 37℃ for 20 minutes.<br />
<br />
4. Culture at 30℃ overnight.<br />
<br />
5. Dilute at propriety concentration and spread at plate shown blow.<br />
<br />
(1) tet only: to test whether the Helper plasmid exist.<br />
<br />
(2) one of the antibiotic among the Donor plasmid only (depend on the Donor plasmid) for count.<br />
<br />
(3) tet + IPTG + arabinose + spectinomycin.<br />
<br />
(4) one of the antibiotic + IPTG + arabinose + spectinomycin.<br />
<br />
6. Pick colonies from plate, using well designed primers to detect. The principle of primer design is that the up-stream primer is on the genome next to the recombination site and the down-stream one is on the combination gene. Calculate the rate of recombination last.<br />
<br />
<br />
<br />
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<h1>Outline</h1><br />
<br />
<div class="content_block"><br />
<br />
<br/><br />
In order to realize the production of antibodies in prokaryotic cells, we firstly investigate the whole process of antibody production in mammalian immune system and try to simulate the whole process via the methods and concepts of synthetic biology. Finally, we successfully constructed the desired system. The comparison between the key steps involved in antibody generation and corresponding synthetic methods are shown in the chart below.<br />
<br/><br />
<br />
<table border="2" bordercolor="maroon" bgcolor="silver"><br />
<tbody><br />
<tr><br />
<th colspan=3>Antibody Production</th><br />
<th width=50px></th><br />
<th colspan=2>E Coli. Production System</th></tr><br />
<tr><br />
<td rowspan=4 width=70px><a href="#VDJ_Recombination_vs_Lading_Pad_Recombination">VDJ Recombination</a></td><br />
<td rowspan=3 width=70px>Preparation</td><br />
<td width=70px>RSS Sequence</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">Landing Pad Insertion</a></td><br />
<td rowspan=4 width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1">Module I: Recombination System</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Recombinase</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">Helper Plasmid Insertion</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Library</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">Donor Plasmid Construction</a></td><br />
</tr><br />
<tr><br />
<td>Recombination</td><br />
<td colspan=2><img src="https://static.igem.org/mediawiki/2010/d/d6/THUarrow2.png" width=180></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">DP Insertion and Recombination Induction</a></td><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="#Somatic_hyper-mutation_vs_Junctional_mutation">Somatic Hypermutation</a></td><br />
<td rowspan=2 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
<!---------<td>Junctional Diversity</td>----------------------><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">CBD-based Microarray</a></td><br />
<td rowspan=4><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2">Module II: Selection System</a></td><br />
</tr><br />
<tr><br />
<td rowspan=3><a href="#Antigen-specific_Selection_vs_CBD-Based_Microarray_and_ToxR-Based_Transmembrane_pathway_method">Antigen Selection</a></td><br />
<td rowspan=3 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">ToxR-based Transmembrane Pathway</a></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">Cooperation With Macquarie_Australia</a></td><br />
</tr><br />
</tbody><br />
</table><br />
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<br/><br />
Generally speaking, antibody production in our project can be divided into two modules.<br />
<br />
<html><br />
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<br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1"><font face="Comic Sans MS" size="4">Module I: Generation of antibody library</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2"><font face="Comic Sans MS" size="4">Module II: Selection of specific antibodies</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
</html><br />
<br />
==Project Design==<br />
The first module is carried out by a novel method called E coli in vivo recombination system. The theoretical and experimental basis comes from the work of Thomas E. Kuhlman and Edward C. Cox. We managed to modify their system, wishing to utilize it for simulation of antibody recombination.<br />
<br />
Firstly, we try to insert a DNA segment called”Landing Pad(LP)” into the specific sites of E coli via Att recombination. Landing pad sequence consists of the following parts(from 5’ to 3’ in the sequence):<br />
<br />
1)25bp-long random sequence<br />
<br />
2)15bp-long recognition sequence of restriction enzyme I-scel<br />
<br />
3) antibiotic resistance gene used for antibody selection<br />
<br />
4) 15bp-long recognition sequence of restriction enzyme I-scel(corresponding to 2)<br />
<br />
5) 25bp-long random sequence (corresponding to 1)<br />
<br />
After integrating DNA segment of Landing pad into the genome of E coli, we completed the genetic engineering of the genome of E coli. All the five parts of landing pad are designed for subsequent recombination. <br />
<br />
Besides, in order to achieve different recombination goals, we designed several landing pads of different sequences.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion"><font face="Comic Sans MS" size=3>'Landing Pad Construction and Insertion'</font></a></html> to learn about the details of Landing Pad design. <br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#att"><font face="Comic Sans MS" size=3>'link'</font></a></html> to learn more about ATT recombination.<br />
<br />
After the integration of landing pad, we have to insert another DNA fragment called Helper Plasmid(HP), which provides necessary tools for in vivo recombination. <br />
<br />
In this plasmid(Helper Plasmid), there are two genes used for recombination. <br />
<br />
The first one encodes restriction enzyme I-scel after induction of L-arabinose. I-scel is able to recognize the 15bp-length sequence flanking a target DNA fragment and cut out the fragment, therefore providing DNA segments for recombination mediated by other components.<br />
<br />
The second one encodes the enzyme responsible for recombination called Lamda-Red. With the presence of IPTG, Lamda-Red will recombine DNA fragments flanked by the same DNA sequence recognized by Lamda-Red.<br />
<br />
In addition, Helper Plasmid contains a specific temperature-sensitive replicon, which is used to control the replication via temperature changes. We can remove the replicon when necessary.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi"><font face="Comic Sans MS" size=3>'HP insertion'</font></a></html> to learn more about Helper Plasmid.<br />
<br />
Up till now, we successfully engineered a ‘recepient strain’. Then we have to transform the ‘recepient strain’ with the Donor Plasmid(DP) containing recombination fragments. Donor Plasmid contains several Insertion Fragments and each Insertion Fragment contains following five parts:<br />
<br />
1) one 15bp-long restriction enzyme I-scel recognition site<br />
<br />
2) one 25bp-long random sequence<br />
<br />
3) one fragment for insertion and recombination<br />
<br />
4) one 25bp-long random sequence<br />
<br />
5) one 15bp-long restriction enzyme I-scel recognition site(in correspondence with 1)<br />
<br />
(Note: the order of the five parts differ from that of landing pad)<br />
<br />
As we previously mentioned, Donor plasmid contains several Insertion Fragments. Based on the flanking landing pad sequence, we can ascribe Insertion Fragments to the same group as long as the flanking landing pad sequences of those Insertion Fragments are the same.<br />
<br />
In order to achieve different goals, we design different Donor Plasmids, different Donor Plasmids contain different number of Insertion Fragments, which belong to different groups.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc"><font face="Comic Sans MS" size=3>‘DP construction’</font></a></html> to learn about the details of Donor Plasmid and Insertion Fragments and the strategies employed in our design. <br />
<br />
As we all know, antibody diversity mainly lean on the number of the fragments used for recombination. Therefore, in order to mimic the antibody generation process, we need to construct Donor Plasmid which contains large sum of Insertion Fragments. Theoretically, we can construct Donor Plasmid of this kind. However, due to the time limit of the competition, we didn’t try to construct DP containing many Insertion Fragments.<br />
<br />
In spite of this, in order to meet the needs, we designed two methods to construct plasmids on a large scale and attempted the usage in parts of our Donor Plasmids.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Strategies"><font face="Comic Sans MS" size=3>'DP construction and Strategies'</font></a></html> for the methods of rapid plasmid construction. <br />
<br />
Besides, we have applied for two BBFRFC, which are BBFRFC61 and BBFRFC 62 respectively.<br />
<br />
Then, after transformation of the prepared ‘recepient strain’ with Donor Plasmid and the induction of corresponding agents, I-scel enzyme cuts out DNA fragments from DP and LP, generating numerous Insertion Fragments. Besides, antibiotic resistance gene integrated into E coli genome is also cut out, providing recombination sites for Insertion Fragments.<br />
<br />
Provided with Insertion Fragments mentioned above and Lamda-Red expressed by Donor Plasmid, Insertion Fragments recombine with the corresponding landing pad sequence in the genome and thus get inserted into the correct site of the genome. Based on our design of Landing pad, we can ensure that only one Insertion Fragment from one group is randomly inserted into the genome, therefore mimicking the VDJ recombination process.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri"><font face="Comic Sans MS" size=3>‘DP Insertion and Recombination Induction’</font></a></html> to learn more about this part. <br />
<br />
Engineered E coli cells which have undergone successful recombination can be selected out of the pool through specific screening methods. After tests on different strains, our project achieve a recombination rate above 50% based on our current techniques.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/experiments#res"><font face="Comic Sans MS" size=3>‘Result’</font></a></html> to learn more about the identification of recombination rate. <br />
<br />
The goal of the second module in our project is aimed at selection of specific antibodies. Due to the maturity of existing antibody screening technique, our work mainly focuses on imitation of mammalian antibody selection, thus providing possible alternatives for existing methods.<br />
<br />
The outline of our ideas is to find mechanisms similar to in vivo antibody selection and take in,to account the industry production costs, controllability and reliability, thus developing new methods for antibody selection.<br />
In fact, in mammalian system, antibody selection is achieved through the activation of B lymphocytes and thus the rapid proliferation of the B lymphocytes that express the specific antibodies that bind to antigen. Simply put, the whole process relies on the interaction between antigen epitopes and membrane integral immunoglobins.<br />
<br />
To mimic the activation mediated by membrane integral immunoglobins, we develop ToxR-mediated transmembrane activation signal system. In this system, the interaction between antibodies and antigen triggered downstream expression of reporter genes, thus providing signals for selection.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2"><font face="Comic Sans MS" size=3>‘ToxR-based Transmembrane Signaling Pathway Method’</font></a></html> for detailed description of this method.<br />
<br />
In addition, we develop a technique called ‘Bacterial based Microarray’ for selection purpose. In this method, we combine membrane display technique and high throughput microarray technique, that is, a bacterial based microarray method.<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1"><font face="Comic Sans MS" size=3>’Bacterial based microarray’</font></a></html> for details.<br />
<br />
In order to find methods for antibody selection, we cooperate with other iGEM teams. For example, we talked with Macquarie_Australia iGEM team about the project. The techniques involved in their project might be useful for our selection methods. Therefore, members from both teams worked together to research on this problem.<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3"><font face="Comic Sans MS" size=3>'link'</font></a></html> to learn more about our cooperation.<br />
<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens. <br />
Our system is aiming at imitating this recombination process, using E.coli as the gene carrier.<br />
It is known to all that regional genes (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and the recombination occurs when VDJ recombinase are expressed. We choose RecA enzyme to induce the recombination, while use I-Sel enzyme to cut the genome, just mimicking the process happened in B cells.<br />
<br/><br />
<br/><br />
<br />
==Comparison between natural antibody production and E. Coli system==<br />
<br/><br />
===VDJ Recombination vs Lading Pad Recombination===<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is the mechanism by which immunoglobulin (Ig) and T cell receptors (TCR) are generated in the immune system. Through V(D)J recombination, Variable, Diverse, and Joining gene segments are randomly combined, thus encoding various kinds of immunoglobins and T cell receptors to recognize potential antigens. <br />
During the process of B cell maturation, gene segments are rearranged and thus transcribe mRNA that can produce diverse types of antibody for recognition of billions of potential epitopes. The rearrangement brings three gene segments (termed V, D, and J) in close proximity, which are then joined together head to tail, the process of which is known as V(D)J recombination based on the V,D,J gene segments involved. Multiple copies of the V, D, and J genes in the human genome are shuffled in the process, and a specific antibody will be generate among millions of other possible combinations. Because gene are joined together permanently, one mature B cell will produce only one specific antibody.<br />
<br />
Our system is aimed to mimic this recombination process, using E.coli as the carrier.<br />
Through previous research in immunology field, each kind of gene segments (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and recombination occurs when VDJ recombinase recognize the specific signal sequence and mediate the recombination process. In our project, we design a sequence, which carries separated parts of one complete gene and integrate synthesized DNA fragment into the genome of E coli via landing pad method. Then we use I-Sel enzyme to cut the gene segments out of E coli genome, into which gene segments are inserted and choose RecA enzyme to recognize the previously designed signal sequence flanking our target sequence and recombine the gene segments, just like what recombinase accomplish in B cells. The whole process is called landing pad recombination.<br />
<br />
===Somatic hyper-mutation vs Junctional mutation===<br />
<br />
Somatic hypermutation involves a programmed process of mutation affecting the variable regions of immunoglobulin genes. During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is at least 105-106 fold greater than the normal rate of mutation across the genome. Variation is mainly in the form of single base substitutions, with insertions and deletions being less common. These mutations occur mostly at “hotspots” in the DNA, known as hypervariable regions. These regions correspond to the complementarity determining regions; the sites involved in antigen recognition on the immunoglobulin. This directed hypermutation allows for the selection of B cells that express immunoglobulin receptors possessing an enhanced ability to recognize and bind a specific foreign antigen.<br />
<br />
In addition, the cross ligation, namely Landing pad recombination, has higher opportunity to mutate in the link sequence. The introduction of 4 junctional shuffler 64 will meet the need of the rest 107 diversity, perfectly undertaking the work in the antibody diversity construction that hyper-mutation does in the highly variable region. In this way, we have finished a building library, waiting for the specialized antigen to select.<br />
<br />
===Antigen-specific Selection vs CBD-Based Microarray and ToxR-Based Transmembrane pathway method===<br />
In mammalian immune system, billions of antibodies will be generated. Then how can the immune system find out specific antibody that indiscriminately recognize the specific antigen that comes in. Generally speaking, due to the fact that one mature B cell only generates one specific immunoglobin, once the membrane integral immunoglobin bind to the specific antigen, a series of intracellular response, such as gene activation and cell differetiation will be triggered in B cell. Then, B cells expressing the specific antibodies that recognize the specific antigen will proliferate and generate enormous amount of antibodies that help eliminate the specific antigen.<br />
<br />
Simply put, antibody selection is accomplished through the specific interaction between the membrane immunoglobin and the specific antigen. Therefore, in our project, we also plan to mimic this process through interaction between the antibodies generated through landing pad recombination and the antigen we target. CBD-based Microarray and ToxR-based Transmembrane pathway are the two methods by which we achieve this goal.<br />
CBD domain, which interacts with cellulose, potentially provides us way to locate our E.coli to the microarray coated with cellulose. OmpA (outer membrane protein A), displays the antibody and antigen out to the membrane surface. Then by applying the antibodies generated through landing pad recombination to the microarray, antibodies that bind to the antigen will be retained and thus selected out of the pool of antibodies.<br />
<br />
Alternatively, the ToxR-based transmembrane pathway method shows a different strategy for us to seek the right folded antibody. Once the antigen comes, ToxR outer part will form dimer shape and the downstream signal occur, supplying the approach of reporters, such as GFP, to examine and pick out what we want.<br />
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</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/project/outlineTeam:Tsinghua/project/outline2010-10-27T14:58:46Z<p>Gumilton: /* Project Design */</p>
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<h1>Outline</h1><br />
<br />
<div class="content_block"><br />
<br />
<br/><br />
In order to realize the production of antibodies in prokaryotic cells, we firstly investigate the whole process of antibody production in mammalian immune system and try to simulate the whole process via the methods and concepts of synthetic biology. Finally, we successfully constructed the desired system. The comparison between the key steps involved in antibody generation and corresponding synthetic methods are shown in the chart below.<br />
<br/><br />
<br />
<table border="2" bordercolor="maroon" bgcolor="silver"><br />
<tbody><br />
<tr><br />
<th colspan=3>Antibody Production</th><br />
<th width=50px></th><br />
<th colspan=2>E Coli. Production System</th></tr><br />
<tr><br />
<td rowspan=4 width=70px><a href="#VDJ_Recombination_vs_Lading_Pad_Recombination">VDJ Recombination</a></td><br />
<td rowspan=3 width=70px>Preparation</td><br />
<td width=70px>RSS Sequence</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">Landing Pad Insertion</a></td><br />
<td rowspan=4 width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1">Module I: Recombination System</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Recombinase</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">Helper Plasmid Insertion</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Library</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">Donor Plasmid Construction</a></td><br />
</tr><br />
<tr><br />
<td>Recombination</td><br />
<td colspan=2><img src="https://static.igem.org/mediawiki/2010/d/d6/THUarrow2.png" width=180></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">DP Insertion and Recombination Induction</a></td><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="#Somatic_hyper-mutation_vs_Junctional_mutation">Somatic Hypermutation</a></td><br />
<td rowspan=2 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
<!---------<td>Junctional Diversity</td>----------------------><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">CBD-based Microarray</a></td><br />
<td rowspan=4><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2">Module II: Selection System</a></td><br />
</tr><br />
<tr><br />
<td rowspan=3><a href="#Antigen-specific_Selection_vs_CBD-Based_Microarray_and_ToxR-Based_Transmembrane_pathway_method">Antigen Selection</a></td><br />
<td rowspan=3 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">ToxR-based Transmembrane Pathway</a></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">Cooperation With Macquarie_Australia</a></td><br />
</tr><br />
</tbody><br />
</table><br />
</html><br />
<br/><br />
Generally speaking, antibody production in our project can be divided into two modules.<br />
<br />
<html><br />
<body bgcolor="#336699" text="#ffffff" link="#60a179"><br />
<br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1"><font face="Comic Sans MS" size="4">Module I: Generation of antibody library</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2"><font face="Comic Sans MS" size="4">Module II: Selection of specific antibodies</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
</html><br />
<br />
==Project Design==<br />
The first module is carried out by a novel method called E coli in vivo recombination system. The theoretical and experimental basis comes from the work of Thomas E. Kuhlman and Edward C. Cox. We managed to modify their system, wishing to utilize it for simulation of antibody recombination.<br />
<br />
Firstly, we try to insert a DNA segment called”Landing Pad(LP)” into the specific sites of E coli via Att recombination. Landing pad sequence consists of the following parts(from 5’ to 3’ in the sequence):<br />
<br />
1)25bp-long random sequence<br />
<br />
2)15bp-long recognition sequence of restriction enzyme I-scel<br />
<br />
3) antibiotic resistance gene used for antibody selection<br />
<br />
4) 15bp-long recognition sequence of restriction enzyme I-scel(corresponding to 2)<br />
<br />
5) 25bp-long random sequence (corresponding to 1)<br />
<br />
After integrating DNA segment of Landing pad into the genome of E coli, we completed the genetic engineering of the genome of E coli. All the five parts of landing pad are designed for subsequent recombination. <br />
<br />
Besides, in order to achieve different recombination goals, we designed several landing pads of different sequences.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion"><font face="Comic Sans MS" size=2>'Landing Pad Construction and Insertion'</font></a></html> to learn about the details of Landing Pad design. <br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#att">'link'</a></html> to learn more about ATT recombination.<br />
<br />
After the integration of landing pad, we have to insert another DNA fragment called Helper Plasmid(HP), which provides necessary tools for in vivo recombination. <br />
<br />
In this plasmid(Helper Plasmid), there are two genes used for recombination. <br />
<br />
The first one encodes restriction enzyme I-scel after induction of L-arabinose. I-scel is able to recognize the 15bp-length sequence flanking a target DNA fragment and cut out the fragment, therefore providing DNA segments for recombination mediated by other components.<br />
<br />
The second one encodes the enzyme responsible for recombination called Lamda-Red. With the presence of IPTG, Lamda-Red will recombine DNA fragments flanked by the same DNA sequence recognized by Lamda-Red.<br />
<br />
In addition, Helper Plasmid contains a specific temperature-sensitive replicon, which is used to control the replication via temperature changes. We can remove the replicon when necessary.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">'HP insertion'</a></html> to learn more about Helper Plasmid.<br />
<br />
Up till now, we successfully engineered a ‘recepient strain’. Then we have to transform the ‘recepient strain’ with the Donor Plasmid(DP) containing recombination fragments. Donor Plasmid contains several Insertion Fragments and each Insertion Fragment contains following five parts:<br />
<br />
1) one 15bp-long restriction enzyme I-scel recognition site<br />
<br />
2) one 25bp-long random sequence<br />
<br />
3) one fragment for insertion and recombination<br />
<br />
4) one 25bp-long random sequence<br />
<br />
5) one 15bp-long restriction enzyme I-scel recognition site(in correspondence with 1)<br />
<br />
(Note: the order of the five parts differ from that of landing pad)<br />
<br />
As we previously mentioned, Donor plasmid contains several Insertion Fragments. Based on the flanking landing pad sequence, we can ascribe Insertion Fragments to the same group as long as the flanking landing pad sequences of those Insertion Fragments are the same.<br />
<br />
In order to achieve different goals, we design different Donor Plasmids, different Donor Plasmids contain different number of Insertion Fragments, which belong to different groups.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">‘DP construction’</a></html> to learn about the details of Donor Plasmid and Insertion Fragments and the strategies employed in our design. <br />
<br />
As we all know, antibody diversity mainly lean on the number of the fragments used for recombination. Therefore, in order to mimic the antibody generation process, we need to construct Donor Plasmid which contains large sum of Insertion Fragments. Theoretically, we can construct Donor Plasmid of this kind. However, due to the time limit of the competition, we didn’t try to construct DP containing many Insertion Fragments.<br />
<br />
In spite of this, in order to meet the needs, we designed two methods to construct plasmids on a large scale and attempted the usage in parts of our Donor Plasmids.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Strategies">'DP construction and Strategies'</a></html> for the methods of rapid plasmid construction. <br />
<br />
Besides, we have applied for two BBFRFC, which are BBFRFC61 and BBFRFC 62 respectively.<br />
<br />
Then, after transformation of the prepared ‘recepient strain’ with Donor Plasmid and the induction of corresponding agents, I-scel enzyme cuts out DNA fragments from DP and LP, generating numerous Insertion Fragments. Besides, antibiotic resistance gene integrated into E coli genome is also cut out, providing recombination sites for Insertion Fragments.<br />
<br />
Provided with Insertion Fragments mentioned above and Lamda-Red expressed by Donor Plasmid, Insertion Fragments recombine with the corresponding landing pad sequence in the genome and thus get inserted into the correct site of the genome. Based on our design of Landing pad, we can ensure that only one Insertion Fragment from one group is randomly inserted into the genome, therefore mimicking the VDJ recombination process.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">‘DP Insertion and Recombination Induction’</a></html> to learn more about this part. <br />
<br />
Engineered E coli cells which have undergone successful recombination can be selected out of the pool through specific screening methods. After tests on different strains, our project achieve a recombination rate above 50% based on our current techniques.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/experiments#res">‘Result’</a></html> to learn more about the identification of recombination rate. <br />
<br />
The goal of the second module in our project is aimed at selection of specific antibodies. Due to the maturity of existing antibody screening technique, our work mainly focuses on imitation of mammalian antibody selection, thus providing possible alternatives for existing methods.<br />
<br />
The outline of our ideas is to find mechanisms similar to in vivo antibody selection and take in,to account the industry production costs, controllability and reliability, thus developing new methods for antibody selection.<br />
In fact, in mammalian system, antibody selection is achieved through the activation of B lymphocytes and thus the rapid proliferation of the B lymphocytes that express the specific antibodies that bind to antigen. Simply put, the whole process relies on the interaction between antigen epitopes and membrane integral immunoglobins.<br />
<br />
To mimic the activation mediated by membrane integral immunoglobins, we develop ToxR-mediated transmembrane activation signal system. In this system, the interaction between antibodies and antigen triggered downstream expression of reporter genes, thus providing signals for selection.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">‘ToxR-based Transmembrane Signaling Pathway Method’</a></html> for detailed description of this method.<br />
<br />
In addition, we develop a technique called ‘Bacterial based Microarray’ for selection purpose. In this method, we combine membrane display technique and high throughput microarray technique, that is, a bacterial based microarray method.<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">’Bacterial based microarray’</a></html> for details.<br />
<br />
In order to find methods for antibody selection, we cooperate with other iGEM teams. For example, we talked with Macquarie_Australia iGEM team about the project. The techniques involved in their project might be useful for our selection methods. Therefore, members from both teams worked together to research on this problem.<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">'link'</a></html> to learn more about our cooperation.<br />
<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens. <br />
Our system is aiming at imitating this recombination process, using E.coli as the gene carrier.<br />
It is known to all that regional genes (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and the recombination occurs when VDJ recombinase are expressed. We choose RecA enzyme to induce the recombination, while use I-Sel enzyme to cut the genome, just mimicking the process happened in B cells.<br />
<br/><br />
<br/><br />
<br />
==Comparison between natural antibody production and E. Coli system==<br />
<br/><br />
===VDJ Recombination vs Lading Pad Recombination===<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is the mechanism by which immunoglobulin (Ig) and T cell receptors (TCR) are generated in the immune system. Through V(D)J recombination, Variable, Diverse, and Joining gene segments are randomly combined, thus encoding various kinds of immunoglobins and T cell receptors to recognize potential antigens. <br />
During the process of B cell maturation, gene segments are rearranged and thus transcribe mRNA that can produce diverse types of antibody for recognition of billions of potential epitopes. The rearrangement brings three gene segments (termed V, D, and J) in close proximity, which are then joined together head to tail, the process of which is known as V(D)J recombination based on the V,D,J gene segments involved. Multiple copies of the V, D, and J genes in the human genome are shuffled in the process, and a specific antibody will be generate among millions of other possible combinations. Because gene are joined together permanently, one mature B cell will produce only one specific antibody.<br />
<br />
Our system is aimed to mimic this recombination process, using E.coli as the carrier.<br />
Through previous research in immunology field, each kind of gene segments (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and recombination occurs when VDJ recombinase recognize the specific signal sequence and mediate the recombination process. In our project, we design a sequence, which carries separated parts of one complete gene and integrate synthesized DNA fragment into the genome of E coli via landing pad method. Then we use I-Sel enzyme to cut the gene segments out of E coli genome, into which gene segments are inserted and choose RecA enzyme to recognize the previously designed signal sequence flanking our target sequence and recombine the gene segments, just like what recombinase accomplish in B cells. The whole process is called landing pad recombination.<br />
<br />
===Somatic hyper-mutation vs Junctional mutation===<br />
<br />
Somatic hypermutation involves a programmed process of mutation affecting the variable regions of immunoglobulin genes. During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is at least 105-106 fold greater than the normal rate of mutation across the genome. Variation is mainly in the form of single base substitutions, with insertions and deletions being less common. These mutations occur mostly at “hotspots” in the DNA, known as hypervariable regions. These regions correspond to the complementarity determining regions; the sites involved in antigen recognition on the immunoglobulin. This directed hypermutation allows for the selection of B cells that express immunoglobulin receptors possessing an enhanced ability to recognize and bind a specific foreign antigen.<br />
<br />
In addition, the cross ligation, namely Landing pad recombination, has higher opportunity to mutate in the link sequence. The introduction of 4 junctional shuffler 64 will meet the need of the rest 107 diversity, perfectly undertaking the work in the antibody diversity construction that hyper-mutation does in the highly variable region. In this way, we have finished a building library, waiting for the specialized antigen to select.<br />
<br />
===Antigen-specific Selection vs CBD-Based Microarray and ToxR-Based Transmembrane pathway method===<br />
In mammalian immune system, billions of antibodies will be generated. Then how can the immune system find out specific antibody that indiscriminately recognize the specific antigen that comes in. Generally speaking, due to the fact that one mature B cell only generates one specific immunoglobin, once the membrane integral immunoglobin bind to the specific antigen, a series of intracellular response, such as gene activation and cell differetiation will be triggered in B cell. Then, B cells expressing the specific antibodies that recognize the specific antigen will proliferate and generate enormous amount of antibodies that help eliminate the specific antigen.<br />
<br />
Simply put, antibody selection is accomplished through the specific interaction between the membrane immunoglobin and the specific antigen. Therefore, in our project, we also plan to mimic this process through interaction between the antibodies generated through landing pad recombination and the antigen we target. CBD-based Microarray and ToxR-based Transmembrane pathway are the two methods by which we achieve this goal.<br />
CBD domain, which interacts with cellulose, potentially provides us way to locate our E.coli to the microarray coated with cellulose. OmpA (outer membrane protein A), displays the antibody and antigen out to the membrane surface. Then by applying the antibodies generated through landing pad recombination to the microarray, antibodies that bind to the antigen will be retained and thus selected out of the pool of antibodies.<br />
<br />
Alternatively, the ToxR-based transmembrane pathway method shows a different strategy for us to seek the right folded antibody. Once the antigen comes, ToxR outer part will form dimer shape and the downstream signal occur, supplying the approach of reporters, such as GFP, to examine and pick out what we want.<br />
<br />
<html><br />
&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a><br />
</body><br />
</div><br />
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</div><br />
</div></div></body><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/project/outlineTeam:Tsinghua/project/outline2010-10-27T14:58:29Z<p>Gumilton: /* Project Design */</p>
<hr />
<div>__NOTOC__<br />
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<script><br />
navlist=new Array("Background", "Project Outline", "Module I", "Module II", "Future");<br />
linkl = new Array("/Team:Tsinghua/project", "#outline", "/Team:Tsinghua/project/outline/m1", "/Team:Tsinghua/project/outline/m2", "future");<br />
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<div id="main_content"><a name="outline"></a><br />
<h1>Outline</h1><br />
<br />
<div class="content_block"><br />
<br />
<br/><br />
In order to realize the production of antibodies in prokaryotic cells, we firstly investigate the whole process of antibody production in mammalian immune system and try to simulate the whole process via the methods and concepts of synthetic biology. Finally, we successfully constructed the desired system. The comparison between the key steps involved in antibody generation and corresponding synthetic methods are shown in the chart below.<br />
<br/><br />
<br />
<table border="2" bordercolor="maroon" bgcolor="silver"><br />
<tbody><br />
<tr><br />
<th colspan=3>Antibody Production</th><br />
<th width=50px></th><br />
<th colspan=2>E Coli. Production System</th></tr><br />
<tr><br />
<td rowspan=4 width=70px><a href="#VDJ_Recombination_vs_Lading_Pad_Recombination">VDJ Recombination</a></td><br />
<td rowspan=3 width=70px>Preparation</td><br />
<td width=70px>RSS Sequence</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">Landing Pad Insertion</a></td><br />
<td rowspan=4 width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1">Module I: Recombination System</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Recombinase</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">Helper Plasmid Insertion</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Library</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">Donor Plasmid Construction</a></td><br />
</tr><br />
<tr><br />
<td>Recombination</td><br />
<td colspan=2><img src="https://static.igem.org/mediawiki/2010/d/d6/THUarrow2.png" width=180></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">DP Insertion and Recombination Induction</a></td><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="#Somatic_hyper-mutation_vs_Junctional_mutation">Somatic Hypermutation</a></td><br />
<td rowspan=2 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
<!---------<td>Junctional Diversity</td>----------------------><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">CBD-based Microarray</a></td><br />
<td rowspan=4><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2">Module II: Selection System</a></td><br />
</tr><br />
<tr><br />
<td rowspan=3><a href="#Antigen-specific_Selection_vs_CBD-Based_Microarray_and_ToxR-Based_Transmembrane_pathway_method">Antigen Selection</a></td><br />
<td rowspan=3 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">ToxR-based Transmembrane Pathway</a></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">Cooperation With Macquarie_Australia</a></td><br />
</tr><br />
</tbody><br />
</table><br />
</html><br />
<br/><br />
Generally speaking, antibody production in our project can be divided into two modules.<br />
<br />
<html><br />
<body bgcolor="#336699" text="#ffffff" link="#60a179"><br />
<br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1"><font face="Comic Sans MS" size="4">Module I: Generation of antibody library</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2"><font face="Comic Sans MS" size="4">Module II: Selection of specific antibodies</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
</html><br />
<br />
==Project Design==<br />
The first module is carried out by a novel method called E coli in vivo recombination system. The theoretical and experimental basis comes from the work of Thomas E. Kuhlman and Edward C. Cox. We managed to modify their system, wishing to utilize it for simulation of antibody recombination.<br />
<br />
Firstly, we try to insert a DNA segment called”Landing Pad(LP)” into the specific sites of E coli via Att recombination. Landing pad sequence consists of the following parts(from 5’ to 3’ in the sequence):<br />
<br />
1)25bp-long random sequence<br />
<br />
2)15bp-long recognition sequence of restriction enzyme I-scel<br />
<br />
3) antibiotic resistance gene used for antibody selection<br />
<br />
4) 15bp-long recognition sequence of restriction enzyme I-scel(corresponding to 2)<br />
<br />
5) 25bp-long random sequence (corresponding to 1)<br />
<br />
After integrating DNA segment of Landing pad into the genome of E coli, we completed the genetic engineering of the genome of E coli. All the five parts of landing pad are designed for subsequent recombination. <br />
<br />
Besides, in order to achieve different recombination goals, we designed several landing pads of different sequences.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion"><font face="Comic Sans MS" size=3>'Landing Pad Construction and Insertion'</font></a></html> to learn about the details of Landing Pad design. <br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#att">'link'</a></html> to learn more about ATT recombination.<br />
<br />
After the integration of landing pad, we have to insert another DNA fragment called Helper Plasmid(HP), which provides necessary tools for in vivo recombination. <br />
<br />
In this plasmid(Helper Plasmid), there are two genes used for recombination. <br />
<br />
The first one encodes restriction enzyme I-scel after induction of L-arabinose. I-scel is able to recognize the 15bp-length sequence flanking a target DNA fragment and cut out the fragment, therefore providing DNA segments for recombination mediated by other components.<br />
<br />
The second one encodes the enzyme responsible for recombination called Lamda-Red. With the presence of IPTG, Lamda-Red will recombine DNA fragments flanked by the same DNA sequence recognized by Lamda-Red.<br />
<br />
In addition, Helper Plasmid contains a specific temperature-sensitive replicon, which is used to control the replication via temperature changes. We can remove the replicon when necessary.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">'HP insertion'</a></html> to learn more about Helper Plasmid.<br />
<br />
Up till now, we successfully engineered a ‘recepient strain’. Then we have to transform the ‘recepient strain’ with the Donor Plasmid(DP) containing recombination fragments. Donor Plasmid contains several Insertion Fragments and each Insertion Fragment contains following five parts:<br />
<br />
1) one 15bp-long restriction enzyme I-scel recognition site<br />
<br />
2) one 25bp-long random sequence<br />
<br />
3) one fragment for insertion and recombination<br />
<br />
4) one 25bp-long random sequence<br />
<br />
5) one 15bp-long restriction enzyme I-scel recognition site(in correspondence with 1)<br />
<br />
(Note: the order of the five parts differ from that of landing pad)<br />
<br />
As we previously mentioned, Donor plasmid contains several Insertion Fragments. Based on the flanking landing pad sequence, we can ascribe Insertion Fragments to the same group as long as the flanking landing pad sequences of those Insertion Fragments are the same.<br />
<br />
In order to achieve different goals, we design different Donor Plasmids, different Donor Plasmids contain different number of Insertion Fragments, which belong to different groups.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">‘DP construction’</a></html> to learn about the details of Donor Plasmid and Insertion Fragments and the strategies employed in our design. <br />
<br />
As we all know, antibody diversity mainly lean on the number of the fragments used for recombination. Therefore, in order to mimic the antibody generation process, we need to construct Donor Plasmid which contains large sum of Insertion Fragments. Theoretically, we can construct Donor Plasmid of this kind. However, due to the time limit of the competition, we didn’t try to construct DP containing many Insertion Fragments.<br />
<br />
In spite of this, in order to meet the needs, we designed two methods to construct plasmids on a large scale and attempted the usage in parts of our Donor Plasmids.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Strategies">'DP construction and Strategies'</a></html> for the methods of rapid plasmid construction. <br />
<br />
Besides, we have applied for two BBFRFC, which are BBFRFC61 and BBFRFC 62 respectively.<br />
<br />
Then, after transformation of the prepared ‘recepient strain’ with Donor Plasmid and the induction of corresponding agents, I-scel enzyme cuts out DNA fragments from DP and LP, generating numerous Insertion Fragments. Besides, antibiotic resistance gene integrated into E coli genome is also cut out, providing recombination sites for Insertion Fragments.<br />
<br />
Provided with Insertion Fragments mentioned above and Lamda-Red expressed by Donor Plasmid, Insertion Fragments recombine with the corresponding landing pad sequence in the genome and thus get inserted into the correct site of the genome. Based on our design of Landing pad, we can ensure that only one Insertion Fragment from one group is randomly inserted into the genome, therefore mimicking the VDJ recombination process.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">‘DP Insertion and Recombination Induction’</a></html> to learn more about this part. <br />
<br />
Engineered E coli cells which have undergone successful recombination can be selected out of the pool through specific screening methods. After tests on different strains, our project achieve a recombination rate above 50% based on our current techniques.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/experiments#res">‘Result’</a></html> to learn more about the identification of recombination rate. <br />
<br />
The goal of the second module in our project is aimed at selection of specific antibodies. Due to the maturity of existing antibody screening technique, our work mainly focuses on imitation of mammalian antibody selection, thus providing possible alternatives for existing methods.<br />
<br />
The outline of our ideas is to find mechanisms similar to in vivo antibody selection and take in,to account the industry production costs, controllability and reliability, thus developing new methods for antibody selection.<br />
In fact, in mammalian system, antibody selection is achieved through the activation of B lymphocytes and thus the rapid proliferation of the B lymphocytes that express the specific antibodies that bind to antigen. Simply put, the whole process relies on the interaction between antigen epitopes and membrane integral immunoglobins.<br />
<br />
To mimic the activation mediated by membrane integral immunoglobins, we develop ToxR-mediated transmembrane activation signal system. In this system, the interaction between antibodies and antigen triggered downstream expression of reporter genes, thus providing signals for selection.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">‘ToxR-based Transmembrane Signaling Pathway Method’</a></html> for detailed description of this method.<br />
<br />
In addition, we develop a technique called ‘Bacterial based Microarray’ for selection purpose. In this method, we combine membrane display technique and high throughput microarray technique, that is, a bacterial based microarray method.<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">’Bacterial based microarray’</a></html> for details.<br />
<br />
In order to find methods for antibody selection, we cooperate with other iGEM teams. For example, we talked with Macquarie_Australia iGEM team about the project. The techniques involved in their project might be useful for our selection methods. Therefore, members from both teams worked together to research on this problem.<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">'link'</a></html> to learn more about our cooperation.<br />
<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens. <br />
Our system is aiming at imitating this recombination process, using E.coli as the gene carrier.<br />
It is known to all that regional genes (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and the recombination occurs when VDJ recombinase are expressed. We choose RecA enzyme to induce the recombination, while use I-Sel enzyme to cut the genome, just mimicking the process happened in B cells.<br />
<br/><br />
<br/><br />
<br />
==Comparison between natural antibody production and E. Coli system==<br />
<br/><br />
===VDJ Recombination vs Lading Pad Recombination===<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is the mechanism by which immunoglobulin (Ig) and T cell receptors (TCR) are generated in the immune system. Through V(D)J recombination, Variable, Diverse, and Joining gene segments are randomly combined, thus encoding various kinds of immunoglobins and T cell receptors to recognize potential antigens. <br />
During the process of B cell maturation, gene segments are rearranged and thus transcribe mRNA that can produce diverse types of antibody for recognition of billions of potential epitopes. The rearrangement brings three gene segments (termed V, D, and J) in close proximity, which are then joined together head to tail, the process of which is known as V(D)J recombination based on the V,D,J gene segments involved. Multiple copies of the V, D, and J genes in the human genome are shuffled in the process, and a specific antibody will be generate among millions of other possible combinations. Because gene are joined together permanently, one mature B cell will produce only one specific antibody.<br />
<br />
Our system is aimed to mimic this recombination process, using E.coli as the carrier.<br />
Through previous research in immunology field, each kind of gene segments (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and recombination occurs when VDJ recombinase recognize the specific signal sequence and mediate the recombination process. In our project, we design a sequence, which carries separated parts of one complete gene and integrate synthesized DNA fragment into the genome of E coli via landing pad method. Then we use I-Sel enzyme to cut the gene segments out of E coli genome, into which gene segments are inserted and choose RecA enzyme to recognize the previously designed signal sequence flanking our target sequence and recombine the gene segments, just like what recombinase accomplish in B cells. The whole process is called landing pad recombination.<br />
<br />
===Somatic hyper-mutation vs Junctional mutation===<br />
<br />
Somatic hypermutation involves a programmed process of mutation affecting the variable regions of immunoglobulin genes. During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is at least 105-106 fold greater than the normal rate of mutation across the genome. Variation is mainly in the form of single base substitutions, with insertions and deletions being less common. These mutations occur mostly at “hotspots” in the DNA, known as hypervariable regions. These regions correspond to the complementarity determining regions; the sites involved in antigen recognition on the immunoglobulin. This directed hypermutation allows for the selection of B cells that express immunoglobulin receptors possessing an enhanced ability to recognize and bind a specific foreign antigen.<br />
<br />
In addition, the cross ligation, namely Landing pad recombination, has higher opportunity to mutate in the link sequence. The introduction of 4 junctional shuffler 64 will meet the need of the rest 107 diversity, perfectly undertaking the work in the antibody diversity construction that hyper-mutation does in the highly variable region. In this way, we have finished a building library, waiting for the specialized antigen to select.<br />
<br />
===Antigen-specific Selection vs CBD-Based Microarray and ToxR-Based Transmembrane pathway method===<br />
In mammalian immune system, billions of antibodies will be generated. Then how can the immune system find out specific antibody that indiscriminately recognize the specific antigen that comes in. Generally speaking, due to the fact that one mature B cell only generates one specific immunoglobin, once the membrane integral immunoglobin bind to the specific antigen, a series of intracellular response, such as gene activation and cell differetiation will be triggered in B cell. Then, B cells expressing the specific antibodies that recognize the specific antigen will proliferate and generate enormous amount of antibodies that help eliminate the specific antigen.<br />
<br />
Simply put, antibody selection is accomplished through the specific interaction between the membrane immunoglobin and the specific antigen. Therefore, in our project, we also plan to mimic this process through interaction between the antibodies generated through landing pad recombination and the antigen we target. CBD-based Microarray and ToxR-based Transmembrane pathway are the two methods by which we achieve this goal.<br />
CBD domain, which interacts with cellulose, potentially provides us way to locate our E.coli to the microarray coated with cellulose. OmpA (outer membrane protein A), displays the antibody and antigen out to the membrane surface. Then by applying the antibodies generated through landing pad recombination to the microarray, antibodies that bind to the antigen will be retained and thus selected out of the pool of antibodies.<br />
<br />
Alternatively, the ToxR-based transmembrane pathway method shows a different strategy for us to seek the right folded antibody. Once the antigen comes, ToxR outer part will form dimer shape and the downstream signal occur, supplying the approach of reporters, such as GFP, to examine and pick out what we want.<br />
<br />
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<h1>Outline</h1><br />
<br />
<div class="content_block"><br />
<br />
<br/><br />
In order to realize the production of antibodies in prokaryotic cells, we firstly investigate the whole process of antibody production in mammalian immune system and try to simulate the whole process via the methods and concepts of synthetic biology. Finally, we successfully constructed the desired system. The comparison between the key steps involved in antibody generation and corresponding synthetic methods are shown in the chart below.<br />
<br/><br />
<br />
<table border="2" bordercolor="maroon" bgcolor="silver"><br />
<tbody><br />
<tr><br />
<th colspan=3>Antibody Production</th><br />
<th width=50px></th><br />
<th colspan=2>E Coli. Production System</th></tr><br />
<tr><br />
<td rowspan=4 width=70px><a href="#VDJ_Recombination_vs_Lading_Pad_Recombination">VDJ Recombination</a></td><br />
<td rowspan=3 width=70px>Preparation</td><br />
<td width=70px>RSS Sequence</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">Landing Pad Insertion</a></td><br />
<td rowspan=4 width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1">Module I: Recombination System</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Recombinase</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">Helper Plasmid Insertion</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Library</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">Donor Plasmid Construction</a></td><br />
</tr><br />
<tr><br />
<td>Recombination</td><br />
<td colspan=2><img src="https://static.igem.org/mediawiki/2010/d/d6/THUarrow2.png" width=180></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">DP Insertion and Recombination Induction</a></td><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="#Somatic_hyper-mutation_vs_Junctional_mutation">Somatic Hypermutation</a></td><br />
<td rowspan=2 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
<!---------<td>Junctional Diversity</td>----------------------><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">CBD-based Microarray</a></td><br />
<td rowspan=4><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2">Module II: Selection System</a></td><br />
</tr><br />
<tr><br />
<td rowspan=3><a href="#Antigen-specific_Selection_vs_CBD-Based_Microarray_and_ToxR-Based_Transmembrane_pathway_method">Antigen Selection</a></td><br />
<td rowspan=3 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">ToxR-based Transmembrane Pathway</a></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">Cooperation With Macquarie_Australia</a></td><br />
</tr><br />
</tbody><br />
</table><br />
</html><br />
<br/><br />
Generally speaking, antibody production in our project can be divided into two modules.<br />
<br />
<html><br />
<body bgcolor="#336699" text="#ffffff" link="#60a179"><br />
<br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1"><font face="Comic Sans MS" size="4">Module I: Generation of antibody library</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2"><font face="Comic Sans MS" size="4">Module II: Selection of specific antibodies</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
</html><br />
<br />
==Project Design==<br />
The first module is carried out by a novel method called E coli in vivo recombination system. The theoretical and experimental basis comes from the work of Thomas E. Kuhlman and Edward C. Cox. We managed to modify their system, wishing to utilize it for simulation of antibody recombination.<br />
<br />
Firstly, we try to insert a DNA segment called”Landing Pad(LP)” into the specific sites of E coli via Att recombination. Landing pad sequence consists of the following parts(from 5’ to 3’ in the sequence):<br />
<br />
1)25bp-long random sequence<br />
<br />
2)15bp-long recognition sequence of restriction enzyme I-scel<br />
<br />
3) antibiotic resistance gene used for antibody selection<br />
<br />
4) 15bp-long recognition sequence of restriction enzyme I-scel(corresponding to 2)<br />
<br />
5) 25bp-long random sequence (corresponding to 1)<br />
<br />
After integrating DNA segment of Landing pad into the genome of E coli, we completed the genetic engineering of the genome of E coli. All the five parts of landing pad are designed for subsequent recombination. <br />
<br />
Besides, in order to achieve different recombination goals, we designed several landing pads of different sequences.<br/> <br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion"><font face="Comic Sans MS">'Landing Pad Construction and Insertion'</font></a></html> to learn about the details of Landing Pad design. <br />
<br/><br />
<br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#att">'link'</a></html> to learn more about ATT recombination.<br />
<br />
After the integration of landing pad, we have to insert another DNA fragment called Helper Plasmid(HP), which provides necessary tools for in vivo recombination. <br />
<br />
In this plasmid(Helper Plasmid), there are two genes used for recombination. <br />
<br />
The first one encodes restriction enzyme I-scel after induction of L-arabinose. I-scel is able to recognize the 15bp-length sequence flanking a target DNA fragment and cut out the fragment, therefore providing DNA segments for recombination mediated by other components.<br />
<br />
The second one encodes the enzyme responsible for recombination called Lamda-Red. With the presence of IPTG, Lamda-Red will recombine DNA fragments flanked by the same DNA sequence recognized by Lamda-Red.<br />
<br />
In addition, Helper Plasmid contains a specific temperature-sensitive replicon, which is used to control the replication via temperature changes. We can remove the replicon when necessary.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">'HP insertion'</a></html> to learn more about Helper Plasmid.<br />
<br />
Up till now, we successfully engineered a ‘recepient strain’. Then we have to transform the ‘recepient strain’ with the Donor Plasmid(DP) containing recombination fragments. Donor Plasmid contains several Insertion Fragments and each Insertion Fragment contains following five parts:<br />
<br />
1) one 15bp-long restriction enzyme I-scel recognition site<br />
<br />
2) one 25bp-long random sequence<br />
<br />
3) one fragment for insertion and recombination<br />
<br />
4) one 25bp-long random sequence<br />
<br />
5) one 15bp-long restriction enzyme I-scel recognition site(in correspondence with 1)<br />
<br />
(Note: the order of the five parts differ from that of landing pad)<br />
<br />
As we previously mentioned, Donor plasmid contains several Insertion Fragments. Based on the flanking landing pad sequence, we can ascribe Insertion Fragments to the same group as long as the flanking landing pad sequences of those Insertion Fragments are the same.<br />
<br />
In order to achieve different goals, we design different Donor Plasmids, different Donor Plasmids contain different number of Insertion Fragments, which belong to different groups.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">‘DP construction’</a></html> to learn about the details of Donor Plasmid and Insertion Fragments and the strategies employed in our design. <br />
<br />
As we all know, antibody diversity mainly lean on the number of the fragments used for recombination. Therefore, in order to mimic the antibody generation process, we need to construct Donor Plasmid which contains large sum of Insertion Fragments. Theoretically, we can construct Donor Plasmid of this kind. However, due to the time limit of the competition, we didn’t try to construct DP containing many Insertion Fragments.<br />
<br />
In spite of this, in order to meet the needs, we designed two methods to construct plasmids on a large scale and attempted the usage in parts of our Donor Plasmids.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Strategies">'DP construction and Strategies'</a></html> for the methods of rapid plasmid construction. <br />
<br />
Besides, we have applied for two BBFRFC, which are BBFRFC61 and BBFRFC 62 respectively.<br />
<br />
Then, after transformation of the prepared ‘recepient strain’ with Donor Plasmid and the induction of corresponding agents, I-scel enzyme cuts out DNA fragments from DP and LP, generating numerous Insertion Fragments. Besides, antibiotic resistance gene integrated into E coli genome is also cut out, providing recombination sites for Insertion Fragments.<br />
<br />
Provided with Insertion Fragments mentioned above and Lamda-Red expressed by Donor Plasmid, Insertion Fragments recombine with the corresponding landing pad sequence in the genome and thus get inserted into the correct site of the genome. Based on our design of Landing pad, we can ensure that only one Insertion Fragment from one group is randomly inserted into the genome, therefore mimicking the VDJ recombination process.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">‘DP Insertion and Recombination Induction’</a></html> to learn more about this part. <br />
<br />
Engineered E coli cells which have undergone successful recombination can be selected out of the pool through specific screening methods. After tests on different strains, our project achieve a recombination rate above 50% based on our current techniques.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/experiments#res">‘Result’</a></html> to learn more about the identification of recombination rate. <br />
<br />
The goal of the second module in our project is aimed at selection of specific antibodies. Due to the maturity of existing antibody screening technique, our work mainly focuses on imitation of mammalian antibody selection, thus providing possible alternatives for existing methods.<br />
<br />
The outline of our ideas is to find mechanisms similar to in vivo antibody selection and take in,to account the industry production costs, controllability and reliability, thus developing new methods for antibody selection.<br />
In fact, in mammalian system, antibody selection is achieved through the activation of B lymphocytes and thus the rapid proliferation of the B lymphocytes that express the specific antibodies that bind to antigen. Simply put, the whole process relies on the interaction between antigen epitopes and membrane integral immunoglobins.<br />
<br />
To mimic the activation mediated by membrane integral immunoglobins, we develop ToxR-mediated transmembrane activation signal system. In this system, the interaction between antibodies and antigen triggered downstream expression of reporter genes, thus providing signals for selection.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">‘ToxR-based Transmembrane Signaling Pathway Method’</a></html> for detailed description of this method.<br />
<br />
In addition, we develop a technique called ‘Bacterial based Microarray’ for selection purpose. In this method, we combine membrane display technique and high throughput microarray technique, that is, a bacterial based microarray method.<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">’Bacterial based microarray’</a></html> for details.<br />
<br />
In order to find methods for antibody selection, we cooperate with other iGEM teams. For example, we talked with Macquarie_Australia iGEM team about the project. The techniques involved in their project might be useful for our selection methods. Therefore, members from both teams worked together to research on this problem.<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">'link'</a></html> to learn more about our cooperation.<br />
<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens. <br />
Our system is aiming at imitating this recombination process, using E.coli as the gene carrier.<br />
It is known to all that regional genes (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and the recombination occurs when VDJ recombinase are expressed. We choose RecA enzyme to induce the recombination, while use I-Sel enzyme to cut the genome, just mimicking the process happened in B cells.<br />
<br/><br />
<br/><br />
<br />
==Comparison between natural antibody production and E. Coli system==<br />
<br/><br />
===VDJ Recombination vs Lading Pad Recombination===<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is the mechanism by which immunoglobulin (Ig) and T cell receptors (TCR) are generated in the immune system. Through V(D)J recombination, Variable, Diverse, and Joining gene segments are randomly combined, thus encoding various kinds of immunoglobins and T cell receptors to recognize potential antigens. <br />
During the process of B cell maturation, gene segments are rearranged and thus transcribe mRNA that can produce diverse types of antibody for recognition of billions of potential epitopes. The rearrangement brings three gene segments (termed V, D, and J) in close proximity, which are then joined together head to tail, the process of which is known as V(D)J recombination based on the V,D,J gene segments involved. Multiple copies of the V, D, and J genes in the human genome are shuffled in the process, and a specific antibody will be generate among millions of other possible combinations. Because gene are joined together permanently, one mature B cell will produce only one specific antibody.<br />
<br />
Our system is aimed to mimic this recombination process, using E.coli as the carrier.<br />
Through previous research in immunology field, each kind of gene segments (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and recombination occurs when VDJ recombinase recognize the specific signal sequence and mediate the recombination process. In our project, we design a sequence, which carries separated parts of one complete gene and integrate synthesized DNA fragment into the genome of E coli via landing pad method. Then we use I-Sel enzyme to cut the gene segments out of E coli genome, into which gene segments are inserted and choose RecA enzyme to recognize the previously designed signal sequence flanking our target sequence and recombine the gene segments, just like what recombinase accomplish in B cells. The whole process is called landing pad recombination.<br />
<br />
===Somatic hyper-mutation vs Junctional mutation===<br />
<br />
Somatic hypermutation involves a programmed process of mutation affecting the variable regions of immunoglobulin genes. During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is at least 105-106 fold greater than the normal rate of mutation across the genome. Variation is mainly in the form of single base substitutions, with insertions and deletions being less common. These mutations occur mostly at “hotspots” in the DNA, known as hypervariable regions. These regions correspond to the complementarity determining regions; the sites involved in antigen recognition on the immunoglobulin. This directed hypermutation allows for the selection of B cells that express immunoglobulin receptors possessing an enhanced ability to recognize and bind a specific foreign antigen.<br />
<br />
In addition, the cross ligation, namely Landing pad recombination, has higher opportunity to mutate in the link sequence. The introduction of 4 junctional shuffler 64 will meet the need of the rest 107 diversity, perfectly undertaking the work in the antibody diversity construction that hyper-mutation does in the highly variable region. In this way, we have finished a building library, waiting for the specialized antigen to select.<br />
<br />
===Antigen-specific Selection vs CBD-Based Microarray and ToxR-Based Transmembrane pathway method===<br />
In mammalian immune system, billions of antibodies will be generated. Then how can the immune system find out specific antibody that indiscriminately recognize the specific antigen that comes in. Generally speaking, due to the fact that one mature B cell only generates one specific immunoglobin, once the membrane integral immunoglobin bind to the specific antigen, a series of intracellular response, such as gene activation and cell differetiation will be triggered in B cell. Then, B cells expressing the specific antibodies that recognize the specific antigen will proliferate and generate enormous amount of antibodies that help eliminate the specific antigen.<br />
<br />
Simply put, antibody selection is accomplished through the specific interaction between the membrane immunoglobin and the specific antigen. Therefore, in our project, we also plan to mimic this process through interaction between the antibodies generated through landing pad recombination and the antigen we target. CBD-based Microarray and ToxR-based Transmembrane pathway are the two methods by which we achieve this goal.<br />
CBD domain, which interacts with cellulose, potentially provides us way to locate our E.coli to the microarray coated with cellulose. OmpA (outer membrane protein A), displays the antibody and antigen out to the membrane surface. Then by applying the antibodies generated through landing pad recombination to the microarray, antibodies that bind to the antigen will be retained and thus selected out of the pool of antibodies.<br />
<br />
Alternatively, the ToxR-based transmembrane pathway method shows a different strategy for us to seek the right folded antibody. Once the antigen comes, ToxR outer part will form dimer shape and the downstream signal occur, supplying the approach of reporters, such as GFP, to examine and pick out what we want.<br />
<br />
<html><br />
&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a><br />
</body><br />
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<br />
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</div></div></body><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/project/outlineTeam:Tsinghua/project/outline2010-10-27T14:57:16Z<p>Gumilton: /* Project Design */</p>
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<h1>Outline</h1><br />
<br />
<div class="content_block"><br />
<br />
<br/><br />
In order to realize the production of antibodies in prokaryotic cells, we firstly investigate the whole process of antibody production in mammalian immune system and try to simulate the whole process via the methods and concepts of synthetic biology. Finally, we successfully constructed the desired system. The comparison between the key steps involved in antibody generation and corresponding synthetic methods are shown in the chart below.<br />
<br/><br />
<br />
<table border="2" bordercolor="maroon" bgcolor="silver"><br />
<tbody><br />
<tr><br />
<th colspan=3>Antibody Production</th><br />
<th width=50px></th><br />
<th colspan=2>E Coli. Production System</th></tr><br />
<tr><br />
<td rowspan=4 width=70px><a href="#VDJ_Recombination_vs_Lading_Pad_Recombination">VDJ Recombination</a></td><br />
<td rowspan=3 width=70px>Preparation</td><br />
<td width=70px>RSS Sequence</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">Landing Pad Insertion</a></td><br />
<td rowspan=4 width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1">Module I: Recombination System</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Recombinase</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">Helper Plasmid Insertion</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Library</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">Donor Plasmid Construction</a></td><br />
</tr><br />
<tr><br />
<td>Recombination</td><br />
<td colspan=2><img src="https://static.igem.org/mediawiki/2010/d/d6/THUarrow2.png" width=180></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">DP Insertion and Recombination Induction</a></td><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="#Somatic_hyper-mutation_vs_Junctional_mutation">Somatic Hypermutation</a></td><br />
<td rowspan=2 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
<!---------<td>Junctional Diversity</td>----------------------><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">CBD-based Microarray</a></td><br />
<td rowspan=4><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2">Module II: Selection System</a></td><br />
</tr><br />
<tr><br />
<td rowspan=3><a href="#Antigen-specific_Selection_vs_CBD-Based_Microarray_and_ToxR-Based_Transmembrane_pathway_method">Antigen Selection</a></td><br />
<td rowspan=3 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">ToxR-based Transmembrane Pathway</a></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">Cooperation With Macquarie_Australia</a></td><br />
</tr><br />
</tbody><br />
</table><br />
</html><br />
<br/><br />
Generally speaking, antibody production in our project can be divided into two modules.<br />
<br />
<html><br />
<body bgcolor="#336699" text="#ffffff" link="#60a179"><br />
<br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1"><font face="Comic Sans MS" size="4">Module I: Generation of antibody library</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2"><font face="Comic Sans MS" size="4">Module II: Selection of specific antibodies</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
</html><br />
<br />
==Project Design==<br />
The first module is carried out by a novel method called E coli in vivo recombination system. The theoretical and experimental basis comes from the work of Thomas E. Kuhlman and Edward C. Cox. We managed to modify their system, wishing to utilize it for simulation of antibody recombination.<br />
<br />
Firstly, we try to insert a DNA segment called”Landing Pad(LP)” into the specific sites of E coli via Att recombination. Landing pad sequence consists of the following parts(from 5’ to 3’ in the sequence):<br />
<br />
1)25bp-long random sequence<br />
<br />
2)15bp-long recognition sequence of restriction enzyme I-scel<br />
<br />
3) antibiotic resistance gene used for antibody selection<br />
<br />
4) 15bp-long recognition sequence of restriction enzyme I-scel(corresponding to 2)<br />
<br />
5) 25bp-long random sequence (corresponding to 1)<br />
<br />
After integrating DNA segment of Landing pad into the genome of E coli, we completed the genetic engineering of the genome of E coli. All the five parts of landing pad are designed for subsequent recombination. <br />
<br />
Besides, in order to achieve different recombination goals, we designed several landing pads of different sequences. <br />
<br />
<br/><br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion"><font face="Comic Sans MS">'Landing Pad Construction and Insertion'</font></a></html> to learn about the details of Landing Pad design. <br />
<br/><br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#att">'link'</a></html> to learn more about ATT recombination.<br />
<br />
After the integration of landing pad, we have to insert another DNA fragment called Helper Plasmid(HP), which provides necessary tools for in vivo recombination. <br />
<br />
In this plasmid(Helper Plasmid), there are two genes used for recombination. <br />
<br />
The first one encodes restriction enzyme I-scel after induction of L-arabinose. I-scel is able to recognize the 15bp-length sequence flanking a target DNA fragment and cut out the fragment, therefore providing DNA segments for recombination mediated by other components.<br />
<br />
The second one encodes the enzyme responsible for recombination called Lamda-Red. With the presence of IPTG, Lamda-Red will recombine DNA fragments flanked by the same DNA sequence recognized by Lamda-Red.<br />
<br />
In addition, Helper Plasmid contains a specific temperature-sensitive replicon, which is used to control the replication via temperature changes. We can remove the replicon when necessary.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">'HP insertion'</a></html> to learn more about Helper Plasmid.<br />
<br />
Up till now, we successfully engineered a ‘recepient strain’. Then we have to transform the ‘recepient strain’ with the Donor Plasmid(DP) containing recombination fragments. Donor Plasmid contains several Insertion Fragments and each Insertion Fragment contains following five parts:<br />
<br />
1) one 15bp-long restriction enzyme I-scel recognition site<br />
<br />
2) one 25bp-long random sequence<br />
<br />
3) one fragment for insertion and recombination<br />
<br />
4) one 25bp-long random sequence<br />
<br />
5) one 15bp-long restriction enzyme I-scel recognition site(in correspondence with 1)<br />
<br />
(Note: the order of the five parts differ from that of landing pad)<br />
<br />
As we previously mentioned, Donor plasmid contains several Insertion Fragments. Based on the flanking landing pad sequence, we can ascribe Insertion Fragments to the same group as long as the flanking landing pad sequences of those Insertion Fragments are the same.<br />
<br />
In order to achieve different goals, we design different Donor Plasmids, different Donor Plasmids contain different number of Insertion Fragments, which belong to different groups.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">‘DP construction’</a></html> to learn about the details of Donor Plasmid and Insertion Fragments and the strategies employed in our design. <br />
<br />
As we all know, antibody diversity mainly lean on the number of the fragments used for recombination. Therefore, in order to mimic the antibody generation process, we need to construct Donor Plasmid which contains large sum of Insertion Fragments. Theoretically, we can construct Donor Plasmid of this kind. However, due to the time limit of the competition, we didn’t try to construct DP containing many Insertion Fragments.<br />
<br />
In spite of this, in order to meet the needs, we designed two methods to construct plasmids on a large scale and attempted the usage in parts of our Donor Plasmids.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Strategies">'DP construction and Strategies'</a></html> for the methods of rapid plasmid construction. <br />
<br />
Besides, we have applied for two BBFRFC, which are BBFRFC61 and BBFRFC 62 respectively.<br />
<br />
Then, after transformation of the prepared ‘recepient strain’ with Donor Plasmid and the induction of corresponding agents, I-scel enzyme cuts out DNA fragments from DP and LP, generating numerous Insertion Fragments. Besides, antibiotic resistance gene integrated into E coli genome is also cut out, providing recombination sites for Insertion Fragments.<br />
<br />
Provided with Insertion Fragments mentioned above and Lamda-Red expressed by Donor Plasmid, Insertion Fragments recombine with the corresponding landing pad sequence in the genome and thus get inserted into the correct site of the genome. Based on our design of Landing pad, we can ensure that only one Insertion Fragment from one group is randomly inserted into the genome, therefore mimicking the VDJ recombination process.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">‘DP Insertion and Recombination Induction’</a></html> to learn more about this part. <br />
<br />
Engineered E coli cells which have undergone successful recombination can be selected out of the pool through specific screening methods. After tests on different strains, our project achieve a recombination rate above 50% based on our current techniques.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/experiments#res">‘Result’</a></html> to learn more about the identification of recombination rate. <br />
<br />
The goal of the second module in our project is aimed at selection of specific antibodies. Due to the maturity of existing antibody screening technique, our work mainly focuses on imitation of mammalian antibody selection, thus providing possible alternatives for existing methods.<br />
<br />
The outline of our ideas is to find mechanisms similar to in vivo antibody selection and take in,to account the industry production costs, controllability and reliability, thus developing new methods for antibody selection.<br />
In fact, in mammalian system, antibody selection is achieved through the activation of B lymphocytes and thus the rapid proliferation of the B lymphocytes that express the specific antibodies that bind to antigen. Simply put, the whole process relies on the interaction between antigen epitopes and membrane integral immunoglobins.<br />
<br />
To mimic the activation mediated by membrane integral immunoglobins, we develop ToxR-mediated transmembrane activation signal system. In this system, the interaction between antibodies and antigen triggered downstream expression of reporter genes, thus providing signals for selection.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">‘ToxR-based Transmembrane Signaling Pathway Method’</a></html> for detailed description of this method.<br />
<br />
In addition, we develop a technique called ‘Bacterial based Microarray’ for selection purpose. In this method, we combine membrane display technique and high throughput microarray technique, that is, a bacterial based microarray method.<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">’Bacterial based microarray’</a></html> for details.<br />
<br />
In order to find methods for antibody selection, we cooperate with other iGEM teams. For example, we talked with Macquarie_Australia iGEM team about the project. The techniques involved in their project might be useful for our selection methods. Therefore, members from both teams worked together to research on this problem.<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">'link'</a></html> to learn more about our cooperation.<br />
<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens. <br />
Our system is aiming at imitating this recombination process, using E.coli as the gene carrier.<br />
It is known to all that regional genes (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and the recombination occurs when VDJ recombinase are expressed. We choose RecA enzyme to induce the recombination, while use I-Sel enzyme to cut the genome, just mimicking the process happened in B cells.<br />
<br/><br />
<br/><br />
<br />
==Comparison between natural antibody production and E. Coli system==<br />
<br/><br />
===VDJ Recombination vs Lading Pad Recombination===<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is the mechanism by which immunoglobulin (Ig) and T cell receptors (TCR) are generated in the immune system. Through V(D)J recombination, Variable, Diverse, and Joining gene segments are randomly combined, thus encoding various kinds of immunoglobins and T cell receptors to recognize potential antigens. <br />
During the process of B cell maturation, gene segments are rearranged and thus transcribe mRNA that can produce diverse types of antibody for recognition of billions of potential epitopes. The rearrangement brings three gene segments (termed V, D, and J) in close proximity, which are then joined together head to tail, the process of which is known as V(D)J recombination based on the V,D,J gene segments involved. Multiple copies of the V, D, and J genes in the human genome are shuffled in the process, and a specific antibody will be generate among millions of other possible combinations. Because gene are joined together permanently, one mature B cell will produce only one specific antibody.<br />
<br />
Our system is aimed to mimic this recombination process, using E.coli as the carrier.<br />
Through previous research in immunology field, each kind of gene segments (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and recombination occurs when VDJ recombinase recognize the specific signal sequence and mediate the recombination process. In our project, we design a sequence, which carries separated parts of one complete gene and integrate synthesized DNA fragment into the genome of E coli via landing pad method. Then we use I-Sel enzyme to cut the gene segments out of E coli genome, into which gene segments are inserted and choose RecA enzyme to recognize the previously designed signal sequence flanking our target sequence and recombine the gene segments, just like what recombinase accomplish in B cells. The whole process is called landing pad recombination.<br />
<br />
===Somatic hyper-mutation vs Junctional mutation===<br />
<br />
Somatic hypermutation involves a programmed process of mutation affecting the variable regions of immunoglobulin genes. During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is at least 105-106 fold greater than the normal rate of mutation across the genome. Variation is mainly in the form of single base substitutions, with insertions and deletions being less common. These mutations occur mostly at “hotspots” in the DNA, known as hypervariable regions. These regions correspond to the complementarity determining regions; the sites involved in antigen recognition on the immunoglobulin. This directed hypermutation allows for the selection of B cells that express immunoglobulin receptors possessing an enhanced ability to recognize and bind a specific foreign antigen.<br />
<br />
In addition, the cross ligation, namely Landing pad recombination, has higher opportunity to mutate in the link sequence. The introduction of 4 junctional shuffler 64 will meet the need of the rest 107 diversity, perfectly undertaking the work in the antibody diversity construction that hyper-mutation does in the highly variable region. In this way, we have finished a building library, waiting for the specialized antigen to select.<br />
<br />
===Antigen-specific Selection vs CBD-Based Microarray and ToxR-Based Transmembrane pathway method===<br />
In mammalian immune system, billions of antibodies will be generated. Then how can the immune system find out specific antibody that indiscriminately recognize the specific antigen that comes in. Generally speaking, due to the fact that one mature B cell only generates one specific immunoglobin, once the membrane integral immunoglobin bind to the specific antigen, a series of intracellular response, such as gene activation and cell differetiation will be triggered in B cell. Then, B cells expressing the specific antibodies that recognize the specific antigen will proliferate and generate enormous amount of antibodies that help eliminate the specific antigen.<br />
<br />
Simply put, antibody selection is accomplished through the specific interaction between the membrane immunoglobin and the specific antigen. Therefore, in our project, we also plan to mimic this process through interaction between the antibodies generated through landing pad recombination and the antigen we target. CBD-based Microarray and ToxR-based Transmembrane pathway are the two methods by which we achieve this goal.<br />
CBD domain, which interacts with cellulose, potentially provides us way to locate our E.coli to the microarray coated with cellulose. OmpA (outer membrane protein A), displays the antibody and antigen out to the membrane surface. Then by applying the antibodies generated through landing pad recombination to the microarray, antibodies that bind to the antigen will be retained and thus selected out of the pool of antibodies.<br />
<br />
Alternatively, the ToxR-based transmembrane pathway method shows a different strategy for us to seek the right folded antibody. Once the antigen comes, ToxR outer part will form dimer shape and the downstream signal occur, supplying the approach of reporters, such as GFP, to examine and pick out what we want.<br />
<br />
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&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a><br />
</body><br />
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</div></div></body><br />
</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/project/outlineTeam:Tsinghua/project/outline2010-10-27T14:56:49Z<p>Gumilton: /* Project Design */</p>
<hr />
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<div id="main_content"><a name="outline"></a><br />
<h1>Outline</h1><br />
<br />
<div class="content_block"><br />
<br />
<br/><br />
In order to realize the production of antibodies in prokaryotic cells, we firstly investigate the whole process of antibody production in mammalian immune system and try to simulate the whole process via the methods and concepts of synthetic biology. Finally, we successfully constructed the desired system. The comparison between the key steps involved in antibody generation and corresponding synthetic methods are shown in the chart below.<br />
<br/><br />
<br />
<table border="2" bordercolor="maroon" bgcolor="silver"><br />
<tbody><br />
<tr><br />
<th colspan=3>Antibody Production</th><br />
<th width=50px></th><br />
<th colspan=2>E Coli. Production System</th></tr><br />
<tr><br />
<td rowspan=4 width=70px><a href="#VDJ_Recombination_vs_Lading_Pad_Recombination">VDJ Recombination</a></td><br />
<td rowspan=3 width=70px>Preparation</td><br />
<td width=70px>RSS Sequence</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">Landing Pad Insertion</a></td><br />
<td rowspan=4 width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1">Module I: Recombination System</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Recombinase</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">Helper Plasmid Insertion</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Library</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">Donor Plasmid Construction</a></td><br />
</tr><br />
<tr><br />
<td>Recombination</td><br />
<td colspan=2><img src="https://static.igem.org/mediawiki/2010/d/d6/THUarrow2.png" width=180></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">DP Insertion and Recombination Induction</a></td><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="#Somatic_hyper-mutation_vs_Junctional_mutation">Somatic Hypermutation</a></td><br />
<td rowspan=2 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
<!---------<td>Junctional Diversity</td>----------------------><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">CBD-based Microarray</a></td><br />
<td rowspan=4><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2">Module II: Selection System</a></td><br />
</tr><br />
<tr><br />
<td rowspan=3><a href="#Antigen-specific_Selection_vs_CBD-Based_Microarray_and_ToxR-Based_Transmembrane_pathway_method">Antigen Selection</a></td><br />
<td rowspan=3 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">ToxR-based Transmembrane Pathway</a></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">Cooperation With Macquarie_Australia</a></td><br />
</tr><br />
</tbody><br />
</table><br />
</html><br />
<br/><br />
Generally speaking, antibody production in our project can be divided into two modules.<br />
<br />
<html><br />
<body bgcolor="#336699" text="#ffffff" link="#60a179"><br />
<br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1"><font face="Comic Sans MS" size="4">Module I: Generation of antibody library</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2"><font face="Comic Sans MS" size="4">Module II: Selection of specific antibodies</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
</html><br />
<br />
==Project Design==<br />
The first module is carried out by a novel method called E coli in vivo recombination system. The theoretical and experimental basis comes from the work of Thomas E. Kuhlman and Edward C. Cox. We managed to modify their system, wishing to utilize it for simulation of antibody recombination.<br />
<br />
Firstly, we try to insert a DNA segment called”Landing Pad(LP)” into the specific sites of E coli via Att recombination. Landing pad sequence consists of the following parts(from 5’ to 3’ in the sequence):<br />
<br />
1)25bp-long random sequence<br />
<br />
2)15bp-long recognition sequence of restriction enzyme I-scel<br />
<br />
3) antibiotic resistance gene used for antibody selection<br />
<br />
4) 15bp-long recognition sequence of restriction enzyme I-scel(corresponding to 2)<br />
<br />
5) 25bp-long random sequence (corresponding to 1)<br />
<br />
After integrating DNA segment of Landing pad into the genome of E coli, we completed the genetic engineering of the genome of E coli. All the five parts of landing pad are designed for subsequent recombination. <br />
<br />
Besides, in order to achieve different recombination goals, we designed several landing pads of different sequences. <br />
<br/><br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion"><font face="Comic Sans MS">'Landing Pad Construction and Insertion'</font></a></html> to learn about the details of Landing Pad design. <br />
<br/><br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#att">'link'</a></html> to learn more about ATT recombination.<br />
<br />
After the integration of landing pad, we have to insert another DNA fragment called Helper Plasmid(HP), which provides necessary tools for in vivo recombination. <br />
<br />
In this plasmid(Helper Plasmid), there are two genes used for recombination. <br />
<br />
The first one encodes restriction enzyme I-scel after induction of L-arabinose. I-scel is able to recognize the 15bp-length sequence flanking a target DNA fragment and cut out the fragment, therefore providing DNA segments for recombination mediated by other components.<br />
<br />
The second one encodes the enzyme responsible for recombination called Lamda-Red. With the presence of IPTG, Lamda-Red will recombine DNA fragments flanked by the same DNA sequence recognized by Lamda-Red.<br />
<br />
In addition, Helper Plasmid contains a specific temperature-sensitive replicon, which is used to control the replication via temperature changes. We can remove the replicon when necessary.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">'HP insertion'</a></html> to learn more about Helper Plasmid.<br />
<br />
Up till now, we successfully engineered a ‘recepient strain’. Then we have to transform the ‘recepient strain’ with the Donor Plasmid(DP) containing recombination fragments. Donor Plasmid contains several Insertion Fragments and each Insertion Fragment contains following five parts:<br />
<br />
1) one 15bp-long restriction enzyme I-scel recognition site<br />
<br />
2) one 25bp-long random sequence<br />
<br />
3) one fragment for insertion and recombination<br />
<br />
4) one 25bp-long random sequence<br />
<br />
5) one 15bp-long restriction enzyme I-scel recognition site(in correspondence with 1)<br />
<br />
(Note: the order of the five parts differ from that of landing pad)<br />
<br />
As we previously mentioned, Donor plasmid contains several Insertion Fragments. Based on the flanking landing pad sequence, we can ascribe Insertion Fragments to the same group as long as the flanking landing pad sequences of those Insertion Fragments are the same.<br />
<br />
In order to achieve different goals, we design different Donor Plasmids, different Donor Plasmids contain different number of Insertion Fragments, which belong to different groups.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">‘DP construction’</a></html> to learn about the details of Donor Plasmid and Insertion Fragments and the strategies employed in our design. <br />
<br />
As we all know, antibody diversity mainly lean on the number of the fragments used for recombination. Therefore, in order to mimic the antibody generation process, we need to construct Donor Plasmid which contains large sum of Insertion Fragments. Theoretically, we can construct Donor Plasmid of this kind. However, due to the time limit of the competition, we didn’t try to construct DP containing many Insertion Fragments.<br />
<br />
In spite of this, in order to meet the needs, we designed two methods to construct plasmids on a large scale and attempted the usage in parts of our Donor Plasmids.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Strategies">'DP construction and Strategies'</a></html> for the methods of rapid plasmid construction. <br />
<br />
Besides, we have applied for two BBFRFC, which are BBFRFC61 and BBFRFC 62 respectively.<br />
<br />
Then, after transformation of the prepared ‘recepient strain’ with Donor Plasmid and the induction of corresponding agents, I-scel enzyme cuts out DNA fragments from DP and LP, generating numerous Insertion Fragments. Besides, antibiotic resistance gene integrated into E coli genome is also cut out, providing recombination sites for Insertion Fragments.<br />
<br />
Provided with Insertion Fragments mentioned above and Lamda-Red expressed by Donor Plasmid, Insertion Fragments recombine with the corresponding landing pad sequence in the genome and thus get inserted into the correct site of the genome. Based on our design of Landing pad, we can ensure that only one Insertion Fragment from one group is randomly inserted into the genome, therefore mimicking the VDJ recombination process.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">‘DP Insertion and Recombination Induction’</a></html> to learn more about this part. <br />
<br />
Engineered E coli cells which have undergone successful recombination can be selected out of the pool through specific screening methods. After tests on different strains, our project achieve a recombination rate above 50% based on our current techniques.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/experiments#res">‘Result’</a></html> to learn more about the identification of recombination rate. <br />
<br />
The goal of the second module in our project is aimed at selection of specific antibodies. Due to the maturity of existing antibody screening technique, our work mainly focuses on imitation of mammalian antibody selection, thus providing possible alternatives for existing methods.<br />
<br />
The outline of our ideas is to find mechanisms similar to in vivo antibody selection and take in,to account the industry production costs, controllability and reliability, thus developing new methods for antibody selection.<br />
In fact, in mammalian system, antibody selection is achieved through the activation of B lymphocytes and thus the rapid proliferation of the B lymphocytes that express the specific antibodies that bind to antigen. Simply put, the whole process relies on the interaction between antigen epitopes and membrane integral immunoglobins.<br />
<br />
To mimic the activation mediated by membrane integral immunoglobins, we develop ToxR-mediated transmembrane activation signal system. In this system, the interaction between antibodies and antigen triggered downstream expression of reporter genes, thus providing signals for selection.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">‘ToxR-based Transmembrane Signaling Pathway Method’</a></html> for detailed description of this method.<br />
<br />
In addition, we develop a technique called ‘Bacterial based Microarray’ for selection purpose. In this method, we combine membrane display technique and high throughput microarray technique, that is, a bacterial based microarray method.<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">’Bacterial based microarray’</a></html> for details.<br />
<br />
In order to find methods for antibody selection, we cooperate with other iGEM teams. For example, we talked with Macquarie_Australia iGEM team about the project. The techniques involved in their project might be useful for our selection methods. Therefore, members from both teams worked together to research on this problem.<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">'link'</a></html> to learn more about our cooperation.<br />
<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens. <br />
Our system is aiming at imitating this recombination process, using E.coli as the gene carrier.<br />
It is known to all that regional genes (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and the recombination occurs when VDJ recombinase are expressed. We choose RecA enzyme to induce the recombination, while use I-Sel enzyme to cut the genome, just mimicking the process happened in B cells.<br />
<br/><br />
<br/><br />
<br />
==Comparison between natural antibody production and E. Coli system==<br />
<br/><br />
===VDJ Recombination vs Lading Pad Recombination===<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is the mechanism by which immunoglobulin (Ig) and T cell receptors (TCR) are generated in the immune system. Through V(D)J recombination, Variable, Diverse, and Joining gene segments are randomly combined, thus encoding various kinds of immunoglobins and T cell receptors to recognize potential antigens. <br />
During the process of B cell maturation, gene segments are rearranged and thus transcribe mRNA that can produce diverse types of antibody for recognition of billions of potential epitopes. The rearrangement brings three gene segments (termed V, D, and J) in close proximity, which are then joined together head to tail, the process of which is known as V(D)J recombination based on the V,D,J gene segments involved. Multiple copies of the V, D, and J genes in the human genome are shuffled in the process, and a specific antibody will be generate among millions of other possible combinations. Because gene are joined together permanently, one mature B cell will produce only one specific antibody.<br />
<br />
Our system is aimed to mimic this recombination process, using E.coli as the carrier.<br />
Through previous research in immunology field, each kind of gene segments (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and recombination occurs when VDJ recombinase recognize the specific signal sequence and mediate the recombination process. In our project, we design a sequence, which carries separated parts of one complete gene and integrate synthesized DNA fragment into the genome of E coli via landing pad method. Then we use I-Sel enzyme to cut the gene segments out of E coli genome, into which gene segments are inserted and choose RecA enzyme to recognize the previously designed signal sequence flanking our target sequence and recombine the gene segments, just like what recombinase accomplish in B cells. The whole process is called landing pad recombination.<br />
<br />
===Somatic hyper-mutation vs Junctional mutation===<br />
<br />
Somatic hypermutation involves a programmed process of mutation affecting the variable regions of immunoglobulin genes. During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is at least 105-106 fold greater than the normal rate of mutation across the genome. Variation is mainly in the form of single base substitutions, with insertions and deletions being less common. These mutations occur mostly at “hotspots” in the DNA, known as hypervariable regions. These regions correspond to the complementarity determining regions; the sites involved in antigen recognition on the immunoglobulin. This directed hypermutation allows for the selection of B cells that express immunoglobulin receptors possessing an enhanced ability to recognize and bind a specific foreign antigen.<br />
<br />
In addition, the cross ligation, namely Landing pad recombination, has higher opportunity to mutate in the link sequence. The introduction of 4 junctional shuffler 64 will meet the need of the rest 107 diversity, perfectly undertaking the work in the antibody diversity construction that hyper-mutation does in the highly variable region. In this way, we have finished a building library, waiting for the specialized antigen to select.<br />
<br />
===Antigen-specific Selection vs CBD-Based Microarray and ToxR-Based Transmembrane pathway method===<br />
In mammalian immune system, billions of antibodies will be generated. Then how can the immune system find out specific antibody that indiscriminately recognize the specific antigen that comes in. Generally speaking, due to the fact that one mature B cell only generates one specific immunoglobin, once the membrane integral immunoglobin bind to the specific antigen, a series of intracellular response, such as gene activation and cell differetiation will be triggered in B cell. Then, B cells expressing the specific antibodies that recognize the specific antigen will proliferate and generate enormous amount of antibodies that help eliminate the specific antigen.<br />
<br />
Simply put, antibody selection is accomplished through the specific interaction between the membrane immunoglobin and the specific antigen. Therefore, in our project, we also plan to mimic this process through interaction between the antibodies generated through landing pad recombination and the antigen we target. CBD-based Microarray and ToxR-based Transmembrane pathway are the two methods by which we achieve this goal.<br />
CBD domain, which interacts with cellulose, potentially provides us way to locate our E.coli to the microarray coated with cellulose. OmpA (outer membrane protein A), displays the antibody and antigen out to the membrane surface. Then by applying the antibodies generated through landing pad recombination to the microarray, antibodies that bind to the antigen will be retained and thus selected out of the pool of antibodies.<br />
<br />
Alternatively, the ToxR-based transmembrane pathway method shows a different strategy for us to seek the right folded antibody. Once the antigen comes, ToxR outer part will form dimer shape and the downstream signal occur, supplying the approach of reporters, such as GFP, to examine and pick out what we want.<br />
<br />
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&nbsp;&nbsp;<a href="#top">&nbsp;<img src="https://static.igem.org/mediawiki/2010/d/dd/TOP.png" />TOP</a><br />
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</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/project/outlineTeam:Tsinghua/project/outline2010-10-27T14:54:25Z<p>Gumilton: /* Project Design */</p>
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<h1>Outline</h1><br />
<br />
<div class="content_block"><br />
<br />
<br/><br />
In order to realize the production of antibodies in prokaryotic cells, we firstly investigate the whole process of antibody production in mammalian immune system and try to simulate the whole process via the methods and concepts of synthetic biology. Finally, we successfully constructed the desired system. The comparison between the key steps involved in antibody generation and corresponding synthetic methods are shown in the chart below.<br />
<br/><br />
<br />
<table border="2" bordercolor="maroon" bgcolor="silver"><br />
<tbody><br />
<tr><br />
<th colspan=3>Antibody Production</th><br />
<th width=50px></th><br />
<th colspan=2>E Coli. Production System</th></tr><br />
<tr><br />
<td rowspan=4 width=70px><a href="#VDJ_Recombination_vs_Lading_Pad_Recombination">VDJ Recombination</a></td><br />
<td rowspan=3 width=70px>Preparation</td><br />
<td width=70px>RSS Sequence</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">Landing Pad Insertion</a></td><br />
<td rowspan=4 width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1">Module I: Recombination System</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Recombinase</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">Helper Plasmid Insertion</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Library</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">Donor Plasmid Construction</a></td><br />
</tr><br />
<tr><br />
<td>Recombination</td><br />
<td colspan=2><img src="https://static.igem.org/mediawiki/2010/d/d6/THUarrow2.png" width=180></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">DP Insertion and Recombination Induction</a></td><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="#Somatic_hyper-mutation_vs_Junctional_mutation">Somatic Hypermutation</a></td><br />
<td rowspan=2 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
<!---------<td>Junctional Diversity</td>----------------------><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">CBD-based Microarray</a></td><br />
<td rowspan=4><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2">Module II: Selection System</a></td><br />
</tr><br />
<tr><br />
<td rowspan=3><a href="#Antigen-specific_Selection_vs_CBD-Based_Microarray_and_ToxR-Based_Transmembrane_pathway_method">Antigen Selection</a></td><br />
<td rowspan=3 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">ToxR-based Transmembrane Pathway</a></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">Cooperation With Macquarie_Australia</a></td><br />
</tr><br />
</tbody><br />
</table><br />
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<br/><br />
Generally speaking, antibody production in our project can be divided into two modules.<br />
<br />
<html><br />
<body bgcolor="#336699" text="#ffffff" link="#60a179"><br />
<br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1"><font face="Comic Sans MS" size="4">Module I: Generation of antibody library</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2"><font face="Comic Sans MS" size="4">Module II: Selection of specific antibodies</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
</html><br />
<br />
==Project Design==<br />
The first module is carried out by a novel method called E coli in vivo recombination system. The theoretical and experimental basis comes from the work of Thomas E. Kuhlman and Edward C. Cox. We managed to modify their system, wishing to utilize it for simulation of antibody recombination.<br />
<br />
Firstly, we try to insert a DNA segment called”Landing Pad(LP)” into the specific sites of E coli via Att recombination. Landing pad sequence consists of the following parts(from 5’ to 3’ in the sequence):<br />
<br />
1)25bp-long random sequence<br />
<br />
2)15bp-long recognition sequence of restriction enzyme I-scel<br />
<br />
3) antibiotic resistance gene used for antibody selection<br />
<br />
4) 15bp-long recognition sequence of restriction enzyme I-scel(corresponding to 2)<br />
<br />
5) 25bp-long random sequence (corresponding to 1)<br />
<br />
After integrating DNA segment of Landing pad into the genome of E coli, we completed the genetic engineering of the genome of E coli. All the five parts of landing pad are designed for subsequent recombination. <br />
<br />
Besides, in order to achieve different recombination goals, we designed several landing pads of different sequences. <br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">'Landing Pad Construction and Insertion'</a></html> to learn about the details of Landing Pad design. <br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#att">'link'</a></html> to learn more about ATT recombination.<br />
<br />
After the integration of landing pad, we have to insert another DNA fragment called Helper Plasmid(HP), which provides necessary tools for in vivo recombination. <br />
<br />
In this plasmid(Helper Plasmid), there are two genes used for recombination. <br />
<br />
The first one encodes restriction enzyme I-scel after induction of L-arabinose. I-scel is able to recognize the 15bp-length sequence flanking a target DNA fragment and cut out the fragment, therefore providing DNA segments for recombination mediated by other components.<br />
<br />
The second one encodes the enzyme responsible for recombination called Lamda-Red. With the presence of IPTG, Lamda-Red will recombine DNA fragments flanked by the same DNA sequence recognized by Lamda-Red.<br />
<br />
In addition, Helper Plasmid contains a specific temperature-sensitive replicon, which is used to control the replication via temperature changes. We can remove the replicon when necessary.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">'HP insertion'</a></html> to learn more about Helper Plasmid.<br />
<br />
Up till now, we successfully engineered a ‘recepient strain’. Then we have to transform the ‘recepient strain’ with the Donor Plasmid(DP) containing recombination fragments. Donor Plasmid contains several Insertion Fragments and each Insertion Fragment contains following five parts:<br />
<br />
1) one 15bp-long restriction enzyme I-scel recognition site<br />
<br />
2) one 25bp-long random sequence<br />
<br />
3) one fragment for insertion and recombination<br />
<br />
4) one 25bp-long random sequence<br />
<br />
5) one 15bp-long restriction enzyme I-scel recognition site(in correspondence with 1)<br />
<br />
(Note: the order of the five parts differ from that of landing pad)<br />
<br />
As we previously mentioned, Donor plasmid contains several Insertion Fragments. Based on the flanking landing pad sequence, we can ascribe Insertion Fragments to the same group as long as the flanking landing pad sequences of those Insertion Fragments are the same.<br />
<br />
In order to achieve different goals, we design different Donor Plasmids, different Donor Plasmids contain different number of Insertion Fragments, which belong to different groups.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">‘DP construction’</a></html> to learn about the details of Donor Plasmid and Insertion Fragments and the strategies employed in our design. <br />
<br />
As we all know, antibody diversity mainly lean on the number of the fragments used for recombination. Therefore, in order to mimic the antibody generation process, we need to construct Donor Plasmid which contains large sum of Insertion Fragments. Theoretically, we can construct Donor Plasmid of this kind. However, due to the time limit of the competition, we didn’t try to construct DP containing many Insertion Fragments.<br />
<br />
In spite of this, in order to meet the needs, we designed two methods to construct plasmids on a large scale and attempted the usage in parts of our Donor Plasmids.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Strategies">'DP construction and Strategies'</a></html> for the methods of rapid plasmid construction. <br />
<br />
Besides, we have applied for two BBFRFC, which are BBFRFC61 and BBFRFC 62 respectively.<br />
<br />
Then, after transformation of the prepared ‘recepient strain’ with Donor Plasmid and the induction of corresponding agents, I-scel enzyme cuts out DNA fragments from DP and LP, generating numerous Insertion Fragments. Besides, antibiotic resistance gene integrated into E coli genome is also cut out, providing recombination sites for Insertion Fragments.<br />
<br />
Provided with Insertion Fragments mentioned above and Lamda-Red expressed by Donor Plasmid, Insertion Fragments recombine with the corresponding landing pad sequence in the genome and thus get inserted into the correct site of the genome. Based on our design of Landing pad, we can ensure that only one Insertion Fragment from one group is randomly inserted into the genome, therefore mimicking the VDJ recombination process.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">‘DP Insertion and Recombination Induction’</a></html> to learn more about this part. <br />
<br />
Engineered E coli cells which have undergone successful recombination can be selected out of the pool through specific screening methods. After tests on different strains, our project achieve a recombination rate above 50% based on our current techniques.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/experiments#res">‘Result’</a></html> to learn more about the identification of recombination rate. <br />
<br />
The goal of the second module in our project is aimed at selection of specific antibodies. Due to the maturity of existing antibody screening technique, our work mainly focuses on imitation of mammalian antibody selection, thus providing possible alternatives for existing methods.<br />
<br />
The outline of our ideas is to find mechanisms similar to in vivo antibody selection and take in,to account the industry production costs, controllability and reliability, thus developing new methods for antibody selection.<br />
In fact, in mammalian system, antibody selection is achieved through the activation of B lymphocytes and thus the rapid proliferation of the B lymphocytes that express the specific antibodies that bind to antigen. Simply put, the whole process relies on the interaction between antigen epitopes and membrane integral immunoglobins.<br />
<br />
To mimic the activation mediated by membrane integral immunoglobins, we develop ToxR-mediated transmembrane activation signal system. In this system, the interaction between antibodies and antigen triggered downstream expression of reporter genes, thus providing signals for selection.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">‘ToxR-based Transmembrane Signaling Pathway Method’</a></html> for detailed description of this method.<br />
<br />
In addition, we develop a technique called ‘Bacterial based Microarray’ for selection purpose. In this method, we combine membrane display technique and high throughput microarray technique, that is, a bacterial based microarray method.<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">’Bacterial based microarray’</a></html> for details.<br />
<br />
In order to find methods for antibody selection, we cooperate with other iGEM teams. For example, we talked with Macquarie_Australia iGEM team about the project. The techniques involved in their project might be useful for our selection methods. Therefore, members from both teams worked together to research on this problem.<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">'link'</a></html> to learn more about our cooperation.<br />
<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens. <br />
Our system is aiming at imitating this recombination process, using E.coli as the gene carrier.<br />
It is known to all that regional genes (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and the recombination occurs when VDJ recombinase are expressed. We choose RecA enzyme to induce the recombination, while use I-Sel enzyme to cut the genome, just mimicking the process happened in B cells.<br />
<br/><br />
<br/><br />
==Comparison between natural antibody production and E. Coli system==<br />
<br/><br />
===VDJ Recombination vs Lading Pad Recombination===<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is the mechanism by which immunoglobulin (Ig) and T cell receptors (TCR) are generated in the immune system. Through V(D)J recombination, Variable, Diverse, and Joining gene segments are randomly combined, thus encoding various kinds of immunoglobins and T cell receptors to recognize potential antigens. <br />
During the process of B cell maturation, gene segments are rearranged and thus transcribe mRNA that can produce diverse types of antibody for recognition of billions of potential epitopes. The rearrangement brings three gene segments (termed V, D, and J) in close proximity, which are then joined together head to tail, the process of which is known as V(D)J recombination based on the V,D,J gene segments involved. Multiple copies of the V, D, and J genes in the human genome are shuffled in the process, and a specific antibody will be generate among millions of other possible combinations. Because gene are joined together permanently, one mature B cell will produce only one specific antibody.<br />
<br />
Our system is aimed to mimic this recombination process, using E.coli as the carrier.<br />
Through previous research in immunology field, each kind of gene segments (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and recombination occurs when VDJ recombinase recognize the specific signal sequence and mediate the recombination process. In our project, we design a sequence, which carries separated parts of one complete gene and integrate synthesized DNA fragment into the genome of E coli via landing pad method. Then we use I-Sel enzyme to cut the gene segments out of E coli genome, into which gene segments are inserted and choose RecA enzyme to recognize the previously designed signal sequence flanking our target sequence and recombine the gene segments, just like what recombinase accomplish in B cells. The whole process is called landing pad recombination.<br />
<br />
===Somatic hyper-mutation vs Junctional mutation===<br />
<br />
Somatic hypermutation involves a programmed process of mutation affecting the variable regions of immunoglobulin genes. During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is at least 105-106 fold greater than the normal rate of mutation across the genome. Variation is mainly in the form of single base substitutions, with insertions and deletions being less common. These mutations occur mostly at “hotspots” in the DNA, known as hypervariable regions. These regions correspond to the complementarity determining regions; the sites involved in antigen recognition on the immunoglobulin. This directed hypermutation allows for the selection of B cells that express immunoglobulin receptors possessing an enhanced ability to recognize and bind a specific foreign antigen.<br />
<br />
In addition, the cross ligation, namely Landing pad recombination, has higher opportunity to mutate in the link sequence. The introduction of 4 junctional shuffler 64 will meet the need of the rest 107 diversity, perfectly undertaking the work in the antibody diversity construction that hyper-mutation does in the highly variable region. In this way, we have finished a building library, waiting for the specialized antigen to select.<br />
<br />
===Antigen-specific Selection vs CBD-Based Microarray and ToxR-Based Transmembrane pathway method===<br />
In mammalian immune system, billions of antibodies will be generated. Then how can the immune system find out specific antibody that indiscriminately recognize the specific antigen that comes in. Generally speaking, due to the fact that one mature B cell only generates one specific immunoglobin, once the membrane integral immunoglobin bind to the specific antigen, a series of intracellular response, such as gene activation and cell differetiation will be triggered in B cell. Then, B cells expressing the specific antibodies that recognize the specific antigen will proliferate and generate enormous amount of antibodies that help eliminate the specific antigen.<br />
<br />
Simply put, antibody selection is accomplished through the specific interaction between the membrane immunoglobin and the specific antigen. Therefore, in our project, we also plan to mimic this process through interaction between the antibodies generated through landing pad recombination and the antigen we target. CBD-based Microarray and ToxR-based Transmembrane pathway are the two methods by which we achieve this goal.<br />
CBD domain, which interacts with cellulose, potentially provides us way to locate our E.coli to the microarray coated with cellulose. OmpA (outer membrane protein A), displays the antibody and antigen out to the membrane surface. Then by applying the antibodies generated through landing pad recombination to the microarray, antibodies that bind to the antigen will be retained and thus selected out of the pool of antibodies.<br />
<br />
Alternatively, the ToxR-based transmembrane pathway method shows a different strategy for us to seek the right folded antibody. Once the antigen comes, ToxR outer part will form dimer shape and the downstream signal occur, supplying the approach of reporters, such as GFP, to examine and pick out what we want.<br />
<br />
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</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/project/outlineTeam:Tsinghua/project/outline2010-10-27T14:54:03Z<p>Gumilton: /* Comparison between natural antibody production and E. Coli system */</p>
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<h1>Outline</h1><br />
<br />
<div class="content_block"><br />
<br />
<br/><br />
In order to realize the production of antibodies in prokaryotic cells, we firstly investigate the whole process of antibody production in mammalian immune system and try to simulate the whole process via the methods and concepts of synthetic biology. Finally, we successfully constructed the desired system. The comparison between the key steps involved in antibody generation and corresponding synthetic methods are shown in the chart below.<br />
<br/><br />
<br />
<table border="2" bordercolor="maroon" bgcolor="silver"><br />
<tbody><br />
<tr><br />
<th colspan=3>Antibody Production</th><br />
<th width=50px></th><br />
<th colspan=2>E Coli. Production System</th></tr><br />
<tr><br />
<td rowspan=4 width=70px><a href="#VDJ_Recombination_vs_Lading_Pad_Recombination">VDJ Recombination</a></td><br />
<td rowspan=3 width=70px>Preparation</td><br />
<td width=70px>RSS Sequence</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">Landing Pad Insertion</a></td><br />
<td rowspan=4 width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1">Module I: Recombination System</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Recombinase</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">Helper Plasmid Insertion</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Library</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">Donor Plasmid Construction</a></td><br />
</tr><br />
<tr><br />
<td>Recombination</td><br />
<td colspan=2><img src="https://static.igem.org/mediawiki/2010/d/d6/THUarrow2.png" width=180></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">DP Insertion and Recombination Induction</a></td><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="#Somatic_hyper-mutation_vs_Junctional_mutation">Somatic Hypermutation</a></td><br />
<td rowspan=2 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
<!---------<td>Junctional Diversity</td>----------------------><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">CBD-based Microarray</a></td><br />
<td rowspan=4><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2">Module II: Selection System</a></td><br />
</tr><br />
<tr><br />
<td rowspan=3><a href="#Antigen-specific_Selection_vs_CBD-Based_Microarray_and_ToxR-Based_Transmembrane_pathway_method">Antigen Selection</a></td><br />
<td rowspan=3 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">ToxR-based Transmembrane Pathway</a></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">Cooperation With Macquarie_Australia</a></td><br />
</tr><br />
</tbody><br />
</table><br />
</html><br />
<br/><br />
Generally speaking, antibody production in our project can be divided into two modules.<br />
<br />
<html><br />
<body bgcolor="#336699" text="#ffffff" link="#60a179"><br />
<br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1"><font face="Comic Sans MS" size="4">Module I: Generation of antibody library</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2"><font face="Comic Sans MS" size="4">Module II: Selection of specific antibodies</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
</html><br />
<br />
=Project Design=<br />
The first module is carried out by a novel method called E coli in vivo recombination system. The theoretical and experimental basis comes from the work of Thomas E. Kuhlman and Edward C. Cox. We managed to modify their system, wishing to utilize it for simulation of antibody recombination.<br />
<br />
Firstly, we try to insert a DNA segment called”Landing Pad(LP)” into the specific sites of E coli via Att recombination. Landing pad sequence consists of the following parts(from 5’ to 3’ in the sequence):<br />
<br />
1)25bp-long random sequence<br />
<br />
2)15bp-long recognition sequence of restriction enzyme I-scel<br />
<br />
3) antibiotic resistance gene used for antibody selection<br />
<br />
4) 15bp-long recognition sequence of restriction enzyme I-scel(corresponding to 2)<br />
<br />
5) 25bp-long random sequence (corresponding to 1)<br />
<br />
After integrating DNA segment of Landing pad into the genome of E coli, we completed the genetic engineering of the genome of E coli. All the five parts of landing pad are designed for subsequent recombination. <br />
<br />
Besides, in order to achieve different recombination goals, we designed several landing pads of different sequences. <br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">'Landing Pad Construction and Insertion'</a></html> to learn about the details of Landing Pad design. <br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#att">'link'</a></html> to learn more about ATT recombination.<br />
<br />
After the integration of landing pad, we have to insert another DNA fragment called Helper Plasmid(HP), which provides necessary tools for in vivo recombination. <br />
<br />
In this plasmid(Helper Plasmid), there are two genes used for recombination. <br />
<br />
The first one encodes restriction enzyme I-scel after induction of L-arabinose. I-scel is able to recognize the 15bp-length sequence flanking a target DNA fragment and cut out the fragment, therefore providing DNA segments for recombination mediated by other components.<br />
<br />
The second one encodes the enzyme responsible for recombination called Lamda-Red. With the presence of IPTG, Lamda-Red will recombine DNA fragments flanked by the same DNA sequence recognized by Lamda-Red.<br />
<br />
In addition, Helper Plasmid contains a specific temperature-sensitive replicon, which is used to control the replication via temperature changes. We can remove the replicon when necessary.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">'HP insertion'</a></html> to learn more about Helper Plasmid.<br />
<br />
Up till now, we successfully engineered a ‘recepient strain’. Then we have to transform the ‘recepient strain’ with the Donor Plasmid(DP) containing recombination fragments. Donor Plasmid contains several Insertion Fragments and each Insertion Fragment contains following five parts:<br />
<br />
1) one 15bp-long restriction enzyme I-scel recognition site<br />
<br />
2) one 25bp-long random sequence<br />
<br />
3) one fragment for insertion and recombination<br />
<br />
4) one 25bp-long random sequence<br />
<br />
5) one 15bp-long restriction enzyme I-scel recognition site(in correspondence with 1)<br />
<br />
(Note: the order of the five parts differ from that of landing pad)<br />
<br />
As we previously mentioned, Donor plasmid contains several Insertion Fragments. Based on the flanking landing pad sequence, we can ascribe Insertion Fragments to the same group as long as the flanking landing pad sequences of those Insertion Fragments are the same.<br />
<br />
In order to achieve different goals, we design different Donor Plasmids, different Donor Plasmids contain different number of Insertion Fragments, which belong to different groups.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">‘DP construction’</a></html> to learn about the details of Donor Plasmid and Insertion Fragments and the strategies employed in our design. <br />
<br />
As we all know, antibody diversity mainly lean on the number of the fragments used for recombination. Therefore, in order to mimic the antibody generation process, we need to construct Donor Plasmid which contains large sum of Insertion Fragments. Theoretically, we can construct Donor Plasmid of this kind. However, due to the time limit of the competition, we didn’t try to construct DP containing many Insertion Fragments.<br />
<br />
In spite of this, in order to meet the needs, we designed two methods to construct plasmids on a large scale and attempted the usage in parts of our Donor Plasmids.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Strategies">'DP construction and Strategies'</a></html> for the methods of rapid plasmid construction. <br />
<br />
Besides, we have applied for two BBFRFC, which are BBFRFC61 and BBFRFC 62 respectively.<br />
<br />
Then, after transformation of the prepared ‘recepient strain’ with Donor Plasmid and the induction of corresponding agents, I-scel enzyme cuts out DNA fragments from DP and LP, generating numerous Insertion Fragments. Besides, antibiotic resistance gene integrated into E coli genome is also cut out, providing recombination sites for Insertion Fragments.<br />
<br />
Provided with Insertion Fragments mentioned above and Lamda-Red expressed by Donor Plasmid, Insertion Fragments recombine with the corresponding landing pad sequence in the genome and thus get inserted into the correct site of the genome. Based on our design of Landing pad, we can ensure that only one Insertion Fragment from one group is randomly inserted into the genome, therefore mimicking the VDJ recombination process.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">‘DP Insertion and Recombination Induction’</a></html> to learn more about this part. <br />
<br />
Engineered E coli cells which have undergone successful recombination can be selected out of the pool through specific screening methods. After tests on different strains, our project achieve a recombination rate above 50% based on our current techniques.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/experiments#res">‘Result’</a></html> to learn more about the identification of recombination rate. <br />
<br />
The goal of the second module in our project is aimed at selection of specific antibodies. Due to the maturity of existing antibody screening technique, our work mainly focuses on imitation of mammalian antibody selection, thus providing possible alternatives for existing methods.<br />
<br />
The outline of our ideas is to find mechanisms similar to in vivo antibody selection and take in,to account the industry production costs, controllability and reliability, thus developing new methods for antibody selection.<br />
In fact, in mammalian system, antibody selection is achieved through the activation of B lymphocytes and thus the rapid proliferation of the B lymphocytes that express the specific antibodies that bind to antigen. Simply put, the whole process relies on the interaction between antigen epitopes and membrane integral immunoglobins.<br />
<br />
To mimic the activation mediated by membrane integral immunoglobins, we develop ToxR-mediated transmembrane activation signal system. In this system, the interaction between antibodies and antigen triggered downstream expression of reporter genes, thus providing signals for selection.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">‘ToxR-based Transmembrane Signaling Pathway Method’</a></html> for detailed description of this method.<br />
<br />
In addition, we develop a technique called ‘Bacterial based Microarray’ for selection purpose. In this method, we combine membrane display technique and high throughput microarray technique, that is, a bacterial based microarray method.<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">’Bacterial based microarray’</a></html> for details.<br />
<br />
In order to find methods for antibody selection, we cooperate with other iGEM teams. For example, we talked with Macquarie_Australia iGEM team about the project. The techniques involved in their project might be useful for our selection methods. Therefore, members from both teams worked together to research on this problem.<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">'link'</a></html> to learn more about our cooperation.<br />
<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens. <br />
Our system is aiming at imitating this recombination process, using E.coli as the gene carrier.<br />
It is known to all that regional genes (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and the recombination occurs when VDJ recombinase are expressed. We choose RecA enzyme to induce the recombination, while use I-Sel enzyme to cut the genome, just mimicking the process happened in B cells.<br />
<br/><br />
<br/><br />
==Comparison between natural antibody production and E. Coli system==<br />
<br/><br />
===VDJ Recombination vs Lading Pad Recombination===<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is the mechanism by which immunoglobulin (Ig) and T cell receptors (TCR) are generated in the immune system. Through V(D)J recombination, Variable, Diverse, and Joining gene segments are randomly combined, thus encoding various kinds of immunoglobins and T cell receptors to recognize potential antigens. <br />
During the process of B cell maturation, gene segments are rearranged and thus transcribe mRNA that can produce diverse types of antibody for recognition of billions of potential epitopes. The rearrangement brings three gene segments (termed V, D, and J) in close proximity, which are then joined together head to tail, the process of which is known as V(D)J recombination based on the V,D,J gene segments involved. Multiple copies of the V, D, and J genes in the human genome are shuffled in the process, and a specific antibody will be generate among millions of other possible combinations. Because gene are joined together permanently, one mature B cell will produce only one specific antibody.<br />
<br />
Our system is aimed to mimic this recombination process, using E.coli as the carrier.<br />
Through previous research in immunology field, each kind of gene segments (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and recombination occurs when VDJ recombinase recognize the specific signal sequence and mediate the recombination process. In our project, we design a sequence, which carries separated parts of one complete gene and integrate synthesized DNA fragment into the genome of E coli via landing pad method. Then we use I-Sel enzyme to cut the gene segments out of E coli genome, into which gene segments are inserted and choose RecA enzyme to recognize the previously designed signal sequence flanking our target sequence and recombine the gene segments, just like what recombinase accomplish in B cells. The whole process is called landing pad recombination.<br />
<br />
===Somatic hyper-mutation vs Junctional mutation===<br />
<br />
Somatic hypermutation involves a programmed process of mutation affecting the variable regions of immunoglobulin genes. During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is at least 105-106 fold greater than the normal rate of mutation across the genome. Variation is mainly in the form of single base substitutions, with insertions and deletions being less common. These mutations occur mostly at “hotspots” in the DNA, known as hypervariable regions. These regions correspond to the complementarity determining regions; the sites involved in antigen recognition on the immunoglobulin. This directed hypermutation allows for the selection of B cells that express immunoglobulin receptors possessing an enhanced ability to recognize and bind a specific foreign antigen.<br />
<br />
In addition, the cross ligation, namely Landing pad recombination, has higher opportunity to mutate in the link sequence. The introduction of 4 junctional shuffler 64 will meet the need of the rest 107 diversity, perfectly undertaking the work in the antibody diversity construction that hyper-mutation does in the highly variable region. In this way, we have finished a building library, waiting for the specialized antigen to select.<br />
<br />
===Antigen-specific Selection vs CBD-Based Microarray and ToxR-Based Transmembrane pathway method===<br />
In mammalian immune system, billions of antibodies will be generated. Then how can the immune system find out specific antibody that indiscriminately recognize the specific antigen that comes in. Generally speaking, due to the fact that one mature B cell only generates one specific immunoglobin, once the membrane integral immunoglobin bind to the specific antigen, a series of intracellular response, such as gene activation and cell differetiation will be triggered in B cell. Then, B cells expressing the specific antibodies that recognize the specific antigen will proliferate and generate enormous amount of antibodies that help eliminate the specific antigen.<br />
<br />
Simply put, antibody selection is accomplished through the specific interaction between the membrane immunoglobin and the specific antigen. Therefore, in our project, we also plan to mimic this process through interaction between the antibodies generated through landing pad recombination and the antigen we target. CBD-based Microarray and ToxR-based Transmembrane pathway are the two methods by which we achieve this goal.<br />
CBD domain, which interacts with cellulose, potentially provides us way to locate our E.coli to the microarray coated with cellulose. OmpA (outer membrane protein A), displays the antibody and antigen out to the membrane surface. Then by applying the antibodies generated through landing pad recombination to the microarray, antibodies that bind to the antigen will be retained and thus selected out of the pool of antibodies.<br />
<br />
Alternatively, the ToxR-based transmembrane pathway method shows a different strategy for us to seek the right folded antibody. Once the antigen comes, ToxR outer part will form dimer shape and the downstream signal occur, supplying the approach of reporters, such as GFP, to examine and pick out what we want.<br />
<br />
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</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/project/outlineTeam:Tsinghua/project/outline2010-10-27T14:53:45Z<p>Gumilton: /* Comparison between natural antibody production and E. Coli system */</p>
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<h1>Outline</h1><br />
<br />
<div class="content_block"><br />
<br />
<br/><br />
In order to realize the production of antibodies in prokaryotic cells, we firstly investigate the whole process of antibody production in mammalian immune system and try to simulate the whole process via the methods and concepts of synthetic biology. Finally, we successfully constructed the desired system. The comparison between the key steps involved in antibody generation and corresponding synthetic methods are shown in the chart below.<br />
<br/><br />
<br />
<table border="2" bordercolor="maroon" bgcolor="silver"><br />
<tbody><br />
<tr><br />
<th colspan=3>Antibody Production</th><br />
<th width=50px></th><br />
<th colspan=2>E Coli. Production System</th></tr><br />
<tr><br />
<td rowspan=4 width=70px><a href="#VDJ_Recombination_vs_Lading_Pad_Recombination">VDJ Recombination</a></td><br />
<td rowspan=3 width=70px>Preparation</td><br />
<td width=70px>RSS Sequence</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">Landing Pad Insertion</a></td><br />
<td rowspan=4 width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1">Module I: Recombination System</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Recombinase</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">Helper Plasmid Insertion</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Library</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">Donor Plasmid Construction</a></td><br />
</tr><br />
<tr><br />
<td>Recombination</td><br />
<td colspan=2><img src="https://static.igem.org/mediawiki/2010/d/d6/THUarrow2.png" width=180></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">DP Insertion and Recombination Induction</a></td><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="#Somatic_hyper-mutation_vs_Junctional_mutation">Somatic Hypermutation</a></td><br />
<td rowspan=2 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
<!---------<td>Junctional Diversity</td>----------------------><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">CBD-based Microarray</a></td><br />
<td rowspan=4><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2">Module II: Selection System</a></td><br />
</tr><br />
<tr><br />
<td rowspan=3><a href="#Antigen-specific_Selection_vs_CBD-Based_Microarray_and_ToxR-Based_Transmembrane_pathway_method">Antigen Selection</a></td><br />
<td rowspan=3 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">ToxR-based Transmembrane Pathway</a></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">Cooperation With Macquarie_Australia</a></td><br />
</tr><br />
</tbody><br />
</table><br />
</html><br />
<br/><br />
Generally speaking, antibody production in our project can be divided into two modules.<br />
<br />
<html><br />
<body bgcolor="#336699" text="#ffffff" link="#60a179"><br />
<br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1"><font face="Comic Sans MS" size="4">Module I: Generation of antibody library</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2"><font face="Comic Sans MS" size="4">Module II: Selection of specific antibodies</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
</html><br />
<br />
=Project Design=<br />
The first module is carried out by a novel method called E coli in vivo recombination system. The theoretical and experimental basis comes from the work of Thomas E. Kuhlman and Edward C. Cox. We managed to modify their system, wishing to utilize it for simulation of antibody recombination.<br />
<br />
Firstly, we try to insert a DNA segment called”Landing Pad(LP)” into the specific sites of E coli via Att recombination. Landing pad sequence consists of the following parts(from 5’ to 3’ in the sequence):<br />
<br />
1)25bp-long random sequence<br />
<br />
2)15bp-long recognition sequence of restriction enzyme I-scel<br />
<br />
3) antibiotic resistance gene used for antibody selection<br />
<br />
4) 15bp-long recognition sequence of restriction enzyme I-scel(corresponding to 2)<br />
<br />
5) 25bp-long random sequence (corresponding to 1)<br />
<br />
After integrating DNA segment of Landing pad into the genome of E coli, we completed the genetic engineering of the genome of E coli. All the five parts of landing pad are designed for subsequent recombination. <br />
<br />
Besides, in order to achieve different recombination goals, we designed several landing pads of different sequences. <br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">'Landing Pad Construction and Insertion'</a></html> to learn about the details of Landing Pad design. <br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#att">'link'</a></html> to learn more about ATT recombination.<br />
<br />
After the integration of landing pad, we have to insert another DNA fragment called Helper Plasmid(HP), which provides necessary tools for in vivo recombination. <br />
<br />
In this plasmid(Helper Plasmid), there are two genes used for recombination. <br />
<br />
The first one encodes restriction enzyme I-scel after induction of L-arabinose. I-scel is able to recognize the 15bp-length sequence flanking a target DNA fragment and cut out the fragment, therefore providing DNA segments for recombination mediated by other components.<br />
<br />
The second one encodes the enzyme responsible for recombination called Lamda-Red. With the presence of IPTG, Lamda-Red will recombine DNA fragments flanked by the same DNA sequence recognized by Lamda-Red.<br />
<br />
In addition, Helper Plasmid contains a specific temperature-sensitive replicon, which is used to control the replication via temperature changes. We can remove the replicon when necessary.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">'HP insertion'</a></html> to learn more about Helper Plasmid.<br />
<br />
Up till now, we successfully engineered a ‘recepient strain’. Then we have to transform the ‘recepient strain’ with the Donor Plasmid(DP) containing recombination fragments. Donor Plasmid contains several Insertion Fragments and each Insertion Fragment contains following five parts:<br />
<br />
1) one 15bp-long restriction enzyme I-scel recognition site<br />
<br />
2) one 25bp-long random sequence<br />
<br />
3) one fragment for insertion and recombination<br />
<br />
4) one 25bp-long random sequence<br />
<br />
5) one 15bp-long restriction enzyme I-scel recognition site(in correspondence with 1)<br />
<br />
(Note: the order of the five parts differ from that of landing pad)<br />
<br />
As we previously mentioned, Donor plasmid contains several Insertion Fragments. Based on the flanking landing pad sequence, we can ascribe Insertion Fragments to the same group as long as the flanking landing pad sequences of those Insertion Fragments are the same.<br />
<br />
In order to achieve different goals, we design different Donor Plasmids, different Donor Plasmids contain different number of Insertion Fragments, which belong to different groups.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">‘DP construction’</a></html> to learn about the details of Donor Plasmid and Insertion Fragments and the strategies employed in our design. <br />
<br />
As we all know, antibody diversity mainly lean on the number of the fragments used for recombination. Therefore, in order to mimic the antibody generation process, we need to construct Donor Plasmid which contains large sum of Insertion Fragments. Theoretically, we can construct Donor Plasmid of this kind. However, due to the time limit of the competition, we didn’t try to construct DP containing many Insertion Fragments.<br />
<br />
In spite of this, in order to meet the needs, we designed two methods to construct plasmids on a large scale and attempted the usage in parts of our Donor Plasmids.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Strategies">'DP construction and Strategies'</a></html> for the methods of rapid plasmid construction. <br />
<br />
Besides, we have applied for two BBFRFC, which are BBFRFC61 and BBFRFC 62 respectively.<br />
<br />
Then, after transformation of the prepared ‘recepient strain’ with Donor Plasmid and the induction of corresponding agents, I-scel enzyme cuts out DNA fragments from DP and LP, generating numerous Insertion Fragments. Besides, antibiotic resistance gene integrated into E coli genome is also cut out, providing recombination sites for Insertion Fragments.<br />
<br />
Provided with Insertion Fragments mentioned above and Lamda-Red expressed by Donor Plasmid, Insertion Fragments recombine with the corresponding landing pad sequence in the genome and thus get inserted into the correct site of the genome. Based on our design of Landing pad, we can ensure that only one Insertion Fragment from one group is randomly inserted into the genome, therefore mimicking the VDJ recombination process.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">‘DP Insertion and Recombination Induction’</a></html> to learn more about this part. <br />
<br />
Engineered E coli cells which have undergone successful recombination can be selected out of the pool through specific screening methods. After tests on different strains, our project achieve a recombination rate above 50% based on our current techniques.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/experiments#res">‘Result’</a></html> to learn more about the identification of recombination rate. <br />
<br />
The goal of the second module in our project is aimed at selection of specific antibodies. Due to the maturity of existing antibody screening technique, our work mainly focuses on imitation of mammalian antibody selection, thus providing possible alternatives for existing methods.<br />
<br />
The outline of our ideas is to find mechanisms similar to in vivo antibody selection and take in,to account the industry production costs, controllability and reliability, thus developing new methods for antibody selection.<br />
In fact, in mammalian system, antibody selection is achieved through the activation of B lymphocytes and thus the rapid proliferation of the B lymphocytes that express the specific antibodies that bind to antigen. Simply put, the whole process relies on the interaction between antigen epitopes and membrane integral immunoglobins.<br />
<br />
To mimic the activation mediated by membrane integral immunoglobins, we develop ToxR-mediated transmembrane activation signal system. In this system, the interaction between antibodies and antigen triggered downstream expression of reporter genes, thus providing signals for selection.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">‘ToxR-based Transmembrane Signaling Pathway Method’</a></html> for detailed description of this method.<br />
<br />
In addition, we develop a technique called ‘Bacterial based Microarray’ for selection purpose. In this method, we combine membrane display technique and high throughput microarray technique, that is, a bacterial based microarray method.<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">’Bacterial based microarray’</a></html> for details.<br />
<br />
In order to find methods for antibody selection, we cooperate with other iGEM teams. For example, we talked with Macquarie_Australia iGEM team about the project. The techniques involved in their project might be useful for our selection methods. Therefore, members from both teams worked together to research on this problem.<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">'link'</a></html> to learn more about our cooperation.<br />
<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens. <br />
Our system is aiming at imitating this recombination process, using E.coli as the gene carrier.<br />
It is known to all that regional genes (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and the recombination occurs when VDJ recombinase are expressed. We choose RecA enzyme to induce the recombination, while use I-Sel enzyme to cut the genome, just mimicking the process happened in B cells.<br />
<br/><br />
==Comparison between natural antibody production and E. Coli system==<br />
<br/><br/><br />
===VDJ Recombination vs Lading Pad Recombination===<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is the mechanism by which immunoglobulin (Ig) and T cell receptors (TCR) are generated in the immune system. Through V(D)J recombination, Variable, Diverse, and Joining gene segments are randomly combined, thus encoding various kinds of immunoglobins and T cell receptors to recognize potential antigens. <br />
During the process of B cell maturation, gene segments are rearranged and thus transcribe mRNA that can produce diverse types of antibody for recognition of billions of potential epitopes. The rearrangement brings three gene segments (termed V, D, and J) in close proximity, which are then joined together head to tail, the process of which is known as V(D)J recombination based on the V,D,J gene segments involved. Multiple copies of the V, D, and J genes in the human genome are shuffled in the process, and a specific antibody will be generate among millions of other possible combinations. Because gene are joined together permanently, one mature B cell will produce only one specific antibody.<br />
<br />
Our system is aimed to mimic this recombination process, using E.coli as the carrier.<br />
Through previous research in immunology field, each kind of gene segments (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and recombination occurs when VDJ recombinase recognize the specific signal sequence and mediate the recombination process. In our project, we design a sequence, which carries separated parts of one complete gene and integrate synthesized DNA fragment into the genome of E coli via landing pad method. Then we use I-Sel enzyme to cut the gene segments out of E coli genome, into which gene segments are inserted and choose RecA enzyme to recognize the previously designed signal sequence flanking our target sequence and recombine the gene segments, just like what recombinase accomplish in B cells. The whole process is called landing pad recombination.<br />
<br />
===Somatic hyper-mutation vs Junctional mutation===<br />
<br />
Somatic hypermutation involves a programmed process of mutation affecting the variable regions of immunoglobulin genes. During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is at least 105-106 fold greater than the normal rate of mutation across the genome. Variation is mainly in the form of single base substitutions, with insertions and deletions being less common. These mutations occur mostly at “hotspots” in the DNA, known as hypervariable regions. These regions correspond to the complementarity determining regions; the sites involved in antigen recognition on the immunoglobulin. This directed hypermutation allows for the selection of B cells that express immunoglobulin receptors possessing an enhanced ability to recognize and bind a specific foreign antigen.<br />
<br />
In addition, the cross ligation, namely Landing pad recombination, has higher opportunity to mutate in the link sequence. The introduction of 4 junctional shuffler 64 will meet the need of the rest 107 diversity, perfectly undertaking the work in the antibody diversity construction that hyper-mutation does in the highly variable region. In this way, we have finished a building library, waiting for the specialized antigen to select.<br />
<br />
===Antigen-specific Selection vs CBD-Based Microarray and ToxR-Based Transmembrane pathway method===<br />
In mammalian immune system, billions of antibodies will be generated. Then how can the immune system find out specific antibody that indiscriminately recognize the specific antigen that comes in. Generally speaking, due to the fact that one mature B cell only generates one specific immunoglobin, once the membrane integral immunoglobin bind to the specific antigen, a series of intracellular response, such as gene activation and cell differetiation will be triggered in B cell. Then, B cells expressing the specific antibodies that recognize the specific antigen will proliferate and generate enormous amount of antibodies that help eliminate the specific antigen.<br />
<br />
Simply put, antibody selection is accomplished through the specific interaction between the membrane immunoglobin and the specific antigen. Therefore, in our project, we also plan to mimic this process through interaction between the antibodies generated through landing pad recombination and the antigen we target. CBD-based Microarray and ToxR-based Transmembrane pathway are the two methods by which we achieve this goal.<br />
CBD domain, which interacts with cellulose, potentially provides us way to locate our E.coli to the microarray coated with cellulose. OmpA (outer membrane protein A), displays the antibody and antigen out to the membrane surface. Then by applying the antibodies generated through landing pad recombination to the microarray, antibodies that bind to the antigen will be retained and thus selected out of the pool of antibodies.<br />
<br />
Alternatively, the ToxR-based transmembrane pathway method shows a different strategy for us to seek the right folded antibody. Once the antigen comes, ToxR outer part will form dimer shape and the downstream signal occur, supplying the approach of reporters, such as GFP, to examine and pick out what we want.<br />
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</html></div>Gumiltonhttp://2010.igem.org/Team:Tsinghua/project/outlineTeam:Tsinghua/project/outline2010-10-27T14:53:28Z<p>Gumilton: </p>
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<div>__NOTOC__<br />
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<script><br />
navlist=new Array("Background", "Project Outline", "Module I", "Module II", "Future");<br />
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<div id="main_content"><a name="outline"></a><br />
<h1>Outline</h1><br />
<br />
<div class="content_block"><br />
<br />
<br/><br />
In order to realize the production of antibodies in prokaryotic cells, we firstly investigate the whole process of antibody production in mammalian immune system and try to simulate the whole process via the methods and concepts of synthetic biology. Finally, we successfully constructed the desired system. The comparison between the key steps involved in antibody generation and corresponding synthetic methods are shown in the chart below.<br />
<br/><br />
<br />
<table border="2" bordercolor="maroon" bgcolor="silver"><br />
<tbody><br />
<tr><br />
<th colspan=3>Antibody Production</th><br />
<th width=50px></th><br />
<th colspan=2>E Coli. Production System</th></tr><br />
<tr><br />
<td rowspan=4 width=70px><a href="#VDJ_Recombination_vs_Lading_Pad_Recombination">VDJ Recombination</a></td><br />
<td rowspan=3 width=70px>Preparation</td><br />
<td width=70px>RSS Sequence</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">Landing Pad Insertion</a></td><br />
<td rowspan=4 width=70px><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1">Module I: Recombination System</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Recombinase</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">Helper Plasmid Insertion</a></td><br />
</tr><br />
<tr><br />
<td>VDJ Library</td><br />
<td><img src="https://static.igem.org/mediawiki/2010/6/69/THUarrow1.png" width=50 /></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">Donor Plasmid Construction</a></td><br />
</tr><br />
<tr><br />
<td>Recombination</td><br />
<td colspan=2><img src="https://static.igem.org/mediawiki/2010/d/d6/THUarrow2.png" width=180></td><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">DP Insertion and Recombination Induction</a></td><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="#Somatic_hyper-mutation_vs_Junctional_mutation">Somatic Hypermutation</a></td><br />
<td rowspan=2 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
<!---------<td>Junctional Diversity</td>----------------------><br />
</tr><br />
<tr><br />
<td rowspan=2><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">CBD-based Microarray</a></td><br />
<td rowspan=4><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2">Module II: Selection System</a></td><br />
</tr><br />
<tr><br />
<td rowspan=3><a href="#Antigen-specific_Selection_vs_CBD-Based_Microarray_and_ToxR-Based_Transmembrane_pathway_method">Antigen Selection</a></td><br />
<td rowspan=3 colspan=3><img src="https://static.igem.org/mediawiki/2010/4/48/THUarrow.png" width=325 ></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">ToxR-based Transmembrane Pathway</a></td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">Cooperation With Macquarie_Australia</a></td><br />
</tr><br />
</tbody><br />
</table><br />
</html><br />
<br/><br />
Generally speaking, antibody production in our project can be divided into two modules.<br />
<br />
<html><br />
<body bgcolor="#336699" text="#ffffff" link="#60a179"><br />
<br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1"><font face="Comic Sans MS" size="4">Module I: Generation of antibody library</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
<span class="rightfloat"><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2"><font face="Comic Sans MS" size="4">Module II: Selection of specific antibodies</font><img src="https://static.igem.org/mediawiki/2010/3/3c/Right_Arrow.png" width="30px"></a></span><br />
<br/><br/><br />
</html><br />
<br />
=Project Design=<br />
The first module is carried out by a novel method called E coli in vivo recombination system. The theoretical and experimental basis comes from the work of Thomas E. Kuhlman and Edward C. Cox. We managed to modify their system, wishing to utilize it for simulation of antibody recombination.<br />
<br />
Firstly, we try to insert a DNA segment called”Landing Pad(LP)” into the specific sites of E coli via Att recombination. Landing pad sequence consists of the following parts(from 5’ to 3’ in the sequence):<br />
<br />
1)25bp-long random sequence<br />
<br />
2)15bp-long recognition sequence of restriction enzyme I-scel<br />
<br />
3) antibiotic resistance gene used for antibody selection<br />
<br />
4) 15bp-long recognition sequence of restriction enzyme I-scel(corresponding to 2)<br />
<br />
5) 25bp-long random sequence (corresponding to 1)<br />
<br />
After integrating DNA segment of Landing pad into the genome of E coli, we completed the genetic engineering of the genome of E coli. All the five parts of landing pad are designed for subsequent recombination. <br />
<br />
Besides, in order to achieve different recombination goals, we designed several landing pads of different sequences. <br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Landing_Pad_Construction_and_Insertion">'Landing Pad Construction and Insertion'</a></html> to learn about the details of Landing Pad design. <br />
<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#att">'link'</a></html> to learn more about ATT recombination.<br />
<br />
After the integration of landing pad, we have to insert another DNA fragment called Helper Plasmid(HP), which provides necessary tools for in vivo recombination. <br />
<br />
In this plasmid(Helper Plasmid), there are two genes used for recombination. <br />
<br />
The first one encodes restriction enzyme I-scel after induction of L-arabinose. I-scel is able to recognize the 15bp-length sequence flanking a target DNA fragment and cut out the fragment, therefore providing DNA segments for recombination mediated by other components.<br />
<br />
The second one encodes the enzyme responsible for recombination called Lamda-Red. With the presence of IPTG, Lamda-Red will recombine DNA fragments flanked by the same DNA sequence recognized by Lamda-Red.<br />
<br />
In addition, Helper Plasmid contains a specific temperature-sensitive replicon, which is used to control the replication via temperature changes. We can remove the replicon when necessary.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#hpi">'HP insertion'</a></html> to learn more about Helper Plasmid.<br />
<br />
Up till now, we successfully engineered a ‘recepient strain’. Then we have to transform the ‘recepient strain’ with the Donor Plasmid(DP) containing recombination fragments. Donor Plasmid contains several Insertion Fragments and each Insertion Fragment contains following five parts:<br />
<br />
1) one 15bp-long restriction enzyme I-scel recognition site<br />
<br />
2) one 25bp-long random sequence<br />
<br />
3) one fragment for insertion and recombination<br />
<br />
4) one 25bp-long random sequence<br />
<br />
5) one 15bp-long restriction enzyme I-scel recognition site(in correspondence with 1)<br />
<br />
(Note: the order of the five parts differ from that of landing pad)<br />
<br />
As we previously mentioned, Donor plasmid contains several Insertion Fragments. Based on the flanking landing pad sequence, we can ascribe Insertion Fragments to the same group as long as the flanking landing pad sequences of those Insertion Fragments are the same.<br />
<br />
In order to achieve different goals, we design different Donor Plasmids, different Donor Plasmids contain different number of Insertion Fragments, which belong to different groups.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpc">‘DP construction’</a></html> to learn about the details of Donor Plasmid and Insertion Fragments and the strategies employed in our design. <br />
<br />
As we all know, antibody diversity mainly lean on the number of the fragments used for recombination. Therefore, in order to mimic the antibody generation process, we need to construct Donor Plasmid which contains large sum of Insertion Fragments. Theoretically, we can construct Donor Plasmid of this kind. However, due to the time limit of the competition, we didn’t try to construct DP containing many Insertion Fragments.<br />
<br />
In spite of this, in order to meet the needs, we designed two methods to construct plasmids on a large scale and attempted the usage in parts of our Donor Plasmids.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#Strategies">'DP construction and Strategies'</a></html> for the methods of rapid plasmid construction. <br />
<br />
Besides, we have applied for two BBFRFC, which are BBFRFC61 and BBFRFC 62 respectively.<br />
<br />
Then, after transformation of the prepared ‘recepient strain’ with Donor Plasmid and the induction of corresponding agents, I-scel enzyme cuts out DNA fragments from DP and LP, generating numerous Insertion Fragments. Besides, antibiotic resistance gene integrated into E coli genome is also cut out, providing recombination sites for Insertion Fragments.<br />
<br />
Provided with Insertion Fragments mentioned above and Lamda-Red expressed by Donor Plasmid, Insertion Fragments recombine with the corresponding landing pad sequence in the genome and thus get inserted into the correct site of the genome. Based on our design of Landing pad, we can ensure that only one Insertion Fragment from one group is randomly inserted into the genome, therefore mimicking the VDJ recombination process.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m1#dpiri">‘DP Insertion and Recombination Induction’</a></html> to learn more about this part. <br />
<br />
Engineered E coli cells which have undergone successful recombination can be selected out of the pool through specific screening methods. After tests on different strains, our project achieve a recombination rate above 50% based on our current techniques.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/experiments#res">‘Result’</a></html> to learn more about the identification of recombination rate. <br />
<br />
The goal of the second module in our project is aimed at selection of specific antibodies. Due to the maturity of existing antibody screening technique, our work mainly focuses on imitation of mammalian antibody selection, thus providing possible alternatives for existing methods.<br />
<br />
The outline of our ideas is to find mechanisms similar to in vivo antibody selection and take in,to account the industry production costs, controllability and reliability, thus developing new methods for antibody selection.<br />
In fact, in mammalian system, antibody selection is achieved through the activation of B lymphocytes and thus the rapid proliferation of the B lymphocytes that express the specific antibodies that bind to antigen. Simply put, the whole process relies on the interaction between antigen epitopes and membrane integral immunoglobins.<br />
<br />
To mimic the activation mediated by membrane integral immunoglobins, we develop ToxR-mediated transmembrane activation signal system. In this system, the interaction between antibodies and antigen triggered downstream expression of reporter genes, thus providing signals for selection.<br />
<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s2">‘ToxR-based Transmembrane Signaling Pathway Method’</a></html> for detailed description of this method.<br />
<br />
In addition, we develop a technique called ‘Bacterial based Microarray’ for selection purpose. In this method, we combine membrane display technique and high throughput microarray technique, that is, a bacterial based microarray method.<br />
PLEASE refer to <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#m2s1">’Bacterial based microarray’</a></html> for details.<br />
<br />
In order to find methods for antibody selection, we cooperate with other iGEM teams. For example, we talked with Macquarie_Australia iGEM team about the project. The techniques involved in their project might be useful for our selection methods. Therefore, members from both teams worked together to research on this problem.<br />
PLEASE refer to this <html><a href="https://2010.igem.org/Team:Tsinghua/project/outline/m2#Strategy_3">'link'</a></html> to learn more about our cooperation.<br />
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Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is a mechanism of genetic recombination in the early stages of immunoglobulin (Ig) and T cell receptors (TCR) production of the immune system. V(D)J recombination nearly-randomly combines Variable, Diverse, and Joining gene segments of vertebrates, and because of its randomness in choosing different genes, is able to diversely encode proteins to match antigens. <br />
Our system is aiming at imitating this recombination process, using E.coli as the gene carrier.<br />
It is known to all that regional genes (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and the recombination occurs when VDJ recombinase are expressed. We choose RecA enzyme to induce the recombination, while use I-Sel enzyme to cut the genome, just mimicking the process happened in B cells.<br />
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=Comparison between natural antibody production and E. Coli system=<br />
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===VDJ Recombination vs Lading Pad Recombination===<br />
Antibody Coding Gene Recombination, also known as V(D)J recombination, somatic recombination, is the mechanism by which immunoglobulin (Ig) and T cell receptors (TCR) are generated in the immune system. Through V(D)J recombination, Variable, Diverse, and Joining gene segments are randomly combined, thus encoding various kinds of immunoglobins and T cell receptors to recognize potential antigens. <br />
During the process of B cell maturation, gene segments are rearranged and thus transcribe mRNA that can produce diverse types of antibody for recognition of billions of potential epitopes. The rearrangement brings three gene segments (termed V, D, and J) in close proximity, which are then joined together head to tail, the process of which is known as V(D)J recombination based on the V,D,J gene segments involved. Multiple copies of the V, D, and J genes in the human genome are shuffled in the process, and a specific antibody will be generate among millions of other possible combinations. Because gene are joined together permanently, one mature B cell will produce only one specific antibody.<br />
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Our system is aimed to mimic this recombination process, using E.coli as the carrier.<br />
Through previous research in immunology field, each kind of gene segments (V, D, J) are flanked by Recombination Signal Sequences (RSSs), and recombination occurs when VDJ recombinase recognize the specific signal sequence and mediate the recombination process. In our project, we design a sequence, which carries separated parts of one complete gene and integrate synthesized DNA fragment into the genome of E coli via landing pad method. Then we use I-Sel enzyme to cut the gene segments out of E coli genome, into which gene segments are inserted and choose RecA enzyme to recognize the previously designed signal sequence flanking our target sequence and recombine the gene segments, just like what recombinase accomplish in B cells. The whole process is called landing pad recombination.<br />
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===Somatic hyper-mutation vs Junctional mutation===<br />
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Somatic hypermutation involves a programmed process of mutation affecting the variable regions of immunoglobulin genes. During proliferation, the B cell receptor locus undergoes an extremely high rate of somatic mutation that is at least 105-106 fold greater than the normal rate of mutation across the genome. Variation is mainly in the form of single base substitutions, with insertions and deletions being less common. These mutations occur mostly at “hotspots” in the DNA, known as hypervariable regions. These regions correspond to the complementarity determining regions; the sites involved in antigen recognition on the immunoglobulin. This directed hypermutation allows for the selection of B cells that express immunoglobulin receptors possessing an enhanced ability to recognize and bind a specific foreign antigen.<br />
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In addition, the cross ligation, namely Landing pad recombination, has higher opportunity to mutate in the link sequence. The introduction of 4 junctional shuffler 64 will meet the need of the rest 107 diversity, perfectly undertaking the work in the antibody diversity construction that hyper-mutation does in the highly variable region. In this way, we have finished a building library, waiting for the specialized antigen to select.<br />
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===Antigen-specific Selection vs CBD-Based Microarray and ToxR-Based Transmembrane pathway method===<br />
In mammalian immune system, billions of antibodies will be generated. Then how can the immune system find out specific antibody that indiscriminately recognize the specific antigen that comes in. Generally speaking, due to the fact that one mature B cell only generates one specific immunoglobin, once the membrane integral immunoglobin bind to the specific antigen, a series of intracellular response, such as gene activation and cell differetiation will be triggered in B cell. Then, B cells expressing the specific antibodies that recognize the specific antigen will proliferate and generate enormous amount of antibodies that help eliminate the specific antigen.<br />
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Simply put, antibody selection is accomplished through the specific interaction between the membrane immunoglobin and the specific antigen. Therefore, in our project, we also plan to mimic this process through interaction between the antibodies generated through landing pad recombination and the antigen we target. CBD-based Microarray and ToxR-based Transmembrane pathway are the two methods by which we achieve this goal.<br />
CBD domain, which interacts with cellulose, potentially provides us way to locate our E.coli to the microarray coated with cellulose. OmpA (outer membrane protein A), displays the antibody and antigen out to the membrane surface. Then by applying the antibodies generated through landing pad recombination to the microarray, antibodies that bind to the antigen will be retained and thus selected out of the pool of antibodies.<br />
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Alternatively, the ToxR-based transmembrane pathway method shows a different strategy for us to seek the right folded antibody. Once the antigen comes, ToxR outer part will form dimer shape and the downstream signal occur, supplying the approach of reporters, such as GFP, to examine and pick out what we want.<br />
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