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2024-03-29T02:03:11Z
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http://2010.igem.org/Team:NYU/Programming
Team:NYU/Programming
2010-10-28T03:59:26Z
<p>RussellDurrett: </p>
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<br />
=====About this Page=====<br />
* Competent programming skills are quickly becoming a necessity for working in biology. Because most of our team members had never had exposure to a programming environment, we decided to use the obstacles presented in our iGEM experiments as cause for learning the Perl programming language and produced some simple scripts that could do routine iGEM labors for us. Here are some examples:<br />
<br />
<br />
=====Biobricking Primers=====<br />
* This script allows the user to input a DNA sequence and outputs the primers necessary for biobricking that sequence via PCR. The script calculates Tm but does not (yet) consider primer dimers. <br />
<br />
*Finally got the CGI working - click below or go [http://openwetware.org/images/a/a7/Biobrickprimers.txt here for the source code]<br />
<br />
<html><br />
<a href="http://russelldurrett.com/biobrickprimers.html"><img src="https://static.igem.org/mediawiki/2010/7/77/Biobricking_Primers_Logo.gif"/></a><br />
</html><br />
<br />
<br />
<br />
=====Overlap Oligo Maker=====<br />
<br />
* Some very fast and efficient DNA assembly methods require parts to overlap on the ends of their sequences. This script will take two DNA sequences and output the oligos (and their Tms) one would need to PCR constructs with overlapping ends. <br />
<br />
*I'm a CGI machine! [http://openwetware.org/images/6/67/Overlapoligos.txt Here is the source code.] or click below for an interactive version. <br />
<br />
<br />
<html><br />
<a href="http://www.russelldurrett.com/overlapoligos.html"><img src="https://static.igem.org/mediawiki/2010/5/5c/Overlaplogo.gif"/></a></html><br />
<br />
<br />
=====Manipulation of Protein Structures=====<br />
<br />
<br />
[[Image:Screen_shot_2010-10-27_at_10.19.59_PM.png|200px|left]]<br />
<br />
In order to more fully understand certain parts of our system and to use the ridiculously amazing CAVE system at Cornell Med School, we decided to explore the 3D structures of the proteins involved in our system. Because the mechanism of Cre recombination is difficult to understand, we dove into the structures available on the Protein DataBase (PDB) and explored details of the mechanism. We were able to locate the active sites of the protein, isolate the catalytic residues, locate the vanadium cofactors and, in all, gain a better understanding of the protein's function. Check out some pictures below!<br />
<br />
<br />
[[Image:Nyu_composite.png|400px|center]]<br />
<br />
<br />
<br />
<br />
<br />
=====Processing Visualization=====<br />
<br />
<br />
Sung has also made a [http://www.processing.org Processing] language[[Image:Processing.jpg|200px|left]]-based visual aid describing the Immunoyeast system. You can go [http://www.russelldurrett.com/processing here] or click the icon below to interact with it to learn more about ImmunoYeast - don't forget to press all the buttons! <br />
<br />
Also feel free to play around with the open source code! <br />
<br />
<br />
We will also have an interactive touch-based system near our poster installation at the Jamboree, you won't want to miss it!<br />
<br />
<br />
<html><br />
<br />
<a href="http://www.russelldurrett.com/processing" align="center"><img src="https://static.igem.org/mediawiki/2010/e/e5/Nyu_visualization.png"/></a></html></div>
RussellDurrett
http://2010.igem.org/Team:NYU/Programming
Team:NYU/Programming
2010-10-28T03:56:25Z
<p>RussellDurrett: </p>
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<br />
[[Image:NYU_OverviewSideF_2.png|200px|right]]<br />
<br />
=====About this Page=====<br />
* Competent programming skills are quickly becoming a necessity for working in biology. Because most of our team members had never had exposure to a programming environment, we decided to use the obstacles presented in our iGEM experiments as cause for learning the Perl programming language and produced some simple scripts that could do routine iGEM labors for us. Here are some examples:<br />
<br />
<br />
=====Biobricking Primers=====<br />
* This script allows the user to input a DNA sequence and outputs the primers necessary for biobricking that sequence via PCR. The script calculates Tm but does not (yet) consider primer dimers. <br />
<br />
*Finally got the CGI working - click below or go [http://openwetware.org/images/a/a7/Biobrickprimers.txt here for the source code]<br />
<br />
<html><br />
<a href="http://russelldurrett.com/biobrickprimers.html"><img src="https://static.igem.org/mediawiki/2010/7/77/Biobricking_Primers_Logo.gif"/></a><br />
</html><br />
<br />
<br />
<br />
=====Overlap Oligo Maker=====<br />
<br />
* Some very fast and efficient DNA assembly methods require parts to overlap on the ends of their sequences. This script will take two DNA sequences and output the oligos (and their Tms) one would need to PCR constructs with overlapping ends. <br />
<br />
*I'm a CGI machine! [http://openwetware.org/images/6/67/Overlapoligos.txt Here is the source code.] or click below for an interactive version. <br />
<br />
<br />
<html><br />
<a href="http://www.russelldurrett.com/overlapoligos.html"><img src="https://static.igem.org/mediawiki/2010/5/5c/Overlaplogo.gif"/></a></html><br />
<br />
<br />
=====Manipulation of Protein Structures=====<br />
<br />
<br />
[[Image:Screen_shot_2010-10-27_at_10.19.59_PM.png|200px|left]]<br />
<br />
In order to more fully understand certain parts of our system and to use the ridiculously amazing CAVE system at Cornell Med School, we decided to explore the 3D structures of the proteins involved in our system. Because the mechanism of Cre recombination is difficult to understand, we dove into the structures available on the Protein DataBase (PDB) and explored details of the mechanism. We were able to locate the active sites of the protein, isolate the catalytic residues, locate the vanadium cofactors and, in all, gain a better understanding of the protein's function. Check out some pictures below!<br />
<br />
<br />
[[Image:Nyu_composite.png|400px|center]]<br />
<br />
<br />
<br />
<br />
<br />
=====Processing Visualization=====<br />
<br />
<br />
Sung also made a [http://www.processing.org Processing] language[[Image:Processing.jpg|200px|left]]-based visual aid describing the Immunoyeast system. Go [http://www.russelldurrett.com/processing here] or click the icon below to interact with it, and feel free to play around with the code too! <br />
<br />
We will also have an interactive touch-based system near our poster installation for you to play with at the jamboree. <br />
<br />
<br />
<br />
<html><br />
<br />
<a href="http://www.russelldurrett.com/processing"><img src="https://static.igem.org/mediawiki/2010/e/e5/Nyu_visualization.png"/></a></html></div>
RussellDurrett
http://2010.igem.org/Team:NYU/Experiments
Team:NYU/Experiments
2010-10-28T03:37:54Z
<p>RussellDurrett: </p>
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<br />
=====Antibody Screening=====<br />
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*To see which antibodies in our diverse population are binding the antigen, we put our population through a modified yeast two-hybrid screen. As you see below, the LexBD domain binds lexO just upstream of the Ura3 CDS, anchoring the bait antigen to the DNA at that point. If the antibody binds the antigen, it localizes the fused VP16 domain to that site as well. This causes an increase of transcription of Ura3 which allows the cells with positive binding antibodies to survive in media without Uracil while those with antibodies that do not bind the antigen die off and are cleared from the population. <br />
[[image:NYU_bind1.png|300px|left]]<br />
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[[image:NYU_bind2.png|300px|left]]<br />
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*Because iGEM is such a [[Image:NYU_binding4.png|300px|right]] short competition and biology takes time to engineer we decided to simulate this two-hybrid screen and ensure that a uracil selection system such as this would actually work. To do this, we physically joined the antibody and antigen constructs together by replacing those parts with a flexible (Gly3Ser)4 linker and nuclear localization sequences. The translated protein then becomes LexBD:NLS:linker:NLS:VP16. Because VP16 is now physically linked to the Lex DNA binding domain, as long as this construct is localized in the nucleus then VP16 will be anchored to the lex operator, increasing transcription through any promoter system that is in lexO's proximity. Because the screening no longer relies on antibody:antigen interactions, expression of this construct can serve as a positive control for the screen. We linked this construct to the pGAL promoter so it would only be expressed in galactose-containing media. To actually test if the system is working or not, we plate these cells on multiple -ura plates, some containing galactose and others containing only glucose. If the screening functions properly, the cells on the galactose plate should grow while the cells on the glucose plate die. <br />
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=====CRE Recombination=====<br />
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*To test for functionality of our Cre recombination constructs we built a reporter plasmid that can function as a positive control. [[image:NYU_plasmid.png|300px|left]] Because our system is all about the protein localization, secretion and surface display of proteins of interest we decided that our control plasmid should involve the same parts. We built a plasmid nearly identical to the [[image:NYU_creillustr.png|300px|right]] antibody plasmid in our system but substituted a yeast-optimized eCFP biobrick for the antibody gene.<br />
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At the outset of this system we observe the eCFP protein localized into the nucleus. Upon addition of galactose Cre recombinase linked to a nuclear localization sequence should be transcribed off of the Gal promoter, translated in the cytoplasm and then imported into the nucleus of the cell. There it recombines the plasmid between the two loxP sites. This brings the eCFP into a fusion with the secretion tag rather than the nuclear localization tag, so we should observe an eventual exclusion of CFP from the nucleus and secretion from the cell.</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Team
Team:NYU/Team
2010-10-28T02:41:22Z
<p>RussellDurrett: </p>
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<br />
=Students=<br />
{|align="center"<br />
|<br />
|'''[http://openwetware.org/wiki/User:Russell_Durrett Russell Durrett], the Man with the Plan'''<br />
|[[Image:NYU_Russsmall.jpg|100px|left|]]<br />
|-<br />
|<br />
|'''John Phair, the antibody authority'''<br />
|[[Image:NYU_John_Phairsmall.jpg|100px|left|]]<br />
|-<br />
|<br />
|'''Harris Kaplan, the lab dude'''<br />
|[[Image:NYU_Harris_Kaplansmall.jpg|100px|left|]]<br />
|-<br />
|<br />
|'''Sung won Lim, in a test tube'''<br />
|[[Image:Testtube2NYU.png|100px|left|]]<br />
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|<br />
|align="center"|<br />
|-<br />
|<br />
|}<br />
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=Advisor=<br />
{|align="center"<br />
|<br />
|'''David Gresham, master of yeast and generous lab space donor'''<br />
|[[Image:NYU_David_GreshamSmall.png|100px|left|]]<br />
|-<br />
|<br />
|align="center"|<br />
|-<br />
|<br />
|}</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Programming
Team:NYU/Programming
2010-10-28T02:32:16Z
<p>RussellDurrett: </p>
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[[Image:NYU_OverviewSideF_2.png|200px|right]]<br />
<br />
=====About this Page=====<br />
* Competent programming skills are quickly becoming a necessity for working in biology. Because most of our team members had never had exposure to a programming environment, we decided to use the obstacles presented in our iGEM experiments as cause for learning the Perl programming language and produced some simple scripts that could do routine iGEM labors for us. Here are some examples:<br />
<br />
<br />
=====Biobricking Primers=====<br />
* This script allows the user to input a DNA sequence and outputs the primers necessary for biobricking that sequence via PCR. The script calculates Tm but does not (yet) consider primer dimers. <br />
<br />
*Finally got the CGI working - click below or go [http://openwetware.org/images/a/a7/Biobrickprimers.txt here for the source code]<br />
<br />
<html><br />
<a href="http://russelldurrett.com/biobrickprimers.html"><img src="https://static.igem.org/mediawiki/2010/7/77/Biobricking_Primers_Logo.gif"/></a><br />
</html><br />
<br />
<br />
<br />
=====Overlap Oligo Maker=====<br />
<br />
* Some very fast and efficient DNA assembly methods require parts to overlap on the ends of their sequences. This script will take two DNA sequences and output the oligos (and their Tms) one would need to PCR constructs with overlapping ends. <br />
<br />
*I'm a CGI machine! [http://openwetware.org/images/6/67/Overlapoligos.txt Here is the source code.] or click below for an interactive version. <br />
<br />
<br />
<html><br />
<a href="http://www.russelldurrett.com/overlapoligos.html"><img src="https://static.igem.org/mediawiki/2010/5/5c/Overlaplogo.gif"/></a></html><br />
<br />
<br />
=====Manipulation of Protein Structures=====<br />
<br />
<br />
[[Image:Screen_shot_2010-10-27_at_10.19.59_PM.png|200px|left]]<br />
<br />
In order to more fully understand certain parts of our system and to use the ridiculously amazing CAVE system at Cornell Med School, we decided to explore the 3D structures of the proteins involved in our system. Because the mechanism of Cre recombination is difficult to understand, we dove into the structures available on the Protein DataBase (PDB) and explored details of the mechanism. We were able to locate the active sites of the protein, isolate the catalytic residues, locate the vanadium cofactors and, in all, gain a better understanding of the protein's function. Check out some pictures below!<br />
<br />
<br />
[Image:https://static.igem.org/mediawiki/igem.org/4/47/Cave_composite.png]</div>
RussellDurrett
http://2010.igem.org/File:Screen_shot_2010-10-27_at_10.19.59_PM.png
File:Screen shot 2010-10-27 at 10.19.59 PM.png
2010-10-28T02:22:26Z
<p>RussellDurrett: </p>
<hr />
<div></div>
RussellDurrett
http://2010.igem.org/File:Cre_Recombinase_GIF_2.gif
File:Cre Recombinase GIF 2.gif
2010-10-28T02:06:08Z
<p>RussellDurrett: </p>
<hr />
<div></div>
RussellDurrett
http://2010.igem.org/Team:NYU/Safety
Team:NYU/Safety
2010-10-28T01:43:18Z
<p>RussellDurrett: </p>
<hr />
<div>{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"<br />
!align="center"|[[Team:NYU|Home]]<br />
!align="center"|[[Team:NYU/Team|Team]]<br />
!align="center"|[[Team:NYU/Project|Project]]<br />
!align="center"|[[Team:NYU/Parts|BioBricks]]<br />
!align="center"|[[Team:NYU/Modeling|Modeling]]<br />
!align="center"|[[Team:NYU/Notebook|Notebook]]<br />
!align="center"|[[Team:NYU/Safety|Safety]]<br />
|}<br />
<br />
<br />
<br />
{|align="justify"<br />
|Safety considerations are very important when conducting any research with recombinant DNA technologies to control or prevent possible adverse outcomes of the research. When designing our project, we asked ourselves if our project ideas would raise safety issues in terms of researcher's safety, public safety or environmental safety. While it is not necessary to steer completely clear of research agendas that might pose safety risks because of their possible positive benefits, it is important to account for those risks by implementing safety protocols to protect the health of the researchers, public and environment. <br />
|[[Image:NYU_logo.png|200px|right]]<br />
|-<br />
|<br />
Because no organism or biobrick part we used is from an infectious organism, we use no human cell lines or cultures, no biological toxins or toxin coding regions and do not release our organisms into the environment for testing, the safety considerations of our project are less than the projects of many other teams. <br />
<br />
Our project is done in two phases. First, the construction of genetic constructs through biobrick parts in DH5alpha bacteria and Second, the transformation of the completed constructs into trp- FY4 yeast for intrabody library screening and production. While some strains of E. coli are more suitable to recombinant DNA work because of genomic deletions that make them less stable in the wild, the DH5alpha strain is important to our work because of its very high transformation efficiency. Because this strain is slightly more hardy, certain safety protocols have been instituted to prevent escape of biological agents into the environment: All petri plates deposited in biohazard waste collection which is incinerated before disposal, all liquid cultures are kept under 5 mL and are combined with 10% bleach and left for 30 minutes to kill all biological agents before disposal. The trp- FY4 strain of yeast is unable to survive in the wild due to its incapacity to synthesize tryptophan, but the same safety procedures are still used.<br />
<br />
The inherent danger of the genetic constructs in our system is minimal. The constructs entailing the most thought of biological safety are the gene expression control elements, the diversified iDab library constructs, which affect transcription of the yeast genome. Because these constructs are only active in the genome of the organism with Lex operators, it is very unlikely that even direct human exposure to yeast translating these proteins will have any effect on the cellular metabolism. Regardless, safety protocols for dealing with recombinant organisms are observed including gloves worn at all times in the laboratory and proper disposal of biological cultures as previously described.''</div>
RussellDurrett
http://2010.igem.org/Team:NYU/CornellMed
Team:NYU/CornellMed
2010-10-28T01:16:50Z
<p>RussellDurrett: /* Advisor */</p>
<hr />
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<br />
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<br />
[[Image:NYU_cavewall_v.png|150px|right]]<br />
<br />
{|align="center"<br />
|<br />
|''Over the summer and fall researchers at Cornell Medical School's Computational Biomedicine Department were also a part of the team. We used the department's 3D visualization room dubbed the CAVE to explore the structures of the proteins involved in our project and really get inside of our system, so to speak.''<br />
|-<br />
|}<br />
<br />
=Students=<br />
{|align="center"<br />
|<br />
|'''Yossi Steinberger, powerpoint animation connoisseur'''<br />
|[[Image:NYU_Yossele.png|100px|left|]]<br />
|-<br />
|<br />
|align="center"|<br />
|-<br />
|<br />
|}<br />
<br />
=Advisor=<br />
{|align="center"<br />
|<br />
|'''Chris Mason, champion of genetics and sequencing samurai, PI of the Mason lab'''<br />
|[[Image:ChrisMasonRNYU.jpg|100px|left|]]<br />
|-<br />
|<br />
|align="center"|<br />
|-<br />
|<br />
|}</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Programming
Team:NYU/Programming
2010-10-28T01:10:59Z
<p>RussellDurrett: /* Manipulation of Protein Structures */</p>
<hr />
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[[Image:NYU_OverviewSideF_2.png|200px|right]]<br />
<br />
=====About this Page=====<br />
* Competent programming skills are quickly becoming a necessity for working in biology. Because most of our team members had never had exposure to a programming environment, we decided to use the obstacles presented in our iGEM experiments as cause for learning the Perl programming language and produced some simple scripts that could do routine iGEM labors for us. Here are some examples:<br />
<br />
<br />
=====Biobricking Primers=====<br />
* This script allows the user to input a DNA sequence and outputs the primers necessary for biobricking that sequence via PCR. The script calculates Tm but does not (yet) consider primer dimers. <br />
<br />
*Finally got the CGI working - click below or go [http://openwetware.org/images/a/a7/Biobrickprimers.txt here for the source code]<br />
<br />
<html><br />
<a href="http://russelldurrett.com/biobrickprimers.html"><img src="https://static.igem.org/mediawiki/2010/7/77/Biobricking_Primers_Logo.gif"/></a><br />
</html><br />
<br />
<br />
<br />
=====Overlap Oligo Maker=====<br />
<br />
* Some very fast and efficient DNA assembly methods require parts to overlap on the ends of their sequences. This script will take two DNA sequences and output the oligos (and their Tms) one would need to PCR constructs with overlapping ends. <br />
<br />
*I'm a CGI machine! [http://openwetware.org/images/6/67/Overlapoligos.txt Here is the source code.] or click below for an interactive version. <br />
<br />
<br />
<html><br />
<a href="http://www.russelldurrett.com/overlapoligos.html"><img src="https://static.igem.org/mediawiki/2010/5/5c/Overlaplogo.gif"/></a></html><br />
<br />
<br />
=====Manipulation of Protein Structures=====<br />
<br />
*In order to more fully understand certain parts of our system and to use the ridiculously amazing CAVE system at Cornell Med School, we decided to explore the 3D structures of the proteins involved in our system. Because the mechanism of Cre recombination is difficult to understand, we dove into the structures available on the Protein DataBase (PDB) and explored details of the mechanism. We were able to locate the active sites of the protein, isolate the catalytic residues and vanadium cofactors and, in all, gain a better understanding of the protein's function.<br />
<br />
<br />
[Image:https://static.igem.org/mediawiki/igem.org/4/47/Cave_composite.png]</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Parts
Team:NYU/Parts
2010-10-28T01:09:37Z
<p>RussellDurrett: </p>
<hr />
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<br />
When choosing what simple parts to make for our project we had two main goals: 1) to improve the quality of parts already in the registry by making them more accessible to all users and 2) to augment the current set of yeast-compatible biobricks to harness the power of this synthetic biology-friendly model organism. Incorporating these goals with the 'Quality not Quantity' mantra, we decided to focus our efforts on certain areas of advancement that we believe future researchers will like to see in the registry.<br />
<br />
<br />
1. To improve parts already in the registry by lessening the constrictions on their use and making them more adaptable. <br />
<br />
[[Image:NYU_Cre.jpg|300px|right]]<br />
*'''36bp LoxP site''': the LoxP site is the naturally-occurring substrate for the Cre recombinase. In nature (and in previous biobricks) it is a palindromic sequence 34 base pairs long. Because this is not a multiple of three, when used inside a transcribed ORF the 34bp LoxP site can throw off the frame of the translated parts. To get around this, previous teams had to either add or take away bases from surrounding parts to keep their systems in frame (painstakingly and sometimes unsuccessfully). To get around this restriction, we designed and Biobricked a 36bp LoxP site that Registry users can include in transcribed regions without worrying about keeping everything in frame. <br />
<br />
*'''pGAL-RBS''': This promoter is induced by the presence of galactose and repressed by the presence of glucose. Because most future projects will involve fusing a Ribosome Binding Site to the promoter we decided to simplify things and submit this construct to the Registry. <br />
<br />
*'''pTEF-RBS''': This promoter is a medium-strength constitutive promoter. We submitted the part fused to the RBS.<br />
<br />
*'''lexOpCYC-RBS''': This pCYC promoter has a very limited amount of constitutive transcription. Just upstream is the lex operator to which the LexA DNA binding domain will bind. This can be useful for a number of constructs including our yeast two-hybrid screen. Again, we submitted this part with the RBS attached. <br />
<br />
<br />
2. Yeast strains are more rarely used in iGEM projects but contain certain parameters that are ideal for certain projects. Many teams are beginning to use yeast this year and are submitting useful biobricks, so we decided to make a collection of biobricks parts aimed at protein localization within the cell (known as 'yeast functional tags'):<br />
<br />
[[Image:NYU_Aga2-2.gif|200px|right]]<br />
*'''Aga2''': The Aga2 protein is screted from the yeast cell and attaches itself through disulfide bonds to the Aga1 protein on the cellular surface. If the Aga2 protein fused to a part of interest, the fusion is secreted, Aga2 complexes with Aga1 on the cellular surface, and the part of interest is displayed on the surface of the cell. While we did not end up using this part in our official construct, biobricking it from a plasmid we already had in our lab will surely benefit future teams.<br />
<br />
*'''Aga2+Linker''': Most everyone will want a linker attached to the Aga2 protein, so we went ahead and submitted this construct as well. COLLABORATION: We also sent this part along with the secretion tag to iGEM Osaka to aid them in the wetwork for their own project.<br />
<br />
*'''Secretion Tag''': Another 'yeast functional tag', the secretion tag does just what you think when fused to the C terminus of a protein of interest - directs secretion of the fused protein. Cleverly, this tag is cleaved from the fusion along the secretion pathway, resulting secretion of the protein of interest without the tag attached. <br />
<br />
*'''Ura3''': This is one of the proteins in the pathway for Uracil biosynthesis. It is incredibly useful as a selectable marker in yeast genetics because it can be selected for and against. We use it in our system as the nutritional marker in our yeast two-hybrid screen. <br />
<br />
<br />
3. To construct and submit certain ubiquitously useful biobricks that we would be using.<br />
<br />
*'''Flexible (Gly4Ser)3 peptide linker''': for use with most any peptide fusions<br />
<br />
<br />
4. To provide reporter constructs as positive controls of commonly used devices<br />
<br />
*'''pGal-Aga2-eCFP''': This reporter secretes Aga2:eCFP when the yeast is grown in galactose media. It is used as a positive control for the function of the surface display system. This construct was used by our team to test whether the Aga2 protein could direct secretion of a fusion and if the the Aga1 protein was present on the extracellular surface of our strain. <br />
<br />
*'''pGal-LexBD-NLS-VP16''':This construct should produce a nucleus-localized LexBD:VP16 fusion protein when grown in galactose media. This can be used as a positive control for any yeast two-hybrid system that uses the Lex operator and Lex binding domain. <br />
===Parts===<br />
<br />
New for iGEM 2010 is the ''groupparts'' tag. This tag will generate a table with all of the parts that your team adds to your team sandbox. Note that if you want to document a part you need to document it on the [http://partsregistry.org Registry], not on your team wiki.<br />
<br />
<groupparts>iGEM010 NYU</groupparts></div>
RussellDurrett
http://2010.igem.org/Team:NYU/Parts
Team:NYU/Parts
2010-10-27T23:45:18Z
<p>RussellDurrett: </p>
<hr />
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</html><br />
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<br />
<br />
<br />
<br />
When choosing what simple parts to make for our project we had two main goals: 1) to improve the quality of parts already in the registry by making them more accessible to all users and 2) to augment the current set of yeast-compatible biobricks to harness the power of this synthetic biology-friendly model organism. Incorporating these goals with the 'Quality not Quantity' mantra, we decided to focus our efforts on certain areas of advancement that we believe future researchers will like to see in the registry.<br />
<br />
<br />
1. To improve parts already in the registry by lessening the constrictions on their use and making them more adaptable. <br />
<br />
[[Image:NYU_Cre.jpg|300px|right]]<br />
*'''36bp LoxP site''': the LoxP site is the naturally-occurring substrate for the Cre recombinase. In nature (and in previous biobricks) it is a palindromic sequence 34 base pairs long. Because this is not a multiple of three, when used inside a transcribed ORF the 34bp LoxP site can throw off the frame of the translated parts. To get around this, previous teams had to either add or take away bases from surrounding parts to keep their systems in frame (painstakingly and sometimes unsuccessfully). To get around this restriction, we designed and Biobricked a 36bp LoxP site that Registry users can include in transcribed regions without worrying about keeping everything in frame. <br />
<br />
*'''pGAL-RBS''': This promoter is induced by the presence of galactose and repressed by the presence of glucose. Because most future projects will involve fusing a Ribosome Binding Site to the promoter we decided to simplify things and submit this construct to the Registry. <br />
<br />
*'''pTEF-RBS''': This promoter is a medium-strength constitutive promoter. We submitted the part fused to the RBS.<br />
<br />
*'''lexOpCYC-RBS''': This pCYC promoter has a very limited amount of constitutive transcription. Just upstream is the lex operator to which the LexA DNA binding domain will bind. This can be useful for a number of constructs including our yeast two-hybrid screen. Again, we submitted this part with the RBS attached. <br />
<br />
<br />
2. Yeast strains are more rarely used in iGEM projects but contain certain parameters that are ideal for certain projects. Many teams are beginning to use yeast this year and are submitting useful biobricks, so we decided to make a collection of biobricks parts aimed at protein localization within the cell (known as 'yeast functional tags'):<br />
<br />
[[Image:NYU_Aga2-2.gif|200px|right]]<br />
*'''Aga2''': The Aga2 protein is screted from the yeast cell and attaches itself through disulfide bonds to the Aga1 protein on the cellular surface. If the Aga2 protein fused to a part of interest, the fusion is secreted, Aga2 complexes with Aga1 on the cellular surface, and the part of interest is displayed on the surface of the cell. While we did not end up using this part in our official construct, biobricking it from a plasmid we already had in our lab will surely benefit future teams.<br />
<br />
*'''Aga2+Linker''': Most everyone will want a linker attached to the Aga2 protein, so we went ahead and submitted this construct as well. COLLABORATION: We also sent this part along with the secretion tag to iGEM Osaka to aid them in the wetwork for their own project.<br />
<br />
*'''Secretion Tag''': Another 'yeast functional tag', the secretion tag does just what you think when fused to the C terminus of a protein of interest - directs secretion of the fused protein. Cleverly, this tag is cleaved from the fusion along the secretion pathway, resulting secretion of the protein of interest without the tag attached. <br />
<br />
*'''Ura3''': This is one of the proteins in the pathway for Uracil biosynthesis. It is incredibly useful as a selectable marker in yeast genetics because it can be selected for and against. We use it in our system as the nutritional marker in our yeast two-hybrid screen. <br />
<br />
<br />
3. To construct and submit certain ubiquitously useful biobricks that we would be using.<br />
<br />
*'''Flexible (Gly4Ser)3 peptide linker''': for use with most any peptide fusions<br />
<br />
<br />
===Parts===<br />
<br />
New for iGEM 2010 is the ''groupparts'' tag. This tag will generate a table with all of the parts that your team adds to your team sandbox. Note that if you want to document a part you need to document it on the [http://partsregistry.org Registry], not on your team wiki.<br />
<br />
<groupparts>iGEM010 NYU</groupparts></div>
RussellDurrett
http://2010.igem.org/Team:NYU/Programming
Team:NYU/Programming
2010-10-27T20:55:25Z
<p>RussellDurrett: </p>
<hr />
<div><html><br />
<br />
<div id="allcontent"><br />
<img class="bannerimage" src="https://static.igem.org/mediawiki/2010/c/ce/NYU_cavebanner_1.png" height="200px" width="950px"><br /><br />
<br />
<br />
<div id="headerlinks"><br />
<ul id="nav"><br />
<li><br />
<a class="bannertoplinks" href="https://2010.igem.org/Team:NYU">Home</a><br />
</li><br />
<br />
<li><a class="bannertoplinks" href="https://2010.igem.org/Team:NYU/Project">Project</a><br />
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<li><a class="bannerlinks" href="https://2010.igem.org/Team:NYU/Project">Overview</a></li><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:NYU/Experiments">Experiments</a></li><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:NYU/Assembly">Overlap Assembly</a></li><br />
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<li><a class="bannerlinks" href="http://www.nysynbio.org">NY synbio</a></li><br />
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<li><a class="bannertoplinks" href="https://2010.igem.org/Team:NYU/Contact">Contact Us</a><br />
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[[Image:NYU_OverviewSideF_2.png|200px|right]]<br />
<br />
=====About this Page=====<br />
* Competent programming skills are quickly becoming a necessity for working in biology. Because most of our team members had never had exposure to a programming environment, we decided to use the obstacles presented in our iGEM experiments as cause for learning the Perl programming language and produced some simple scripts that could do routine iGEM labors for us. Here are some examples:<br />
<br />
<br />
=====Biobricking Primers=====<br />
* This script allows the user to input a DNA sequence and outputs the primers necessary for biobricking that sequence via PCR. The script calculates Tm but does not (yet) consider primer dimers. <br />
<br />
*Finally got the CGI working - click below or go [http://openwetware.org/images/a/a7/Biobrickprimers.txt here for the source code]<br />
<br />
<html><br />
<a href="http://russelldurrett.com/biobrickprimers.html"><img src="https://static.igem.org/mediawiki/2010/7/77/Biobricking_Primers_Logo.gif"/></a><br />
</html><br />
<br />
<br />
<br />
=====Overlap Oligo Maker=====<br />
<br />
* Some very fast and efficient DNA assembly methods require parts to overlap on the ends of their sequences. This script will take two DNA sequences and output the oligos (and their Tms) one would need to PCR constructs with overlapping ends. <br />
<br />
*I'm a CGI machine! [http://openwetware.org/images/6/67/Overlapoligos.txt Here is the source code.] or click below for an interactive version. <br />
<br />
<br />
<html><br />
<a href="http://www.russelldurrett.com/overlapoligos.html"><img src="https://static.igem.org/mediawiki/2010/5/5c/Overlaplogo.gif"/></a></html><br />
<br />
<br />
=====Manipulation of Protein Structures=====<br />
<br />
*In order to more fully understand certain parts of our system and to use the ridiculously amazing CAVE system at Cornell Med School, we decided to explore the 3D structures of the proteins involved in our system. Because the mechanism of Cre recombination is difficult to understand, we dove into the structures available on the Protein DataBase (PDB) and explored details of the mechanism. We were able to locate the active sites of the protein, isolate the catalytic residues and Vanadium cofactors and, in all, gain a better understanding of the protein's function.<br />
<br />
<br />
[Image:https://static.igem.org/mediawiki/igem.org/4/47/Cave_composite.png]</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Programming
Team:NYU/Programming
2010-10-27T20:38:51Z
<p>RussellDurrett: </p>
<hr />
<div><html><br />
<br />
<div id="allcontent"><br />
<img class="bannerimage" src="https://static.igem.org/mediawiki/2010/c/ce/NYU_cavebanner_1.png" height="200px" width="950px"><br /><br />
<br />
<br />
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<li><br />
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<li><a class="bannerlinks" href="https://2010.igem.org/Team:NYU/Assembly">Overlap Assembly</a></li><br />
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</ul> <br />
</li> <br />
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font-family: Helvetica, Arial, sans-serif;<br />
width: 965px;<br />
line-height: 1.2em;<br />
<br />
}<br />
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a {<br />
text-decoration: none;<br />
color: #7F5217;<br />
}<br />
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color: #A0C544;<br />
text-decoration: none;<br />
}<br />
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h1, h2, h3, h4, h5, h6 {<br />
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margin-left: -20px;<br />
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list-style: none;<br />
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</html><br />
<br />
[[Image:NYU_OverviewSideF_2.png|200px|right]]<br />
<br />
=====About this Page=====<br />
* Competent programming skills are quickly becoming a necessity for working in biology. Because most of our team members had never had exposure to a programming environment, we decided to use the obstacles presented in our iGEM experiments as cause for learning the Perl programming language and produced some simple scripts that could do routine iGEM labors for us. Here are some examples:<br />
<br />
<br />
=====Biobricking Primers=====<br />
* This script allows the user to input a DNA sequence and outputs the primers necessary for biobricking that sequence via PCR. The script calculates Tm but does not (yet) consider primer dimers. <br />
<br />
*Finally got the CGI working - click below or go [http://openwetware.org/images/a/a7/Biobrickprimers.txt here for the source code]<br />
<br />
<html><br />
<a href="http://russelldurrett.com/biobrickprimers.html"><img src="https://static.igem.org/mediawiki/2010/7/77/Biobricking_Primers_Logo.gif"/></a><br />
</html><br />
<br />
<br />
<br />
=====Overlap Oligo Maker=====<br />
<br />
* Some very fast and efficient DNA assembly methods require parts to overlap on the ends of their sequences. This script will take two DNA sequences and output the oligos (and their Tms) one would need to PCR constructs with overlapping ends. <br />
<br />
*I'm a CGI machine! [http://openwetware.org/images/6/67/Overlapoligos.txt Here is the source code.] or click below for an interactive version. <br />
<br />
<br />
<html><br />
<a href="http://www.russelldurrett.com/overlapoligos.html"><img src="https://static.igem.org/mediawiki/2010/5/5c/Overlaplogo.gif"/></a></html><br />
<br />
<br />
=====Programming of Crystal Structures=====<br />
<br />
<br />
<br />
[Image:https://static.igem.org/mediawiki/igem.org/4/47/Cave_composite.png]</div>
RussellDurrett
http://2010.igem.org/File:CAVE1.JPG
File:CAVE1.JPG
2010-10-27T19:58:27Z
<p>RussellDurrett: </p>
<hr />
<div></div>
RussellDurrett
http://2010.igem.org/Team:NYU/Project
Team:NYU/Project
2010-10-27T09:29:01Z
<p>RussellDurrett: </p>
<hr />
<div><html><br />
<br />
<div id="allcontent"><br />
<img class="bannerimage" src="https://static.igem.org/mediawiki/2010/2/2d/NYU_bannerv_6.png" height="200px" width="950px"><br /><br />
<br />
<br />
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<ul id="nav"><br />
<li><br />
<a class="bannertoplinks" href="https://2010.igem.org/Team:NYU">Home</a><br />
</li><br />
<br />
<li><a class="bannertoplinks" href="https://2010.igem.org/Team:NYU/Project">Project</a><br />
<ul style="z-index:1"><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:NYU/Project">Overview</a></li><br />
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<br />
[[Image:NYU_OverviewSideF_1.png|200px|right]]<br />
<br />
<br />
[[Image:NYU_logo.png|220px|left]]<br />
<br />
<br />
Our goal is to use the tools of synthetic biology to make a biological machine capable of antibody discovery. To do this, we have decided to learn from our own immune system by porting its own antibody discovery strategies into an engineered strain of yeast. This yeast will be capable of screening a library of antibodies against a target antigen, recombining the antibody gene ''in vivo'' and then either secreting or surface displaying the resulting antibody protein for a variety of purposes. Our hope is to demonstrate the feasibility of using the yeast cell not only as a vessel for antibody discovery but as a streamlined processing unit that can discover and also begin production of new antibodies - all in one test tube!<br />
<br />
<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
<br />
Our plan for this year's competition is to construct an easy-to-use yeast strain capable of intracellular antibody discovery. The current dominant mode of microbial antibody discovery requires cellular surface display and high-throughput fluorescent screening. Instead of using this method, we wanted our cells to be able to sense if the antibody they are translating will bind the target antigen. <br />
<br />
<br />
<br />
[[Image:NYU_bind3.png|180px|left]]<br />
<br />
<br />
Using a modified form of the yeast two-hybrid screening system, the yeast cells are able to sense the amount of antibody::antigen interaction inside the cell. When the antibody binds the target antigen it anchors the VP16 transcriptional activator near our response system. VP16 then increases the level of transcription of a reporter gene. If this gene encodes a nutritional marker, such as Ura3, only cells experiencing antibody:antigen interactions can be selected using selective media. Using this method the original population of our yeast strain will be able to, in essence, screen itself and our resulting yeast population will contain only antibodies that bind our antigen.<br />
<br />
<br />
<br />
<br />
<br />
In a research setting, once you have discovered the antibody you wish to use you must then reclone the gene for it into a secretion vector for production of pure antibody protein. This involves taking the plasmid out of the yeast cell, excising the antibody gene, ligating it into the second vector and then transforming it back into a suitable yeast strain.<br />
<br />
<br />
[[Image:NYU_Cre.jpg|350px|left]]<br />
To make this process faster and more easily accomplished, we have incorporated a recombination-based architecture into our system that will allow the cells to modify the antibody plasmid ''in vivo''. Using this system transcription of the antibody gene can go straight from screening to protein production in a matter of minutes. This concept has the capability to not only shave weeks off of current antibody discovery protocols, but opens the door to cellular programming for other methods, such as restructuring of antibody genes or performing more complex screenings for different purposes.<br />
<br />
<br />
<br />
<br />
== The Experiments ==<br />
<br />
<br />
In the spectrum of molecular biology, iGEM is a very short time frame. Because of this short time frame, our wet lab team having only two members and our extremely limited research budget, we felt that radical simplification of our experiments would allow us to demonstrate the feasibility of our ideas without requiring resources we simply did not have. Because many of the individual aspects that this project has brought together have been investigated with success, we were able to perform some experiments using proxies for the 'real thing'.<br />
<br />
Please click [https://2010.igem.org/Team:NYU/Experiments Here] to see how we simulated each of the systems.</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Project
Team:NYU/Project
2010-10-27T09:21:08Z
<p>RussellDurrett: /* Project Details */</p>
<hr />
<div><html><br />
<br />
<div id="allcontent"><br />
<img class="bannerimage" src="https://static.igem.org/mediawiki/2010/2/2d/NYU_bannerv_6.png" height="200px" width="950px"><br /><br />
<br />
<br />
<div id="headerlinks"><br />
<ul id="nav"><br />
<li><br />
<a class="bannertoplinks" href="https://2010.igem.org/Team:NYU">Home</a><br />
</li><br />
<br />
<li><a class="bannertoplinks" href="https://2010.igem.org/Team:NYU/Project">Project</a><br />
<ul style="z-index:1"><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:NYU/Project">Overview</a></li><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:NYU/Experiments">Experiments</a></li><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:NYU/Assembly">Overlap Assembly</a></li><br />
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<br />
<br />
[[Image:NYU_logo.png|220px|left]]<br />
<br />
Our goal is to use the tools of synthetic biology to make a biological machine capable of antibody discovery. To do this, we have decided to learn from our own immune system by porting its own antibody discovery strategies into an engineered strain of yeast. This yeast will be capable of screening a library of antibodies against a target antigen, recombining the antibody gene ''in vivo'' and then either secreting or surface displaying the resulting antibody protein for a variety of purposes. Our hope is to demonstrate the feasibility of using the yeast cell not only as a vessel for antibody discovery but as a streamlined processing unit that can discover and also begin production of new antibodies - all in one test tube!<br />
<br />
<br />
<br />
== Project Details==<br />
<br />
<br />
[[Image:NYU_bind3.png|260px|right]] <br />
Our plan for this year's competition is to construct an easy-to-use yeast strain capable of intracellular antibody discovery. The current dominant mode of microbial antibody discovery requires cellular surface display and high-throughput fluorescent screening. Instead of using this method, we wanted our cells to be able to sense if the antibody they are translating will bind the target antigen. Using a modified form of the yeast two-hybrid screening system, the yeast cells are able to sense the amount of antibody::antigen interaction inside the cell. When the antibody binds the target antigen it anchors the VP16 transcriptional activator near our response system. VP16 then increases the level of transcription of a reporter gene. If this gene encodes a nutritional marker, such as Ura3, only cells experiencing antibody:antigen interactions can be selected using selective media. Using this method the original population of our yeast strain will be able to, in essence, screen itself and our resulting yeast population will contain only antibodies that bind our antigen.<br />
<br />
<br />
<br />
<br />
In a research setting, once you have discovered the antibody you wish to use you must then reclone the gene for it into a secretion vector for production of pure antibody protein. This involves taking the plasmid out of the yeast cell, excising the antibody gene, ligating it into the second vector and then transforming it back into a suitable yeast strain.<br />
[[Image:NYU_Cre.jpg|350px|left]]<br />
To make this process faster and more easily accomplished, we have incorporated a recombination-based architecture into our system that will allow the cells to modify the antibody plasmid ''in vivo''. Using this system transcription of the antibody gene can go straight from screening to protein production in a matter of minutes. This concept has the capability to not only shave weeks off of current antibody discovery protocols, but opens the door to cellular programming for other methods, such as restructuring of antibody genes or performing more complex screenings for different purposes.<br />
<br />
== The Experiments ==<br />
<br />
<br />
In the spectrum of molecular biology, iGEM is a very short time frame. Because of this short time frame, our wet lab team having only two members and our extremely limited research budget, we felt that radical simplification of our experiments would allow us to demonstrate the feasibility of our ideas without requiring resources we simply did not have. Because many of the individual aspects that this project has brought together have been investigated with success, we were able to perform some experiments using proxies for the 'real thing'.<br />
<br />
Please click [https://2010.igem.org/Team:NYU/Experiments Here] to see how we simulated each of the systems.</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Project
Team:NYU/Project
2010-10-27T09:03:43Z
<p>RussellDurrett: </p>
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<br />
[[Image:NYU_OverviewSideF_1.png|200px|right]]<br />
<br />
<br />
[[Image:NYU_logo.png|220px|left]]<br />
<br />
Our goal is to use the tools of synthetic biology to make a biological machine capable of antibody discovery. To do this, we have decided to learn from our own immune system by porting its own antibody discovery strategies into an engineered strain of yeast. This yeast will be capable of screening a library of antibodies against a target antigen, recombining the antibody gene ''in vivo'' and then either secreting or surface displaying the resulting antibody protein for a variety of purposes. Our hope is to demonstrate the feasibility of using the yeast cell not only as a vessel for antibody discovery but as a streamlined processing unit that can discover and also begin production of new antibodies - all in one test tube!<br />
<br />
<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
Our game plan for this year's competition is to construct an easy-to-use yeast strain capable of intracellular antibody discovery. The current dominant mode of microbial antibody discovery requires cellular surface display and high-throughput fluorescent screening. Instead of using this method, we wanted our cells to be able to sense if the antibody they are translating will bind the target antigen. Using a modified form of the yeast two-hybrid screening system, the yeast cells will sense the amount of antibody::antigen interaction because it will increase the level of transcription of a reporter gene. If this gene encodes a nutritional marker, such as Ura3, only cells experiencing antibody:antigen interactions will grow in selective media (i.e. without the nutrient such as Uracil). Using this method the original population of our yeast strain will be able to, in essence, screen itself and our resulting yeast population will contain only antibodies that bind our antigen.<br />
<br />
<br />
In a research setting, once you have discovered the antibody you wish to use you must then reclone the gene for it into a secretion vector for production of pure antibody protein. This involves taking the plasmid out of the yeast cell, excising the antibody gene, ligating it into the second vector and then transforming it back into a suitable yeast strain.<br />
[[Image:NYU_Cre.jpg|350px|left]]<br />
To make this process faster and more easily accomplished, we have incorporated a recombination-based architecture into our system that will allow the cells to modify the antibody plasmid ''in vivo''. Using this system transcription of the antibody gene can go straight from screening to protein production in a matter of minutes. This concept has the capability to not only shave weeks off of current antibody discovery protocols, but opens the door to cellular programming for other methods, such as restructuring of antibody genes or performing more complex screenings for different purposes.<br />
<br />
<br />
== The Experiments ==<br />
<br />
<br />
In the spectrum of molecular biology, iGEM is a very short time frame. Because of this short time frame, our wet lab team having only two members and our extremely limited research budget, we felt that radical simplification of our experiments would allow us to demonstrate the feasibility of our ideas without requiring resources we simply did not have. Because many of the individual aspects that this project has brought together have been investigated with success, we were able to perform some experiments using proxies for the 'real thing'.<br />
<br />
Please click [https://2010.igem.org/Team:NYU/Experiments Here] to see how we simulated each of the systems.</div>
RussellDurrett
http://2010.igem.org/Team:NYU/CornellMed
Team:NYU/CornellMed
2010-10-27T09:02:53Z
<p>RussellDurrett: </p>
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{|align="center"<br />
|<br />
|''Over the summer and fall researchers at Cornell Medical School's Computational Biomedicine Department were also a part of the team. We used the department's 3D visualization room dubbed the CAVE to explore the structures of the proteins involved in our project and really get inside of our system, so to speak.''<br />
|-<br />
|}<br />
<br />
=Students=<br />
{|align="center"<br />
|<br />
|'''Yossi Steinberger, powerpoint animation connoisseur'''<br />
|[[Image:NYU_Yossele.png|100px|left|]]<br />
|-<br />
|<br />
|align="center"|<br />
|-<br />
|<br />
|}<br />
<br />
=Advisor=<br />
{|align="center"<br />
|<br />
|'''Chris Mason, bionformatics guru and PI of the Mason lab'''<br />
|[[Image:ChrisMasonRNYU.jpg|100px|left|]]<br />
|-<br />
|<br />
|align="center"|<br />
|-<br />
|<br />
|}</div>
RussellDurrett
http://2010.igem.org/Team:NYU/CornellMed
Team:NYU/CornellMed
2010-10-27T09:01:25Z
<p>RussellDurrett: /* Advisor */</p>
<hr />
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<br />
</html><br />
<br />
[[Image:NYU_cavewall_v.png|150px|right]]<br />
<br />
{|align="center"<br />
|<br />
|''Researchers at Cornell Medical School's Computational Biomedicine Department were also a part of the team. We decided to use the department's 3D visualization room dubbed the CAVE to explore the structures of the proteins involved in our project and make some 3D animations of the various processes.''<br />
|-<br />
|}<br />
<br />
=Students=<br />
{|align="center"<br />
|<br />
|'''Yossi Steinberger, powerpoint animation connoisseur'''<br />
|[[Image:NYU_Yossele.png|100px|left|]]<br />
|-<br />
|<br />
|align="center"|<br />
|-<br />
|<br />
|}<br />
<br />
=Advisor=<br />
{|align="center"<br />
|<br />
|'''Chris Mason, bionformatics guru and PI of the Mason lab'''<br />
|[[Image:ChrisMasonRNYU.jpg|100px|left|]]<br />
|-<br />
|<br />
|align="center"|<br />
|-<br />
|<br />
|}</div>
RussellDurrett
http://2010.igem.org/Team:NYU/CornellMed
Team:NYU/CornellMed
2010-10-27T09:00:23Z
<p>RussellDurrett: /* Students */</p>
<hr />
<div><html><br />
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<div id="allcontent"><br />
<img class="bannerimage" src="https://static.igem.org/mediawiki/2010/c/ce/NYU_cavebanner_1.png" height="200px" width="950px"><br /><br />
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</style><br />
<!-- !END NYU CSS- Thanks Harvard --><br />
<br />
</html><br />
<br />
[[Image:NYU_cavewall_v.png|150px|right]]<br />
<br />
{|align="center"<br />
|<br />
|''Researchers at Cornell Medical School's Computational Biomedicine Department were also a part of the team. We decided to use the department's 3D visualization room dubbed the CAVE to explore the structures of the proteins involved in our project and make some 3D animations of the various processes.''<br />
|-<br />
|}<br />
<br />
=Students=<br />
{|align="center"<br />
|<br />
|'''Yossi Steinberger, powerpoint animation connoisseur'''<br />
|[[Image:NYU_Yossele.png|100px|left|]]<br />
|-<br />
|<br />
|align="center"|<br />
|-<br />
|<br />
|}<br />
<br />
=Advisor=<br />
{|align="center"<br />
|<br />
|'''Chris Mason, from the Mason lab'''<br />
|[[Image:ChrisMasonRNYU.jpg|100px|left|]]<br />
|-<br />
|<br />
|align="center"|<br />
|-<br />
|<br />
|}</div>
RussellDurrett
http://2010.igem.org/Team:NYU/CornellMed
Team:NYU/CornellMed
2010-10-27T08:59:53Z
<p>RussellDurrett: /* Adviser */</p>
<hr />
<div><html><br />
<br />
<div id="allcontent"><br />
<img class="bannerimage" src="https://static.igem.org/mediawiki/2010/c/ce/NYU_cavebanner_1.png" height="200px" width="950px"><br /><br />
<br />
<br />
<div id="headerlinks"><br />
<ul id="nav"><br />
<li><br />
<a class="bannertoplinks" href="https://2010.igem.org/Team:NYU">Home</a><br />
</li><br />
<br />
<li><a class="bannertoplinks" href="https://2010.igem.org/Team:NYU/Project">Project</a><br />
<ul style="z-index:1"><br />
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<li><a class="bannerlinks" href="https://2010.igem.org/Team:NYU/Experiments">Experiments</a></li><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:NYU/Assembly">Overlap Assembly</a></li><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:NYU/Notebook">Notebook</a></li><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:NYU/Parts">Biobricks</a></li><br />
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<li><a class="bannerlinks" href="http://www.sciencehouse.com">ScienceHouse</a></li><br />
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<br />
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<br />
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#allcontent {<br />
width: 950px;<br />
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<br />
<br />
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<br />
</html><br />
<br />
[[Image:NYU_cavewall_v.png|150px|right]]<br />
<br />
{|align="center"<br />
|<br />
|''Researchers at Cornell Medical School's Computational Biomedicine Department were also a part of the team. We decided to use the department's 3D visualization room dubbed the CAVE to explore the structures of the proteins involved in our project and make some 3D animations of the various processes.''<br />
|-<br />
|}<br />
<br />
=Students=<br />
{|align="center"<br />
|<br />
|''' CAVE '''<br />
|[[Image:NYU_Yossele.png|100px|left|]]<br />
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|<br />
|align="center"|<br />
|-<br />
|<br />
|}<br />
<br />
=Advisor=<br />
{|align="center"<br />
|<br />
|'''Chris Mason, from the Mason lab'''<br />
|[[Image:ChrisMasonRNYU.jpg|100px|left|]]<br />
|-<br />
|<br />
|align="center"|<br />
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|<br />
|}</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Team
Team:NYU/Team
2010-10-27T08:58:20Z
<p>RussellDurrett: /* Adviser */</p>
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<br />
=Students=<br />
{|align="center"<br />
|<br />
|'''[http://openwetware.org/wiki/User:Russell_Durrett Russell Durrett], the Man with the Plan'''<br />
|[[Image:NYU_Russsmall.jpg|100px|left|]]<br />
|-<br />
|<br />
|'''John Phair, the antibody authority'''<br />
|[[Image:NYU_John_Phairsmall.jpg|100px|left|]]<br />
|-<br />
|<br />
|'''Harris Kaplan, the lab dude'''<br />
|[[Image:NYU_Harris_Kaplansmall.jpg|100px|left|]]<br />
|-<br />
|<br />
|'''Sung won Lim, in a test tube'''<br />
|[[Image:Testtube2NYU.png|100px|left|]]<br />
|-<br />
|<br />
|align="center"|<br />
|-<br />
|<br />
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=Advisor=<br />
{|align="center"<br />
|<br />
|'''David Gresham, gracious lab space donor'''<br />
|[[Image:NYU_David_GreshamSmall.png|100px|left|]]<br />
|-<br />
|<br />
|align="center"|<br />
|-<br />
|<br />
|}</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Team
Team:NYU/Team
2010-10-27T08:58:00Z
<p>RussellDurrett: /* Students */</p>
<hr />
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<br />
=Students=<br />
{|align="center"<br />
|<br />
|'''[http://openwetware.org/wiki/User:Russell_Durrett Russell Durrett], the Man with the Plan'''<br />
|[[Image:NYU_Russsmall.jpg|100px|left|]]<br />
|-<br />
|<br />
|'''John Phair, the antibody authority'''<br />
|[[Image:NYU_John_Phairsmall.jpg|100px|left|]]<br />
|-<br />
|<br />
|'''Harris Kaplan, the lab dude'''<br />
|[[Image:NYU_Harris_Kaplansmall.jpg|100px|left|]]<br />
|-<br />
|<br />
|'''Sung won Lim, in a test tube'''<br />
|[[Image:Testtube2NYU.png|100px|left|]]<br />
|-<br />
|<br />
|align="center"|<br />
|-<br />
|<br />
|}<br />
<br />
=Adviser=<br />
{|align="center"<br />
|<br />
|'''David Gresham, gracious lab space donor'''<br />
|[[Image:NYU_David_GreshamSmall.png|100px|left|]]<br />
|-<br />
|<br />
|align="center"|<br />
|-<br />
|<br />
|}</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Parts
Team:NYU/Parts
2010-10-27T08:53:58Z
<p>RussellDurrett: </p>
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<br />
When choosing what simple parts to make for our project we had two main goals: 1) to improve the quality of parts already in the registry by making them more accessible to all users and 2) to augment the current set of yeast-compatible biobricks to harness the power of this synthetic biology-friendly model organism. Incorporating these goals with the 'Quality not Quantity' mantra, we decided to focus our efforts on certain areas of advancement that we believe future researchers will like to see in the registry.<br />
<br />
<br />
1. To improve parts already in the registry by lessening the constrictions on their use and making them more adaptable. <br />
<br />
[[Image:NYU_Cre.jpg|300px|right]]<br />
*'''36bp LoxP site''': the LoxP site is the naturally-occurring substrate for the Cre recombinase. In nature (and in previous biobricks) it is a palindromic sequence 34 base pairs long. Because this is not a multiple of three, when used inside a transcribed ORF the 34bp LoxP site can throw off the frame of the translated parts. To get around this, previous teams had to either add or take away bases from surrounding parts to keep their systems in frame (painstakingly and sometimes unsuccessfully). To get around this restriction, we designed and Biobricked a 36bp LoxP site that Registry users can include in transcribed regions without worrying about keeping everything in frame. <br />
<br />
*'''pGAL-RBS''': This promoter is induced by the presence of galactose and repressed by the presence of glucose. Because most future projects will involve fusing a Ribosome Binding Site to the promoter we decided to simplify things and submit this construct to the Registry. <br />
<br />
*'''pTEF-RBS''': This promoter is a medium-strength constitutive promoter. We submitted the part fused to the RBS.<br />
<br />
*'''lexOpCYC-RBS''': This pCYC promoter has a very limited amount of constitutive transcription. Just upstream is the lex operator to which the LexA DNA binding domain will bind. This can be useful for a number of constructs including our yeast two-hybrid screen. Again, we submitted this part with the RBS attached. <br />
<br />
<br />
2. Yeast strains are more rarely used in iGEM projects but contain certain parameters that are ideal for certain projects. Many teams are beginning to use yeast this year and are submitting useful biobricks, so we decided to make a collection of biobricks parts aimed at protein localization within the cell (known as 'yeast functional tags'):<br />
<br />
[[Image:NYU_Aga2-2.gif|200px|right]]<br />
*'''Aga2''': The Aga2 protein is screted from the yeast cell and attaches itself through disulfide bonds to the Aga1 protein on the cellular surface. If the Aga2 protein fused to a part of interest, the fusion is secreted, Aga2 complexes with Aga1 on the cellular surface, and the part of interest is displayed on the surface of the cell. While we did not end up using this part in our official construct, biobricking it from a plasmid we already had in our lab will surely benefit future teams.<br />
<br />
*'''Aga2+Linker''': Most everyone will want a linker attached to the Aga2 protein, so we went ahead and submitted this construct as well. <br />
<br />
*'''Secretion Tag''': Another 'yeast functional tag', the secretion tag does just what you think when fused to the C terminus of a protein of interest - directs secretion of the fused protein. Cleverly, this tag is cleaved from the fusion along the secretion pathway, resulting secretion of the protein of interest without the tag attached. <br />
<br />
*'''Ura3''': This is one of the proteins in the pathway for Uracil biosynthesis. It is incredibly useful as a selectable marker in yeast genetics because it can be selected for and against. We use it in our system as the nutritional marker in our yeast two-hybrid screen. <br />
<br />
<br />
3. To construct and submit certain ubiquitously useful biobricks that we would be using.<br />
<br />
*'''Flexible (Gly4Ser)3 peptide linker''': for use with most any peptide fusions<br />
<br />
<br />
===Parts===<br />
<br />
New for iGEM 2010 is the ''groupparts'' tag. This tag will generate a table with all of the parts that your team adds to your team sandbox. Note that if you want to document a part you need to document it on the [http://partsregistry.org Registry], not on your team wiki.<br />
<br />
<groupparts>iGEM010 NYU</groupparts></div>
RussellDurrett
http://2010.igem.org/Team:NYU/Assembly
Team:NYU/Assembly
2010-10-27T05:51:30Z
<p>RussellDurrett: </p>
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<!-- !END NYU HYBRID CSS- Thanks Harvard --><br />
<br />
</html><br />
<br />
[[Image:NYU_OverviewSideF_3.png|200px|right]]<br />
<br />
While the 3A assembly method is well suited for stepwise building of devices, larger complex constructs can take time to make. Being the impatient New Yorkers we are, as soon as we came up with the constructs for our project we had to have them immediately. Because of this, we began exploring other methods to piece together our system. <br />
<br />
We decided to use the Overlap Assembly Method most famously published in this JCVI paper: <br />
<br />
<br />
[http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1318.html Gibson et al, Nature Methods 6: 343-345]<br />
<br />
<br />
This method allows researchers to assemble complex systems of many parts all at once rather than in a stepwise fashion. By combining your overlapping DNA parts with the required enzymes and buffers you can easily assemble a complete system in one day - perfect for the impatient New Yorkers that we are. Here's the general scheme of this reaction:<br />
<br />
[[Image:NYU_Overlapassembly.jpg|300px|center]]<br />
<br />
<br />
<br />
The downside of this system is that the parts to be assembled must overlap one another in their sequence, so you must order DNA oligos to PCR your parts into an overlapping form. To help us out with this, Russell programmed an overlap oligo maker that did much of the work for us - check it out by clicking this link:<br />
<br />
<html><br />
<a href="http://www.russelldurrett.com/overlapoligos.html"><img src="https://static.igem.org/mediawiki/2010/5/5c/Overlaplogo.gif" /></a></html><br />
<br />
After getting the oligos in the lab we combined equimolar amounts of each part in a single reaction and added ExoIII, Phusion polymerase and Taq Ligase. We opted for a 2-step thermocycled reaction method for simplicity and cost-effectiveness and the relative ease of making a reaction mix. Using the reaction mix suggested in the Gibson et. al. paper, Russell designed a simplified version by using the Phusion and Ligase prepared buffers. We were able to get successful reactions from this simple mix:<br />
<br />
50uL Assembly Reaction:<br />
<br />
Equimolar amounts of overlapping DNA (~100ng) from PCR cleanup - up to 18uL<br />
+<br />
10uL 5x Phusion HF Polymerase Buffer<br />
5uL 10x Taq Ligase Buffer<br />
8uL 50mM MgCl2<br />
4uL 2.5mM dNTPs<br />
+<br />
.5uL ExoIII diluted to 10 U/uL<br />
2uL Phusion HF Polymerase (courtesy of Finnzyme)@ 2 U/uL<br />
4uL Taq Ligase @ 40 U/uL<br />
+<br />
Water to 50uL<br />
<br />
<br />
We kept this reaction mix at 50C for 60 seconds to let the ExoIII digest our DNA, then heat inactivated it by keeping it at 75C for 20 minutes. When cooled down to 60C at a very slow ramp rate the now-single-stranded parts anneal to one another, allowing the Phusion polymerase and Taq ligase to fill in and closed the gaps. Because the yields are too low to clone directly from this reaction we PCRed full constructs using BB prefix and suffix primers and high fidelity polymerase which could then be cloned into a biobrick-compatible plasmid.<br />
<br />
<br />
[[Image:Overlap_gel.jpg|400px|right]]<br />
To ensure that we actually had our construct of interest we ran a diagnostic PCR to see what parts had actually ligated together in our reaction. Because we already obtained forward and reverse PCR oligos for each part, it was relatively easy to visualize the stepwise progression of the assembly by making PCR reactions with a Biobrick forward primer and each part reverse primer. A typical (successful) diagnostic PCR looks like this:<br />
<br />
Then all you need to do is make a larger PCR reaction using the Biobrick forward and reverse primers and you're set!</div>
RussellDurrett
http://2010.igem.org/File:Overlap_gel.jpg
File:Overlap gel.jpg
2010-10-27T05:34:33Z
<p>RussellDurrett: uploaded a new version of "Image:Overlap gel.jpg"</p>
<hr />
<div></div>
RussellDurrett
http://2010.igem.org/File:Overlap_gel.jpg
File:Overlap gel.jpg
2010-10-27T05:33:22Z
<p>RussellDurrett: </p>
<hr />
<div></div>
RussellDurrett
http://2010.igem.org/File:Overlapgel.tif
File:Overlapgel.tif
2010-10-27T05:32:16Z
<p>RussellDurrett: </p>
<hr />
<div></div>
RussellDurrett
http://2010.igem.org/Team:NYU/Assembly
Team:NYU/Assembly
2010-10-27T04:36:17Z
<p>RussellDurrett: </p>
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<!-- !END NYU HYBRID CSS- Thanks Harvard --><br />
<br />
</html><br />
<br />
[[Image:NYU_OverviewSideF_3.png|200px|right]]<br />
<br />
While the 3A assembly method is well suited for stepwise building of devices, larger complex constructs can take time to make. Being the impatient New Yorkers we are, as soon as we came up with the constructs for our project we had to have them immediately. Because of this, we began exploring other methods to piece together our system. <br />
<br />
We decided to use the Overlap Assembly Method most famously published in this JCVI paper: <br />
<br />
[http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1318.html Gibson et al, Nature Methods 6: 343-345]<br />
<br />
<br />
This method allows researchers to assemble complex systems of many parts all at once rather than in a stepwise fashion. By combining your overlapping DNA parts with the required enzymes and buffers you can easily assemble a complete system in one day - perfect for the impatient New Yorkers that we are. Here's the general schematic:<br />
<br />
[[Image:NYU_Overlapassembly.jpg|300px|center]]<br />
<br />
<br />
<br />
The downside of this system is that the parts to be assembled must overlap one another in their sequence, so you must order DNA oligos to PCR constructs into overlapping forms. To help us out with this, Russell programmed an overlap oligo maker that did much of the work for us - check it out by clicking this link:<br />
<br />
<html><br />
<a href="http://www.russelldurrett.com/overlapoligos.html"><img src="https://static.igem.org/mediawiki/2010/5/5c/Overlaplogo.gif" /></a></html><br />
<br />
After getting the oligos in the lab we combined equimolar amounts of each part in a single reaction and added ExoIII, Phusion polymerase and Taq Ligase. We opted for a 2-step thermocycled reaction method for simplicity and cost-effectiveness and the relative ease of making a reaction mix. Using the reaction mix suggested in the Gibson et. al. paper, Russell designed a simplified version by using the Phusion and Ligase prepared buffers. We were able to get successful reactions from this simple mix:<br />
<br />
50uL Assembly Reaction:<br />
<br />
Equimolar amounts of overlapping DNA (~100ng) from PCR cleanup - up to 18uL<br />
+<br />
10uL 5x Phusion HF Polymerase Buffer<br />
5uL 10x Taq Ligase Buffer<br />
8uL 50mM MgCl2<br />
4uL 2.5mM dNTPs<br />
+<br />
.5uL ExoIII diluted to 10 U/uL<br />
2uL Phusion HF Polymerase (courtesy of Finnzyme)@ 2 U/uL<br />
4uL Taq Ligase @ 40 U/uL<br />
+<br />
Water to 50uL<br />
<br />
<br />
We kept this reaction mix at 50C for 60 seconds to let the ExoIII digest our DNA, then heat inactivated it by keeping it at 75C for 20 minutes. When cooled down to 60C at a very slow ramp rate the now-single-stranded parts anneal to one another, allowing the Phusion polymerase and Taq ligase to fill in and closed the gaps. Because the yields are too low to clone directly from this reaction we PCRed full constructs using BB prefix and suffix primers and high fidelity polymerase which could then be cloned into a biobrick-compatible plasmid.</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Programming
Team:NYU/Programming
2010-10-26T06:00:45Z
<p>RussellDurrett: </p>
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[[Image:NYU_OverviewSideF_2.png|200px|right]]<br />
<br />
=====About this Page=====<br />
* Competent programming skills are quickly becoming a necessity for working in biology. Because most of our team members had never had exposure to a programming environment, we decided to use the obstacles presented in our iGEM experiments as cause for learning the Perl programming language and produced some simple scripts that could do routine iGEM labors for us. Here are some examples:<br />
<br />
<br />
=====Biobricking Primers=====<br />
* This script allows the user to input a DNA sequence and outputs the primers necessary for biobricking that sequence via PCR. The script calculates Tm but does not (yet) consider primer dimers. <br />
<br />
*Finally got the CGI working - click below or go [http://openwetware.org/images/a/a7/Biobrickprimers.txt here for the source code]<br />
<br />
<html><br />
<a href="http://russelldurrett.com/biobrickprimers.html"><img src="https://static.igem.org/mediawiki/2010/7/77/Biobricking_Primers_Logo.gif"/></a><br />
</html><br />
<br />
<br />
<br />
=====Overlap Oligo Maker=====<br />
<br />
* Some very fast and efficient DNA assembly methods require parts to overlap on the ends of their sequences. This script will take two DNA sequences and output the oligos (and their Tms) one would need to PCR constructs with overlapping ends. <br />
<br />
*I'm a CGI machine! [http://openwetware.org/images/6/67/Overlapoligos.txt Here is the source code.] or click below for an interactive version. <br />
<br />
<br />
<html><br />
<a href="http://www.russelldurrett.com/overlapoligos.html"><img src="https://static.igem.org/mediawiki/2010/5/5c/Overlaplogo.gif"/></a></html></div>
RussellDurrett
http://2010.igem.org/Team:NYU/Project
Team:NYU/Project
2010-10-26T05:57:51Z
<p>RussellDurrett: </p>
<hr />
<div><html><br />
<br />
<div id="allcontent"><br />
<img class="bannerimage" src="https://static.igem.org/mediawiki/2010/d/d8/NYU_logo.png" height="200px" width="950px"><br /><br />
<br />
<br />
<div id="headerlinks"><br />
<ul id="nav"><br />
<li><br />
<a class="bannertoplinks" href="https://2010.igem.org/Team:NYU">Home</a><br />
</li><br />
<br />
<li><a class="bannertoplinks" href="#">Project</a><br />
<ul style="z-index:1"><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:NYU/Project">Overview</a></li><br />
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<br />
<br />
<br />
{|align="left"<br />
|Our goal is to use the tools of synthetic biology to make a biological machine capable of antibody discovery. To do this, we have decided to learn from our own immune system by porting its own antibody discovery strategies into an engineered strain of yeast. This yeast will be capable of screening a library of antibodies against a target antigen, recombining the antibody gene ''in vivo'' and then either secreting or surface displaying the resulting antibody protein for a variety of purposes. Our hope is to demonstrate the feasibility of using the yeast cell not only as a vessel for antibody discovery but as a streamlined processing unit that can discover and also begin production of new antibodies - all in one test tube! <br />
<br />
|[[Image:NYU_logo.png|220px|left]]<br />
|-<br />
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|align="left"|<br />
|-<br />
|<br />
|}<br />
<br />
<br />
[[Image:NYU_OverviewSideF_1.png|200px|right]]<br />
<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
Our game plan for this year's competition is to construct an easy-to-use yeast strain capable of intracellular antibody discovery. The current dominant mode of microbial antibody discovery requires cellular surface display and high-throughput fluorescent screening. Instead of using this method, we wanted our cells to be able to sense if the antibody they are translating will bind the target antigen. Using a modified form of the yeast two-hybrid screening system, the yeast cells will sense the amount of antibody::antigen interaction because it will increase the level of transcription of a reporter gene. If this gene encodes a nutritional marker, such as Ura3, only cells experiencing antibody:antigen interactions will grow in selective media (i.e. without the nutrient such as Uracil). Using this method the original population of our yeast strain will be able to, in essence, screen itself and our resulting yeast population will contain only antibodies that bind our antigen.<br />
<br />
<br />
<br />
In a research setting, once you have discovered the antibody you wish to use you must then reclone the gene for it into a secretion vector for production of pure antibody protein. This involves taking the plasmid out of the yeast cell, excising the antibody gene, ligating it into the second vector and then transforming it back into a suitable yeast strain. To make this process faster and more easily accomplished, we have incorporated a recombination-based architecture into our system that will allow the cells to modify the antibody plasmid ''in vivo''. Using this system transcription of the antibody gene can go straight from screening to protein production in a matter of minutes. This concept has the capability to not only shave weeks off of current antibody discovery protocols, but opens the door to cellular programming for other methods, such as restructuring of antibody genes or performing more complex screenings for different purposes.<br />
<br />
== The Experiments ==<br />
<br />
In the spectrum of molecular biology, iGEM is a very short time frame. Because of this short time frame, our wet lab team having only two members and our extremely limited research budget, we felt that radical simplification of our experiments would allow us to demonstrate the feasibility of our ideas without requiring resources we simply did not have. Because many of the individual aspects that this project has brought together have been investigated with success, we were able to perform some experiments using proxies for the 'real thing'. <br />
<br />
== Results ==</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Project
Team:NYU/Project
2010-10-26T05:57:23Z
<p>RussellDurrett: </p>
<hr />
<div><html><br />
<br />
<div id="allcontent"><br />
<img class="bannerimage" src="https://static.igem.org/mediawiki/2010/d/d8/NYU_logo.png" height="200px" width="950px"><br /><br />
<br />
<br />
<div id="headerlinks"><br />
<ul id="nav"><br />
<li><br />
<a class="bannertoplinks" href="https://2010.igem.org/Team:NYU">Home</a><br />
</li><br />
<br />
<li><a class="bannertoplinks" href="#">Project</a><br />
<ul style="z-index:1"><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:NYU/Project">Overview</a></li><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:NYU/Experiments">Experiments</a></li><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:NYU/Assembly">Assembly Methods</a></li><br />
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<ul style="z-index:1"><br />
<li><a class="bannerlinks" href="https://2010.igem.org/Team:NYU/Sponsors">Sponsors</a></li><br />
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|Our goal is to use the tools of synthetic biology to make a biological machine capable of antibody discovery. To do this, we have decided to learn from our own immune system by porting its own antibody discovery strategies into an engineered strain of yeast. This yeast will be capable of screening a library of antibodies against a target antigen, recombining the antibody gene ''in vivo'' and then either secreting or surface displaying the resulting antibody protein for a variety of purposes. Our hope is to demonstrate the feasibility of using the yeast cell not only as a vessel for antibody discovery but as a streamlined processing unit that can discover and also begin production of new antibodies - all in one test tube! <br />
<br />
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<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
Our game plan for this year's competition is to construct an easy-to-use yeast strain capable of intracellular antibody discovery. The current dominant mode of microbial antibody discovery requires cellular surface display and high-throughput fluorescent screening. Instead of using this method, we wanted our cells to be able to sense if the antibody they are translating will bind the target antigen. Using a modified form of the yeast two-hybrid screening system, the yeast cells will sense the amount of antibody::antigen interaction because it will increase the level of transcription of a reporter gene. If this gene encodes a nutritional marker, such as Ura3, only cells experiencing antibody:antigen interactions will grow in selective media (i.e. without the nutrient such as Uracil). Using this method the original population of our yeast strain will be able to, in essence, screen itself and our resulting yeast population will contain only antibodies that bind our antigen.<br />
<br />
<br />
<br />
In a research setting, once you have discovered the antibody you wish to use you must then reclone the gene for it into a secretion vector for production of pure antibody protein. This involves taking the plasmid out of the yeast cell, excising the antibody gene, ligating it into the second vector and then transforming it back into a suitable yeast strain. To make this process faster and more easily accomplished, we have incorporated a recombination-based architecture into our system that will allow the cells to modify the antibody plasmid ''in vivo''. Using this system transcription of the antibody gene can go straight from screening to protein production in a matter of minutes. This concept has the capability to not only shave weeks off of current antibody discovery protocols, but opens the door to cellular programming for other methods, such as restructuring of antibody genes or performing more complex screenings for different purposes.<br />
<br />
== The Experiments ==<br />
<br />
In the spectrum of molecular biology, iGEM is a very short time frame. Because of this short time frame, our wet lab team having only two members and our extremely limited research budget, we felt that radical simplification of our experiments would allow us to demonstrate the feasibility of our ideas without requiring resources we simply did not have. Because many of the individual aspects that this project has brought together have been investigated with success, we were able to perform some experiments using proxies for the 'real thing'. <br />
<br />
== Results ==</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Project
Team:NYU/Project
2010-10-26T05:56:34Z
<p>RussellDurrett: </p>
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|Our goal is to use the tools of synthetic biology to make a biological machine capable of antibody discovery. To do this, we have decided to learn from our own immune system by porting its own antibody discovery strategies into an engineered strain of yeast. This yeast will be capable of screening a library of antibodies against a target antigen, recombining the antibody gene ''in vivo'' and then either secreting or surface displaying the resulting antibody protein for a variety of purposes. Our hope is to demonstrate the feasibility of using the yeast cell not only as a vessel for antibody discovery but as a streamlined processing unit that can discover and also begin production of new antibodies - all in one test tube! <br />
<br />
|[[Image:NYU_logo.png|220px|right]]<br />
|-<br />
|<br />
|<br />
|-<br />
|<br />
|align="right"|<br />
|-<br />
|<br />
|}<br />
<br />
<br />
[[Image:NYU_OverviewSideF_1.png|200px|right]]<br />
<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
Our game plan for this year's competition is to construct an easy-to-use yeast strain capable of intracellular antibody discovery. The current dominant mode of microbial antibody discovery requires cellular surface display and high-throughput fluorescent screening. Instead of using this method, we wanted our cells to be able to sense if the antibody they are translating will bind the target antigen. Using a modified form of the yeast two-hybrid screening system, the yeast cells will sense the amount of antibody::antigen interaction because it will increase the level of transcription of a reporter gene. If this gene encodes a nutritional marker, such as Ura3, only cells experiencing antibody:antigen interactions will grow in selective media (i.e. without the nutrient such as Uracil). Using this method the original population of our yeast strain will be able to, in essence, screen itself and our resulting yeast population will contain only antibodies that bind our antigen.<br />
<br />
<br />
<br />
In a research setting, once you have discovered the antibody you wish to use you must then reclone the gene for it into a secretion vector for production of pure antibody protein. This involves taking the plasmid out of the yeast cell, excising the antibody gene, ligating it into the second vector and then transforming it back into a suitable yeast strain. To make this process faster and more easily accomplished, we have incorporated a recombination-based architecture into our system that will allow the cells to modify the antibody plasmid ''in vivo''. Using this system transcription of the antibody gene can go straight from screening to protein production in a matter of minutes. This concept has the capability to not only shave weeks off of current antibody discovery protocols, but opens the door to cellular programming for other methods, such as restructuring of antibody genes or performing more complex screenings for different purposes.<br />
<br />
== The Experiments ==<br />
<br />
In the spectrum of molecular biology, iGEM is a very short time frame. Because of this short time frame, our wet lab team having only two members and our extremely limited research budget, we felt that radical simplification of our experiments would allow us to demonstrate the feasibility of our ideas without requiring resources we simply did not have. Because many of the individual aspects that this project has brought together have been investigated with success, we were able to perform some experiments using proxies for the 'real thing'. <br />
<br />
== Results ==</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Assembly
Team:NYU/Assembly
2010-10-25T08:23:15Z
<p>RussellDurrett: </p>
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While the 3A assembly method is well suited for stepwise building of devices, larger complex constructs can take time to make. Being the impatient New Yorkers we are, as soon as we came up with the constructs for our project we had to have them immediately. Because of this, we began exploring other methods to piece together our system. <br />
<br />
We decided to use the Overlap Assembly Method most famously published in this JCVI paper: <br />
<br />
[http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1318.html Gibson et al, Nature Methods 6: 343-345]<br />
<br />
This method allows researchers to assemble complex systems of many parts all at once rather than in a stepwise fashion. The downside of this system is that the parts to be assembled must overlap one another in their sequence, so you must order DNA oligos to PCR constructs into overlapping forms. To help us out with this, Russell programmed an overlap oligo maker that did much of the work for us - check it out by clicking this link:<br />
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<br />
After getting the oligos in the lab we combined equimolar amounts of each part in a single reaction and added ExoIII, Phusion polymerase and Taq Ligase. We opted for a 2-step thermocycled reaction method for simplicity and cost-effectiveness and the relative ease of making a reaction mix. Using the reaction mix suggested in the Gibson et. al. paper, we designed our own simplified version by using the Phusion and Ligase prepared buffers. We were able to get successful reactions from this mix:<br />
<br />
ENTER REACTION MIX HERE<br />
<br />
We kept this reaction mix at 50C for 3 minutes to let the ExoIII digest our DNA, then heat inactivated it by keeping it at 75C for 20 minutes. When cooled down to 60C, the now single-stranded parts annealed to one another and the Phusion polymerase and Taq ligase filled in and closed the gaps. The yields were too low to clone directly from this reaction so instead we PCRed full constructs using BB prefix and suffix reverse complement primers and high fidelity polymerase.</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Assembly
Team:NYU/Assembly
2010-10-25T08:17:57Z
<p>RussellDurrett: </p>
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<br />
While the 3A assembly method is well suited for stepwise building of devices, larger complex constructs can take time to make. Being the impatient New Yorkers we are, as soon as we came up with the constructs for our project we had to have them immediately. Because of this, we began exploring other methods to piece together our system. <br />
<br />
We decided to use the Overlap Assembly Method most famously published in this JCVI paper: <br />
<br />
[http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1318.html Gibson et al, Nature Methods 6: 343-345]<br />
<br />
This method allows researchers to assemble complex systems of many parts all at once rather than in a stepwise fashion. The downside of this system is that the parts to be assembled must overlap one another in their sequence, so you must order DNA oligos to PCR constructs into overlapping forms. To help us out with this, Russell programmed an overlap oligo maker that did much of the work for us - check it out by clicking this link:<br />
<br />
<html><br />
<a href="http://www.russelldurrett.com/overlapoligos.html"><img src="https://static.igem.org/mediawiki/2010/5/5c/Overlaplogo.gif" /></a></html><br />
<br />
<br />
After getting the oligos in the lab we combined equimolar amounts of each part in a single reaction and added ExoIII, Phusion polymerase and Taq Ligase. We opted for a 2-step thermocycled reaction method for simplicity and cost-effectiveness and the relative ease of making a reaction mix. Using the reaction mix suggested in the Gibson et. al. paper, we designed our own simplified version by using the Phusion and Ligase prepared buffers. We were able to get successful reactions from this mix:<br />
<br />
ENTER REACTION MIX HERE<br />
<br />
We kept this reaction mix at 50C for 3 minutes to let the ExoIII digest our DNA, then heat inactivated it by keeping it at 75C for 20 minutes. When cooled down to 60C, the now single-stranded parts annealed to one another and the Phusion polymerase and Taq ligase filled in and closed the gaps. The yields were too low to clone directly from this reaction so instead we PCRed full constructs using BB prefix and suffix reverse complement primers and high fidelity polymerase.</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Assembly
Team:NYU/Assembly
2010-10-25T08:17:35Z
<p>RussellDurrett: </p>
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<br />
While the 3A assembly method is well suited for stepwise building of devices, larger complex constructs can take time to make. Being the impatient New Yorkers we are, as soon as we came up with the constructs for our project we had to have them immediately. Because of this, we began exploring other methods to piece together our system. <br />
<br />
We decided to use the Overlap Assembly Method most famously published in this JCVI paper: <br />
<br />
[http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1318.html Gibson et al, Nature Methods 6: 343-345]<br />
<br />
This method allows researchers to assemble complex systems of many parts all at once rather than in a stepwise fashion. The downside of this system is that the parts to be assembled must overlap one another in their sequence, so you must order DNA oligos to PCR constructs into overlapping forms. To help us out with this, Russell programmed an overlap oligo maker that did much of the work for us - check it out by clicking this link:<br />
<br />
<html><br />
<a href="http://www.russelldurrett.com/overlapoligos.html"><img src="https://static.igem.org/mediawiki/2010/5/5c/Overlaplogo.gif" width="300" height="200" /></a></html><br />
<br />
<br />
After getting the oligos in the lab we combined equimolar amounts of each part in a single reaction and added ExoIII, Phusion polymerase and Taq Ligase. We opted for a 2-step thermocycled reaction method for simplicity and cost-effectiveness and the relative ease of making a reaction mix. Using the reaction mix suggested in the Gibson et. al. paper, we designed our own simplified version by using the Phusion and Ligase prepared buffers. We were able to get successful reactions from this mix:<br />
<br />
ENTER REACTION MIX HERE<br />
<br />
We kept this reaction mix at 50C for 3 minutes to let the ExoIII digest our DNA, then heat inactivated it by keeping it at 75C for 20 minutes. When cooled down to 60C, the now single-stranded parts annealed to one another and the Phusion polymerase and Taq ligase filled in and closed the gaps. The yields were too low to clone directly from this reaction so instead we PCRed full constructs using BB prefix and suffix reverse complement primers and high fidelity polymerase.</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Programming
Team:NYU/Programming
2010-10-25T08:17:13Z
<p>RussellDurrett: </p>
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<br />
<br />
=====About this Page=====<br />
* Competent programming skills are quickly becoming a necessity for working in biology. Because most of our team members had never had exposure to a programming environment, we decided to use the obstacles presented in our iGEM experiments as cause for learning the Perl programming language and produced some simple scripts that could do routine iGEM labors for us. Here are some examples:<br />
<br />
=====Biobricking Primers=====<br />
* This script allows the user to input a DNA sequence and outputs the primers necessary for biobricking that sequence via PCR. The script calculates Tm but does not (yet) consider primer dimers. <br />
<br />
*Finally got the CGI working - click below or go [http://openwetware.org/images/a/a7/Biobrickprimers.txt here for the source code]<br />
<br />
<html><br />
<a href="http://russelldurrett.com/biobrickprimers.html"><img src="https://static.igem.org/mediawiki/2010/7/77/Biobricking_Primers_Logo.gif"/></a><br />
</html><br />
<br />
=====Overlap Oligo Maker=====<br />
<br />
* Some very fast and efficient DNA assembly methods require parts to overlap on the ends of their sequences. This script will take two DNA sequences and output the oligos (and their Tms) one would need to PCR constructs with overlapping ends. <br />
<br />
*I'm a CGI machine! [http://openwetware.org/images/6/67/Overlapoligos.txt Here is the source code.] or click below for an interactive version. <br />
<br />
<br />
<html><br />
<a href="http://www.russelldurrett.com/overlapoligos.html"><img src="https://static.igem.org/mediawiki/2010/5/5c/Overlaplogo.gif"/></a></html></div>
RussellDurrett
http://2010.igem.org/File:Overlaplogo.gif
File:Overlaplogo.gif
2010-10-25T08:13:42Z
<p>RussellDurrett: </p>
<hr />
<div></div>
RussellDurrett
http://2010.igem.org/Team:NYU/Assembly
Team:NYU/Assembly
2010-10-25T07:55:04Z
<p>RussellDurrett: </p>
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<br />
Overlap Assembly Method<br />
<br />
<br />
Because we are impatient New Yorkers, as soon as we came up with the constructs for our project we had to have them immediately. While the 3A assembly method is well suited for stepwise building of devices, larger complex constructs can take time to make. Because of this, we began exploring other methods to piece together our system. <br />
<br />
We decided to use the Overlap Assembly Method most famously published in this JCVI paper: <br />
<br />
Gibson et. al. Nature Methods 6: 343-345<br />
<br />
http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1318.html<br />
<br />
<br />
This method allows researchers to assemble complex systems of many parts all at once rather than in a stepwise fashion. The downside of this system is that the parts to be assembled must overlap one another in their sequence, so you must order DNA oligos to PCR constructs into overlapping forms. To help us out with this, Russell programmed an overlap oligo maker that did much of the work for us - check it out by clicking this link:<br />
<br />
<br />
<br />
After getting the oligos in the lab we combined equimolar amounts of each part in a single reaction and added ExoIII, Phusion polymerase and Taq Ligase. We opted for a 2-step thermocycled reaction method for simplicity and cost-effectiveness and the relative ease of making a reaction mix. Using the reaction mix suggested in the Gibson et. al. paper, we designed our own simplified version by using the Phusion and Ligase prepared buffers. We were able to get successful reactions from this mix:<br />
<br />
ENTER REACTION MIX HERE<br />
<br />
We kept this reaction mix at 50C for 3 minutes to let the ExoIII digest our DNA, then heat inactivated it by keeping it at 75C for 20 minutes. When cooled down to 60C, the now single-stranded parts annealed to one another and the Phusion polymerase and Taq ligase filled in and closed the gaps. The yields were too low to clone directly from this reaction so instead we PCRed full constructs using BB prefix and suffix reverse complement primers and high fidelity polymerase.</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Programming
Team:NYU/Programming
2010-10-25T06:59:45Z
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<!-- !END NYU HYBRID CSS- Thanks Harvard --><br />
<br />
</html><br />
<br />
<br />
<br />
=====About this Page=====<br />
* Competent programming skills are quickly becoming a necessity for working in biology. Because most of our team members had never had exposure to a programming environment, we decided to use the obstacles presented in our iGEM experiments as cause for learning the Perl programming language and produced some simple scripts that could do routine iGEM labors for us. Here are some examples:<br />
<br />
=====Biobricking Primers=====<br />
* This script allows the user to input a DNA sequence and outputs the primers necessary for biobricking that sequence via PCR. The script calculates Tm but does not (yet) consider primer dimers. <br />
<br />
*Finally got the CGI working - click below or go [http://openwetware.org/images/a/a7/Biobrickprimers.txt here for the source code]<br />
<br />
<html><br />
<a href="http://russelldurrett.com/biobrickprimers.html"><img src="https://static.igem.org/mediawiki/2010/7/77/Biobricking_Primers_Logo.gif"/></a><br />
</html><br />
<br />
=====Overlap Oligo Maker=====<br />
<br />
* Some very fast and efficient DNA assembly methods require parts to overlap on the ends of their sequences. This script will take two DNA sequences and output the oligos (and their Tms) one would need to PCR constructs with overlapping ends. <br />
<br />
*I'm a CGI machine! [http://openwetware.org/images/6/67/Overlapoligos.txt Here is the source code.] or click below for an interactive version. <br />
<br />
<html><br />
<a href="http://www.russelldurrett.com/overlapoligos.html"><img src="https://static.igem.org/mediawiki/2010/7/74/Overlap_Oligos_Logo.gif" width="300" height="200" /></a></html></div>
RussellDurrett
http://2010.igem.org/File:Overlap_Oligos_Logo.gif
File:Overlap Oligos Logo.gif
2010-10-25T06:55:14Z
<p>RussellDurrett: </p>
<hr />
<div></div>
RussellDurrett
http://2010.igem.org/File:Biobricking_Primers_Logo.gif
File:Biobricking Primers Logo.gif
2010-10-25T06:48:31Z
<p>RussellDurrett: </p>
<hr />
<div></div>
RussellDurrett
http://2010.igem.org/Team:NYU/CornellMed
Team:NYU/CornellMed
2010-10-23T21:31:31Z
<p>RussellDurrett: </p>
<hr />
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</style><br />
<!-- !END NYU HYBRID CSS- Thanks Harvard --><br />
<br />
</html><br />
<br />
{|align="center"<br />
|<br />
|''Researchers at Cornell Medical School's Computational Biomedicine Department were also a part of the team. We decided to use the department's 3D visualization room dubbed the CAVE to explore the structures of the proteins involved in our project and make some 3D animations of the various processes.''<br />
|-<br />
|}<br />
<br />
=Adviser=<br />
{|align="center"<br />
|<br />
|'''Chris Mason, from the Mason lab'''<br />
|[[Image:ChrisMasonRNYU.jpg|100px|left|]]<br />
|-<br />
|<br />
|align="center"|<br />
|-<br />
|<br />
|}</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Project
Team:NYU/Project
2010-10-14T00:46:07Z
<p>RussellDurrett: </p>
<hr />
<div><html><br />
<br />
<div id="allcontent"><br />
<img class="bannerimage" src="https://static.igem.org/mediawiki/2010/d/d8/NYU_logo.png" height="200px" width="950px"><br /><br />
<br />
<br />
<div id="headerlinks"><br />
<ul id="nav"><br />
<li><br />
<a class="bannertoplinks" href="https://2010.igem.org/Team:NYU">Home</a><br />
</li><br />
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<li><a class="bannertoplinks" href="#">Project</a><br />
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|Our goal is to use the tools of synthetic biology to improve the field of antibody engineering. To do this, we have decided to learn from our own immune system by porting its own antibody discovery strategies into an engineered strain of yeast. This yeast will be capable of screening a library of antibodies against a target antigen, recombining the antibody gene ''in vivo'' and then either secreting or surface displaying the resulting antibody protein for a variety of purposes. Our hope is to demonstrate the feasibility of using the yeast cell not only as a vessel for antibody discovery but as a streamlined processing unit that can discover and begin production of new antibodies - all in one test tube! This will reduce the cost and time required for antibody discovery and enable many new technologies in this field.<br />
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== Project Details==<br />
<br />
<br />
<br />
Our game plan for this year's competition is to construct an easy-to-use yeast strain capable of intracellular antibody discovery. The current dominant mode of antibody discovery using microbes requires cellular surface display and high-throughput fluorescent screening. Instead of using this method, we wanted our cells to be able to sense if the antibody they are translating will bind the target antigen. Using a modified form of the yeast two-hybrid screening system, the yeast cells will sense the amount of antibody::antigen interaction based on the level of transcription of a reporter gene. If this gene encodes a nutritional marker, such as Ura3, then when grown on Ura3 selective media only those yeast strains that are experienceing antibody::antigen interactions will survive. This way the strain will be able to, in essence, screen themselves. <br />
<br />
In a research setting, once you have discovered the antibody you wish to use you must then reclone the gene for it into a secretion vector for production of pure antibody protein. This involves taking the plasmid out of the yeast cell, excising the antibody gene, ligating it into the second vector and then transforming it back into a suitable yeast strain. To make this process faster and more easily accomplished, we have incorporated a recombination-based architecture into our system that will allow the cells to modify the antibody plasmid ''in vivo''. Using this system transcription of the antibody gene can go straight from screening to protein production in a matter of minutes. This concept has the capability to not only shave weeks off of current antibody discovery protocols, but opens the door to cellular programming for other methods, such as restructuring of antibody genes or performing more complex screenings for different purposes.<br />
<br />
== The Experiments ==<br />
<br />
In the spectrum of molecular biology, iGEM is a very short time frame. Because of this short time frame, our wet lab team having only two members and our extremely limited research budget, we felt that radical simplification of our experiments would allow us to demonstrate the feasibility of our ideas without requiring resources we simply did not have. Because many of the individual aspects that this project has brought together have been investigated with success, we were able to perform some experiments using proxies for the 'real thing'. <br />
<br />
== Results ==</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Notebook
Team:NYU/Notebook
2010-10-13T06:10:29Z
<p>RussellDurrett: </p>
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In order to keep everyone (including you) up to date on the team's progress over the course of the summer and fall we've decided to keep an ongoing lab notebook on our wiki. In that light, here are the thoughts, opinions, trials and tribulations of the 2010 NYU iGEM team:<br />
<br />
Check out our OpenWetware Lab Notebook to see the protocols optimized over the summer : [http://openwetware.org/wiki/IGEM:New_York_University/2009/Notebook/ImmunoYeast_2010 ImmunoYeast2010]<br />
<br />
==Notebook==<br />
<br />
<br />
=====7/20/10=====<br />
*Russell:<br />
Welcome to the NYU iGEM Lab Notebook! Today we initiate writing in this notebook in order for each of us on the team to know the trials and tribulations everyone else is going through, and for our advisers to know what's going on with the team without having to be in the lab all the time. While it may not have the formatting of some of the more computer-literate teams, it will certainly work for us!<br />
<br />
*John:<br />
Performed digestion of 32 plasmids using either EcoRI, PstI, or both. These plasmids included RBS, Flag, E1, F1, sectag (multiple), NUB, and Strep. This was performed to see if our digestion enzymes were working correctly. After digestion, these solutions were run in 4 1.5 gels with large sized wells to verify if the enzymes were cutting.<br />
<br />
=====7/21/10=====<br />
*John:<br />
Ran 1.5 % agarose gel to verify whether primers worked on the Nub-Fusion construct. Previously, we PCRed NUB-Fusion plasmid using verification primers. After, we used this product as a template for PCR with the NUB specific primers that we designed. Sec-tag was also PCRed and run on the gel. Additionally, old PCR cleanup NUB and Sectag were run on the gel to see if they can be used or disposed.<br />
<br />
*Harris:<br />
Transformations from the day before of ADH1-Aga2Linker and ECFP-Terminator in the Biobrick Chloramphenicol plasmid failed, although the retransformation of Aga2 (in the Amp plasmid) alongside them worked. Ligations left overnight so today I am retransforming them alongside BBa_K098994, which is 5B from Plate 3 of the initial distribution, and is in a Chloramphenicol resistance plasmid. This should grow up fine, and if it does, then we know that the problem is with the ligations, but if it doesn’t, then there is a problem with the Chloramphenicol plates.<br />
<br />
=====7/22/10=====<br />
*Russell:<br />
PCR results from 7/20 were disappointing but not unforeseen. Previous sec tag PCRs show band of appropriate size, but biobricking attempts have been unsuccessful with cause unknown. Will perform one more biobricking procedure on the sec tag under careful scrutiny before troubleshooting for larger problems. <br />
<br />
Transformed the N-terminal and C-terminal GFP biobricks from the initial distribution. These will be used to show that the test scFv and test antigen are, in fact, complexing in the cytoplasm (and maybe on the surface). <br />
<br />
Miniprepped the Gal4 and VP16 biobricks to use as a Gal1-10 promoter activator for the response system.<br />
<br />
=====7/25/10=====<br />
*Russell:<br />
Harris’ transformations of pADH1:Aga2-Linker and ECFP:Terminator assembly were successful. I inoculated 4 colonies from each assembly product in LB+Chloramphenicol and performed colony PCRs. <br />
<br />
Unfortunately I REALIZED THERE IS NO RBS BETWEEN THE pADH1 PROMOTER AND AGA2! This simple mistake prevented the MIT team last year from having a successful system so we need to watch out for this. We may want to ligate an RBS onto each of the promoters we want to use and use each of those instead so we definitely do not make this mistake.<br />
<br />
Retransformation of the secretion tag biobricking ligation was also successful. I inoculated 6 colonies from the two plates and performed colony PCR. <br />
<br />
Transformation of the split-GFP biobricks (N-terminal and C-terminal halves of GFP) from the iGEM initial distribution was successful – inoculated 3 colonies of each biobrick but had already started thermal cycler, so colony PCR will be done later. <br />
<br />
Ordered second set of Nub primers as well as biobrick-compatible primers for manipulating the backbone of pCTCON.<br />
<br />
=====7/26/10=====<br />
*Russell:<br />
Got sequencing results from Genewiz for both of the supposed N-ub fusions we received from iGEM – neither sequence contained Nub, apparently both plasmids are void. <br />
<br />
Instead of excising from a plasmid, we get to build the part via overlapping PCR (which, luckily, I’ve been wanting to try for awhile). So I broke the ~140bp Nub gene down into 3 overlapping sequences and ordered the appropriate oligos for the PCR.<br />
<br />
=====7/28/10=====<br />
*Russell:<br />
Received both orders from IDT. Performed two overlap PCRs with different concentrations of oligos. Will run on a gel tomorrow with some Biobrick verification PCRs. <br />
<br />
*John:<br />
Research potential providers of scFv and antigen parts. Made transformation media and SOB.<br />
<br />
=====7/29/10=====<br />
*Russell:<br />
Constructed the pCTCON plasmid backbone with BIOBRICK ends through PCR amplification. [extremely long cycle - 5:30 ext. time]. Also verified biobrick Aga2:Linker:LoxP via VerF/R2 primers and Aga2F/VerR2.<br />
<br />
*John:<br />
Email out requests for potential scFv and antigen parts.<br />
<br />
=====7/30/10=====<br />
*Russell:<br />
I am trying to develop a couple of new (at least for me) protocols to use with biobricks. <br />
<br />
The first is overlap PCR of the N-ubiquitin biobrick. This biobrick is only 140 bases long, so it is not viable to synthesize it via GeneArt (even though we still have all our basebucks) because of the much larger minimum order requirement. Instead, I broke the part down into three overlapping oligos and ordered them from IDT. The first oligo is just the first 60bp of the CDS. The second and third are the next 60bps (with 10bp overlap) but they are taken from the reverse complement of the sequence. I am currently writing a perl script to do this automatically and will post it as soon as I have it. <br />
<br />
The second protocol I’m developing will help with maintaining stocks of biobricks. We are in full swing at the moment and keeping our stocks of soluble biobrick DNA is troublesome. Instead of using a good amount of each of our biobrick minipreps in an assembly, it may be possible to use a PCR product of the VerF/R2 primers and a biobrick template. That way we can just amplify the biobrick region from biobrick template and use the PCR product (with or without purification) directly in our assembly digestions and ligations. I just started the PCR cycle and will post results when I have them. <br />
<br />
=====8/02/10=====<br />
*John:<br />
Emailed Dr. Marasco about anti-Tat ScFv, potential for this ScFv to work in the reducing environment of the cytoplasm. Also, researched potential sources for other “intrabodies,” stumbling on “Single Domain intracellular Antibodies: A Minimal Fragment for Direct In Vivo Selction of Antigen-specific Intrabodies”: Further thought required on the applicability of intracellular single variable domain (IDab).<br />
<br />
*Russell:<br />
N-ubiquitin biobricking transformation resulted in only one colony on the plate with 200uL of transformant. I inoculated it, but such a low efficiency may mean that it is a self-ligated plasmid (although self-ligation control resulted in no colonies). <br />
<br />
pTEF:RBS assemblies from PCR amplicons resulted in many colonies. I inoculated 4 colonies and performed VerF/R PCR test to see if inserts are the expected size. Alongside this PCR I am making another 50uL stock of pCTCON backbone, so the cycle will be overnight and will run the gel tomorrow. <br />
<br />
Literature research has turned a new leaf. Research into intrabody scFv has yielded a lot of great information and previous studies similar to the functional aspect of our system. Previous studies, however, do not use the split ubiquitin system nor encourage transcription of nutritional markers for directed evolution toward antibody binding (rather, they merely mutate via error-prone PCR and scan for binding antibodies through a system more like the two-hybrid). John is writing to researchers requesting scFv intrabodies and antigens, as well as a library of scFvs mutagenized from the original intrabody scaffold. Hopefully these requests will result in useful materials and advice!<br />
<br />
*Harris:<br />
Miniprepped pTEF-RBS from ligation of PCR product digested pieces, along with minipreps of the FLAG tag grown up and transformed from the initial distribution. <br />
<br />
=====8/03/10=====<br />
<br />
*Harris<br />
Ran gel for pTEF-RBS and Aga2Linker-ECFPTerminator, and found that pTEF-RBS isn’t correct, probably only RBS was cut out. Innoculated pTEF-RBS from plate from last week, where the ligation was performed from a Miniprepped digest, rather than from a PCR (from which the current minipreps of pTEF-RBS are). Aga2Linker-ECFPTerm had a band at the correct size (1.3 kb) but also had a smaller band around 400-500 bp, need to think about this and make sure the insert is correct. <br />
<br />
=====8/09/10=====<br />
<br />
*Russell<br />
Today I transformed biobricks necessary for the second half of our system construction. Over the weekend I've been brainstorming ways to construct the response system circuits and have decided that we'll be using the LexA and LacI DNA binding proteins. The Initial Distribution this year came with most of the parts we'll need including some very useful composite reporter devices. For a time I also organized our growing collection of liquid and glycerol biobricks to maintain smaller stocks, something that will probably be an ongoing battle in the war against disorganization. <br />
<br />
We are having some trouble assembling very small parts onto larger ones, case in point: the RBS onto a large promoter. Harris believes that the dichotomy of sizes of these parts makes it hard to relatively represent each in reaction solutions. We thought that the PCR product assembly method would work for this but have been disappointed thus far. For the sake of time I am going to order oligos and PCR-ligate the RBS onto pGAL and pTEF, as well as the RBS onto LacO onto pTEF for our reporter constructs. Once this is finished we'll hopefully be able to work smoothly through the assembly procedure. At this point I would probably give up a kidney for the ability to synthesize all of these constructs. <br />
<br />
Also sent ECFP:Terminator I and II to sequencing to be 100% certain they are of the correct sequence. <br />
<br />
=====8/10/10/=====<br />
<br />
*Russell<br />
I ordered the rest of the primers we will need for the construction of our system (except for genomic integration, which I will order when I know what loci we want to use). Because we are having so much trouble assembling two parts of great size differences (the RBS – 18bp and a promoter – 1000bp) we are going to biobrick the RBS onto the promoter using PCR assembly. For that method I ordered two primer constructs for an inducible Gal promoter and the constitutive Tef promoter. <br />
<br />
Although the Biobrick Verification primers that the registry uses work for sequencing and larger parts, PCR gel verification of biobrick assemblies or basic parts would be easier if we could amplify just the insert and not the ~200bp outside the insert. In that light, I constructed primers based on the biobrick prefix and suffix, including 6bp 5’ GCG clamps so that, if we so choose, we can use the PCR products to do assemblies.<br />
<br />
<br />
=====8/16/10=====<br />
<br />
*Russell<br />
Weekend trip to LA’s over – let’s dive right in:<br />
<br />
In order to circumvent the difficulty of assembling biobricks of very different size (namely a ribosome binding site, 18bp, and a promoter, ~1000bp) we PCRed and biobricked the inducible pGAL and the consititutive pTEF promoters with RBSs on the reverse primers. Today we actually did the main portion of this biobricking. We’ll submit these promoters to the Registry and hopefully this will save some time and stress in future assemblies. <br />
<br />
In order to use the backbone of the pCTCON plasmid (which has a trp1 selectable marker) we need a strain of yeast that does not have the trp1 gene. After talking with Dr. Gresham we’ve decided to make our own yeast knockout strain – something I’m excited about because I’ll get to learn something new! We’re going to use the FY4 wild-type strain because the entire genomic sequence is available online, making it very easy to manipulate the genome thanks to yeast DSB recombination pathways. To this end I designed and ordered primers for the KanMX gene (an antibiotic-selectable gene) with trp1 extensions flanking the KanMX coding region. After PCRing the KanMX CDS, we can transform the PCR product and the yeast will take it from there, inserting the KanMX CDS into the trp1 locus. Because this will konckout trp1 function, we’ll have to grow this strain with tryptophan as well as G148 to select for KanMX transcription. <br />
<br />
I also ordered primers for genomic integration at the Ura3 locus of any biobrick part. Using sequences complementary to the biobrick prefix and suffix, these oligos will make it possible to insert any fully-assembled biobrick construct into this locus. We will post these sequences to the Registry after we get a successful integration. <br />
<br />
*Harris<br />
Biobricking PCR assemblies of pTEF-RBS and pGAL-RBS, along with Ura3 from PCR product clean up as well, all into Amp plasmid. Assembling ECFP-Terminator into Tet plasmid, also from PCR product clean ups. Digested more Amp plasmid for this purpose, 50 uL digest at 10 ng/uL.<br />
<br />
=====8/30/10=====<br />
<br />
*Russell<br />
<br />
We’ve had some success with the PCR amplification of biobrick parts for assembly. Instead of growing up large quantities of a 2.5kb plasmid and using ~100bp, we’ve decided to amplify the part via PCR and then use the PCR product in our assemblies. This has helped us manage stocks of biobrick parts because each team member can have their own PCR product to do their assemblies while working off of the Biobrick reservoir that I maintain. Because each person has their own PCR product from the original stock PCR-induced mutations are less likely to occur, but they are still a possibility. Because of this we are maintaining a rigorous (and costly) Quality Control program by sequencing every assembly.<br />
<br />
In an effort to conserve funds we have moved from Qiagen spin columns to those offered by Epoch Life Sciences. We have begun to make our own buffers (or use those leftover from Qiagen kits) to do our purifications. We chose this tack because it is cheaper and easier to do large quantities of minipreps, but instead we could recycle our spin columns to be a bit more environmentally friendly [like Harvard’s team this year].<br />
<br />
=====9/1/10=====<br />
<br />
*Russell<br />
<br />
Successful assembly of the Aga2….linker…..eCFP! Stay tuned for pictures of cyan fluorescing yeast cell surfaces.</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Notebook
Team:NYU/Notebook
2010-10-13T06:07:51Z
<p>RussellDurrett: </p>
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In order to keep everyone (including you) up to date on the team's progress over the course of the summer and fall we've decided to keep an ongoing lab notebook on our wiki. In that light, here are the thoughts, opinions, trials and tribulations of the 2010 NYU iGEM team:<br />
<br />
Check out our OpenWetware Lab Notebook to see the protocols optimized over the summer : [http://openwetware.org/wiki/IGEM:New_York_University/2009/Notebook/ImmunoYeast_2010 ImmunoYeast 2010<br />
==Notebook==<br />
<br />
<br />
=====7/20/10=====<br />
*Russell:<br />
Welcome to the NYU iGEM Lab Notebook! Today we initiate writing in this notebook in order for each of us on the team to know the trials and tribulations everyone else is going through, and for our advisers to know what's going on with the team without having to be in the lab all the time. While it may not have the formatting of some of the more computer-literate teams, it will certainly work for us!<br />
<br />
*John:<br />
Performed digestion of 32 plasmids using either EcoRI, PstI, or both. These plasmids included RBS, Flag, E1, F1, sectag (multiple), NUB, and Strep. This was performed to see if our digestion enzymes were working correctly. After digestion, these solutions were run in 4 1.5 gels with large sized wells to verify if the enzymes were cutting.<br />
<br />
=====7/21/10=====<br />
*John:<br />
Ran 1.5 % agarose gel to verify whether primers worked on the Nub-Fusion construct. Previously, we PCRed NUB-Fusion plasmid using verification primers. After, we used this product as a template for PCR with the NUB specific primers that we designed. Sec-tag was also PCRed and run on the gel. Additionally, old PCR cleanup NUB and Sectag were run on the gel to see if they can be used or disposed.<br />
<br />
*Harris:<br />
Transformations from the day before of ADH1-Aga2Linker and ECFP-Terminator in the Biobrick Chloramphenicol plasmid failed, although the retransformation of Aga2 (in the Amp plasmid) alongside them worked. Ligations left overnight so today I am retransforming them alongside BBa_K098994, which is 5B from Plate 3 of the initial distribution, and is in a Chloramphenicol resistance plasmid. This should grow up fine, and if it does, then we know that the problem is with the ligations, but if it doesn’t, then there is a problem with the Chloramphenicol plates.<br />
<br />
=====7/22/10=====<br />
*Russell:<br />
PCR results from 7/20 were disappointing but not unforeseen. Previous sec tag PCRs show band of appropriate size, but biobricking attempts have been unsuccessful with cause unknown. Will perform one more biobricking procedure on the sec tag under careful scrutiny before troubleshooting for larger problems. <br />
<br />
Transformed the N-terminal and C-terminal GFP biobricks from the initial distribution. These will be used to show that the test scFv and test antigen are, in fact, complexing in the cytoplasm (and maybe on the surface). <br />
<br />
Miniprepped the Gal4 and VP16 biobricks to use as a Gal1-10 promoter activator for the response system.<br />
<br />
=====7/25/10=====<br />
*Russell:<br />
Harris’ transformations of pADH1:Aga2-Linker and ECFP:Terminator assembly were successful. I inoculated 4 colonies from each assembly product in LB+Chloramphenicol and performed colony PCRs. <br />
<br />
Unfortunately I REALIZED THERE IS NO RBS BETWEEN THE pADH1 PROMOTER AND AGA2! This simple mistake prevented the MIT team last year from having a successful system so we need to watch out for this. We may want to ligate an RBS onto each of the promoters we want to use and use each of those instead so we definitely do not make this mistake.<br />
<br />
Retransformation of the secretion tag biobricking ligation was also successful. I inoculated 6 colonies from the two plates and performed colony PCR. <br />
<br />
Transformation of the split-GFP biobricks (N-terminal and C-terminal halves of GFP) from the iGEM initial distribution was successful – inoculated 3 colonies of each biobrick but had already started thermal cycler, so colony PCR will be done later. <br />
<br />
Ordered second set of Nub primers as well as biobrick-compatible primers for manipulating the backbone of pCTCON.<br />
<br />
=====7/26/10=====<br />
*Russell:<br />
Got sequencing results from Genewiz for both of the supposed N-ub fusions we received from iGEM – neither sequence contained Nub, apparently both plasmids are void. <br />
<br />
Instead of excising from a plasmid, we get to build the part via overlapping PCR (which, luckily, I’ve been wanting to try for awhile). So I broke the ~140bp Nub gene down into 3 overlapping sequences and ordered the appropriate oligos for the PCR.<br />
<br />
=====7/28/10=====<br />
*Russell:<br />
Received both orders from IDT. Performed two overlap PCRs with different concentrations of oligos. Will run on a gel tomorrow with some Biobrick verification PCRs. <br />
<br />
*John:<br />
Research potential providers of scFv and antigen parts. Made transformation media and SOB.<br />
<br />
=====7/29/10=====<br />
*Russell:<br />
Constructed the pCTCON plasmid backbone with BIOBRICK ends through PCR amplification. [extremely long cycle - 5:30 ext. time]. Also verified biobrick Aga2:Linker:LoxP via VerF/R2 primers and Aga2F/VerR2.<br />
<br />
*John:<br />
Email out requests for potential scFv and antigen parts.<br />
<br />
=====7/30/10=====<br />
*Russell:<br />
I am trying to develop a couple of new (at least for me) protocols to use with biobricks. <br />
<br />
The first is overlap PCR of the N-ubiquitin biobrick. This biobrick is only 140 bases long, so it is not viable to synthesize it via GeneArt (even though we still have all our basebucks) because of the much larger minimum order requirement. Instead, I broke the part down into three overlapping oligos and ordered them from IDT. The first oligo is just the first 60bp of the CDS. The second and third are the next 60bps (with 10bp overlap) but they are taken from the reverse complement of the sequence. I am currently writing a perl script to do this automatically and will post it as soon as I have it. <br />
<br />
The second protocol I’m developing will help with maintaining stocks of biobricks. We are in full swing at the moment and keeping our stocks of soluble biobrick DNA is troublesome. Instead of using a good amount of each of our biobrick minipreps in an assembly, it may be possible to use a PCR product of the VerF/R2 primers and a biobrick template. That way we can just amplify the biobrick region from biobrick template and use the PCR product (with or without purification) directly in our assembly digestions and ligations. I just started the PCR cycle and will post results when I have them. <br />
<br />
=====8/02/10=====<br />
*John:<br />
Emailed Dr. Marasco about anti-Tat ScFv, potential for this ScFv to work in the reducing environment of the cytoplasm. Also, researched potential sources for other “intrabodies,” stumbling on “Single Domain intracellular Antibodies: A Minimal Fragment for Direct In Vivo Selction of Antigen-specific Intrabodies”: Further thought required on the applicability of intracellular single variable domain (IDab).<br />
<br />
*Russell:<br />
N-ubiquitin biobricking transformation resulted in only one colony on the plate with 200uL of transformant. I inoculated it, but such a low efficiency may mean that it is a self-ligated plasmid (although self-ligation control resulted in no colonies). <br />
<br />
pTEF:RBS assemblies from PCR amplicons resulted in many colonies. I inoculated 4 colonies and performed VerF/R PCR test to see if inserts are the expected size. Alongside this PCR I am making another 50uL stock of pCTCON backbone, so the cycle will be overnight and will run the gel tomorrow. <br />
<br />
Literature research has turned a new leaf. Research into intrabody scFv has yielded a lot of great information and previous studies similar to the functional aspect of our system. Previous studies, however, do not use the split ubiquitin system nor encourage transcription of nutritional markers for directed evolution toward antibody binding (rather, they merely mutate via error-prone PCR and scan for binding antibodies through a system more like the two-hybrid). John is writing to researchers requesting scFv intrabodies and antigens, as well as a library of scFvs mutagenized from the original intrabody scaffold. Hopefully these requests will result in useful materials and advice!<br />
<br />
*Harris:<br />
Miniprepped pTEF-RBS from ligation of PCR product digested pieces, along with minipreps of the FLAG tag grown up and transformed from the initial distribution. <br />
<br />
=====8/03/10=====<br />
<br />
*Harris<br />
Ran gel for pTEF-RBS and Aga2Linker-ECFPTerminator, and found that pTEF-RBS isn’t correct, probably only RBS was cut out. Innoculated pTEF-RBS from plate from last week, where the ligation was performed from a Miniprepped digest, rather than from a PCR (from which the current minipreps of pTEF-RBS are). Aga2Linker-ECFPTerm had a band at the correct size (1.3 kb) but also had a smaller band around 400-500 bp, need to think about this and make sure the insert is correct. <br />
<br />
=====8/09/10=====<br />
<br />
*Russell<br />
Today I transformed biobricks necessary for the second half of our system construction. Over the weekend I've been brainstorming ways to construct the response system circuits and have decided that we'll be using the LexA and LacI DNA binding proteins. The Initial Distribution this year came with most of the parts we'll need including some very useful composite reporter devices. For a time I also organized our growing collection of liquid and glycerol biobricks to maintain smaller stocks, something that will probably be an ongoing battle in the war against disorganization. <br />
<br />
We are having some trouble assembling very small parts onto larger ones, case in point: the RBS onto a large promoter. Harris believes that the dichotomy of sizes of these parts makes it hard to relatively represent each in reaction solutions. We thought that the PCR product assembly method would work for this but have been disappointed thus far. For the sake of time I am going to order oligos and PCR-ligate the RBS onto pGAL and pTEF, as well as the RBS onto LacO onto pTEF for our reporter constructs. Once this is finished we'll hopefully be able to work smoothly through the assembly procedure. At this point I would probably give up a kidney for the ability to synthesize all of these constructs. <br />
<br />
Also sent ECFP:Terminator I and II to sequencing to be 100% certain they are of the correct sequence. <br />
<br />
=====8/10/10/=====<br />
<br />
*Russell<br />
I ordered the rest of the primers we will need for the construction of our system (except for genomic integration, which I will order when I know what loci we want to use). Because we are having so much trouble assembling two parts of great size differences (the RBS – 18bp and a promoter – 1000bp) we are going to biobrick the RBS onto the promoter using PCR assembly. For that method I ordered two primer constructs for an inducible Gal promoter and the constitutive Tef promoter. <br />
<br />
Although the Biobrick Verification primers that the registry uses work for sequencing and larger parts, PCR gel verification of biobrick assemblies or basic parts would be easier if we could amplify just the insert and not the ~200bp outside the insert. In that light, I constructed primers based on the biobrick prefix and suffix, including 6bp 5’ GCG clamps so that, if we so choose, we can use the PCR products to do assemblies.<br />
<br />
<br />
=====8/16/10=====<br />
<br />
*Russell<br />
Weekend trip to LA’s over – let’s dive right in:<br />
<br />
In order to circumvent the difficulty of assembling biobricks of very different size (namely a ribosome binding site, 18bp, and a promoter, ~1000bp) we PCRed and biobricked the inducible pGAL and the consititutive pTEF promoters with RBSs on the reverse primers. Today we actually did the main portion of this biobricking. We’ll submit these promoters to the Registry and hopefully this will save some time and stress in future assemblies. <br />
<br />
In order to use the backbone of the pCTCON plasmid (which has a trp1 selectable marker) we need a strain of yeast that does not have the trp1 gene. After talking with Dr. Gresham we’ve decided to make our own yeast knockout strain – something I’m excited about because I’ll get to learn something new! We’re going to use the FY4 wild-type strain because the entire genomic sequence is available online, making it very easy to manipulate the genome thanks to yeast DSB recombination pathways. To this end I designed and ordered primers for the KanMX gene (an antibiotic-selectable gene) with trp1 extensions flanking the KanMX coding region. After PCRing the KanMX CDS, we can transform the PCR product and the yeast will take it from there, inserting the KanMX CDS into the trp1 locus. Because this will konckout trp1 function, we’ll have to grow this strain with tryptophan as well as G148 to select for KanMX transcription. <br />
<br />
I also ordered primers for genomic integration at the Ura3 locus of any biobrick part. Using sequences complementary to the biobrick prefix and suffix, these oligos will make it possible to insert any fully-assembled biobrick construct into this locus. We will post these sequences to the Registry after we get a successful integration. <br />
<br />
*Harris<br />
Biobricking PCR assemblies of pTEF-RBS and pGAL-RBS, along with Ura3 from PCR product clean up as well, all into Amp plasmid. Assembling ECFP-Terminator into Tet plasmid, also from PCR product clean ups. Digested more Amp plasmid for this purpose, 50 uL digest at 10 ng/uL.<br />
<br />
=====8/30/10=====<br />
<br />
*Russell<br />
<br />
We’ve had some success with the PCR amplification of biobrick parts for assembly. Instead of growing up large quantities of a 2.5kb plasmid and using ~100bp, we’ve decided to amplify the part via PCR and then use the PCR product in our assemblies. This has helped us manage stocks of biobrick parts because each team member can have their own PCR product to do their assemblies while working off of the Biobrick reservoir that I maintain. Because each person has their own PCR product from the original stock PCR-induced mutations are less likely to occur, but they are still a possibility. Because of this we are maintaining a rigorous (and costly) Quality Control program by sequencing every assembly.<br />
<br />
In an effort to conserve funds we have moved from Qiagen spin columns to those offered by Epoch Life Sciences. We have begun to make our own buffers (or use those leftover from Qiagen kits) to do our purifications. We chose this tack because it is cheaper and easier to do large quantities of minipreps, but instead we could recycle our spin columns to be a bit more environmentally friendly [like Harvard’s team this year].<br />
<br />
=====9/1/10=====<br />
<br />
*Russell<br />
<br />
Successful assembly of the Aga2….linker…..eCFP! Stay tuned for pictures of cyan fluorescing yeast cell surfaces.</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Project
Team:NYU/Project
2010-10-11T18:22:47Z
<p>RussellDurrett: </p>
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{|align="right"<br />
|Our goal is to use the tools of synthetic biology to improve the field of antibody engineering. To do this, we have decided to learn from our own immune system by employing its own strategies in an engineered strain of yeast. This yeast will be capable of screening a library of antibodies against a target antigen, recombining the antibody gene in vivo and then either secreting or surface displaying the resulting antibody protein for a variety of purposes. Our hope is to demonstrate the feasibility of using the yeast cell not only as a vessel for antibody discovery but as a streamlined processing unit that can discover and begin production of new antibodies - all in one test tube! This will reduce the cost and time required for antibody discovery and enable many new technologies in this field.<br />
<br />
|[[Image:NYU_logo.png|220px|right]]<br />
|-<br />
|<br />
|<br />
|-<br />
|<br />
|align="right"|<br />
|-<br />
|<br />
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<br />
<br />
<br />
<br />
== Project Details==<br />
<br />
<br />
<br />
Our game plan for this year's competition is to construct an easy-to-use yeast strain capable of intracellular antibody discovery. The current dominant mode of antibody discovery using microbes requires cellular surface display and high-throughput fluorescent screening. Instead of using this method, we wanted our cells to be able to sense when the antibody they are translating will bind the target antigen. Using a modified form of the yeast two-hybrid screening system, the yeast cells will sense the amount of antibody::antigen interaction and will, in essence, screen themselves. <br />
<br />
In a research setting, once you have discovered the antibody you wish to use you must then reclone the coding region into a secretion vector for production of pure antibody protein. To make this process faster and more easily accomplished, we are also building a recombination-based architecture into our system that will allow the cells to go straight from screening to protein production in a matter of minutes. This concept has the capability to not only shave weeks off of current antibody discovery protocols, but opens the door to programmed cellular restructuring of antibody genes for different purposes.<br />
<br />
== The Experiments ==<br />
<br />
<br />
<br />
<br />
== Results ==</div>
RussellDurrett
http://2010.igem.org/Team:NYU/Parts
Team:NYU/Parts
2010-10-11T02:22:30Z
<p>RussellDurrett: </p>
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<br />
When choosing what simple parts to make for our project we had two main goals: 1) to improve the quality of parts already in the registry by making them more accessible to all users and 2) to augment the current set of yeast-compatible biobricks to harness the power of this synthetic biology-friendly model organism. Incorporating these goals with the 'Quality not Quantity' mantra, we decided to focus our efforts on certain areas of advancement that we believe future researchers will like to see in the registry.<br />
<br />
<br />
1. To improve parts already in the registry by lessening the constrictions on their use and making them more adaptable. <br />
<br />
*'''36bp LoxP site''': the LoxP site is the naturally-occurring substrate for the Cre recombinase. In nature (and in previous biobricks) it is 34 base pairs long. Because it is not a multiple of three, when used inside a transcribed ORF the 34bp LoxP site can throw off the frame of the translated parts. To get around this, previous teams had to either add or take away bases from surrounding parts to keep their systems in frame (painstakingly and sometimes unsuccessfully). To get around this restriction, we designed and Biobricked a 36bp LoxP site that Registry users can include in transcribed regions without worrying about keeping everything in frame. <br />
<br />
*'''pGAL-RBS''':<br />
<br />
*'''pTEF-RBS''':<br />
<br />
*'''lexOpCYC-RBS''':<br />
<br />
<br />
2. Yeast strains are more rarely used in iGEM projects but contain certain parameters that are ideal for certain projects. Many teams are beginning to use yeast this year and are submitting useful biobricks, so we decided to make a collection of biobricks parts aimed at protein localization within the cell (known as 'yeast functional tags'):<br />
<br />
*'''Aga2''': When the Aga2 protein is fused to a part of interest, the fusion is secreted and Aga2 complexes with a protein on the cellular surface - leaving the part of interest displayed on the surface of the cell. While we did not end up using this part in our official construct, biobricking it from a plasmid we already had in our lab will surely benefit future teams.<br />
<br />
*'''Aga2+Linker''': Most everyone will want a linker attached to the Aga2 protein, so we went ahead and submitted this construct as well. <br />
<br />
*'''Secretion Tag''': Another 'yeast functional tag', the secretion tag does just what you think when fused to the C terminus of a protein of interest. <br />
<br />
*'''Ura3''':<br />
<br />
<br />
3. To construct and submit certain ubiquitously useful biobricks that we would be using.<br />
<br />
*'''Flexible (Gly4Ser)3 peptide linker''': for use with most any peptide fusions<br />
<br />
<br />
===Parts===<br />
<br />
New for iGEM 2010 is the ''groupparts'' tag. This tag will generate a table with all of the parts that your team adds to your team sandbox. Note that if you want to document a part you need to document it on the [http://partsregistry.org Registry], not on your team wiki.<br />
<br />
<groupparts>iGEM010 NYU</groupparts></div>
RussellDurrett
http://2010.igem.org/Team:NYU/Parts
Team:NYU/Parts
2010-10-11T02:22:10Z
<p>RussellDurrett: </p>
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<br />
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<br />
<br />
<br />
When choosing what simple parts to make for our project we had two main goals: 1) to improve the quality of parts already in the registry by making them more accessible to all users and 2) to augment the current set of yeast-compatible biobricks to harness the power of this synthetic biology-friendly model organism. Incorporating these goals with the 'Quality not Quantity' mantra, we decided to focus our efforts on certain areas of advancement that we believe future researchers will like to see in the registry.<br />
<br />
<br />
1. To improve parts already in the registry by lessening the constrictions on their use and making them more adaptable. <br />
<br />
*'''36bp LoxP site''': the LoxP site is the naturally-occurring substrate for the Cre recombinase. In nature (and in previous biobricks) it is 34 base pairs long. Because it is not a multiple of three, when used inside a transcribed ORF the 34bp LoxP site can throw off the frame of the translated parts. To get around this, previous teams had to either add or take away bases from surrounding parts to keep their systems in frame (painstakingly and sometimes unsuccessfully). To get around this restriction, we designed and Biobricked a 36bp LoxP site that Registry users can include in transcribed regions without worrying about keeping everything in frame. <br />
<br />
*'''pGAL-RBS''':<br />
<br />
*'''pTEF-RBS''':<br />
<br />
*'''lexOpCYC-RBS''':<br />
<br />
<br />
2. Yeast strains are more rarely used in iGEM projects but contain certain parameters that are ideal for certain projects. Many teams are beginning to use yeast this year and are submitting useful biobricks, so we decided to make a collection of biobricks parts aimed at protein localization within the cell (known as 'yeast functional tags'):<br />
<br />
*'''Aga2''': When the Aga2 protein is fused to a part of interest, the fusion is secreted and Aga2 complexes with a protein on the cellular surface - leaving the part of interest displayed on the surface of the cell. While we did not end up using this part in our official construct, biobricking it from a plasmid we already had in our lab will surely benefit future teams.<br />
<br />
*'''Aga2+Linker''': Most everyone will want a linker attached to the Aga2 protein, so we went ahead and submitted this construct as well. <br />
<br />
*'''Secretion Tag''': Another 'yeast functional tag', the secretion tag does just what you think when fused to the C terminus of a protein of interest. <br />
<br />
*'''Ura3''':<br />
<br />
3. To construct and submit certain ubiquitously useful biobricks that we would be using.<br />
<br />
*'''Flexible (Gly4Ser)3 peptide linker''': for use with most any peptide fusions<br />
<br />
<br />
===Parts===<br />
<br />
New for iGEM 2010 is the ''groupparts'' tag. This tag will generate a table with all of the parts that your team adds to your team sandbox. Note that if you want to document a part you need to document it on the [http://partsregistry.org Registry], not on your team wiki.<br />
<br />
<groupparts>iGEM010 NYU</groupparts></div>
RussellDurrett
http://2010.igem.org/Team:NYU/Parts
Team:NYU/Parts
2010-10-11T02:21:26Z
<p>RussellDurrett: </p>
<hr />
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</style><br />
<!-- !END NYU HYBRID CSS- Thanks Harvard --><br />
<br />
</html><br />
<br />
<br />
<br />
When choosing what simple parts to make for our project we had two main goals: 1) to improve the quality of parts already in the registry by making them more accessible to all users and 2) to augment the current set of yeast-compatible biobricks to harness the power of this synthetic biology-friendly model organism. Incorporating these goals with the 'Quality not Quantity' mantra, we decided to focus our efforts on certain areas of advancement that we believe future researchers will like to see in the registry.<br />
<br />
<br />
1. To improve parts already in the registry by lessening the constrictions on their use and making them more adaptable. <br />
<br />
*'''36bp LoxP site''': the LoxP site is the naturally-occurring substrate for the Cre recombinase. In nature (and in previous biobricks) it is 34 base pairs long. Because it is not a multiple of three, when used inside a transcribed ORF the 34bp LoxP site can throw off the frame of the translated parts. To get around this, previous teams had to either add or take away bases from surrounding parts to keep their systems in frame (painstakingly and sometimes unsuccessfully). To get around this restriction, we designed and Biobricked a 36bp LoxP site that Registry users can include in transcribed regions without worrying about keeping everything in frame. <br />
<br />
*'''pGAL-RBS''':<br />
<br />
*'''pTEF-RBS''':<br />
<br />
*'''lexOpCYC-RBS''':<br />
<br />
<br />
2. Yeast strains are more rarely used in iGEM projects but contain certain parameters that are ideal for certain projects. Many teams are beginning to use yeast this year and are submitting useful biobricks, so we decided to make a collection of biobricks parts aimed at protein localization within the cell (known as 'yeast functional tags'):<br />
<br />
*'''Aga2''': When the Aga2 protein is fused to a part of interest, the fusion is secreted and Aga2 complexes with a protein on the cellular surface - leaving the part of interest displayed on the surface of the cell. While we did not end up using this part in our official construct, biobricking it from a plasmid we already had in our lab will surely benefit future teams.<br />
<br />
*'''Aga2+Linker''': Most everyone will want a linker attached to the Aga2 protein, so we went ahead and submitted this construct as well. <br />
<br />
*'''Secretion Tag''': Another 'yeast functional tag', the secretion tag does just what you think when fused to the C terminus of a protein of interest. <br />
<br />
*'''Ura3''':<br />
<br />
3. To construct and submit certain ubiquitously useful biobricks that we would be using.<br />
<br />
*'''Flexible (Gly4Ser)3 peptide linker''': for use with most any peptide fusions<br />
<br />
<br />
{|align="justify"<br />
|You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing.<br />
|[[Image:NYU_logo.png|200px|right|frame]]<br />
|-<br />
|<br />
''Tell us more about your project. Give us background. Use this is the abstract of your project. Be descriptive but concise (1-2 paragraphs)''<br />
|[[Image:NYU_team.png|right|frame|Your team picture]]<br />
|-<br />
|<br />
|align="center"|[[Team:NYU | Team Example]]<br />
|}<br />
<br />
<br />
<br />
===Parts===<br />
<br />
New for iGEM 2010 is the ''groupparts'' tag. This tag will generate a table with all of the parts that your team adds to your team sandbox. Note that if you want to document a part you need to document it on the [http://partsregistry.org Registry], not on your team wiki.<br />
<br />
<groupparts>iGEM010 NYU</groupparts></div>
RussellDurrett