http://2010.igem.org/wiki/index.php?title=Special:Contributions/Ayjchan&feed=atom&limit=50&target=Ayjchan&year=&month=2010.igem.org - User contributions [en]2024-03-28T15:42:33ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:British_Columbia/Project_OutlookTeam:British Columbia/Project Outlook2010-10-27T23:49:02Z<p>Ayjchan: </p>
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<h3>Project Achievements & Future Directions</h3><br />
<br />
<h4>Biofilm: </h4><p><br />
We have obtained growth curves for <i>S. aureus</i> strains RN4220 and 8325-4 that demonstrate a steady growth phase followed by an oscillatory state of dynamic equilibrium. We have also optimized the existing protocol for biofilm quantification. Additionally, values derived from the biofilm experiments were integral to deriving realistic results from modeling simulations.<br/><br/><br />
The existing curve has demonstrated that 9 hours is the optimal time point for exposure to the matrix-degrading enzyme, DspB, as well as the engineered phage construct with DspB and quorum sensing genes. Future experiments testing the biofilm response to DspB alone as well as DspB incorporated into a phage construct with the quorum sensing P2 promoter will enable the characterization of the construct’s effectiveness against the biofilms of <i>S. aureus</i> strains RN4220 and 8325-4.<br/><br/><br />
<br />
<br />
<h4>Phage Standard: </h4><p><br />
We developed a phage standard that allows for modification of any lysogenic bacteriophage as part of the Biobrick standard. The phage standard works around the problems of illegal cut sites and prohibitively large plasmids. We hope the standard will serve as a foundational advance towards phage research within the iGEM competition, the BioBrick registry and the synthetic biology community as a whole.<br/><br/><br />
In the future we hope to continue developing this standard and optimizing the process of modifying lysogenic phage DNA. We will strive to submit a fully functional set of parts that have been demonstrated to work following the phage standard in the lab. Hopefully we will also be able to include lytic phages under the scope of the phage standard. With some luck we could expand the number of chassis available to iGEM and the BioBrick registry by introducing integration site vectors for multiple species and strains.<br/><br/><br />
<br />
<h4>Quorum Sensing: </h4><p><br />
We have made constructs to characterize the P2 promoter (BBa_I746104) of <i>S. aureus</i> via fluorescent protein production. In order to directly relate AIP to P2 promoter activity, we chose to use an agr null strain. As a next step, genes encoding AgrAC from <i>S. aureus</i> should be put on the same plasmid (the <i>S. aureus</i>/<i>E. coli</i> shuttle vector, pCN33) as the reporter constructs and transformed into agr-null <i>S. aureus</i>. This would allow proper characterization of P2 activity in the presence of AIP. Primers have already been designed and submitted to PCR the genes encoding AgrAC. Additionally, the replicon of the <i>S. aureus</i> pCN33 plasmid can be made into a BioBrick part to facilitate the expression and characterization of BioBrick parts in <i>S. aureus</i>.</p><br/><br />
<h4>DspB: </h4><p><br />
We have contributed to the biobrick parts registry by submitting a new part: DspB, an enzyme that degrades poly-ß-(1,6)-linked N-acetylglucosamine bonds. We have demonstrated that dspB works through a crude cell enzyme activity assay and have added this information to the Registry.<br/><br/><br />
We are currently working on obtaining data from the exposure of DspB protein on a <i>S. aureus</i> biofilm as well as isolating DspB via a histidine tag to attain further characterization data. We hope to gather this data before the presentation. If not fully completed, these components of the sub-team should be future directions.<br />
Future directions include incorporating DspB protein into the phage for exposure to <i>S. aureus</i> biofilms.</p><br />
<br/><br />
<br />
<h4>Modeling: </h4><p><br />
We have developed a mathematical model that describes the dynamics of our genetically engineered phage-assisted biofilm dispersal system. Using this model, we can predict the outcome of introducing a biofilm matrix-degrading phage to a biofilm. We have demonstrated that our model can be used as a tool to help design engineered systems similar to ours and to formulate informed hypotheses for phage-biofilm experiments. We have implemented this model in an easy-to-use Java program. Future work includes the extension of this model to account for components, such as genetic elements, that may impact the system and the development of a GUI with better graphical features.<br />
</p><br/><br />
<br />
<h4>Human Practices: </h4><p><br />
We have gathered hundreds of definitions of synthetic biology from the University of British Columbia community to construct <a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">promoter maps and word clouds</a> representing the prevalent ideas in our different disciplines' awareness of synthetic biology!<br/><br />
<br/>We have started the <a href="https://2010.igem.org/Team:British_Columbia/HP_arts">first iGEM synthetic biology art gallery</a> inviting all iGEM participants, as well as members of the public from Deviantart, IllustratedATCs and ATCsForAll to contribute.<br/><br/>We have forged the <a href="https://2010.igem.org/Team:British_Columbia/HP_nanowrimo">first NaNoWriMo-iGEM collaboration</a> to showcase novels featuring synthetic biology that are written by NaNoWriMo participants.<br/><br/><br />
Our experience communicating with the general public and even students in the sciences and applied sciences has been an enriching one. We have gleaned a lot of insights into public perception of synthetic biology, which still remains a very new and unfamiliar field to the public despite recent press about the first synthetic cell!<br/><br/><br />
Public opinion and risk perception appears to be more informed by controversial topics (e.g. genetically modified organisms and food) and literature featuring synthetic biology (from Frankenstein to Oryx and Crake). So outreach on the part of synthetic biologists still has quite a way to go in order to bring synthetic biology into the schools, workplaces and homes of the public. Our human practices project has generated ripples of thoughts about synthetic biology in various communities, stimulating individuals to find out more about synthetic biology and its recent developments. We hope that this will open up paths of communication between the synthetic biology research community and diverse public communities, which may lead to discussions and collaborations with the purposes of informing the public about synthetic biology and safely expanding its real world applications.<br/><br/><br />
Some specific future directions that address this cause include: (i) Actively inviting more non-science/engineering students to participate in iGEM outreach/projects/teams/Jamboree/fundraising, (ii) Establishing an annual iGEM tradition of stimulating and showcasing works of art or literature by members of iGEM and the general public featuring synthetic biology, and (iii) Investing in other collaborative outreach activities such as elementary/secondary school educational programs and synthetic biology university courses. <br />
</p><br/><br />
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<div id="news" style="height:2500px;"> <br />
<br/><br />
<br />
<center><h3>Quick Links</h3><br />
<a href="https://igem.org/Judging_Form.cgi?id=391">See our Judging Form</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/Parts">See our characterized Biobrick Parts</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/Project_Phage">See our new Phage Standard</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">See our Human Practices Project</a><br />
<br />
<br></br><br />
<br></br><h3>Consideration for Special Awards</h3><br />
<p>Besides striving for a Gold Medal and a place as one of the finalists, our team would also like to be considered for the following special awards:</p><br />
<a href="https://2010.igem.org/Team:British_Columbia/HumanPractices"><img src="https://static.igem.org/mediawiki/2010/f/fa/Bhpa.jpg"></a><br />
<p><b>"What a society deems important is enshrined in its art." -Harry Broudy</b></p><br />
<p>Our <a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">human practices project</a> presents the <b>first iGEM art gallery</b> dedicated to synthetic biology and all its diverse aspects. Sometimes art answers our deepest questions. Sometimes art only deepens the mystery. And sometimes a picture is just worth a thousand words. This is our way of helping human civilization consider, guide and address the impacts of ongoing advances in synthetic biology. Not limited to conventional artwork, our gallery also features synthetic biology <b>promoter maps</b> and poems. We also proudly present the <b>first iGEM collaboration with NaNoWriMo</b> to showcase 50,000 word novels featuring synthetic biology, written from scratch during the month of November!</p><br />
<a href="https://2010.igem.org/Team:British_Columbia/modeling_description"><img src="https://static.igem.org/mediawiki/2010/1/17/Bm.jpg"></a><br />
<p><b>"One of the most insidious and nefarious properties of scientific models is their tendency to take over, and sometimes supplant, reality." -Erwin Chargaff</b></p><br />
<p>Right before your eyes, watch how the population dynamics of a biofilm is affected by the introduction of bacteriophage and a biofilm matrix-degrading enzyme. Using our <a href="https://2010.igem.org/Team:British_Columbia/modeling_description">model</a>, we are able to run simulations that predict outcomes of the system and construct informed hypotheses to test in reality.</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia/Project_Phage"><img src="https://static.igem.org/mediawiki/2010/7/7f/Bs.jpg"></a><br />
<p><b>"Acceptance of prevailing standards often means we have no standards of our own." -Jean Toomer<br />
</b></p><br />
<p>There are standards for prokaryotes and standards for eukaryotes. But what about standards for the living dead? Our new <a href="https://2010.igem.org/Team:British_Columbia/Project_Phage">Phage standard</a> lays down a foundation for future work involving viruses and integrating Biobrick parts into their genome. Prepare to be infected!!!</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia"><img src="https://static.igem.org/mediawiki/2010/3/31/Bw.jpg"></a><br />
<p>We've worked really hard on our wiki to make it accessible, fun and interactive! So we hope that future iGEM teams, students worldwide, and even the general public will visit us here and see what iGEM and synthetic biology is about!</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2010/5/59/Bpbp.jpg"><br />
<p>Come and see our poster and team presentation! It will be a great opportunity to meet our team and learn more about our project. A soft copy of our poster and video of our presentation will be linked here during the Jamboree.</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia"><img src="https://static.igem.org/mediawiki/2010/6/68/Bhmp.jpg"></a><br />
<p>Several diseases and medical conditions are known to be caused by biofilm infections. Pathogens existing in biofilms survive under harsher conditions and are much more difficult to eliminate than free-floating pathogens. Our project aims to engineer a bacteriophage equipped with a biofilm matrix-degrading enzyme to eradicate pathogenic Staphylococcus aureus biofilms. Door knob, we shall fear thee no longer.</p><br />
</center><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/Project_OutlookTeam:British Columbia/Project Outlook2010-10-27T23:47:18Z<p>Ayjchan: </p>
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<div>__NOTOC__<br />
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<img src="https://static.igem.org/mediawiki/2010/a/ac/Medals.jpg"></src><br />
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<h3>Project Achievements & Future Directions</h3><br />
<br />
<p><b>Biofilm: </b><br/><br/><br />
We have obtained growth curves for <i>S. aureus</i> strains RN4220 and 8325-4 that demonstrate a steady growth phase followed by an oscillatory state of dynamic equilibrium. We have also optimized the existing protocol for biofilm quantification. Additionally, values derived from the biofilm experiments were integral to deriving realistic results from modeling simulations.<br/><br/><br />
The existing curve has demonstrated that 9 hours is the optimal time point for exposure to the matrix-degrading enzyme, DspB, as well as the engineered phage construct with DspB and quorum sensing genes. Future experiments testing the biofilm response to DspB alone as well as DspB incorporated into a phage construct with the quorum sensing P2 promoter will enable the characterization of the construct’s effectiveness against the biofilms of <i>S. aureus</i> strains RN4220 and 8325-4.<br/><br/><br />
<br />
<br />
<p><b>Phage Standard: </b><br/><br/><br />
We developed a phage standard that allows for modification of any lysogenic bacteriophage as part of the Biobrick standard. The phage standard works around the problems of illegal cut sites and prohibitively large plasmids. We hope the standard will serve as a foundational advance towards phage research within the iGEM competition, the BioBrick registry and the synthetic biology community as a whole.<br/><br/><br />
In the future we hope to continue developing this standard and optimizing the process of modifying lysogenic phage DNA. We will strive to submit a fully functional set of parts that have been demonstrated to work following the phage standard in the lab. Hopefully we will also be able to include lytic phages under the scope of the phage standard. With some luck we could expand the number of chassis available to iGEM and the BioBrick registry by introducing integration site vectors for multiple species and strains.<br/><br/><br />
<br />
<p><b>Quorum Sensing: </b><br/><br/><br />
We have made constructs to characterize the P2 promoter (BBa_I746104) of <i>S. aureus</i> via fluorescent protein production. In order to directly relate AIP to P2 promoter activity, we chose to use an agr null strain. As a next step, genes encoding AgrAC from <i>S. aureus</i> should be put on the same plasmid (the <i>S. aureus</i>/<i>E. coli</i> shuttle vector, pCN33) as the reporter constructs and transformed into agr-null <i>S. aureus</i>. This would allow proper characterization of P2 activity in the presence of AIP. Primers have already been designed and submitted to PCR the genes encoding AgrAC. Additionally, the replicon of the <i>S. aureus</i> pCN33 plasmid can be made into a BioBrick part to facilitate the expression and characterization of BioBrick parts in <i>S. aureus</i>.</p><br/><br />
<br />
<p><b>DspB: </b><br><br />
We have contributed to the biobrick parts registry by submitting a new part: DspB, an enzyme that degrades poly-ß-(1,6)-linked N-acetylglucosamine bonds. We have demonstrated that dspB works through a crude cell enzyme activity assay and have added this information to the Registry.<br/><br/><br />
We are currently working on obtaining data from the exposure of DspB protein on a <i>S. aureus</i> biofilm as well as isolating DspB via a histidine tag to attain further characterization data. We hope to gather this data before the presentation. If not fully completed, these components of the sub-team should be future directions.<br />
Future directions include incorporating DspB protein into the phage for exposure to <i>S. aureus</i> biofilms.</p><br />
<br/><br />
<br />
<p><b>Modeling: </b><br/><br />
We have developed a mathematical model that describes the dynamics of our genetically engineered phage-assisted biofilm dispersal system. Using this model, we can predict the outcome of introducing a biofilm matrix-degrading phage to a biofilm. We have demonstrated that our model can be used as a tool to help design engineered systems similar to ours and to formulate informed hypotheses for phage-biofilm experiments. We have implemented this model in an easy-to-use Java program. Future work includes the extension of this model to account for components, such as genetic elements, that may impact the system and the development of a GUI with better graphical features.<br />
</p><br/><br />
<br />
<p><b>Human Practices: </b><br/><br/><br />
We have gathered hundreds of definitions of synthetic biology from the University of British Columbia community to construct <a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">promoter maps and word clouds</a> representing the prevalent ideas in our different disciplines' awareness of synthetic biology!<br/><br />
<br/>We have started the <a href="https://2010.igem.org/Team:British_Columbia/HP_arts">first iGEM synthetic biology art gallery</a> inviting all iGEM participants, as well as members of the public from Deviantart, IllustratedATCs and ATCsForAll to contribute.<br/><br/>We have forged the <a href="https://2010.igem.org/Team:British_Columbia/HP_nanowrimo">first NaNoWriMo-iGEM collaboration</a> to showcase novels featuring synthetic biology that are written by NaNoWriMo participants.<br/><br/><br />
Our experience communicating with the general public and even students in the sciences and applied sciences has been an enriching one. We have gleaned a lot of insights into public perception of synthetic biology, which still remains a very new and unfamiliar field to the public despite recent press about the first synthetic cell!<br/><br/><br />
Public opinion and risk perception appears to be more informed by controversial topics (e.g. genetically modified organisms and food) and literature featuring synthetic biology (from Frankenstein to Oryx and Crake). So outreach on the part of synthetic biologists still has quite a way to go in order to bring synthetic biology into the schools, workplaces and homes of the public. Our human practices project has generated ripples of thoughts about synthetic biology in various communities, stimulating individuals to find out more about synthetic biology and its recent developments. We hope that this will open up paths of communication between the synthetic biology research community and diverse public communities, which may lead to discussions and collaborations with the purposes of informing the public about synthetic biology and safely expanding its real world applications.<br/><br/><br />
Some specific future directions that address this cause include: (i) Actively inviting more non-science/engineering students to participate in iGEM outreach/projects/teams/Jamboree/fundraising, (ii) Establishing an annual iGEM tradition of stimulating and showcasing works of art or literature by members of iGEM and the general public featuring synthetic biology, and (iii) Investing in other collaborative outreach activities such as elementary/secondary school educational programs and synthetic biology university courses. <br />
</p><br/><br />
<br />
</div> <!-- end SubWrapper --><br />
<br />
<div id="news" style="height:2500px;"> <br />
<br/><br />
<br />
<center><h3>Quick Links</h3><br />
<a href="https://igem.org/Judging_Form.cgi?id=391">See our Judging Form</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/Parts">See our characterized Biobrick Parts</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/Project_Phage">See our new Phage Standard</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">See our Human Practices Project</a><br />
<br />
<br></br><br />
<br></br><h3>Consideration for Special Awards</h3><br />
<p>Besides striving for a Gold Medal and a place as one of the finalists, our team would also like to be considered for the following special awards:</p><br />
<a href="https://2010.igem.org/Team:British_Columbia/HumanPractices"><img src="https://static.igem.org/mediawiki/2010/f/fa/Bhpa.jpg"></a><br />
<p><b>"What a society deems important is enshrined in its art." -Harry Broudy</b></p><br />
<p>Our <a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">human practices project</a> presents the <b>first iGEM art gallery</b> dedicated to synthetic biology and all its diverse aspects. Sometimes art answers our deepest questions. Sometimes art only deepens the mystery. And sometimes a picture is just worth a thousand words. This is our way of helping human civilization consider, guide and address the impacts of ongoing advances in synthetic biology. Not limited to conventional artwork, our gallery also features synthetic biology <b>promoter maps</b> and poems. We also proudly present the <b>first iGEM collaboration with NaNoWriMo</b> to showcase 50,000 word novels featuring synthetic biology, written from scratch during the month of November!</p><br />
<a href="https://2010.igem.org/Team:British_Columbia/modeling_description"><img src="https://static.igem.org/mediawiki/2010/1/17/Bm.jpg"></a><br />
<p><b>"One of the most insidious and nefarious properties of scientific models is their tendency to take over, and sometimes supplant, reality." -Erwin Chargaff</b></p><br />
<p>Right before your eyes, watch how the population dynamics of a biofilm is affected by the introduction of bacteriophage and a biofilm matrix-degrading enzyme. Using our <a href="https://2010.igem.org/Team:British_Columbia/modeling_description">model</a>, we are able to run simulations that predict outcomes of the system and construct informed hypotheses to test in reality.</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia/Project_Phage"><img src="https://static.igem.org/mediawiki/2010/7/7f/Bs.jpg"></a><br />
<p><b>"Acceptance of prevailing standards often means we have no standards of our own." -Jean Toomer<br />
</b></p><br />
<p>There are standards for prokaryotes and standards for eukaryotes. But what about standards for the living dead? Our new <a href="https://2010.igem.org/Team:British_Columbia/Project_Phage">Phage standard</a> lays down a foundation for future work involving viruses and integrating Biobrick parts into their genome. Prepare to be infected!!!</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia"><img src="https://static.igem.org/mediawiki/2010/3/31/Bw.jpg"></a><br />
<p>We've worked really hard on our wiki to make it accessible, fun and interactive! So we hope that future iGEM teams, students worldwide, and even the general public will visit us here and see what iGEM and synthetic biology is about!</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2010/5/59/Bpbp.jpg"><br />
<p>Come and see our poster and team presentation! It will be a great opportunity to meet our team and learn more about our project. A soft copy of our poster and video of our presentation will be linked here during the Jamboree.</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia"><img src="https://static.igem.org/mediawiki/2010/6/68/Bhmp.jpg"></a><br />
<p>Several diseases and medical conditions are known to be caused by biofilm infections. Pathogens existing in biofilms survive under harsher conditions and are much more difficult to eliminate than free-floating pathogens. Our project aims to engineer a bacteriophage equipped with a biofilm matrix-degrading enzyme to eradicate pathogenic Staphylococcus aureus biofilms. Door knob, we shall fear thee no longer.</p><br />
</center><br />
<br />
<br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/PartsTeam:British Columbia/Parts2010-10-27T22:03:05Z<p>Ayjchan: </p>
<hr />
<div>__NOTOC__<br />
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<br/><div><table align="center" width="90%" cellspacing="0" border="1" cellpadding="1"><caption><b>Favorite British_Columbia 2010 iGEM Team Parts</b></caption><br />
<tr><td colspan="2"><a href="http://partsregistry.org/Help:Availability_and_usefulness"><center><b>?</center></b></a><td><b>Name</b><td><b>Type</b><td><b>Description<td>Designer<td>Length</b></td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391000">BBa_K391000</a><td>Measurement<td>Construct for characterization of P2 activity in the presence of AIP<td>Phillip Chu<td>979</td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391001">BBa_K391001</a><td>Measurement<td>Construct for characterization of P2 activity in the presence of AIP<td>Phillip Chu<td>1018</td></tr><br />
<tr><td><td>W<td><a href="http://partsregistry.org/Part:BBa_K391006">BBa_K391006</a><td>Coding<td>DspB-carbohydrate digesting protein<td>Vicki Ma<td>1086</td></tr><br />
<tr><td><td><td><a href="http://partsregistry.org/Part:BBa_K391007">BBa_K391007</a><td>Composite<td>constitutive promoter-RBS-DspB (J23100:B0034:K391006)<td>Marianne Park<td>1149</td></tr><br />
<tr><td><td><td><a href="http://partsregistry.org/Part:BBa_K391008">BBa_K391008</a><td>Composite<td>constitutive promoter-RBS-DspB-terminator (J23100:B0034:K391006:B0014)<td>Marianne Park<td>1252</td></tr><br />
</table></div><br/><br/><br />
<div><table align="center" width="90%" cellspacing="0" border="1" cellpadding="1"><caption><b>British_Columbia 2010 iGEM Team Parts Sandbox</b></caption><br />
<tr><td colspan="2"><a href="http://partsregistry.org/Help:Availability_and_usefulness"><center><b>?</a></b></center><td>Name<td>Type<td>Description<td>Designer<td>Length</td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391002">BBa_K391002</a><td>Primer<td>Forward Primer to PCR agrCA (with RBS) from S. aureus<td>Phillip Chu<td>30</td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391003">BBa_K391003</a><td>Primer<td>Reverse Primer to PCR agrCA (with RBS) from S. aureus<td>Phillip Chu<td>26</td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391004">BBa_K391004</a><td>Primer<td>Forward primer to PCR S. aureus plasmid replicon<td>Phillip Chu<td>28</td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391005">BBa_K391005</a><td>Primer<td>Reverse primer to PCR S. aureus plasmid replicon<td>Phillip Chu<td>23</td></tr><br />
</table></div><br />
</div> <!-- end SubWrapper --><br />
<br />
<div id="news" style="height:500px;"> <br />
<br/><br />
<br />
<center><h3>Quick Links</h3><br />
<a href="https://igem.org/Judging_Form.cgi?id=391">See our Judging Form</a><br />
<br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/Project_outlook">See our Project Outlook</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/Project_QS">See our Quorum Sensing page</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/Project_DspB">See our DspB page</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/Project_Phage">See our new Phage Standard</a><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/PartsTeam:British Columbia/Parts2010-10-27T22:02:36Z<p>Ayjchan: </p>
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<br/><div><table align="center" width="90%" cellspacing="0" border="1" cellpadding="1"><caption><b>Favorite British_Columbia 2010 iGEM Team Parts</b></caption><br />
<tr><td colspan="2"><a href="http://partsregistry.org/Help:Availability_and_usefulness"><center><b>?</center></b></a><td><b>Name</b><td><b>Type</b><td><b>Description<td>Designer<td>Length</b></td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391000">BBa_K391000</a><td>Measurement<td>Construct for characterization of P2 activity in the presence of AIP<td>Phillip Chu<td>979</td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391001">BBa_K391001</a><td>Measurement<td>Construct for characterization of P2 activity in the presence of AIP<td>Phillip Chu<td>1018</td></tr><br />
<tr><td><td>W<td><a href="http://partsregistry.org/Part:BBa_K391006">BBa_K391006</a><td>Coding<td>DspB-carbohydrate digesting protein<td>Vicki Ma<td>1086</td></tr><br />
<tr><td><td><td><a href="http://partsregistry.org/Part:BBa_K391007">BBa_K391007</a><td>Composite<td>constitutive promoter-RBS-DspB (J23100:B0034:K391006)<td>Marianne Park<td>1149</td></tr><br />
<tr><td><td><td><a href="http://partsregistry.org/Part:BBa_K391008">BBa_K391008</a><td>Composite<td>constitutive promoter-RBS-DspB-terminator (J23100:B0034:K391006:B0014)<td>Marianne Park<td>1252</td></tr><br />
</table></div><br/><br/><br />
<div><table align="center" width="90%" cellspacing="0" border="1" cellpadding="1"><caption><b>British_Columbia 2010 iGEM Team Parts Sandbox</b></caption><br />
<tr><td colspan="2"><a href="http://partsregistry.org/Help:Availability_and_usefulness"><center><b>?</a></b></center><td>Name<td>Type<td>Description<td>Designer<td>Length</td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391002">BBa_K391002</a><td>Primer<td>Forward Primer to PCR agrCA (with RBS) from S. aureus<td>Phillip Chu<td>30</td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391003">BBa_K391003</a><td>Primer<td>Reverse Primer to PCR agrCA (with RBS) from S. aureus<td>Phillip Chu<td>26</td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391004">BBa_K391004</a><td>Primer<td>Forward primer to PCR S. aureus plasmid replicon<td>Phillip Chu<td>28</td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391005">BBa_K391005</a><td>Primer<td>Reverse primer to PCR S. aureus plasmid replicon<td>Phillip Chu<td>23</td></tr><br />
</table></div><br />
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<a href="https://igem.org/Judging_Form.cgi?id=391">See our Judging Form</a><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/PartsTeam:British Columbia/Parts2010-10-27T22:02:17Z<p>Ayjchan: </p>
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<br/><br/><div><table align="center" width="90%" cellspacing="0" border="1" cellpadding="1"><caption><b>Favorite British_Columbia 2010 iGEM Team Parts</b></caption><br />
<tr><td colspan="2"><a href="http://partsregistry.org/Help:Availability_and_usefulness"><center><b>?</center></b></a><td><b>Name</b><td><b>Type</b><td><b>Description<td>Designer<td>Length</b></td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391000">BBa_K391000</a><td>Measurement<td>Construct for characterization of P2 activity in the presence of AIP<td>Phillip Chu<td>979</td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391001">BBa_K391001</a><td>Measurement<td>Construct for characterization of P2 activity in the presence of AIP<td>Phillip Chu<td>1018</td></tr><br />
<tr><td><td>W<td><a href="http://partsregistry.org/Part:BBa_K391006">BBa_K391006</a><td>Coding<td>DspB-carbohydrate digesting protein<td>Vicki Ma<td>1086</td></tr><br />
<tr><td><td><td><a href="http://partsregistry.org/Part:BBa_K391007">BBa_K391007</a><td>Composite<td>constitutive promoter-RBS-DspB (J23100:B0034:K391006)<td>Marianne Park<td>1149</td></tr><br />
<tr><td><td><td><a href="http://partsregistry.org/Part:BBa_K391008">BBa_K391008</a><td>Composite<td>constitutive promoter-RBS-DspB-terminator (J23100:B0034:K391006:B0014)<td>Marianne Park<td>1252</td></tr><br />
</table></div><br/><br/><br />
<div><table align="center" width="90%" cellspacing="0" border="1" cellpadding="1"><caption><b>British_Columbia 2010 iGEM Team Parts Sandbox</b></caption><br />
<tr><td colspan="2"><a href="http://partsregistry.org/Help:Availability_and_usefulness"><center><b>?</a></b></center><td>Name<td>Type<td>Description<td>Designer<td>Length</td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391002">BBa_K391002</a><td>Primer<td>Forward Primer to PCR agrCA (with RBS) from S. aureus<td>Phillip Chu<td>30</td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391003">BBa_K391003</a><td>Primer<td>Reverse Primer to PCR agrCA (with RBS) from S. aureus<td>Phillip Chu<td>26</td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391004">BBa_K391004</a><td>Primer<td>Forward primer to PCR S. aureus plasmid replicon<td>Phillip Chu<td>28</td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391005">BBa_K391005</a><td>Primer<td>Reverse primer to PCR S. aureus plasmid replicon<td>Phillip Chu<td>23</td></tr><br />
</table></div><br />
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<a href="https://igem.org/Judging_Form.cgi?id=391">See our Judging Form</a><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/PartsTeam:British Columbia/Parts2010-10-27T22:01:44Z<p>Ayjchan: </p>
<hr />
<div>__NOTOC__<br />
<br />
{{Template_HD_4}}<br />
<html><body id="parts" onload="setPageSize()"><br />
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<div id="SubWrapper"> <br />
<br/><br/><div><table align="center" width="90%" cellspacing="0" border="1" cellpadding="1"><caption><b>Favorite British_Columbia 2010 iGEM Team Parts</b></caption><br />
<tr><td colspan="2"><a href="http://partsregistry.org/Help:Availability_and_usefulness"><center><b>?</center></b></a><td><b>Name</b><td><b>Type</b><td><b>Description<td>Designer<td>Length</b></td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391000">BBa_K391000</a><td>Measurement<td>Construct for characterization of P2 activity in the presence of AIP<td>Phillip Chu<td>979</td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391001">BBa_K391001</a><td>Measurement<td>Construct for characterization of P2 activity in the presence of AIP<td>Phillip Chu<td>1018</td></tr><br />
<tr><td><td>W<td><a href="http://partsregistry.org/Part:BBa_K391006">BBa_K391006</a><td>Coding<td>DspB-carbohydrate digesting protein<td>Vicki Ma<td>1086</td></tr><br />
<tr><td><td><td><a href="http://partsregistry.org/Part:BBa_K391007">BBa_K391007</a><td>Composite<td>constitutive promoter-RBS-DspB (J23100:B0034:K391006)<td>Marianne Park<td>1149</td></tr><br />
<tr><td><td><td><a href="http://partsregistry.org/Part:BBa_K391008">BBa_K391008</a><td>Composite<td>constitutive promoter-RBS-DspB-terminator (J23100:B0034:K391006:B0014)<td>Marianne Park<td>1252</td></tr><br />
</table></div><br/><br/><br />
<div><table align="center" width="90%" cellspacing="0" border="1" cellpadding="1"><caption><b>British_Columbia 2010 iGEM Team Parts Sandbox</b></caption><br />
<tr><td colspan="2"><a href="http://partsregistry.org/Help:Availability_and_usefulness"><center><b>?</a></b></center><td>Name<td>Type<td>Description<td>Designer<td>Length</td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391002">BBa_K391002</a><td>Primer<td>Forward Primer to PCR agrCA (with RBS) from S. aureus<td>Phillip Chu<td>30</td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391003">BBa_K391003</a><td>Primer<td>Reverse Primer to PCR agrCA (with RBS) from S. aureus<td>Phillip Chu<td>26</td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391004">BBa_K391004</a><td>Primer<td>Forward primer to PCR S. aureus plasmid replicon<td>Phillip Chu<td>28</td></tr><br />
<tr><td>S<td><td><a href="http://partsregistry.org/Part:BBa_K391005">BBa_K391005</a><td>Primer<td>Reverse primer to PCR S. aureus plasmid replicon<td>Phillip Chu<td>23</td></tr><br />
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<br></br><a href="https://2010.igem.org/Team:British_Columbia/Project_QS">See our Quorum Sensing page</a><br />
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<br></br><a href="https://2010.igem.org/Team:British_Columbia/Project_Phage">See our new Phage Standard</a><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/modeling_resultsTeam:British Columbia/modeling results2010-10-27T19:59:21Z<p>Ayjchan: </p>
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<h3>Simulation Results</h3><br />
<p>We ran 1200-generation simulations using the parameters and starting conditions listed in Table 1. To predict the outcome of phage-assisted biofilm dispersal under two different systems-one in which phage particles and DspB are produced, and the other in which only phage particles are generated-we performed simulations by setting the DspB production rate, <i>S</i>, to 1) 0.0001 and 2) 0. To determine whether the phage infection rate and half-life are critical to the design and engineering of biofilm-degradation systems, we applied sensitivity analyses using a reasonably comprehensive range of values for the parameters.</p><br />
<h4>The Binary Outcome</h4><br />
<p><b>Biofilm Death</b></p><br />
<center><img src="https://static.igem.org/mediawiki/2010/d/d2/UBC_main_death.jpg" width="600"></center><br/><br />
<p><b>Figure 1: </b>The simulated behavior of the biofilm and phage populations over 1200 mins (20 hrs). The percent of the total biofilm population remaining after phage introduction (blue) sharply declines at 400 min. This coincides with the sharp increase of the percent of the biofilm population infected by phage (green). The phage population (here, represented by the P-factor) peaks shortly after 400 mins. These features suggest that the accumulation of phage particles in the biofilm between 0 and 400 min triggers mass host cell lysis leading to the destruction of the biofilm at the 1200<sup>th</sup> min.</p><br/><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2010/6/63/UBC_main_death_sub.jpg" width="600"></center><br/><br />
<p><b>Figure 2: </b>The dynamics of the extracellular concentrations of AIP (purple) and DspB (red) over 1200 mins (20 hrs). The maximum level of AIP concentration and the activation threshold level are indicated by the upper and lower dashed lines, respectively. AIP concentration is shown to steadily decrease over time, but does not fall below the threshold level during the 1200 mins. This is due to the relatively high rate of AIP degradation rate arbitrarily set for this simulation. DspB concentration rises rapidly at 400 min, coinciding with decrease in biofilm population and increase in phage population (indicated in Figure 1), and then peaks at 600 min when the biofilm population is substantially reduced.</p><br/><p><br />
<br />
<p><b>Biofilm Survival</b><br/><br />
<center><img src="https://static.igem.org/mediawiki/2010/7/71/UBC_main_survival.jpg" width="600"></center><br/><br />
<p><b>Figure 3: </b>The simulated behavior of the biofilm and phage populations over 1200 mins (20 hrs) in a system where DspB production is absent (<i>i.e.</i> S = 0). The phage population (orange) experiences rapid decline at the beginning, when the phage particles attempt to infect the bacteria at the surface of the biofilm, resulting in a small subpopulation of infected bacteria (green). However, the total biofilm population (blue) is minimally impacted. The relatively constant biofilm and phage populations suggest that the phage is repeatedly attempting infection at the biofilm surface but fails to penetrate into the biofilm structure. This simulation demonstrates that DspB greatly facilitates phage invasion into the biofilm structure, as shown in Collins <i>et al.</i> (2007).</p><br/><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2010/5/57/UBC_main_survival_sub.jpg" width="600"></center><br/><br />
<p><b>Figure 4: </b>The dynamics of the extracellular concentrations of AIP (purple) and DspB (red) over 1200 mins (20 hrs). The maximum level of AIP concentration and the activation threshold level are indicated by the upper and lower dashed lines, respectively. AIP concentration is shown to steadily decrease over time, but does not fall below the threshold level during the 1200 mins. This is due to the relatively high rate of AIP degradation rate arbitrarily set for this simulation. DspB is completely absent.</p><br />
<br />
<h3>Sensitivity Analyses</h3><p><br />
<b>Phage virulence is critical to successful biofilm dispersal</b><br/><br />
<p><b>A</b></p><br />
<center><img src="https://static.igem.org/mediawiki/2010/a/ae/UBC_sen_kappa_1.jpg" width="600"></center><br/><br />
<p><b>B</b></p><br />
<center><img src="https://static.igem.org/mediawiki/2010/f/ff/UBC_sen_kappa_2.jpg" width="600"></center><br/><br />
<p><b>C</b></p><br />
<center><img src="https://static.igem.org/mediawiki/2010/0/0d/UBC_sen_kappa_3.jpg" width="600"></center><br/><p><br />
<p><b>Figure 5: </b>Sensitivity analysis of the phage infection rate parameter, <i>&kappa;</i>. A range of values for the infection rate is used. The infection rate is shown to have some impact on the dynamics of the total biofilm population (panel A), the infected biofilm subpopulation (panel B), and the phage population (panel C). When the rate is high (>=0.4), further rate increase has a diminishing effect on the population dynamics. When the rate is low (<=0.2), further decrease renders the phage particles ineffective for biofilm degradation (panel A and B). This analysis suggests that the phage must be sufficiently virulent for biofilm dispersal to occur. Interaction between the phage and the target biofilm bacteria is an important component in the design of biofilm-degradation systems.</p><br/><br />
<p><b>Half-life is not an outcome-determining factor</b></p><br/><br />
<p><b>A</b></p><br />
<center><img src="https://static.igem.org/mediawiki/2010/5/5b/UBC_sen_half_1.jpg" width="600"></center><br/><br />
<p><b>B</b></p><br />
<center><img src="https://static.igem.org/mediawiki/2010/c/c7/UBC_sen_half_2.jpg" width="600"></center><br/><br />
<p><b>C</b></p><br />
<center><img src="https://static.igem.org/mediawiki/2010/a/ad/UBC_sen_half_3.jpg" width="600"></center><br/><br />
<p><b>Figure 6: </b>Sensitivity analysis of the phage half-life parameter, <i>&eta;</i>. A broad range of values for the phage half-life is investigated. The half-life has little, if any, effect on the dynamics of biofilm (panel A and B) and phage (panel C) populations, and therefore is not a critical factor for biofilm dispersal. Unless the phage has an extremely short lifespan, it is not a major concern in phage-biofilm system design.</p><br />
<br />
<h3>Discussion</h3><br />
<p>Although our model was initially developed to simulate the behavior of our genetically engineered (GE) system, it can be applied to predict the dynamics of other biofilm systems that are introduced to a biofilm matrix-degrading phage. By setting the model parameters to values that represent the properties of a real-world biofilm system, we can simulate scenarios where the biofilms are exposed to a GE phage functionally similar to ours. We can also incorporate information on the properties of the phage by modifying certain parameters. Our model can be used to perform <i>in silico</i> phage-assisted biofilm-degradation experiments. By changing various parameters that represent properties of the biofilm bacteria or phage, we can simulate different phage-biofilm systems. Our model can also assist the designing similar synthetic biology systems. The sensitivity analyses demonstrated here can be performed to weigh the importance of properties that may impact the design of the system.</p><br />
<br></br></p><br />
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<p><b>Table 1: </b>List of parameters and starting conditions used for simulations.</p><br />
<center><img src="https://static.igem.org/mediawiki/2010/1/16/UBC_par_table1.jpg"></center><br />
<ul><br />
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<p>Click <a href="http://openwetware.org/wiki/IGEM:UBC/2009/Notebook/UBC_iGEM_2010">here</a> to view our lab notebook for more details of our experiments and protocols. Listed below are protocols specifically used for the Quorum Sensing Track. Scroll down to see what we learned this summer!</p><br />
<br />
<p><h3>Cell growth and preparation of cells for electroporation</h3><br />
<ol><li>An overnight culture grown in B2 broth (Table 1) with constant aeration at 37C was diluted 1/25 in 25mL of fresh b2 broth in a 300mL flask.</li><br />
<li>The cells were grown with constant aeration at 37C until they reached an OD600 of about 0.4 and were then harvested by centrifugation.</li><br />
<li>Upon harvesting the cells from the B2 broth, the cells were washed three times in an equal volume of deionized water, followed by second washes with 10% glycerol solution.</li><br />
<li>Following resuspension of the cells in the second 10% glycerol wash solution, the cell suspension was incubated for 15 min., centrifuged and the cell pellet resuspended in 800 ul of a l0% glycerol solution.</li><br />
<li>The final cell concentrations obtained were between 1 and 3 x 10^10 cell per mL.</li><br />
<li>All wash solutions and incubation were at 20C. All centrifugation were at 4000 rpm, 5 min, 20oC. The electrocompetent cells were used directly after preparation.</li><br />
<li>Alternatively, 70 ul aliquots of electrocompetent cells were frozen in microfuge tube at -80oC immediately after preparation.</li></ol></p><br />
<br />
<h3>Electroporation protocol for <i>S. aureus</i> RN4220</h3><br />
<ol><li>Remove competent cells (70uL aliquots) from -80C and thaw on ice for 30 minutes</li><br />
<li>Add ligation mix (1ug of DNA)</li><br />
<li>Incubate on ice for 30 minutes</li><br />
<li>Transfer 60uL of cell suspension-DNA mixture to 0.1cm gap electroporation cuvette. Make sure the cuvettes are on ice prior to this.</li><br />
<li>Cells and DNA electroporated at 20C, 100ohm resistance, 25uF capacitance (optimum time constant = 2.5ms), and 2.3kV in a Gene Pulser apparatus with pulse controller.</li><br />
<li>Place cells on ice and immediately resuspend in 940uL of B2 broth.</li><br />
<li>Transfer cells, DNA, and broth to eppendorf microcentrifuge tube. Make sure microcentrifuge tubes were previously on ice.</li><br />
<li>Incubate for at least 2 hours at 37C.</li><br />
<li>Plate on Tryptic Soy Agar (TSA) or NYE agar with appropriate antibiotic. In this case, erthrymycin.</li><br />
<li>Incubate plates at 37C for 48 hours.</li></ol></p><br />
<br />
<div><table align="center" width="65%" cellspacing="0" border="1" cellpadding="1"><caption><b>Table 1.</b> Media Composition</caption><br />
<tr><td>Media<td>Ingredients<td>References/Sources</td></tr><br />
<tr><td>B2<td><ul><li>1.0 % casein hydrolysate</li><li>2.5 % yeast extract</li><li>0.1% K2HPO4</li><li>0.5% glucose</li><li>2.5% NaCl</li><li>adjust pH to 7.5</li></ul><td>Schenk and Laddaga (1992)</td></tr><br />
<tr><td>NYE<td><ul><li>1.0 % casein hydrolysate</li><li>0.5% yeast extract</li><li>0.5% NaCl</li><li>adjust pH to 7.2</li></ul><td>Schenk and Laddaga (1992)</td></tr></table></div><br/><br />
<br />
<p>The above protocols (table included) are from the following paper: Schenk S, Laddaga RA. Improved method for electroporation of Staphylococcus aureus. FEMS Microbiol Lett. 1992 Jul 1;73(1-2):133–138</p><br />
<h3>Lessons Learned</h3><br />
<br />
<p>1. The 3-A method usually works. However, some parts seem harder to join to other parts. In the case of P2 (BBa_I764104), it took 4-5 times as long as expected to join it to 2 different GFP constructs. We haven't found a fix to this problem, even after varying several variables: insert:vector ratio, ligation volume, and changing ligase buffer. </p><br />
<p>2. PCR is a very good technique for verifying inserts. It is not so good for verifying small inserts (e.g. RBS) since the band would barely change. Sometimes, even when the PCR does not show correct bands, further restriction digests and sequencing yields the correct insert. </p><br />
<p>3. It is possible to clone genes directly from non-purified bacterial DNA. Simply pick a colony of cells containing the desired gene in the genome. This is of course harder to do than plasmid DNA. We've found that addition of DMSO (5-10%) helps the PCR (by presumably allowing primers and reagents to better reach the desired gene). This is true in the case of agrAC, where PCR was unsuccessful without the addition of DMSO.</p><br />
<p>4. Don't try to measure PCR product concentration using spectrophotometry right after a PCR. The concentration before and after reaction is basically the same because dNTP's also absorb similarly to strands of DNA. After purification, do measure DNA concentration.</p><br />
<p>5. Don't be afraid of going to the lab and start working. The best way to learn is through practice and troubleshooting. It also helps build up a good work flow.</p><br />
<p>6. Use polymerases with error checking ability (e.g. Phusion) when cloning genes. </p><br />
<p>7. Check antibiotics before using them. </p> <br />
<p>8. DNA is incredibly stable. DNA stocks will last a long time when stored properly, even in water.</p><br />
<p>9. Don't be afraid of asking for help or asking to use other lab groups' equipment.</p><br />
<p>10. DNA purification kits don't always work very well. Do check with a nanodrop or other method. Pay close attention to contaminants absorbing at other wavelengths (large peak), as this can fool the machine into thinking there is DNA. </p><br />
<p>11. Always sequence constructs if possible, even if simply joining 2 registry parts together. It is the only way to be sure the right parts have joined and reduces panic when parts do not work as expected.</p><br />
<br />
<br />
<br></br><br />
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See our <a href="http://openwetware.org/wiki/IGEM:UBC/2009/Notebook/UBC_iGEM_2010">UBC OWW notebook</a>.</p><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/Notebook_QSTeam:British Columbia/Notebook QS2010-10-27T16:22:33Z<p>Ayjchan: </p>
<hr />
<div>__NOTOC__<br />
<br />
{{Template_HD_4}}<br />
{{Template_HD_4}}<br />
<html><body id="notebook" onload="setPageSize()"><br />
<br />
<div id="super_main_wrapper"><br />
<div id="SubWrapper"> <br/><br />
<p>Click <a href="http://openwetware.org/wiki/IGEM:UBC/2009/Notebook/UBC_iGEM_2010">here</a> to view our lab notebook for more details of our experiments and protocols. Listed below are protocols specifically used for the Quorum Sensing Track. Scroll down to see what we learned this summer!</p><br />
<br />
<p><h3>Cell growth and preparation of cells for electroporation</h3><br />
<ol><li>An overnight culture grown in B2 broth (Table 1) with constant aeration at 37C was diluted 1/25 in 25mL of fresh b2 broth in a 300mL flask.</li><br />
<li>The cells were grown with constant aeration at 37C until they reached an OD600 of about 0.4 and were then harvested by centrifugation.</li><br />
<li>Upon harvesting the cells from the B2 broth, the cells were washed three times in an equal volume of deionized water, followed by second washes with 10% glycerol solution.</li><br />
<li>Following resuspension of the cells in the second 10% glycerol wash solution, the cell suspension was incubated for 15 min., centrifuged and the cell pellet resuspended in 800 ul of a l0% glycerol solution.</li><br />
<li>The final cell concentrations obtained were between 1 and 3 x 10^10 cell per mL.</li><br />
<li>All wash solutions and incubation were at 20C. All centrifugation were at 4000 rpm, 5 min, 20oC. The electrocompetent cells were used directly after preparation.</li><br />
<li>Alternatively, 70 ul aliquots of electrocompetent cells were frozen in microfuge tube at -80oC immediately after preparation.</li></ol></p><br />
<br />
<h3>Electroporation protocol for <i>S. aureus</i> RN4220</h3><br />
<ol><li>Remove competent cells (70uL aliquots) from -80C and thaw on ice for 30 minutes</li><br />
<li>Add ligation mix (1ug of DNA)</li><br />
<li>Incubate on ice for 30 minutes</li><br />
<li>Transfer 60uL of cell suspension-DNA mixture to 0.1cm gap electroporation cuvette. Make sure the cuvettes are on ice prior to this.</li><br />
<li>Cells and DNA electroporated at 20C, 100ohm resistance, 25uF capacitance (optimum time constant = 2.5ms), and 2.3kV in a Gene Pulser apparatus with pulse controller.</li><br />
<li>Place cells on ice and immediately resuspend in 940uL of B2 broth.</li><br />
<li>Transfer cells, DNA, and broth to eppendorf microcentrifuge tube. Make sure microcentrifuge tubes were previously on ice.</li><br />
<li>Incubate for at least 2 hours at 37C.</li><br />
<li>Plate on Tryptic Soy Agar (TSA) or NYE agar with appropriate antibiotic. In this case, erthrymycin.</li><br />
<li>Incubate plates at 37C for 48 hours.</li></ol></p><br />
<br />
<div><table align="center" width="65%" cellspacing="0" border="1" cellpadding="1"><caption><b>Table 1.</b> Media Composition</caption><br />
<tr><td>Media<td>Ingredients<td>References/Sources</td></tr><br />
<tr><td>B2<td><ul><li>1.0 % casein hydrolysate</li><li>2.5 % yeast extract</li><li>0.1% K2HPO4</li><li>0.5% glucose</li><li>2.5% NaCl</li><li>adjust pH to 7.5</li></ul><td>Schenk and Laddaga (1992)</td></tr><br />
<tr><td>NYE<td><ul><li>1.0 % casein hydrolysate</li><li>0.5% yeast extract</li><li>0.5% NaCl</li><li>adjust pH to 7.2</li></ul><td>Schenk and Laddaga (1992)</td></tr></table></div><br/><br />
<br />
<p>The above protocols (table included) are from the following paper: Schenk S, Laddaga RA. Improved method for electroporation of Staphylococcus aureus. FEMS Microbiol Lett. 1992 Jul 1;73(1-2):133–138</p><br />
<h3>Lessons Learned</h3><br />
<br />
<p>1. The 3-A method usually works. However, some parts seem harder to join to other parts. In the case of P2 (BBa_I764104), it took 4-5 times as long as expected to join it to 2 different GFP constructs. We haven't found a fix to this problem, even after varying several variables: insert:vector ratio, ligation volume, and changing ligase buffer. </p><br />
<p>2. PCR is a very good technique for verifying inserts. It is not so good for verifying small inserts (e.g. RBS) since the band would barely change. Sometimes, even when the PCR does not show correct bands, further restriction digests and sequencing yields the correct insert. </p><br />
<p>3. It is possible to clone genes directly from non-purified bacterial DNA. Simply pick a colony of cells containing the desired gene in the genome. This is of course harder to do than plasmid DNA. We've found that addition of DMSO (5-10%) helps the PCR (by presumably allowing primers and reagents to better reach the desired gene). This is true in the case of agrAC, where PCR was unsuccessful without the addition of DMSO.</p><br />
<p>4. Don't try to measure PCR product concentration using spectrophotometry right after a PCR. The concentration before and after reaction is basically the same because dNTP's also absorb similarly to strands of DNA. After purification, do measure DNA concentration.</p><br />
<p>5. Don't be afraid of going to the lab and start working. The best way to learn is through practice and troubleshooting. It also helps build up a good work flow.</p><br />
<p>6. Use polymerases with error checking ability (e.g. Phusion) when cloning genes. </p><br />
<p>7. Check antibiotics before using them. </p> <br />
<p>8. DNA is incredibly stable. DNA stocks will last a long time when stored properly, even in water.</p><br />
<p>9. Don't be afraid of asking for help or asking to use other lab groups' equipment.</p><br />
<p>10. DNA purification kits don't always work very well. Do check with a nanodrop or other method. Pay close attention to contaminants absorbing at other wavelengths (large peak), as this can fool the machine into thinking there is DNA. </p><br />
<p>11. Always sequence constructs if possible, even if simply joining 2 registry parts together. It is the only way to be sure the right parts have joined and reduces panic when parts do not work as expected.</p><br />
<br />
<br />
<br></br><br />
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<p><a href="http://openwetware.org/wiki/Main_Page">OpenWetWare</a> (OWW) is an effort to promote the sharing of information, know-how, and wisdom among researchers and groups who are working in biology & biological engineering. OWW hosts lab/research wikis, course wikis, protocol wikis and wiki blogs.<br></br><br/><br />
See our <a href="http://openwetware.org/wiki/IGEM:UBC/2009/Notebook/UBC_iGEM_2010">UBC OWW notebook</a>.</p><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/Notebook_QSTeam:British Columbia/Notebook QS2010-10-27T16:21:21Z<p>Ayjchan: </p>
<hr />
<div>__NOTOC__<br />
<br />
{{Template_HD_4}}<br />
{{Template_HD_4}}<br />
<html><body id="notebook" onload="setPageSize()"><br />
<br />
<div id="super_main_wrapper"><br />
<div id="SubWrapper"> <br/><br />
<p>Click <a href="http://openwetware.org/wiki/IGEM:UBC/2009/Notebook/UBC_iGEM_2010">here</a> to view our lab notebook for more details of our experiments and protocols. Listed below are protocols specifically used for the Quorum Sensing Track. Scroll down to see what we learned this summer!</p><br />
<br />
<p><h3>Cell growth and preparation of cells for electroporation</h3><br />
<ol><li>An overnight culture grown in B2 broth (Table 1) with constant aeration at 37C was diluted 1/25 in 25mL of fresh b2 broth in a 300mL flask.</li><br />
<li>The cells were grown with constant aeration at 37C until they reached an OD600 of about 0.4 and were then harvested by centrifugation.</li><br />
<li>Upon harvesting the cells from the B2 broth, the cells were washed three times in an equal volume of deionized water, followed by second washes with 10% glycerol solution.</li><br />
<li>Following resuspension of the cells in the second 10% glycerol wash solution, the cell suspension was incubated for 15 min., centrifuged and the cell pellet resuspended in 800 ul of a l0% glycerol solution.</li><br />
<li>The final cell concentrations obtained were between 1 and 3 x 10^10 cell per mL.</li><br />
<li>All wash solutions and incubation were at 20C. All centrifugation were at 4000 rpm, 5 min, 20oC. The electrocompetent cells were used directly after preparation.</li><br />
<li>Alternatively, 70 ul aliquots of electrocompetent cells were frozen in microfuge tube at -80oC immediately after preparation.</li></ol></p><br />
<br />
<h3>Electroporation protocol for <i>S. aureus</i> RN4220</h3><br />
<ol><li>Remove competent cells (70uL aliquots) from -80C and thaw on ice for 30 minutes</li><br />
<li>Add ligation mix (1ug of DNA)</li><br />
<li>Incubate on ice for 30 minutes</li><br />
<li>Transfer 60uL of cell suspension-DNA mixture to 0.1cm gap electroporation cuvette. Make sure the cuvettes are on ice prior to this.</li><br />
<li>Cells and DNA electroporated at 20C, 100ohm resistance, 25uF capacitance (optimum time constant = 2.5ms), and 2.3kV in a Gene Pulser apparatus with pulse controller.</li><br />
<li>Place cells on ice and immediately resuspend in 940uL of B2 broth.</li><br />
<li>Transfer cells, DNA, and broth to eppendorf microcentrifuge tube. Make sure microcentrifuge tubes were previously on ice.</li><br />
<li>Incubate for at least 2 hours at 37C.</li><br />
<li>Plate on Tryptic Soy Agar (TSA) or NYE agar with appropriate antibiotic. In this case, erthrymycin.</li><br />
<li>Incubate plates at 37C for 48 hours.</li></ol></p><br />
<br />
<div><table align="center" width="65%" cellspacing="0" border="1" cellpadding="1"><caption> Table 1</caption><br />
<tr><td>Media<td>Ingredients<td>References/Sources</td></tr><br />
<tr><td>B2<td><ul><li>1.0 % casein hydrolysate</li><li>2.5 % yeast extract</li><li>0.1% K2HPO4</li><li>0.5% glucose</li><li>2.5% NaCl</li><li>adjust pH to 7.5</li></ul><td>Schenk and Laddaga (1992)</td></tr><br />
<tr><td>NYE<td><ul><li>1.0 % casein hydrolysate</li><li>0.5% yeast extract</li><li>0.5% NaCl</li><li>adjust pH to 7.2</li></ul><td>Schenk and Laddaga (1992)</td></tr></table></div><br/><br />
<br />
<p>The above protocols (table included) are from the following paper: Schenk S, Laddaga RA. Improved method for electroporation of Staphylococcus aureus. FEMS Microbiol Lett. 1992 Jul 1;73(1-2):133–138</p><br />
<h3>Lessons Learned</h3><br />
<br />
<p>1. The 3-A method usually works. However, some parts seem harder to join to other parts. In the case of P2 (BBa_I764104), it took 4-5 times as long as expected to join it to 2 different GFP constructs. We haven't found a fix to this problem, even after varying several variables: insert:vector ratio, ligation volume, and changing ligase buffer. </p><br />
<p>2. PCR is a very good technique for verifying inserts. It is not so good for verifying small inserts (e.g. RBS) since the band would barely change. Sometimes, even when the PCR does not show correct bands, further restriction digests and sequencing yields the correct insert. </p><br />
<p>3. It is possible to clone genes directly from non-purified bacterial DNA. Simply pick a colony of cells containing the desired gene in the genome. This is of course harder to do than plasmid DNA. We've found that addition of DMSO (5-10%) helps the PCR (by presumably allowing primers and reagents to better reach the desired gene). This is true in the case of agrAC, where PCR was unsuccessful without the addition of DMSO.</p><br />
<p>4. Don't try to measure PCR product concentration using spectrophotometry right after a PCR. The concentration before and after reaction is basically the same because dNTP's also absorb similarly to strands of DNA. After purification, do measure DNA concentration.</p><br />
<p>5. Don't be afraid of going to the lab and start working. The best way to learn is through practice and troubleshooting. It also helps build up a good work flow.</p><br />
<p>6. Use polymerases with error checking ability (e.g. Phusion) when cloning genes. </p><br />
<p>7. Check antibiotics before using them. </p> <br />
<p>8. DNA is incredibly stable. DNA stocks will last a long time when stored properly, even in water.</p><br />
<p>9. Don't be afraid of asking for help or asking to use other lab groups' equipment.</p><br />
<p>10. DNA purification kits don't always work very well. Do check with a nanodrop or other method. Pay close attention to contaminants absorbing at other wavelengths (large peak), as this can fool the machine into thinking there is DNA. </p><br />
<p>11. Always sequence constructs if possible, even if simply joining 2 registry parts together. It is the only way to be sure the right parts have joined and reduces panic when parts do not work as expected.</p><br />
<br />
<br />
<br></br><br />
</div> <!-- end SubWrapper --><br />
<br />
<div id="news" style="height:1800px;"> <br />
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<p><a href="http://openwetware.org/wiki/Main_Page">OpenWetWare</a> (OWW) is an effort to promote the sharing of information, know-how, and wisdom among researchers and groups who are working in biology & biological engineering. OWW hosts lab/research wikis, course wikis, protocol wikis and wiki blogs.<br></br><br/><br />
See our <a href="http://openwetware.org/wiki/IGEM:UBC/2009/Notebook/UBC_iGEM_2010">UBC OWW notebook</a>.</p><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/Notebook_QSTeam:British Columbia/Notebook QS2010-10-27T16:20:56Z<p>Ayjchan: </p>
<hr />
<div>__NOTOC__<br />
<br />
{{Template_HD_4}}<br />
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<html><body id="notebook" onload="setPageSize()"><br />
<br />
<div id="super_main_wrapper"><br />
<div id="SubWrapper"> <br/><br />
<p>Click <a href="http://openwetware.org/wiki/IGEM:UBC/2009/Notebook/UBC_iGEM_2010">here</a> to view our lab notebook for more details of our experiments and protocols. Listed below are protocols specifically used for the Quorum Sensing Track. Scroll down to see what we learned this summer!</p><br />
<br />
<p><h3>Cell growth and preparation of cells for electroporation</h3><br />
<li>An overnight culture grown in B2 broth (Table 1) with constant aeration at 37C was diluted 1/25 in 25mL of fresh b2 broth in a 300mL flask.</li><br />
<li>The cells were grown with constant aeration at 37C until they reached an OD600 of about 0.4 and were then harvested by centrifugation.</li><br />
<li>Upon harvesting the cells from the B2 broth, the cells were washed three times in an equal volume of deionized water, followed by second washes with 10% glycerol solution.</li><br />
<li>Following resuspension of the cells in the second 10% glycerol wash solution, the cell suspension was incubated for 15 min., centrifuged and the cell pellet resuspended in 800 ul of a l0% glycerol solution.</li><br />
<li>The final cell concentrations obtained were between 1 and 3 x 10^10 cell per mL.</li><br />
<li>All wash solutions and incubation were at 20C. All centrifugation were at 4000 rpm, 5 min, 20oC. The electrocompetent cells were used directly after preparation.</li><br />
<li>Alternatively, 70 ul aliquots of electrocompetent cells were frozen in microfuge tube at -80oC immediately after preparation.</li></p></p></p><br />
<br />
<h3>Electroporation protocol for <i>S. aureus</i> RN4220</h3><br />
<ol><li>Remove competent cells (70uL aliquots) from -80C and thaw on ice for 30 minutes</li><br />
<li>Add ligation mix (1ug of DNA)</li><br />
<li>Incubate on ice for 30 minutes</li><br />
<li>Transfer 60uL of cell suspension-DNA mixture to 0.1cm gap electroporation cuvette. Make sure the cuvettes are on ice prior to this.</li><br />
<li>Cells and DNA electroporated at 20C, 100ohm resistance, 25uF capacitance (optimum time constant = 2.5ms), and 2.3kV in a Gene Pulser apparatus with pulse controller.</li><br />
<li>Place cells on ice and immediately resuspend in 940uL of B2 broth.</li><br />
<li>Transfer cells, DNA, and broth to eppendorf microcentrifuge tube. Make sure microcentrifuge tubes were previously on ice.</li><br />
<li>Incubate for at least 2 hours at 37C.</li><br />
<li>Plate on Tryptic Soy Agar (TSA) or NYE agar with appropriate antibiotic. In this case, erthrymycin.</li><br />
<li>Incubate plates at 37C for 48 hours.</li></p><br />
<br />
<div><table align="center" width="65%" cellspacing="0" border="1" cellpadding="1"><caption> Table 1</caption><br />
<tr><td>Media<td>Ingredients<td>References/Sources</td></tr><br />
<tr><td>B2<td><ul><li>1.0 % casein hydrolysate</li><li>2.5 % yeast extract</li><li>0.1% K2HPO4</li><li>0.5% glucose</li><li>2.5% NaCl</li><li>adjust pH to 7.5</li></ul><td>Schenk and Laddaga (1992)</td></tr><br />
<tr><td>NYE<td><ul><li>1.0 % casein hydrolysate</li><li>0.5% yeast extract</li><li>0.5% NaCl</li><li>adjust pH to 7.2</li></ul><td>Schenk and Laddaga (1992)</td></tr></table></div><br/><br />
<br />
<p>The above protocols (table included) are from the following paper: Schenk S, Laddaga RA. Improved method for electroporation of Staphylococcus aureus. FEMS Microbiol Lett. 1992 Jul 1;73(1-2):133–138</p><br />
<h3>Lessons Learned</h3><br />
<br />
<p>1. The 3-A method usually works. However, some parts seem harder to join to other parts. In the case of P2 (BBa_I764104), it took 4-5 times as long as expected to join it to 2 different GFP constructs. We haven't found a fix to this problem, even after varying several variables: insert:vector ratio, ligation volume, and changing ligase buffer. </p><br />
<p>2. PCR is a very good technique for verifying inserts. It is not so good for verifying small inserts (e.g. RBS) since the band would barely change. Sometimes, even when the PCR does not show correct bands, further restriction digests and sequencing yields the correct insert. </p><br />
<p>3. It is possible to clone genes directly from non-purified bacterial DNA. Simply pick a colony of cells containing the desired gene in the genome. This is of course harder to do than plasmid DNA. We've found that addition of DMSO (5-10%) helps the PCR (by presumably allowing primers and reagents to better reach the desired gene). This is true in the case of agrAC, where PCR was unsuccessful without the addition of DMSO.</p><br />
<p>4. Don't try to measure PCR product concentration using spectrophotometry right after a PCR. The concentration before and after reaction is basically the same because dNTP's also absorb similarly to strands of DNA. After purification, do measure DNA concentration.</p><br />
<p>5. Don't be afraid of going to the lab and start working. The best way to learn is through practice and troubleshooting. It also helps build up a good work flow.</p><br />
<p>6. Use polymerases with error checking ability (e.g. Phusion) when cloning genes. </p><br />
<p>7. Check antibiotics before using them. </p> <br />
<p>8. DNA is incredibly stable. DNA stocks will last a long time when stored properly, even in water.</p><br />
<p>9. Don't be afraid of asking for help or asking to use other lab groups' equipment.</p><br />
<p>10. DNA purification kits don't always work very well. Do check with a nanodrop or other method. Pay close attention to contaminants absorbing at other wavelengths (large peak), as this can fool the machine into thinking there is DNA. </p><br />
<p>11. Always sequence constructs if possible, even if simply joining 2 registry parts together. It is the only way to be sure the right parts have joined and reduces panic when parts do not work as expected.</p><br />
<br />
<br />
<br></br><br />
</div> <!-- end SubWrapper --><br />
<br />
<div id="news" style="height:1800px;"> <br />
<br/><center><a href="http://openwetware.org/wiki/Main_Page"><img src="https://static.igem.org/mediawiki/2010/2/21/OWW_Sticker.jpg"></a></center><br/><br />
<p><a href="http://openwetware.org/wiki/Main_Page">OpenWetWare</a> (OWW) is an effort to promote the sharing of information, know-how, and wisdom among researchers and groups who are working in biology & biological engineering. OWW hosts lab/research wikis, course wikis, protocol wikis and wiki blogs.<br></br><br/><br />
See our <a href="http://openwetware.org/wiki/IGEM:UBC/2009/Notebook/UBC_iGEM_2010">UBC OWW notebook</a>.</p><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/Project_OutlookTeam:British Columbia/Project Outlook2010-10-27T16:20:00Z<p>Ayjchan: </p>
<hr />
<div>__NOTOC__<br />
<br />
{{Template_HD_4}}<br />
<html><body id="project" onload="setPageSize()"><br />
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<div id="SubWrapper"> <br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/a/ac/Medals.jpg"></src><br />
</center><br />
<h3>Project Achievements & Future Directions</h3><br />
<br />
<p><b>Biofilm: </b><br/><br/><br />
We have obtained growth curves for <i>S. aureus</i> strains RN4220 and 8325-4 that demonstrate a steady growth phase followed by an oscillatory state of dynamic equilibrium. We have also optimized the existing protocol for biofilm quantification. Additionally, values derived from the biofilm experiments were integral to deriving realistic results from modeling simulations.<br/><br/><br />
The existing curve has demonstrated that 9 hours is the optimal time point for exposure to the matrix-degrading enzyme, DspB, as well as the engineered phage construct with DspB and quorum sensing genes. Future experiments testing the biofilm response to DspB alone as well as DspB incorporated into a phage construct with the quorum sensing P2 promoter will enable the characterization of the construct’s effectiveness against the biofilms of <i>S. aureus</i> strains RN4220 and 8325-4.<br/><br/><br />
<br />
<br />
<p><b>Phage Standard: </b><br/><br/><br />
We developed a phage standard that allows for modification of any lysogenic bacteriophage as part of the Biobrick standard. The phage standard works around the problems of illegal cut sites and prohibitively large plasmids. We hope the standard will serve as a foundational advance towards phage research within the iGEM competition, the BioBrick registry and the synthetic biology community as a whole.</p><br/><br />
<br />
<p><b>Quorum Sensing: </b><br/><br/><br />
We have made constructs to characterize the P2 promoter (BBa_I746104) of <i>S. aureus</i> via fluorescent protein production. In order to directly relate AIP to P2 promoter activity, we chose to use an agr null strain. As a next step, genes encoding AgrAC from <i>S. aureus</i> should be put on the same plasmid (the <i>S. aureus</i>/<i>E. coli</i> shuttle vector, pCN33) as the reporter constructs and transformed into agr-null <i>S. aureus</i>. This would allow proper characterization of P2 activity in the presence of AIP. Primers have already been designed and submitted to PCR the genes encoding AgrAC. Additionally, the replicon of the <i>S. aureus</i> pCN33 plasmid can be made into a BioBrick part to facilitate the expression and characterization of BioBrick parts in <i>S. aureus</i>.</p><br/><br />
<br />
<p><b>DspB: </b><br><br />
We have contributed to the biobrick parts registry by submitting a new part: DspB, an enzyme that degrades poly-ß-(1,6)-linked N-acetylglucosamine bonds. We have demonstrated that dspB works through a crude cell enzyme activity assay and have added this information to the Registry.<br/><br/><br />
We are currently working on obtaining data from the exposure of DspB protein on a <i>S. aureus</i> biofilm as well as isolating DspB via a histidine tag to attain further characterization data. We hope to gather this data before the presentation. If not fully completed, these components of the sub-team should be future directions.<br />
Future directions include incorporating DspB protein into the phage for exposure to <i>S. aureus</i> biofilms.</p><br />
<br/><br />
<br />
<p><b>Modeling: </b><br/><br/><br />
<br />
<br />
<p><b>Human Practices: </b><br/><br/><br />
We have gathered hundreds of definitions of synthetic biology from the University of British Columbia community to construct <a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">promoter maps and word clouds</a> representing the prevalent ideas in our different disciplines' awareness of synthetic biology!<br/><br />
<br/>We have started the <a href="https://2010.igem.org/Team:British_Columbia/HP_arts">first iGEM synthetic biology art gallery</a> inviting all iGEM participants, as well as members of the public from Deviantart, IllustratedATCs and ATCsForAll to contribute.<br/><br/>We have forged the <a href="https://2010.igem.org/Team:British_Columbia/HP_nanowrimo">first NaNoWriMo-iGEM collaboration</a> to showcase novels featuring synthetic biology that are written by NaNoWriMo participants.<br/><br/><br />
Our experience communicating with the general public and even students in the sciences and applied sciences has been an enriching one. We have gleaned a lot of insights into public perception of synthetic biology, which still remains a very new and unfamiliar field to the public despite recent press about the first synthetic cell!<br/><br/><br />
Public opinion and risk perception appears to be more informed by controversial topics (e.g. genetically modified organisms and food) and literature featuring synthetic biology (from Frankenstein to Oryx and Crake). So outreach on the part of synthetic biologists still has quite a way to go in order to bring synthetic biology into the schools, workplaces and homes of the public. Our human practices project has generated ripples of thoughts about synthetic biology in various communities, stimulating individuals to find out more about synthetic biology and its recent developments. We hope that this will open up paths of communication between the synthetic biology research community and diverse public communities, which may lead to discussions and collaborations with the purposes of informing the public about synthetic biology and safely expanding its real world applications.<br/><br/><br />
Some specific future directions that address this cause include: (i) Actively inviting more non-science/engineering students to participate in iGEM outreach/projects/teams/Jamboree/fundraising, (ii) Establishing an annual iGEM tradition of stimulating and showcasing works of art or literature by members of iGEM and the general public featuring synthetic biology, and (iii) Investing in other collaborative outreach activities such as elementary/secondary school educational programs and synthetic biology university courses. <br />
</p><br/><br />
<br />
</div> <!-- end SubWrapper --><br />
<br />
<div id="news" style="height:2500px;"> <br />
<br/><br />
<br />
<center><h3>Quick Links</h3><br />
<a href="https://igem.org/Judging_Form.cgi?id=391">See our Judging Form</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/Parts">See our characterized Biobrick Parts</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/Project_Phage">See our new Phage Standard</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">See our Human Practices Project</a><br />
<br />
<br></br><br />
<br></br><h3>Consideration for Special Awards</h3><br />
<p>Besides striving for a Gold Medal and a place as one of the finalists, our team would also like to be considered for the following special awards:</p><br />
<a href="https://2010.igem.org/Team:British_Columbia/HumanPractices"><img src="https://static.igem.org/mediawiki/2010/f/fa/Bhpa.jpg"></a><br />
<p><b>"What a society deems important is enshrined in its art." -Harry Broudy</b></p><br />
<p>Our <a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">human practices project</a> presents the <b>first iGEM art gallery</b> dedicated to synthetic biology and all its diverse aspects. Sometimes art answers our deepest questions. Sometimes art only deepens the mystery. And sometimes a picture is just worth a thousand words. This is our way of helping human civilization consider, guide and address the impacts of ongoing advances in synthetic biology. Not limited to conventional artwork, our gallery also features synthetic biology <b>promoter maps</b> and poems. We also proudly present the <b>first iGEM collaboration with NaNoWriMo</b> to showcase 50,000 word novels featuring synthetic biology, written from scratch during the month of November!</p><br />
<a href="https://2010.igem.org/Team:British_Columbia/modeling_description"><img src="https://static.igem.org/mediawiki/2010/1/17/Bm.jpg"></a><br />
<p><b>"One of the most insidious and nefarious properties of scientific models is their tendency to take over, and sometimes supplant, reality." -Erwin Chargaff</b></p><br />
<p>Right before your eyes, watch how the population dynamics of a biofilm is affected by the introduction of bacteriophage and a biofilm matrix-degrading enzyme. Using our <a href="https://2010.igem.org/Team:British_Columbia/modeling_description">model</a>, we are able to run simulations that predict outcomes of the system and construct informed hypotheses to test in reality.</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia/Project_Phage"><img src="https://static.igem.org/mediawiki/2010/7/7f/Bs.jpg"></a><br />
<p><b>"Acceptance of prevailing standards often means we have no standards of our own." -Jean Toomer<br />
</b></p><br />
<p>There are standards for prokaryotes and standards for eukaryotes. But what about standards for the living dead? Our new <a href="https://2010.igem.org/Team:British_Columbia/Project_Phage">Phage standard</a> lays down a foundation for future work involving viruses and integrating Biobrick parts into their genome. Prepare to be infected!!!</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia"><img src="https://static.igem.org/mediawiki/2010/3/31/Bw.jpg"></a><br />
<p>We've worked really hard on our wiki to make it accessible, fun and interactive! So we hope that future iGEM teams, students worldwide, and even the general public will visit us here and see what iGEM and synthetic biology is about!</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2010/5/59/Bpbp.jpg"><br />
<p>Come and see our poster and team presentation! It will be a great opportunity to meet our team and learn more about our project. A soft copy of our poster and video of our presentation will be linked here during the Jamboree.</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia"><img src="https://static.igem.org/mediawiki/2010/6/68/Bhmp.jpg"></a><br />
<p>Several diseases and medical conditions are known to be caused by biofilm infections. Pathogens existing in biofilms survive under harsher conditions and are much more difficult to eliminate than free-floating pathogens. Our project aims to engineer a bacteriophage equipped with a biofilm matrix-degrading enzyme to eradicate pathogenic Staphylococcus aureus biofilms. Door knob, we shall fear thee no longer.</p><br />
</center><br />
<br />
<br />
</div> <!-- end news --><br />
</div> <!-- end Super_main_wrapper --><br />
<script type="text/javascript"><br />
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len = document.getElementById('super_main_wrapper').offsetHeight;<br />
document.getElementById('bodyContent').style.height = len + 'px';<br />
document.getElementById('news').style.height = len + 'px';<br />
}<br />
</script><br />
</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/Project_OutlookTeam:British Columbia/Project Outlook2010-10-27T16:16:04Z<p>Ayjchan: </p>
<hr />
<div>__NOTOC__<br />
<br />
{{Template_HD_4}}<br />
<html><body id="project" onload="setPageSize()"><br />
<br />
<div id="super_main_wrapper"><br />
<div id="SubWrapper"> <br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/a/ac/Medals.jpg"></src><br />
</center><br />
<h3>Project Achievements & Future Directions</h3><br />
<br />
<p><b>Biofilm: </b><br/><br/><br />
We have obtained growth curves for <i>S. aureus</i> strains RN4220 and 8325-4 that demonstrate a steady growth phase followed by an oscillatory state of dynamic equilibrium. We have also optimized the existing protocol for biofilm quantification. Additionally, values derived from the biofilm experiments were integral to deriving realistic results from modeling simulations.<br/><br/><br />
The existing curve has demonstrated that 9 hours is the optimal time point for exposure to the matrix-degrading enzyme, DspB, as well as the engineered phage construct with DspB and quorum sensing genes. Future experiments testing the biofilm response to DspB alone as well as DspB incorporated into a phage construct with the quorum sensing P2 promoter will enable the characterization of the construct’s effectiveness against the biofilms of <i>S. aureus</i> strains RN4220 and 8325-4.<br/><br/><br />
<br />
<br />
<p><b>Phage Standard: </b><br/><br/><br />
We developed a phage standard that allows for modification of any lysogenic bacteriophage as part of the Biobrick standard. The phage standard works around the problems of illegal cut sites and prohibitively large plasmids. We hope the standard will serve as a foundational advance towards phage research within the iGEM competition, the BioBrick registry and the synthetic biology community as a whole.</p><br/><br />
<br />
<p><b>Quorum Sensing: </b><br/><br/><br />
We have made constructs to characterize the P2 promoter (BBa_I746104) of <i>S. aureus</i> via fluorescent protein production. In order to directly relate AIP to P2 promoter activity, an agr null strain was used. Primers were designed to PCR off the genes encoding AgrAC from <i>S. aureus</i> in order to allow detection of AIP within the agr null strain. We have also made plans to PCR a <i> S. aureus </i> plasmid replicon to allow BioBrick plasmids to replicate and segregate within a <i> S. aureus </i> host. </p><br/><br />
<br />
<p><b>DspB: </b><br><br />
We have contributed to the biobrick parts registry by submitting a new part: DspB, an enzyme that degrades poly-ß-(1,6)-linked N-acetylglucosamine bonds. We have demonstrated that dspB works through a crude cell enzyme activity assay and have added this information to the Registry.<br/><br/><br />
<p><b>Modeling: </b><br/><br/><br />
<p><b>Human Practices: </b><br/><br/><br />
We have gathered hundreds of definitions of synthetic biology from the University of British Columbia community to construct <a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">promoter maps and word clouds</a> representing the prevalent ideas in our different disciplines' awareness of synthetic biology!<br/><br />
<br/>We have started the <a href="https://2010.igem.org/Team:British_Columbia/HP_arts">first iGEM synthetic biology art gallery</a> inviting all iGEM participants, as well as members of the public from Deviantart, IllustratedATCs and ATCsForAll to contribute.<br/><br/>We have forged the <a href="https://2010.igem.org/Team:British_Columbia/HP_nanowrimo">first NaNoWriMo-iGEM collaboration</a> to showcase novels featuring synthetic biology that are written by NaNoWriMo participants.<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<b>Phage: </b><br/><br/><br />
<p><b>Quorum Sensing: </b><br/><br/><br />
As a next step, genes encoding AgrAC from <i>S. aureus</i> should be put on the same plasmid (the <i>S. aureus</i>/<i>E. coli</i> shuttle vector, pCN33) as the reporter constructs and transformed into agr-null <i>S. aureus</i>. This would allow proper characterization of P2 activity in the presence of AIP. Additionally, the replicon of pCN33 can be cloned off and made into a BioBrick part to facilitate the expression and characterization of BioBrick parts in <i>S. aureus</i>.</p><br/><br />
<p><b>DspB: </b><br />
<p>We are currently working on obtaining data from the exposure of DspB protein on a <i>S. aureus</i> biofilm as well as isolating DspB via a histidine tag to attain further characterization data. We hope to gather this data before the presentation. If not fully completed, these components of the sub-team should be future directions.<br />
Future directions include incorporating DspB protein into the phage for exposure to <i>S. aureus</i> biofilms.</p><br />
<br/><br />
<p><b>Modeling: </b><br/><br/><br />
<p><b>Human Practices: </b><br/><br/><br />
Our experience communicating with the general public and even students in the sciences and applied sciences has been an enriching one. We have gleaned a lot of insights into public perception of synthetic biology, which still remains a very new and unfamiliar field to the public despite recent press about the first synthetic cell!<br/><br/><br />
Public opinion and risk perception appears to be more informed by controversial topics (e.g. genetically modified organisms and food) and literature featuring synthetic biology (from Frankenstein to Oryx and Crake). So outreach on the part of synthetic biologists still has quite a way to go in order to bring synthetic biology into the schools, workplaces and homes of the public. Our human practices project has generated ripples of thoughts about synthetic biology in various communities, stimulating individuals to find out more about synthetic biology and its recent developments. We hope that this will open up paths of communication between the synthetic biology research community and diverse public communities, which may lead to discussions and collaborations with the purposes of informing the public about synthetic biology and safely expanding its real world applications.<br/><br/><br />
Some specific future directions that address this cause include: (i) Actively inviting more non-science/engineering students to participate in iGEM outreach/projects/teams/Jamboree/fundraising, (ii) Establishing an annual iGEM tradition of stimulating and showcasing works of art or literature by members of iGEM and the general public featuring synthetic biology, and (iii) Investing in other collaborative outreach activities such as elementary/secondary school educational programs and synthetic biology university courses. <br />
</p><br />
<br />
</div> <!-- end SubWrapper --><br />
<br />
<div id="news" style="height:2500px;"> <br />
<br/><br />
<br />
<center><h3>Quick Links</h3><br />
<a href="https://igem.org/Judging_Form.cgi?id=391">See our Judging Form</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/Parts">See our characterized Biobrick Parts</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/Project_Phage">See our new Phage Standard</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">See our Human Practices Project</a><br />
<br />
<br></br><br />
<br></br><h3>Consideration for Special Awards</h3><br />
<p>Besides striving for a Gold Medal and a place as one of the finalists, our team would also like to be considered for the following special awards:</p><br />
<a href="https://2010.igem.org/Team:British_Columbia/HumanPractices"><img src="https://static.igem.org/mediawiki/2010/f/fa/Bhpa.jpg"></a><br />
<p><b>"What a society deems important is enshrined in its art." -Harry Broudy</b></p><br />
<p>Our <a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">human practices project</a> presents the <b>first iGEM art gallery</b> dedicated to synthetic biology and all its diverse aspects. Sometimes art answers our deepest questions. Sometimes art only deepens the mystery. And sometimes a picture is just worth a thousand words. This is our way of helping human civilization consider, guide and address the impacts of ongoing advances in synthetic biology. Not limited to conventional artwork, our gallery also features synthetic biology <b>promoter maps</b> and poems. We also proudly present the <b>first iGEM collaboration with NaNoWriMo</b> to showcase 50,000 word novels featuring synthetic biology, written from scratch during the month of November!</p><br />
<a href="https://2010.igem.org/Team:British_Columbia/modeling_description"><img src="https://static.igem.org/mediawiki/2010/1/17/Bm.jpg"></a><br />
<p><b>"One of the most insidious and nefarious properties of scientific models is their tendency to take over, and sometimes supplant, reality." -Erwin Chargaff</b></p><br />
<p>Right before your eyes, watch how the population dynamics of a biofilm is affected by the introduction of bacteriophage and a biofilm matrix-degrading enzyme. Using our <a href="https://2010.igem.org/Team:British_Columbia/modeling_description">model</a>, we are able to run simulations that predict outcomes of the system and construct informed hypotheses to test in reality.</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia/Project_Phage"><img src="https://static.igem.org/mediawiki/2010/7/7f/Bs.jpg"></a><br />
<p><b>"Acceptance of prevailing standards often means we have no standards of our own." -Jean Toomer<br />
</b></p><br />
<p>There are standards for prokaryotes and standards for eukaryotes. But what about standards for the living dead? Our new <a href="https://2010.igem.org/Team:British_Columbia/Project_Phage">Phage standard</a> lays down a foundation for future work involving viruses and integrating Biobrick parts into their genome. Prepare to be infected!!!</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia"><img src="https://static.igem.org/mediawiki/2010/3/31/Bw.jpg"></a><br />
<p>We've worked really hard on our wiki to make it accessible, fun and interactive! So we hope that future iGEM teams, students worldwide, and even the general public will visit us here and see what iGEM and synthetic biology is about!</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2010/5/59/Bpbp.jpg"><br />
<p>Come and see our poster and team presentation! It will be a great opportunity to meet our team and learn more about our project. A soft copy of our poster and video of our presentation will be linked here during the Jamboree.</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia"><img src="https://static.igem.org/mediawiki/2010/6/68/Bhmp.jpg"></a><br />
<p>Several diseases and medical conditions are known to be caused by biofilm infections. Pathogens existing in biofilms survive under harsher conditions and are much more difficult to eliminate than free-floating pathogens. Our project aims to engineer a bacteriophage equipped with a biofilm matrix-degrading enzyme to eradicate pathogenic Staphylococcus aureus biofilms. Door knob, we shall fear thee no longer.</p><br />
</center><br />
<br />
<br />
</div> <!-- end news --><br />
</div> <!-- end Super_main_wrapper --><br />
<script type="text/javascript"><br />
function setPageSize() {<br />
len = document.getElementById('super_main_wrapper').offsetHeight;<br />
document.getElementById('bodyContent').style.height = len + 'px';<br />
document.getElementById('news').style.height = len + 'px';<br />
}<br />
</script><br />
</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/Project_OutlookTeam:British Columbia/Project Outlook2010-10-27T16:14:49Z<p>Ayjchan: </p>
<hr />
<div>__NOTOC__<br />
<br />
{{Template_HD_4}}<br />
<html><body id="project" onload="setPageSize()"><br />
<br />
<div id="super_main_wrapper"><br />
<div id="SubWrapper"> <br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/a/ac/Medals.jpg"></src><br />
</center><br />
<h3>Project Achievements</h3><br />
<br />
<p><b>Biofilm: </b><br/><br/><br />
We have obtained growth curves for <i>S. aureus</i> strains RN4220 and 8325-4 that demonstrate a steady growth phase followed by an oscillatory state of dynamic equilibrium. We have also optimized the existing protocol for biofilm quantification. Additionally, values derived from the biofilm experiments were integral to deriving realistic results from modeling simulations.<br/><br/><br />
<br />
<p><b>Phage Standard: </b><br/><br/><br />
We developed a phage standard that allows for modification of any lysogenic bacteriophage as part of the Biobrick standard. The phage standard works around the problems of illegal cut sites and prohibitively large plasmids. We hope the standard will serve as a foundational advance towards phage research within the iGEM competition, the BioBrick registry and the synthetic biology community as a whole.</p><br/><br />
<br />
<p><b>Quorum Sensing: </b><br/><br/><br />
We have made constructs to characterize the P2 promoter (BBa_I746104) of <i>S. aureus</i> via fluorescent protein production. In order to directly relate AIP to P2 promoter activity, an agr null strain was used. Primers were designed to PCR off the genes encoding AgrAC from <i>S. aureus</i> in order to allow detection of AIP within the agr null strain. We have also made plans to PCR a <i> S. aureus </i> plasmid replicon to allow BioBrick plasmids to replicate and segregate within a <i> S. aureus </i> host. </p><br/><br />
<br />
<p><b>DspB: </b><br><br />
We have contributed to the biobrick parts registry by submitting a new part: DspB, an enzyme that degrades poly-ß-(1,6)-linked N-acetylglucosamine bonds. We have demonstrated that dspB works through a crude cell enzyme activity assay and have added this information to the Registry.<br/><br/><br />
<p><b>Modeling: </b><br/><br/><br />
<p><b>Human Practices: </b><br/><br/><br />
We have gathered hundreds of definitions of synthetic biology from the University of British Columbia community to construct <a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">promoter maps and word clouds</a> representing the prevalent ideas in our different disciplines' awareness of synthetic biology!<br/><br />
<br/>We have started the <a href="https://2010.igem.org/Team:British_Columbia/HP_arts">first iGEM synthetic biology art gallery</a> inviting all iGEM participants, as well as members of the public from Deviantart, IllustratedATCs and ATCsForAll to contribute.<br/><br/>We have forged the <a href="https://2010.igem.org/Team:British_Columbia/HP_nanowrimo">first NaNoWriMo-iGEM collaboration</a> to showcase novels featuring synthetic biology that are written by NaNoWriMo participants.<br />
</p><br />
<h3>Future Directions</h3><br />
<p><b>Biofilm: </b><br/><br/><br />
The existing curve has demonstrated that 9 hours is the optimal time point for exposure to the matrix-degrading enzyme, DspB, as well as the engineered phage construct with DspB and quorum sensing genes. Future experiments testing the biofilm response to DspB alone as well as DspB incorporated into a phage construct with the quorum sensing P2 promoter will enable the characterization of the construct’s effectiveness against the biofilms of <i>S. aureus</i> strains RN4220 and 8325-4.<br/><br/><br />
<b>Phage: </b><br/><br/><br />
<p><b>Quorum Sensing: </b><br/><br/><br />
As a next step, genes encoding AgrAC from <i>S. aureus</i> should be put on the same plasmid (the <i>S. aureus</i>/<i>E. coli</i> shuttle vector, pCN33) as the reporter constructs and transformed into agr-null <i>S. aureus</i>. This would allow proper characterization of P2 activity in the presence of AIP. Additionally, the replicon of pCN33 can be cloned off and made into a BioBrick part to facilitate the expression and characterization of BioBrick parts in <i>S. aureus</i>.</p><br/><br />
<p><b>DspB: </b><br />
<p>We are currently working on obtaining data from the exposure of DspB protein on a <i>S. aureus</i> biofilm as well as isolating DspB via a histidine tag to attain further characterization data. We hope to gather this data before the presentation. If not fully completed, these components of the sub-team should be future directions.<br />
Future directions include incorporating DspB protein into the phage for exposure to <i>S. aureus</i> biofilms.</p><br />
<br/><br />
<p><b>Modeling: </b><br/><br/><br />
<p><b>Human Practices: </b><br/><br/><br />
Our experience communicating with the general public and even students in the sciences and applied sciences has been an enriching one. We have gleaned a lot of insights into public perception of synthetic biology, which still remains a very new and unfamiliar field to the public despite recent press about the first synthetic cell!<br/><br/><br />
Public opinion and risk perception appears to be more informed by controversial topics (e.g. genetically modified organisms and food) and literature featuring synthetic biology (from Frankenstein to Oryx and Crake). So outreach on the part of synthetic biologists still has quite a way to go in order to bring synthetic biology into the schools, workplaces and homes of the public. Our human practices project has generated ripples of thoughts about synthetic biology in various communities, stimulating individuals to find out more about synthetic biology and its recent developments. We hope that this will open up paths of communication between the synthetic biology research community and diverse public communities, which may lead to discussions and collaborations with the purposes of informing the public about synthetic biology and safely expanding its real world applications.<br/><br/><br />
Some specific future directions that address this cause include: (i) Actively inviting more non-science/engineering students to participate in iGEM outreach/projects/teams/Jamboree/fundraising, (ii) Establishing an annual iGEM tradition of stimulating and showcasing works of art or literature by members of iGEM and the general public featuring synthetic biology, and (iii) Investing in other collaborative outreach activities such as elementary/secondary school educational programs and synthetic biology university courses. <br />
</p><br />
<br />
</div> <!-- end SubWrapper --><br />
<br />
<div id="news" style="height:2500px;"> <br />
<br/><br />
<br />
<center><h3>Quick Links</h3><br />
<a href="https://igem.org/Judging_Form.cgi?id=391">See our Judging Form</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/Parts">See our characterized Biobrick Parts</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/Project_Phage">See our new Phage Standard</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">See our Human Practices Project</a><br />
<br />
<br></br><br />
<br></br><h3>Consideration for Special Awards</h3><br />
<p>Besides striving for a Gold Medal and a place as one of the finalists, our team would also like to be considered for the following special awards:</p><br />
<a href="https://2010.igem.org/Team:British_Columbia/HumanPractices"><img src="https://static.igem.org/mediawiki/2010/f/fa/Bhpa.jpg"></a><br />
<p><b>"What a society deems important is enshrined in its art." -Harry Broudy</b></p><br />
<p>Our <a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">human practices project</a> presents the <b>first iGEM art gallery</b> dedicated to synthetic biology and all its diverse aspects. Sometimes art answers our deepest questions. Sometimes art only deepens the mystery. And sometimes a picture is just worth a thousand words. This is our way of helping human civilization consider, guide and address the impacts of ongoing advances in synthetic biology. Not limited to conventional artwork, our gallery also features synthetic biology <b>promoter maps</b> and poems. We also proudly present the <b>first iGEM collaboration with NaNoWriMo</b> to showcase 50,000 word novels featuring synthetic biology, written from scratch during the month of November!</p><br />
<a href="https://2010.igem.org/Team:British_Columbia/modeling_description"><img src="https://static.igem.org/mediawiki/2010/1/17/Bm.jpg"></a><br />
<p><b>"One of the most insidious and nefarious properties of scientific models is their tendency to take over, and sometimes supplant, reality." -Erwin Chargaff</b></p><br />
<p>Right before your eyes, watch how the population dynamics of a biofilm is affected by the introduction of bacteriophage and a biofilm matrix-degrading enzyme. Using our <a href="https://2010.igem.org/Team:British_Columbia/modeling_description">model</a>, we are able to run simulations that predict outcomes of the system and construct informed hypotheses to test in reality.</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia/Project_Phage"><img src="https://static.igem.org/mediawiki/2010/7/7f/Bs.jpg"></a><br />
<p><b>"Acceptance of prevailing standards often means we have no standards of our own." -Jean Toomer<br />
</b></p><br />
<p>There are standards for prokaryotes and standards for eukaryotes. But what about standards for the living dead? Our new <a href="https://2010.igem.org/Team:British_Columbia/Project_Phage">Phage standard</a> lays down a foundation for future work involving viruses and integrating Biobrick parts into their genome. Prepare to be infected!!!</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia"><img src="https://static.igem.org/mediawiki/2010/3/31/Bw.jpg"></a><br />
<p>We've worked really hard on our wiki to make it accessible, fun and interactive! So we hope that future iGEM teams, students worldwide, and even the general public will visit us here and see what iGEM and synthetic biology is about!</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2010/5/59/Bpbp.jpg"><br />
<p>Come and see our poster and team presentation! It will be a great opportunity to meet our team and learn more about our project. A soft copy of our poster and video of our presentation will be linked here during the Jamboree.</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia"><img src="https://static.igem.org/mediawiki/2010/6/68/Bhmp.jpg"></a><br />
<p>Several diseases and medical conditions are known to be caused by biofilm infections. Pathogens existing in biofilms survive under harsher conditions and are much more difficult to eliminate than free-floating pathogens. Our project aims to engineer a bacteriophage equipped with a biofilm matrix-degrading enzyme to eradicate pathogenic Staphylococcus aureus biofilms. Door knob, we shall fear thee no longer.</p><br />
</center><br />
<br />
<br />
</div> <!-- end news --><br />
</div> <!-- end Super_main_wrapper --><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/Project_OutlookTeam:British Columbia/Project Outlook2010-10-27T16:12:23Z<p>Ayjchan: </p>
<hr />
<div>__NOTOC__<br />
<br />
{{Template_HD_4}}<br />
<html><body id="project" onload="setPageSize()"><br />
<br />
<div id="super_main_wrapper"><br />
<div id="SubWrapper"> <br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/a/ac/Medals.jpg"></src><br />
</center><br />
<h3>Project Achievements</h3><br />
<br />
<p><b>Biofilm: </b><br/><br/><br />
We have obtained growth curves for <i>S. aureus</i> strains RN4220 and 8325-4 that demonstrate a steady growth phase followed by an oscillatory state of dynamic equilibrium. We have also optimized the existing protocol for biofilm quantification. Additionally, values derived from the biofilm experiments were integral to deriving realistic results from modeling simulations.<br/><br/><br />
<br />
<p><b>Phage Standard: </b><br/><br/><br />
We developed a phage standard that allows for modification of any lysogenic bacteriophage as part of the Biobrick standard. The phage standard works around the problems of illegal cut sites and prohibitively large plasmids. We hope the standard will serve as a foundational advance towards phage research within the iGEM competition, the BioBrick registry and the synthetic biology community as a whole.</p><br/><br />
<br />
<p><b>Quorum Sensing: </b><br/><br/><br />
We have made constructs to characterize the P2 promoter (BBa_I746104) of <i>S. aureus</i> via fluorescent protein production. In order to directly relate AIP to P2 promoter activity, an agr null strain was used. Primers were designed to PCR off agrAC from <i>S. aureus</i> in order to allow detection of AIP within the agr null strain. We have also made plans to PCR off pT 181, a <i> S. aureus </i> plasmid, to allow BioBrick plasmids to replicate and segregate within a <i> S. aureus </i> host. </p><br/><br />
<p><b>DspB: </b><br><br />
We have contributed to the biobrick parts registry by submitting a new part: DspB, an enzyme that degrades poly-ß-(1,6)-linked N-acetylglucosamine bonds. We have demonstrated that dspB works through a crude cell enzyme activity assay and have added this information to the Registry.<br/><br/><br />
<p><b>Modeling: </b><br/><br/><br />
<p><b>Human Practices: </b><br/><br/><br />
We have gathered hundreds of definitions of synthetic biology from the University of British Columbia community to construct <a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">promoter maps and word clouds</a> representing the prevalent ideas in our different disciplines' awareness of synthetic biology!<br/><br />
<br/>We have started the <a href="https://2010.igem.org/Team:British_Columbia/HP_arts">first iGEM synthetic biology art gallery</a> inviting all iGEM participants, as well as members of the public from Deviantart, IllustratedATCs and ATCsForAll to contribute.<br/><br/>We have forged the <a href="https://2010.igem.org/Team:British_Columbia/HP_nanowrimo">first NaNoWriMo-iGEM collaboration</a> to showcase novels featuring synthetic biology that are written by NaNoWriMo participants.<br />
</p><br />
<h3>Future Directions</h3><br />
<p><b>Biofilm: </b><br/><br/><br />
The existing curve has demonstrated that 9 hours is the optimal time point for exposure to the matrix-degrading enzyme, DspB, as well as the engineered phage construct with DspB and quorum sensing genes. Future experiments testing the biofilm response to DspB alone as well as DspB incorporated into a phage construct with the quorum sensing P2 promoter will enable the characterization of the construct’s effectiveness against the biofilms of <i>S. aureus</i> strains RN4220 and 8325-4.<br/><br/><br />
<b>Phage: </b><br/><br/><br />
<p><b>Quorum Sensing: </b><br/><br/><br />
P2-reporter constructs were successfully made. As a next step, genes encoding AgrAC from <i>S. aureus</i> should be put on the same plasmid (the <i>S. aureus</i>/<i>E. coli</i> shuttle vector, pCN33) as the reporter constructs and transformed into agr-null <i>S. aureus</i>. This would allow proper characterization of P2 activity in the presence of AIP. Additionally, the replicon of pCN33 can be cloned off and made into a BioBrick part to facilitate the expression and characterization of BioBrick parts in <i>S. aureus</i>.</p><br/><br />
<p><b>DspB: </b><br />
<p>We are currently working on obtaining data from the exposure of DspB protein on a <i>S. aureus</i> biofilm as well as isolating DspB via a histidine tag to attain further characterization data. We hope to gather this data before the presentation. If not fully completed, these components of the sub-team should be future directions.<br />
Future directions include incorporating DspB protein into the phage for exposure to <i>S. aureus</i> biofilms.</p><br />
<br/><br />
<p><b>Modeling: </b><br/><br/><br />
<p><b>Human Practices: </b><br/><br/><br />
Our experience communicating with the general public and even students in the sciences and applied sciences has been an enriching one. We have gleaned a lot of insights into public perception of synthetic biology, which still remains a very new and unfamiliar field to the public despite recent press about the first synthetic cell!<br/><br/><br />
Public opinion and risk perception appears to be more informed by controversial topics (e.g. genetically modified organisms and food) and literature featuring synthetic biology (from Frankenstein to Oryx and Crake). So outreach on the part of synthetic biologists still has quite a way to go in order to bring synthetic biology into the schools, workplaces and homes of the public. Our human practices project has generated ripples of thoughts about synthetic biology in various communities, stimulating individuals to find out more about synthetic biology and its recent developments. We hope that this will open up paths of communication between the synthetic biology research community and diverse public communities, which may lead to discussions and collaborations with the purposes of informing the public about synthetic biology and safely expanding its real world applications.<br/><br/><br />
Some specific future directions that address this cause include: (i) Actively inviting more non-science/engineering students to participate in iGEM outreach/projects/teams/Jamboree/fundraising, (ii) Establishing an annual iGEM tradition of stimulating and showcasing works of art or literature by members of iGEM and the general public featuring synthetic biology, and (iii) Investing in other collaborative outreach activities such as elementary/secondary school educational programs and synthetic biology university courses. <br />
</p><br />
<br />
</div> <!-- end SubWrapper --><br />
<br />
<div id="news" style="height:2500px;"> <br />
<br/><br />
<br />
<center><h3>Quick Links</h3><br />
<a href="https://igem.org/Judging_Form.cgi?id=391">See our Judging Form</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/Parts">See our characterized Biobrick Parts</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/Project_Phage">See our new Phage Standard</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">See our Human Practices Project</a><br />
<br />
<br></br><br />
<br></br><h3>Consideration for Special Awards</h3><br />
<p>Besides striving for a Gold Medal and a place as one of the finalists, our team would also like to be considered for the following special awards:</p><br />
<a href="https://2010.igem.org/Team:British_Columbia/HumanPractices"><img src="https://static.igem.org/mediawiki/2010/f/fa/Bhpa.jpg"></a><br />
<p><b>"What a society deems important is enshrined in its art." -Harry Broudy</b></p><br />
<p>Our <a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">human practices project</a> presents the <b>first iGEM art gallery</b> dedicated to synthetic biology and all its diverse aspects. Sometimes art answers our deepest questions. Sometimes art only deepens the mystery. And sometimes a picture is just worth a thousand words. This is our way of helping human civilization consider, guide and address the impacts of ongoing advances in synthetic biology. Not limited to conventional artwork, our gallery also features synthetic biology <b>promoter maps</b> and poems. We also proudly present the <b>first iGEM collaboration with NaNoWriMo</b> to showcase 50,000 word novels featuring synthetic biology, written from scratch during the month of November!</p><br />
<a href="https://2010.igem.org/Team:British_Columbia/modeling_description"><img src="https://static.igem.org/mediawiki/2010/1/17/Bm.jpg"></a><br />
<p><b>"One of the most insidious and nefarious properties of scientific models is their tendency to take over, and sometimes supplant, reality." -Erwin Chargaff</b></p><br />
<p>Right before your eyes, watch how the population dynamics of a biofilm is affected by the introduction of bacteriophage and a biofilm matrix-degrading enzyme. Using our <a href="https://2010.igem.org/Team:British_Columbia/modeling_description">model</a>, we are able to run simulations that predict outcomes of the system and construct informed hypotheses to test in reality.</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia/Project_Phage"><img src="https://static.igem.org/mediawiki/2010/7/7f/Bs.jpg"></a><br />
<p><b>"Acceptance of prevailing standards often means we have no standards of our own." -Jean Toomer<br />
</b></p><br />
<p>There are standards for prokaryotes and standards for eukaryotes. But what about standards for the living dead? Our new <a href="https://2010.igem.org/Team:British_Columbia/Project_Phage">Phage standard</a> lays down a foundation for future work involving viruses and integrating Biobrick parts into their genome. Prepare to be infected!!!</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia"><img src="https://static.igem.org/mediawiki/2010/3/31/Bw.jpg"></a><br />
<p>We've worked really hard on our wiki to make it accessible, fun and interactive! So we hope that future iGEM teams, students worldwide, and even the general public will visit us here and see what iGEM and synthetic biology is about!</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2010/5/59/Bpbp.jpg"><br />
<p>Come and see our poster and team presentation! It will be a great opportunity to meet our team and learn more about our project. A soft copy of our poster and video of our presentation will be linked here during the Jamboree.</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia"><img src="https://static.igem.org/mediawiki/2010/6/68/Bhmp.jpg"></a><br />
<p>Several diseases and medical conditions are known to be caused by biofilm infections. Pathogens existing in biofilms survive under harsher conditions and are much more difficult to eliminate than free-floating pathogens. Our project aims to engineer a bacteriophage equipped with a biofilm matrix-degrading enzyme to eradicate pathogenic Staphylococcus aureus biofilms. Door knob, we shall fear thee no longer.</p><br />
</center><br />
<br />
<br />
</div> <!-- end news --><br />
</div> <!-- end Super_main_wrapper --><br />
<script type="text/javascript"><br />
function setPageSize() {<br />
len = document.getElementById('super_main_wrapper').offsetHeight;<br />
document.getElementById('bodyContent').style.height = len + 'px';<br />
document.getElementById('news').style.height = len + 'px';<br />
}<br />
</script><br />
</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/Project_OutlookTeam:British Columbia/Project Outlook2010-10-27T16:10:29Z<p>Ayjchan: </p>
<hr />
<div>__NOTOC__<br />
<br />
{{Template_HD_4}}<br />
<html><body id="project" onload="setPageSize()"><br />
<br />
<div id="super_main_wrapper"><br />
<div id="SubWrapper"> <br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/a/ac/Medals.jpg"></src><br />
</center><br />
<h3>Project Achievements</h3><br />
<br />
<p><b>Biofilm: </b><br/><br/><br />
We have obtained growth curves for <i>S. aureus</i> strains RN4220 and 8325-4 that demonstrate a steady growth phase followed by an oscillatory state of dynamic equilibrium. We have also optimized the existing protocol for biofilm quantification. Additionally, values derived from the biofilm experiments were integral to deriving realistic results from modeling simulations.<br/><br/><br />
<br />
<p><b>Phage Standard: </b><br/><br/><br />
We developed a phage standard that allows for modification of any lysogenic bacteriophage as part of the Biobrick standard. The phage standard works around the problems of illegal cut sites and prohibitively large plasmids. We hope the standard will serve as a foundational advance towards phage research within the iGEM competition, the BioBrick registry and the synthetic biology community as a whole.</p><br/><br />
<br />
<p><b>Quorum Sensing: </b><br/><br/><br />
We have made constructs to characterize the P2 promoter (BBa_I746104) of <i>S. aureus</i> via fluorescent protein production. In order to directly relate AIP to P2 promoter activity, an agr null strain was used. Primers were designed to PCR off agrAC from <i>S. aureus</i> in order to allow detection of AIP within the agr null strain. We have also made plans to PCR off pT 181, a <i> S. aureus </i> plasmid, to allow BioBrick plasmids to replicate and segregate within a <i> S. aureus </i> host. </p><br/><br />
<p><b>DspB: </b><br><br />
We have contributed to the biobrick parts registry by submitting a new part: DspB, an enzyme that degrades poly-ß-(1,6)-linked N-acetylglucosamine bonds. We have demonstrated that dspB works through a crude cell enzyme activity assay and have added this information to the Registry.<br/><br/><br />
<p><b>Modeling: </b><br/><br/><br />
<p><b>Human Practices: </b><br/><br/><br />
We have gathered hundreds of definitions of synthetic biology from the University of British Columbia community to construct <a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">promoter maps and word clouds</a> representing the prevalent ideas in our different disciplines' awareness of synthetic biology!<br/><br />
<br/>We have started the <a href="https://2010.igem.org/Team:British_Columbia/HP_arts">first iGEM synthetic biology art gallery</a> inviting all iGEM participants, as well as members of the public from Deviantart, IllustratedATCs and ATCsForAll to contribute.<br/><br/>We have forged the <a href="https://2010.igem.org/Team:British_Columbia/HP_nanowrimo">first NaNoWriMo-iGEM collaboration</a> to showcase novels featuring synthetic biology that are written by NaNoWriMo participants.<br />
</p><br />
<h3>Future Directions</h3><br />
<p><b>Biofilm: </b><br/><br/><br />
The existing curve has demonstrated that 9 hours is the optimal time point for exposure to the matrix-degrading enzyme, DspB, as well as the engineered phage construct with DspB and quorum sensing genes. Future experiments testing the biofilm response to DspB alone as well as DspB incorporated into a phage construct with the quorum sensing P2 promoter will enable the characterization of the construct’s effectiveness against the biofilms of <i>S. aureus</i> strains RN4220 and 8325-4.<br/><br/><br />
<b>Phage: </b><br/><br/><br />
<p><b>Quorum Sensing: </b><br/><br/><br />
P2-reporter constructs were successfully made. As a next step, agrAC from <i>S. aureus</i> should be put on the same plasmid (the <i>S. aureus</i>/<i>E. colia</i> shuttle vector, pCN33) as the reporter constructs and transformed into agr-null <i>S. aureus</i>. This would then allow proper characterization of P2 activity in the presence of AIP. The replicon of pCN 33 could be cloned off and made into a BioBrick part. This could allow BioBrick parts to work also in <i>S. aureus</i>.</p><br/><br />
<p><b>DspB: </b><br />
<p>We are currently working on obtaining data from the exposure of DspB protein on a <i>S. aureus</i> biofilm as well as isolating DspB via a histidine tag to attain further characterization data. We hope to gather this data before the presentation. If not fully completed, these components of the sub-team should be future directions.<br />
Future directions include incorporating DspB protein into the phage for exposure to <i>S. aureus</i> biofilms.</p><br />
<br/><br />
<p><b>Modeling: </b><br/><br/><br />
<p><b>Human Practices: </b><br/><br/><br />
Our experience communicating with the general public and even students in the sciences and applied sciences has been an enriching one. We have gleaned a lot of insights into public perception of synthetic biology, which still remains a very new and unfamiliar field to the public despite recent press about the first synthetic cell!<br/><br/><br />
Public opinion and risk perception appears to be more informed by controversial topics (e.g. genetically modified organisms and food) and literature featuring synthetic biology (from Frankenstein to Oryx and Crake). So outreach on the part of synthetic biologists still has quite a way to go in order to bring synthetic biology into the schools, workplaces and homes of the public. Our human practices project has generated ripples of thoughts about synthetic biology in various communities, stimulating individuals to find out more about synthetic biology and its recent developments. We hope that this will open up paths of communication between the synthetic biology research community and diverse public communities, which may lead to discussions and collaborations with the purposes of informing the public about synthetic biology and safely expanding its real world applications.<br/><br/><br />
Some specific future directions that address this cause include: (i) Actively inviting more non-science/engineering students to participate in iGEM outreach/projects/teams/Jamboree/fundraising, (ii) Establishing an annual iGEM tradition of stimulating and showcasing works of art or literature by members of iGEM and the general public featuring synthetic biology, and (iii) Investing in other collaborative outreach activities such as elementary/secondary school educational programs and synthetic biology university courses. <br />
</p><br />
<br />
</div> <!-- end SubWrapper --><br />
<br />
<div id="news" style="height:2500px;"> <br />
<br/><br />
<br />
<center><h3>Quick Links</h3><br />
<a href="https://igem.org/Judging_Form.cgi?id=391">See our Judging Form</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/Parts">See our characterized Biobrick Parts</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/Project_Phage">See our new Phage Standard</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">See our Human Practices Project</a><br />
<br />
<br></br><br />
<br></br><h3>Consideration for Special Awards</h3><br />
<p>Besides striving for a Gold Medal and a place as one of the finalists, our team would also like to be considered for the following special awards:</p><br />
<a href="https://2010.igem.org/Team:British_Columbia/HumanPractices"><img src="https://static.igem.org/mediawiki/2010/f/fa/Bhpa.jpg"></a><br />
<p><b>"What a society deems important is enshrined in its art." -Harry Broudy</b></p><br />
<p>Our <a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">human practices project</a> presents the <b>first iGEM art gallery</b> dedicated to synthetic biology and all its diverse aspects. Sometimes art answers our deepest questions. Sometimes art only deepens the mystery. And sometimes a picture is just worth a thousand words. This is our way of helping human civilization consider, guide and address the impacts of ongoing advances in synthetic biology. Not limited to conventional artwork, our gallery also features synthetic biology <b>promoter maps</b> and poems. We also proudly present the <b>first iGEM collaboration with NaNoWriMo</b> to showcase 50,000 word novels featuring synthetic biology, written from scratch during the month of November!</p><br />
<a href="https://2010.igem.org/Team:British_Columbia/modeling_description"><img src="https://static.igem.org/mediawiki/2010/1/17/Bm.jpg"></a><br />
<p><b>"One of the most insidious and nefarious properties of scientific models is their tendency to take over, and sometimes supplant, reality." -Erwin Chargaff</b></p><br />
<p>Right before your eyes, watch how the population dynamics of a biofilm is affected by the introduction of bacteriophage and a biofilm matrix-degrading enzyme. Using our <a href="https://2010.igem.org/Team:British_Columbia/modeling_description">model</a>, we are able to run simulations that predict outcomes of the system and construct informed hypotheses to test in reality.</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia/Project_Phage"><img src="https://static.igem.org/mediawiki/2010/7/7f/Bs.jpg"></a><br />
<p><b>"Acceptance of prevailing standards often means we have no standards of our own." -Jean Toomer<br />
</b></p><br />
<p>There are standards for prokaryotes and standards for eukaryotes. But what about standards for the living dead? Our new <a href="https://2010.igem.org/Team:British_Columbia/Project_Phage">Phage standard</a> lays down a foundation for future work involving viruses and integrating Biobrick parts into their genome. Prepare to be infected!!!</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia"><img src="https://static.igem.org/mediawiki/2010/3/31/Bw.jpg"></a><br />
<p>We've worked really hard on our wiki to make it accessible, fun and interactive! So we hope that future iGEM teams, students worldwide, and even the general public will visit us here and see what iGEM and synthetic biology is about!</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2010/5/59/Bpbp.jpg"><br />
<p>Come and see our poster and team presentation! It will be a great opportunity to meet our team and learn more about our project. A soft copy of our poster and video of our presentation will be linked here during the Jamboree.</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia"><img src="https://static.igem.org/mediawiki/2010/6/68/Bhmp.jpg"></a><br />
<p>Several diseases and medical conditions are known to be caused by biofilm infections. Pathogens existing in biofilms survive under harsher conditions and are much more difficult to eliminate than free-floating pathogens. Our project aims to engineer a bacteriophage equipped with a biofilm matrix-degrading enzyme to eradicate pathogenic Staphylococcus aureus biofilms. Door knob, we shall fear thee no longer.</p><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/Project_OutlookTeam:British Columbia/Project Outlook2010-10-27T16:09:50Z<p>Ayjchan: </p>
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<h3>Project Achievements</h3><br />
<br />
<p><b>Biofilm: </b><br/><br/><br />
We have obtained growth curves for <i>S. aureus</i> strains RN4220 and 8325-4 that demonstrate a steady growth phase followed by an oscillatory state of dynamic equilibrium. We have also optimized the existing protocol for biofilm quantification. Additionally, values derived from the biofilm experiments were integral to deriving realistic results from modeling simulations.<br/><br/><br />
<br />
<p><b>Phage Standard: </b><br/><br/><br />
We developed a phage standard that allows for modification of any lysogenic bacteriophage as part of the Biobrick standard. The phage standard works around the problems of illegal cut sites and prohibitively large plasmids. We hope the standard will serve as a foundational advance towards phage research within the iGEM competition, the BioBrick registry and the synthetic biology community as a whole.</p><br/><br />
<br />
<p><b>Quorum Sensing: </b><br/><br/><br />
We have made constructs to characterize the P2 promoter (BBa_I746104) of <i>S. aureus</i> via fluorescent protein production. In order to solely relate AIP to P2 promoter activity, an agr null strain was used. Primers were designed to PCR off agrAC from <i>S. aureus</i> in order to allow detection of AIP within the agr null strain. We have also made plans to PCR off pT 181, a <i> S. aureus </i> plasmid, to allow BioBrick plasmids to replicate and segregate within a <i> S. aureus </i> host. </p><br/><br />
<p><b>DspB: </b><br><br />
We have contributed to the biobrick parts registry by submitting a new part: DspB, an enzyme that degrades poly-ß-(1,6)-linked N-acetylglucosamine bonds. We have demonstrated that dspB works through a crude cell enzyme activity assay and have added this information to the Registry.<br/><br/><br />
<p><b>Modeling: </b><br/><br/><br />
<p><b>Human Practices: </b><br/><br/><br />
We have gathered hundreds of definitions of synthetic biology from the University of British Columbia community to construct <a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">promoter maps and word clouds</a> representing the prevalent ideas in our different disciplines' awareness of synthetic biology!<br/><br />
<br/>We have started the <a href="https://2010.igem.org/Team:British_Columbia/HP_arts">first iGEM synthetic biology art gallery</a> inviting all iGEM participants, as well as members of the public from Deviantart, IllustratedATCs and ATCsForAll to contribute.<br/><br/>We have forged the <a href="https://2010.igem.org/Team:British_Columbia/HP_nanowrimo">first NaNoWriMo-iGEM collaboration</a> to showcase novels featuring synthetic biology that are written by NaNoWriMo participants.<br />
</p><br />
<h3>Future Directions</h3><br />
<p><b>Biofilm: </b><br/><br/><br />
The existing curve has demonstrated that 9 hours is the optimal time point for exposure to the matrix-degrading enzyme, DspB, as well as the engineered phage construct with DspB and quorum sensing genes. Future experiments testing the biofilm response to DspB alone as well as DspB incorporated into a phage construct with the quorum sensing P2 promoter will enable the characterization of the construct’s effectiveness against the biofilms of <i>S. aureus</i> strains RN4220 and 8325-4.<br/><br/><br />
<b>Phage: </b><br/><br/><br />
<p><b>Quorum Sensing: </b><br/><br/><br />
P2-reporter constructs were successfully made. As a next step, agrAC from <i>S. aureus</i> should be put on the same plasmid (the <i>S. aureus</i>/<i>E. colia</i> shuttle vector, pCN33) as the reporter constructs and transformed into agr-null <i>S. aureus</i>. This would then allow proper characterization of P2 activity in the presence of AIP. The replicon of pCN 33 could be cloned off and made into a BioBrick part. This could allow BioBrick parts to work also in <i>S. aureus</i>.</p><br/><br />
<p><b>DspB: </b><br />
<p>We are currently working on obtaining data from the exposure of DspB protein on a <i>S. aureus</i> biofilm as well as isolating DspB via a histidine tag to attain further characterization data. We hope to gather this data before the presentation. If not fully completed, these components of the sub-team should be future directions.<br />
Future directions include incorporating DspB protein into the phage for exposure to <i>S. aureus</i> biofilms.</p><br />
<br/><br />
<p><b>Modeling: </b><br/><br/><br />
<p><b>Human Practices: </b><br/><br/><br />
Our experience communicating with the general public and even students in the sciences and applied sciences has been an enriching one. We have gleaned a lot of insights into public perception of synthetic biology, which still remains a very new and unfamiliar field to the public despite recent press about the first synthetic cell!<br/><br/><br />
Public opinion and risk perception appears to be more informed by controversial topics (e.g. genetically modified organisms and food) and literature featuring synthetic biology (from Frankenstein to Oryx and Crake). So outreach on the part of synthetic biologists still has quite a way to go in order to bring synthetic biology into the schools, workplaces and homes of the public. Our human practices project has generated ripples of thoughts about synthetic biology in various communities, stimulating individuals to find out more about synthetic biology and its recent developments. We hope that this will open up paths of communication between the synthetic biology research community and diverse public communities, which may lead to discussions and collaborations with the purposes of informing the public about synthetic biology and safely expanding its real world applications.<br/><br/><br />
Some specific future directions that address this cause include: (i) Actively inviting more non-science/engineering students to participate in iGEM outreach/projects/teams/Jamboree/fundraising, (ii) Establishing an annual iGEM tradition of stimulating and showcasing works of art or literature by members of iGEM and the general public featuring synthetic biology, and (iii) Investing in other collaborative outreach activities such as elementary/secondary school educational programs and synthetic biology university courses. <br />
</p><br />
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<center><h3>Quick Links</h3><br />
<a href="https://igem.org/Judging_Form.cgi?id=391">See our Judging Form</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/Parts">See our characterized Biobrick Parts</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/Project_Phage">See our new Phage Standard</a><br />
<br></br><a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">See our Human Practices Project</a><br />
<br />
<br></br><br />
<br></br><h3>Consideration for Special Awards</h3><br />
<p>Besides striving for a Gold Medal and a place as one of the finalists, our team would also like to be considered for the following special awards:</p><br />
<a href="https://2010.igem.org/Team:British_Columbia/HumanPractices"><img src="https://static.igem.org/mediawiki/2010/f/fa/Bhpa.jpg"></a><br />
<p><b>"What a society deems important is enshrined in its art." -Harry Broudy</b></p><br />
<p>Our <a href="https://2010.igem.org/Team:British_Columbia/HumanPractices">human practices project</a> presents the <b>first iGEM art gallery</b> dedicated to synthetic biology and all its diverse aspects. Sometimes art answers our deepest questions. Sometimes art only deepens the mystery. And sometimes a picture is just worth a thousand words. This is our way of helping human civilization consider, guide and address the impacts of ongoing advances in synthetic biology. Not limited to conventional artwork, our gallery also features synthetic biology <b>promoter maps</b> and poems. We also proudly present the <b>first iGEM collaboration with NaNoWriMo</b> to showcase 50,000 word novels featuring synthetic biology, written from scratch during the month of November!</p><br />
<a href="https://2010.igem.org/Team:British_Columbia/modeling_description"><img src="https://static.igem.org/mediawiki/2010/1/17/Bm.jpg"></a><br />
<p><b>"One of the most insidious and nefarious properties of scientific models is their tendency to take over, and sometimes supplant, reality." -Erwin Chargaff</b></p><br />
<p>Right before your eyes, watch how the population dynamics of a biofilm is affected by the introduction of bacteriophage and a biofilm matrix-degrading enzyme. Using our <a href="https://2010.igem.org/Team:British_Columbia/modeling_description">model</a>, we are able to run simulations that predict outcomes of the system and construct informed hypotheses to test in reality.</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia/Project_Phage"><img src="https://static.igem.org/mediawiki/2010/7/7f/Bs.jpg"></a><br />
<p><b>"Acceptance of prevailing standards often means we have no standards of our own." -Jean Toomer<br />
</b></p><br />
<p>There are standards for prokaryotes and standards for eukaryotes. But what about standards for the living dead? Our new <a href="https://2010.igem.org/Team:British_Columbia/Project_Phage">Phage standard</a> lays down a foundation for future work involving viruses and integrating Biobrick parts into their genome. Prepare to be infected!!!</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia"><img src="https://static.igem.org/mediawiki/2010/3/31/Bw.jpg"></a><br />
<p>We've worked really hard on our wiki to make it accessible, fun and interactive! So we hope that future iGEM teams, students worldwide, and even the general public will visit us here and see what iGEM and synthetic biology is about!</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2010/5/59/Bpbp.jpg"><br />
<p>Come and see our poster and team presentation! It will be a great opportunity to meet our team and learn more about our project. A soft copy of our poster and video of our presentation will be linked here during the Jamboree.</p><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia"><img src="https://static.igem.org/mediawiki/2010/6/68/Bhmp.jpg"></a><br />
<p>Several diseases and medical conditions are known to be caused by biofilm infections. Pathogens existing in biofilms survive under harsher conditions and are much more difficult to eliminate than free-floating pathogens. Our project aims to engineer a bacteriophage equipped with a biofilm matrix-degrading enzyme to eradicate pathogenic Staphylococcus aureus biofilms. Door knob, we shall fear thee no longer.</p><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/ProjectTeam:British Columbia/Project2010-10-27T16:08:47Z<p>Ayjchan: </p>
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<h3>A Multi-prong Approach to Eliminating Staphylococcus aureus Biofilms Using Recombinant Bacteriophages and Biofilm-Degrading Enzymes</h3><br />
<p>Biofilms are ubiquitious microbial communities often with greater resistance and pathogenicity compared to individual microbes. Biofilms commonly cause complications in industrial and medical settings and represent a significant source of morbidity and mortality. A synthetic biology approach to tackling biofilms has only recently been applied to Escherichia coli biofilms. To eliminate the more clinically relevant Staphylococcus aureus biofilms, our team aims to break new ground at iGEM by using S. aureus as a model host and developing a standard for genetically engineering bacteriophages. Our design incorporates DspB, a biofilm matrix-degrading enzyme into the Փ13 phage genome, which is altered to operate under the regulation of the S. aureus agr quorum sensing pathway and thus upon contact with biofilms. As a complement, we have also developed a mathematical model that simulates the dynamics of our system under different conditions.</p><br />
<br/><center><img src="https://static.igem.org/mediawiki/2010/0/04/Introubcigem.png"></src></center><br />
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<center><h3>Glossary</h3><br/><br />
<b>Biofilm</b><br />
<p>A complex structure adhering to surfaces that are regularly in contact with water, consisting of colonies of bacteria and usually other microorganisms such as yeasts, fungi, and protozoa that secrete a mucilaginous protective coating in which they are encased. Biofilms can form on solid or liquid surfaces as well as on soft tissue in living organisms, and are typically resistant to conventional methods of disinfection. Dental plaque, the slimy coating that fouls pipes and tanks, and algal mats on bodies of water are examples of biofilms. While biofilms are generally pathogenic in the body, causing such diseases as cystic fibrosis, they can be used beneficially in treating sewage, industrial waste, and contaminated soil.</p><br/><br />
<b>Phage</b><br/><br />
A virus that infects and destroys bacterial cells. <br></br><br/><br/><br />
<b>Quorum Sensing</b><br />
<p>A type of decision-making process used by decentralized groups to coordinate behavior. Many species of bacteria use quorum sensing to coordinate their gene expression according to the local density of their population. </p><br />
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<h3>A Multi-prong Approach to Eliminating Staphylococcus aureus Biofilms Using Recombinant Bacteriophages and Biofilm-Degrading Enzymes</h3><br />
<p>Biofilms are ubiquitious microbial communities often with greater resistance and pathogenicity compared to individual microbes. Biofilms commonly cause complications in industrial and medical settings and represent a significant source of morbidity and mortality. A synthetic biology approach to tackling biofilms has only recently been applied to Escherichia coli biofilms. To eliminate the more clinically relevant Staphylococcus aureus biofilms, our team aims to break new ground at iGEM by using S. aureus as a model host and developing a standard for genetically engineering bacteriophages. Our design incorporates DspB, a biofilm matrix-degrading enzyme into the Փ13 phage genome, which is altered to operate under the regulation of the S. aureus agr quorum sensing pathway and thus upon contact with biofilms. As a complement, we have also developed a mathematical model that simulates the dynamics of our system under different conditions.</p><br />
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<center><h3>Glossary</h3><br/><br />
<b>Biofilm</b><br />
<p>A complex structure adhering to surfaces that are regularly in contact with water, consisting of colonies of bacteria and usually other microorganisms such as yeasts, fungi, and protozoa that secrete a mucilaginous protective coating in which they are encased. Biofilms can form on solid or liquid surfaces as well as on soft tissue in living organisms, and are typically resistant to conventional methods of disinfection. Dental plaque, the slimy coating that fouls pipes and tanks, and algal mats on bodies of water are examples of biofilms. While biofilms are generally pathogenic in the body, causing such diseases as cystic fibrosis and otitis media, they can be used beneficially in treating sewage, industrial waste, and contaminated soil.</p><br/><br />
<b>Phage</b><br/><br />
A virus that infects and destroys bacterial cells. <br></br><br/><br/><br />
<b>Quorum Sensing</b><br />
<p>A type of decision-making process used by decentralized groups to coordinate behavior. Many species of bacteria use quorum sensing to coordinate their gene expression according to the local density of their population. </p><br />
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<h3>A Multi-prong Approach to Eliminating Staphylococcus aureus Biofilms Using Recombinant Bacteriophages and Biofilm-Degrading Enzymes</h3><br />
<p>Biofilms are ubiquitious microbial communities often with greater resistance and pathogenicity compared to individual microbes. Biofilms commonly cause complications in industrial and medical settings and represent a significant source of morbidity and mortality. A synthetic biology approach to tackling biofilms has only recently been applied to Escherichia coli biofilms. To eliminate the more clinically relevant Staphylococcus aureus biofilms, our team aims to break new ground at iGEM by using S. aureus as a model host and developing a standard for genetically engineering bacteriophages. Our design incorporates DspB, a biofilm matrix-degrading enzyme into the Փ13 phage genome, which is altered to operate under the regulation of the S. aureus agr quorum sensing pathway and thus upon contact with biofilms. As a complement, we have also developed a mathematical model that simulates the dynamics of our system under different conditions.</p><br />
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<center><h3>Glossary</h3><br/><br />
<b>Biofilm</b><br />
<p>A complex structure adhering to surfaces that are regularly in contact with water, consisting of colonies of bacteria and usually other microorganisms such as yeasts, fungi, and protozoa that secrete a mucilaginous protective coating in which they are encased. Biofilms can form on solid or liquid surfaces as well as on soft tissue in living organisms, and are typically resistant to conventional methods of disinfection. Dental plaque, the slimy coating that fouls pipes and tanks, and algal mats on bodies of water are examples of biofilms. While biofilms are generally pathogenic in the body, causing such diseases as cystic fibrosis and otitis media, they can be used beneficially in treating sewage, industrial waste, and contaminated soil.<p><br/><br />
<b>Phage</b><br/><br />
A virus that infects and destroys bacterial cells. <br></br><br/><br/><br />
<b>Quorum Sensing</b><br />
<p>A type of decision-making process used by decentralized groups to coordinate behavior. Many species of bacteria use quorum sensing to coordinate their gene expression according to the local density of their population. </p><br />
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<h3>A Multi-prong Approach to Eliminating Staphylococcus aureus Biofilms Using Recombinant Bacteriophages and Biofilm-Degrading Enzymes</h3><br />
<p>Biofilms are ubiquitious microbial communities often with greater resistance and pathogenicity compared to individual microbes. Biofilms commonly cause complications in industrial and medical settings and represent a significant source of morbidity and mortality. A synthetic biology approach to tackling biofilms has only recently been applied to Escherichia coli biofilms. To eliminate the more clinically relevant Staphylococcus aureus biofilms, our team aims to break new ground at iGEM by using S. aureus as a model host and developing a standard for genetically engineering bacteriophages. Our design incorporates DspB, a biofilm matrix-degrading enzyme into the Փ13 phage genome, which is altered to operate under the regulation of the S. aureus agr quorum sensing pathway and thus upon contact with biofilms. As a complement, we have also developed a mathematical model that simulates the dynamics of our system under different conditions.</p><br />
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<center><h3>Glossary</h3><br/><br />
<b>Biofilm</b><br />
<p>A complex structure adhering to surfaces that are regularly in contact with water, consisting of colonies of bacteria and usually other microorganisms such as yeasts, fungi, and protozoa that secrete a mucilaginous protective coating in which they are encased. Biofilms can form on solid or liquid surfaces as well as on soft tissue in living organisms, and are typically resistant to conventional methods of disinfection. Dental plaque, the slimy coating that fouls pipes and tanks, and algal mats on bodies of water are examples of biofilms. While biofilms are generally pathogenic in the body, causing such diseases as cystic fibrosis and otitis media, they can be used beneficially in treating sewage, industrial waste, and contaminated soil.<br/><br />
<b>Phage</b><br/><br />
A virus that infects and destroys bacterial cells. <br></br><br/><br />
<b>Quorum Sensing</b><br />
<p>A type of decision-making process used by decentralized groups to coordinate behavior. Many species of bacteria use quorum sensing to coordinate their gene expression according to the local density of their population. </p><br />
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<h3>Introduction</h3><br />
<p>We developed a mathematical model that describes the dynamics of biofilm structure (in terms of bacterial population size) and the interactions among major components such as the engineered phage and Dispersin B (DspB) protein. We used numerical simulations to predict the impact of phage and DspB release on the biofilm. We also investigated the weight of each parameter with regards to the design of our system using sensitivity analyses. We implemented our model in a Java program called PhilmIt-v1. Our model can be used as a tool for designing genetically engineered phage-biofilm systems.</p><br/><br />
<h3>Basic Biofilm Geometry</h3><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/2/29/UBC_biofilm_container.jpg" width=250px><br />
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<p><b>Figure 1:</b> Schematic diagram of the biofilm structure. A cylindrical (or rectangular) container of depth, <i>D</i>, and cross-sectional area, <i>A</i>, holds the biofilm mass. Each bacterium occupies a spherical volume of diameter, <i>d</i>, corrected by a constant representing the space filled by surrounding EPS, &alpha; (see zoom-in).</p><br />
<p>The biofilm system assumes a simple planar geometry characterized by depth, <i>D</i>, and cross-sectional area, <i>A</i> (Figure 1). The density and distribution of the biofilm bacterial population (<i>B</i>), extracellular polymeric substance (EPS), and dissolved components (<i>e.g.</i> AIP and metabolites) are uniform throughout the biofilm structure. Assuming that each biofilm bacterium occupies a spherical volume of diameter, <i>d</i>, and the surrounding EPS extends this volume by a constant, α, each bacterium takes up a cubic volume of <i>(α + d)<sup>3</sup></i>, we can estimate the total biofilm population:</p><br />
<p><center><img src="https://static.igem.org/mediawiki/2010/e/e2/UBC_eqn1.jpg"></center></p><br />
<p>The carrying capacity determines the maximum biofilm thickness, and therefore can be coarsely estimated by letting <i>D</i> equal to the maximum thickness in Equation 1.</p><br/><br />
<h3>Biofilm Bacteria</h3><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/f/ff/UBC_flow_diagram.jpg" width=250px><br />
</center><br />
<p><b>Figure 2:</b> Flow diagram indicating the interactions among the core components of the biofilm system. The total biofilm population, <i>B<sub>T</sub></i>, is divided into subpopulations (inside the rounded box). The phage particle population, <i>P</i>, directly interacts with only the subpopulation infected with latent phage, <i>B<sub>L</sub></i>.</p><br />
<p>The total bacterial population, <i>B<sub>T</sub></i>, is divided into two subpopulations: 1) the carrier bacteria, <i>B<sub>i</sub></i>, which are infected with the engineered phage and 2) the non-carrier bacteria, <i>B<sub>u</sub></i>, which are uninfected but susceptible to phage infection upon exposure. The total population, <i>B<sub>T</sub></i>, undergo logistic growth at rate &rho;<sub>T</sub> limited by the carrying capacity, K; the uninfected, <i>B<sub>u</sub></i>, and infected, <i>B<sub>i</sub></i>, subpopulations grow at rates &rho;<sub>u</sub> and &rho;<sub>i</sub>, respectively. We separate the carrier bacteria subpopulation further into two subpopulations: 1) the bacteria infected with the phage in latent phase, <i>B<sub>l</sub></i>, and 2) those infected with the phage in lytic phase, <i>B<sub>L</sub></i>. The flow diagram in Figure 2 summarizes the hierarchical relationships among components of the biofilm population.</p><br/><br />
<h3>Phage Particles</h3><br />
<p>Initially, only the engineered <i>S. aureus</i> bacteria will be introduced to the biofilm. In response to the presence of AIP, they will generate and release the first batch of phage particles. A proportion of these phage particles, &kappa;, will successfully infect and integrate its genetic information into the host genome. Once infected, a bacterium is subject to lysis probability of &lambda;; upon lysis, the bacterium will release <i>R</i> number of phage particles. The dynamics of the phage particles is also governed by half-life, <i>t<sub>1/2</sub></i>, and follows this differential equation, where &delta; is the portion of phage remaining in the biofilm structure after diffusion (described in detail in next section):</p><br />
<p><center><img src="https://static.igem.org/mediawiki/2010/9/9d/UBC_eqn2.jpg"></center></p><br/><br />
<h3>Phage Diffusion and Invasion</h3><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/d/d8/UBC_phage_diffusion.jpg" width=250px><br />
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<p><b>Figure 3:</b> Schematic diagram of the phage invasion process. Phage particles move into the biofilm structure via simple diffusion. Lysis of infected host cells in the phage-infested layer (indicated by a thickness of x) produces new pools of phage particles which undergo diffusion into the biofilm at rate, <i>r<sub>in</sub></i>, or out of biofilm at rate ,<i>r<sub>out</sub></i>. The depth of the biofilm structure is <i>z</i>.</p><br />
<p>Lysis of the infected host cells forms a pool of newly produced phage particles. This phage population diffuses <br />
out 1) towards the bulk liquid at rate <i>r<sub>out</sub></i> or 2) into the biofilm structure through EPS at rate <i>r<sub>in</sub></i>. We assume that the new phage pool is concentrated in a defined layer immediately after lysis. This layer serves as the initial point of diffusion. The diffusion of phage particles into the biofilm can be modeled by Fick’s second law of diffusion, where &phi; is the phage concentration and <i>x</i> the distance from the boundary of the phage-infested layer (here, it is interpreted as the thickness of the new phage-infested layer):</p><br />
<p><center><img src="https://static.igem.org/mediawiki/2010/a/a6/UBC_eqn3.jpg"></center></p><br />
<p>Let us redefine the concentration, &phi;, to be relative to the initial phage concentration, &phi;<sub>0</sub>, at time <i>t</i> = 0 such that &phi;<sub>r</sub> = &phi; / &phi;<sub>0</sub>:</p><br />
<p><center><img src="https://static.igem.org/mediawiki/2010/6/66/UBC_eqn4.jpg"></center></p><br />
<p>Solving for &phi;<sub>r</sub>, we derive:</p><br />
<p><center><img src="https://static.igem.org/mediawiki/2010/d/d7/UBC_eqn5.jpg"></center></p><br />
<p>The distance of phage diffusion within one time step (<i>i.e.</i> from t = 0 to t = 1) can be estimated by letting &phi;<sub>r</sub> = 0 and solving for x, which is dependent on the constant, &theta;, and diffusion coefficient, <i>r<sub>in</sub></i>. Note that the integral cannot be solved analytically in closed form. Numerical methods such as the adaptive Simpson quadrature are required to estimate it.</p><br />
<p><center><img src="https://static.igem.org/mediawiki/2010/a/aa/UBC_eqn6.jpg"></center></p><br />
<p>Since we are treating this as a one-dimensional problem, the distance of phage diffusion can be roughly estimated by the diffusion length for a time period, <i>dt</i>t:</p><br />
<p><center><img src="https://static.igem.org/mediawiki/2010/f/f3/UBC_eqn7.jpg"></center></p><br />
<p>For simplicity, we will implement this equation in our simulations.</p><br />
<p>This distance reflects the depth of the phage-infested layer and is related to the proportion of the total biofilm population susceptible to infection. Details on the incorporation of this equation into the model is discussed in the next section.</p><br />
<p>Assuming that the volume of the bulk liquid is very large, the concentration of the phage particles in the bulk liquid is negligible and therefore the gradient of phage concentration across the bulk liquid and the phage-infested layer does not affect the diffusion rate into the biofilm. This assumption allows us to estimate the portion of phage particles, &delta;, in the phage-infested layer that will diffuse <i>out</i> of the biofilm structure:</p><br />
<p><center><img src="https://static.igem.org/mediawiki/2010/e/ea/UBC_eqn8.jpg"></center></p><br />
<p>This variable influences the decrease term in Equation 2.</p><br/><br />
<h3>Population Dynamics</h3><br />
<p>The phage population introduced to the boundary layer will infect the biofilm starting at the exposed biofilm surface. A portion of the total biofilm population, &epsilon;, is susceptible to infection due to limited phage access to the biofilm biofilm (note that infected bacteria can be re-infected). Because we assume that the composition of biofilm is homogeneous, the portion of susceptible biofilm is related to the depth of phage invasion, <i>z</i>, and the depth of the phage-infested layer, <i>x</i>:</p><br />
<p><center><img src="https://static.igem.org/mediawiki/2010/b/b0/UBC_eqn9.jpg"></center></p><br />
<p>The uninfected bacteria and the infected undergo growth at rates, &rho;<sub>u</sub> and &rho;<sub>i</sub>, respectively. The growth of both subpopulations will contribute to attaining the maximum thickness of the total biofilm. Thus, the dynamics of the two biofilm subpopulations can be described by the following differential equations, <i>where &kappa;P</i> cannot exceed <i>&epsilon;B</i>, the maximum number of biofilm bacteria that can be infected or re-infected:</p><br />
<p><center><img src="https://static.igem.org/mediawiki/2010/b/be/UBC_eqn10and11.jpg"></center></p><br />
<p>The sum of the rate terms of the subpopulations yields the differential equation describing the dynamics of the total bacterial population. Note that the logistic growth component is separated for the subpopulations (Equations 10 and 11). Here, the term for overall biofilm population decline is primarily dependent on the population of bacteria infected with lytic phage, <i>B<sub>l</sub></i><.</p><br />
<p><center><img src-="https://static.igem.org/mediawiki/2010/6/6c/UBC_eqn12.jpg"></center></p><br />
<p>The following differential equations describe the dynamics of the two infected subpopulations, where <i>p</i> is the portion of latent bacteria, &pi; the rate of transition from latent to lytic phase, and &lambda; the rate of host lysis:</p><br />
<p><center><img src="https://static.igem.org/mediawiki/2010/4/44/UBC_eqn13and14.jpg"></center></p><br />
<p>Using a simplified geometry of the biofilm structure, we are easily able to describe the dynamic relationship between the depth of the biofilm structure and the total biofilm population:</p><br />
<p><center><img src="https://static.igem.org/mediawiki/2010/f/f5/UBC_eqn15.jpg"></center></p><br/><br />
<h3>DspB Activity</h3><br />
<p>The production of DspB enzymes is coupled with phage production since they are both activated via the same signal pathway. The concentration of DspB enzymes in the EPS is subject to diffusion forces similar to those acting on the phage particles. The dynamics of extracellular DspB concentration is therefore, where <i>S</i> is the amount of DspB released per host lysis:</p><br />
<p><center><img src="https://static.igem.org/mediawiki/2010/a/a0/UBC_eqn16.jpg"></center></p><br />
<p>The lysis release of DspB enzymes promotes phage invasion by facilitating access into the biofilm structure due to their biofilm EPS-degrading activity. Therefore, DspB increases the diffusion rate of the phage particles into biofilm. The phage particle diffusion rate is related to the concentration of DspB enzymes in the phage-infested layer, where <i>Y</i> is a linear proportionality constant and the rate is constrained by the minimum and maximum values, <i>r<sub>min</sub></i> and <i>r<sub>max</sub></i>, respectively:</p><br />
<p><center><img src="https://static.igem.org/mediawiki/2010/d/d0/UBC_eqn17.jpg"></center></p><br/><br />
<h3>AIP Activity</h3><br />
<p>Here, the biofilm is assumed to maintain its extracellular concentration of AIP at a maximum, [AIP]<sub>max</sub>. AIP is being secreted by the biofilm bacteria at rate which is a function of the total biofilm population and concentration of AIP and degrades at rate &gamma;. The concentration of AIP is assumed to change more or less logistically:</p><br />
<p><center><img src="https://static.igem.org/mediawiki/2010/c/ca/UBC_eqn18.jpg"></center></p><br />
<p>The activation of phage production and <i>dspB</i> transcription is dependent on the extracellular concentration of AIP. We engineered the system so that it responds to a certain threshold level of AIP, [AIP]<sub>thres</sub>, below which phage production and <i>dspB</i> transcription ceases (<i>i.e.</i> <i>R</i> = 0 and <i>S</i> = 0). By modifying Equations 2 and 16, we derive differential equations describing phage population and DspB concentration dynamics conditionally dependent on the concentration of AIP (described by term <i>Z</i> – Equation 21), respectively:</p><br />
<p><center><img src="https://static.igem.org/mediawiki/2010/4/48/UBC_eqn19and20and21.jpg"></center></p><br/><br />
<h3>Output</h3><br />
<p>The progress and outcome of phage invasion can be monitored by tracking the populations of the biofilm bacteria and phage. The output of the model can be summarized using the variables <i>B<sub>T</sub></i>, <i>B<sub>i</sub></i>, and <i>P</i>:</p><br />
<p>1) percent of biofilm population remaining since phage introduction (<i>B<sub>p</sub></i>), where <i>B<sub>0</sub></i> is defined as the initial biofilm population or the carrying capacity:</p><br />
<p><center><img src="https://static.igem.org/mediawiki/2010/7/74/UBC_eqn22.jpg"></center></p><br />
<p>2) percent of the biofilm population uninfected by the phage (<i>B<sub>inf</sub></i>):</p><br />
<p><center><img src="https://static.igem.org/mediawiki/2010/5/50/UBC_eqn23.jpg"></center></p><br />
<p>3) P-factor (<i>P<sub>f</sub></i>), or the log of the ratio between the phage population and the initial phage population, <i>P<sub>0</sub></i>:</p><br />
<p><center><img src="https://static.igem.org/mediawiki/2010/b/b7/UBC_eqn24.jpg"></center></p><br/><br />
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<h3>PhilmIt-v1: Model Implementation</h3><br />
<p>A Java implementation of our model (PhilmIt-v1) is available for download <a href="">here</a>. The program was written to reflect the modularity of our system. Extensions to our model can be done by altering the equations and/or introducing new terms/equations. Modifications to the program can be easily made by changing the code (please note that if you alter the code, we do not guarantee correct results). Our program can be used as a tool for formulating informed hypotheses for future experiments involving genetically engineered biofilm-phage systems.</p><br/><br />
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<h3>References</h3><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/HumanPracticesTeam:British Columbia/HumanPractices2010-10-27T16:05:13Z<p>Ayjchan: </p>
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<div id="orangeBox"><h3>Synthetic Biology & Society</h3><br />
<p>Whether it's agriculture, healthcare, the environment or the economy, synthetic biology has an increasingly important role to play! Join in our discussions!</p><br />
<a href="http://forum2010.ubcigem.com/">Our Forum</a><br />
</div><br />
<div id="greenBox"><h3>NaNoWriMo x iGEM</h3><br />
<p>In our first collaboration ever, iGEM and NaNoWriMo unite to showcase stories featuring synthetic biology! Follow these novelists over the month of November!</p><br />
<a href="https://2010.igem.org/Team:British_Columbia/HP_nanowrimo">Once upon a time...</a><br />
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<div id="blueBox"><h3>Art disturbs, science reassures ~Georges Braque</h3><br />
<p>What can generate more inspiration or discussion than art? And what is not art? Come take a look. Better yet, contribute!</p><br />
<a href="https://2010.igem.org/Team:British_Columbia/HP_arts">Be disturbed!</a><br />
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<h3>Promoter Maps</h3><br />
<p>We collected hundreds of definitions of synthetic biology from the UBC community-undergrads, grad students and faculty from various disciplines. These definitions were sorted by field/occupation as well as level of education in order to synthesize these promoter maps. Each promoter map consists of words arranged from highest to lowest frequency (with implemented cut-off points), representing the prevalent ideas in our community's awareness of synthetic biology!</p></br><center><BR/><br />
<IMG SRC="https://static.igem.org/mediawiki/2010/1/16/LIFESCIPM.jpg"></br></br><br></br><br></br><BR/><br />
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<h3>Word Clouds</h3><br />
<p>Can't get enough? We've got a storm of word clouds headed this way. Each represents definitions of synthetic biology from students in different disciplines!</p><br/><br />
<center><img src="https://static.igem.org/mediawiki/2010/8/81/WordcloudAll.jpg"><br></br><br></br><BR/><br />
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<a href="https://static.igem.org/mediawiki/2010/f/f8/Artscommerce.jpg"><img src="https://static.igem.org/mediawiki/2010/f/f8/Artscommerce.jpg" height=80px></a><br />
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<center><h3>The Synthetic Biology Art Gallery</h3><br />
<a href="https://2010.igem.org/Team:British_Columbia/HP_arts"><img src="https://static.igem.org/mediawiki/2010/9/96/Arts.gif"></a><br />
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<div class="grey"><br></br><p>"Art is the Queen of all sciences communicating knowledge to all the generations of the world." -<b>Leonardo da Vinci</b> <br></br></div><br />
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<p>"Good art is not what it looks like, but what it does to us." -<b>Roy Adzak</b><br />
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<div class="grey"><br></br><p>"Art is literacy of the heart." -<b>Elliot Eisner</b><br></br></div><br />
<br></br><p>"The job of the artist is always to deepen the mystery." -<b>Francis Bacon</b> <br></br><br />
<div class="grey"><br></br><p>"What a society deems important is enshrined in its art." -<b>Harry Broudy</b> <br></br></div><br />
<br></br><p><b>Whatever your thoughts on art are, come visit our <a href="https://2010.igem.org/Team:British_Columbia/HP_arts">gallery</a> and send us your very own artwork on synthetic biology!</b><br></br><br />
<div class="grey"><br/><h3>NaNoWriMo</h3><br />
<img src="http://www.trashionista.com/nanowrimo.jpg" height=120px><br />
<p><a href="http://www.nanowrimo.org/">National Novel Writing Month</a> participants aim to write an astounding 50,000-word novel within the month of November. In our first collaboration ever, iGEM and NaNoWriMo unite to showcase <a href="https://2010.igem.org/Team:British_Columbia/HP_nanowrimo">stories featuring synthetic biology</a>! <br />
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<img src="https://static.igem.org/mediawiki/2010/5/57/Agubc.jpg" height=70px><br />
<b>Augmented Genesis by Tony Southcotte </b><br/><br />
Mankind has undergone a synthetic evolution. So when an electromagnetic storm comes, chaos ensues. <br />
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<img src="https://static.igem.org/mediawiki/2010/6/6a/Bridges_Cover_Art.jpg" height=70px><br />
<b>Bridges by Edrei Zahari </b><br/><br />
In the aftermath of WW3, a genetically enhanced platoon of UN peacekeepers patrol the irradiated countryside...<br></br><br />
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<img src="https://static.igem.org/mediawiki/2010/3/38/Craw.jpg" height=70px><br />
<b>Brought to Life by Richard S. Crawford</b><br/><br />
It's the epic story of the first synthetic person and the challenges he faces as he tries to establish his own identity.<br />
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<img src="https://static.igem.org/mediawiki/2010/3/3f/Catharsisubc.jpg" height=70px><br />
<b>Catharsis by David Litherland </b><br/><br />
A malicious scientist has developed an airborne mutagen which turns rational humans into mindless, violent drones...<br />
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<img src="https://static.igem.org/mediawiki/2010/f/f6/Fledwing.jpg" height=70px><br />
<b>Fledgling Wings by Angelica A.</b><br/><br />
Jonas is a hybrid of human and animal. Now he must discover the secrets behind the disappearance of chimeras.<br />
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<img src="https://static.igem.org/mediawiki/2010/a/a3/NanoKg.jpg" height=70px><br />
<b>Kalopsiac Green by Karen Llamas</b><br/><br />
Thomas and Felix were sold to science, and they know all too well this isn't the destiny sci-fi novelists dreamt of.<br />
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<img src="https://static.igem.org/mediawiki/2010/4/4a/Lyghts.jpg" height=70px><br />
<b>Lyghts by Lyvie Hallman Taylor</b><br/><br />
A scientist finds hope in LYGHT: DNA codes that enhance everything from physical attraction to touch.<br></br><br />
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<img src="https://static.igem.org/mediawiki/2010/3/38/Machmen.jpg" height=70px><br />
<b>Machmen by Kay Proctor</b><br/><br />
Robots turn the tables and program humans to obey the Three Laws of Robotics - for better or for worse.<br />
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<img src="https://static.igem.org/mediawiki/2010/c/c6/Maggie_Burns.jpg" height=70px><br />
<b>Perfect Monster by Maggie Burns</b><br/><br />
A researcher who commits crimes against humanity tries to save the world from the terrible consequences.<br></br><br />
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<img src="https://static.igem.org/mediawiki/2010/4/4c/Progress_unbound.jpg" height=70px><br />
<b>Progress Unbound by David Scheidl</b><br/><br />
Synthetically augmented humans have colonized distant planets and continue to experiment to better survive.<br />
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<img src="https://static.igem.org/mediawiki/2010/e/e9/Twice_cover_again.jpg" height=70px><br />
<b>Twice by Vivian Rivera</b><br/><br />
It isn't safe to say Joseph was murdered because he's still alive. He's just not in his own body.<br />
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<br></br><p><b>Intrigued? Come visit our <a href="https://2010.igem.org/Team:British_Columbia/HP_nanowrimo">NaNoWriMo-iGEM Showcase</a>!</b><br></br><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Template:Template_HD_4Template:Template HD 42010-10-27T16:00:07Z<p>Ayjchan: </p>
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/HumanPracticesTeam:British Columbia/HumanPractices2010-10-27T15:57:46Z<p>Ayjchan: </p>
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<div id="orangeBox"><h3>Synthetic Biology & Society</h3><br />
<p>Whether it's agriculture, healthcare, the environment or the economy, synthetic biology has an increasingly important role to play! Join in our discussions!</p><br />
<a href="http://forum2010.ubcigem.com/">Our Forum</a><br />
</div><br />
<div id="greenBox"><h3>NaNoWriMo x iGEM</h3><br />
<p>In our first collaboration ever, iGEM and NaNoWriMo unite to showcase stories featuring synthetic biology! Follow these NaNoWriMo novelists over the month of November!</p><br />
<a href="https://2010.igem.org/Team:British_Columbia/HP_nanowrimo">Once upon a time...</a><br />
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<div id="blueBox"><h3>Art disturbs, science reassures ~Georges Braque</h3><br />
<p>What can generate more inspiration or discussion than art? And what is not art? Come take a look. Better yet, contribute!</p><br />
<a href="https://2010.igem.org/Team:British_Columbia/HP_arts">Be disturbed!</a><br />
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<h3>Promoter Maps</h3><br />
<p>We collected hundreds of definitions of synthetic biology from the UBC community-undergrads, grad students and faculty from various disciplines. These definitions were sorted by field/occupation as well as level of education in order to synthesize these promoter maps. Each promoter map consists of words arranged from highest to lowest frequency (with implemented cut-off points), representing the prevalent ideas in our community's awareness of synthetic biology!</p></br><center><BR/><br />
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<h3>Word Clouds</h3><br />
<p>Can't get enough? We've got a storm of word clouds headed this way. Each represents definitions of synthetic biology from students in different disciplines!</p><br/><br />
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<center><h3>The Synthetic Biology Art Gallery</h3><br />
<a href="https://2010.igem.org/Team:British_Columbia/HP_arts"><img src="https://static.igem.org/mediawiki/2010/9/96/Arts.gif"></a><br />
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<div class="grey"><br></br><p>"Art is the Queen of all sciences communicating knowledge to all the generations of the world." -<b>Leonardo da Vinci</b> <br></br></div><br />
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<p>"Good art is not what it looks like, but what it does to us." -<b>Roy Adzak</b><br />
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<div class="grey"><br></br><p>"Art is literacy of the heart." -<b>Elliot Eisner</b><br></br></div><br />
<br></br><p>"The job of the artist is always to deepen the mystery." -<b>Francis Bacon</b> <br></br><br />
<div class="grey"><br></br><p>"What a society deems important is enshrined in its art." -<b>Harry Broudy</b> <br></br></div><br />
<br></br><p><b>Whatever your thoughts on art are, come visit our <a href="https://2010.igem.org/Team:British_Columbia/HP_arts">gallery</a> and send us your very own artwork on synthetic biology!</b><br></br><br />
<div class="grey"><br/><h3>NaNoWriMo</h3><br />
<img src="http://www.trashionista.com/nanowrimo.jpg" height=120px><br />
<p><a href="http://www.nanowrimo.org/">National Novel Writing Month</a> participants aim to write an astounding 50,000-word novel within the month of November. In our first collaboration ever, iGEM and NaNoWriMo unite to showcase <a href="https://2010.igem.org/Team:British_Columbia/HP_nanowrimo">stories featuring synthetic biology</a>! <br />
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<img src="https://static.igem.org/mediawiki/2010/5/57/Agubc.jpg" height=70px><br />
<b>Augmented Genesis by Tony Southcotte </b><br/><br />
Mankind has undergone a synthetic evolution. So when an electromagnetic storm comes, chaos ensues. <br />
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<img src="https://static.igem.org/mediawiki/2010/6/6a/Bridges_Cover_Art.jpg" height=70px><br />
<b>Bridges by Edrei Zahari </b><br/><br />
In the aftermath of WW3, a genetically enhanced platoon of UN peacekeepers patrol the irradiated countryside...<br></br><br />
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<img src="https://static.igem.org/mediawiki/2010/3/38/Craw.jpg" height=70px><br />
<b>Brought to Life by Richard S. Crawford</b><br/><br />
It's the epic story of the first synthetic person and the challenges he faces as he tries to establish his own identity.<br />
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<img src="https://static.igem.org/mediawiki/2010/3/3f/Catharsisubc.jpg" height=70px><br />
<b>Catharsis by David Litherland </b><br/><br />
A malicious scientist has developed an airborne mutagen which turns rational humans into mindless, violent drones...<br />
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<img src="https://static.igem.org/mediawiki/2010/f/f6/Fledwing.jpg" height=70px><br />
<b>Fledgling Wings by Angelica A.</b><br/><br />
Jonas is a hybrid of human and animal. Now he must discover the secrets behind the disappearance of chimeras.<br />
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<img src="https://static.igem.org/mediawiki/2010/a/a3/NanoKg.jpg" height=70px><br />
<b>Kalopsiac Green by Karen Llamas</b><br/><br />
Thomas and Felix were sold to science, and they know all too well this isn't the destiny sci-fi novelists dreamt of.<br />
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<img src="https://static.igem.org/mediawiki/2010/4/4a/Lyghts.jpg" height=70px><br />
<b>Lyghts by Lyvie Hallman Taylor</b><br/><br />
A scientist finds hope in LYGHT: DNA codes that enhance everything from physical attraction to touch.<br></br><br />
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<img src="https://static.igem.org/mediawiki/2010/3/38/Machmen.jpg" height=70px><br />
<b>Machmen by Kay Proctor</b><br/><br />
Robots turn the tables and program humans to obey the Three Laws of Robotics - for better or for worse.<br />
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<img src="https://static.igem.org/mediawiki/2010/c/c6/Maggie_Burns.jpg" height=70px><br />
<b>Perfect Monster by Maggie Burns</b><br/><br />
A researcher who commits crimes against humanity tries to save the world from the terrible consequences.<br></br><br />
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<img src="https://static.igem.org/mediawiki/2010/4/4c/Progress_unbound.jpg" height=70px><br />
<b>Progress Unbound by David Scheidl</b><br/><br />
Synthetically augmented humans have colonized distant planets and continue to experiment to better survive.<br />
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<img src="https://static.igem.org/mediawiki/2010/e/e9/Twice_cover_again.jpg" height=70px><br />
<b>Twice by Vivian Rivera</b><br/><br />
It isn't safe to say Joseph was murdered because he's still alive. He's just not in his own body.<br />
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<br></br><p><b>Intrigued? Come visit our <a href="https://2010.igem.org/Team:British_Columbia/HP_nanowrimo">NaNoWriMo-iGEM Showcase</a>!</b><br></br><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/SafetyTeam:British Columbia/Safety2010-10-27T15:55:18Z<p>Ayjchan: </p>
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<h3>1. Would any of your project ideas raise safety issues?</h3></center><br />
<p>Our project idea is to engineer a <i>Staphylococcus aureus</i>-specific phage that contains DspB, a biofilm matrix-degrading enzyme and is controlled by a <i>S. aureus</i> quorum-sensing system. In terms of human or animal safety, this engineered phage should not pose any biohazardous risk since it is specific to bacteria and can already be found in nature albeit without DspB. DspB is also found in nature and not harmful to organisms since it serves only to degrade extracellular carbohydrate polymer bonds in the biofilm matrix. Furthermore, the phage and DspB are expressed/triggered by elements of the <i>S. aureus</i> quorum-sensing system when a notable concentration or biofilm of <i>S. aureus</i> is present. Our project ideas should also not have any severe impact on the environment since the phage targets <i>S. aureus</i> biofilms.<br />
<br/><br/><br />
The phage standard which we are introducing to the iGEM competition allows basic modification of bacteriophage genomes and must be treated with care. Due to a bacteriophage's higher potential for mutation (as high as 10^-6 mutations per base pair compared to eukaryotes at 10^-8 mutations per base pair) there is a greater chance of catastrophic mutations occurring. It should also be considered that if phage DNA mutates to be harmful in some way, the potential spread is greater because every phage is capable of up to 200 progeny. However, since phage genomes generally range from 15 to 150 kilo base pairs so that the genome can fit inside the capsid, the genomes are highly refined and do not contain much redundant DNA available for novel gain-of-function mutations. In summary, the phage standard does not introduce any inherent risk that is not already present when dealing with phages.<br />
<br/><br/>If our phage does mutate to become more promiscuous, there are still many barriers in place to prevent it from effectively eliminating other species' biofilms. The phage will still be under the control of the <i>S. aureus</i> quorum-sensing system, and it will unlikely be expressed/triggered in its new infected host. DspB is also only known to degrade <i>S. aureus</i> and <i>Escherichia coli</i> biofilms. Conversely, if the phage mutates to become unable to infect <i>S. aureus</i>, then that phage will fail to infect or replicate. The probability of this happening is moderate considering the great number of phages produced during each infection, but the results are not hazardous as explained.<br />
<br/><br/>If DspB mutates and gains the ability to degrade a greater variety of biofilms, the mutation may not be uncommon in nature although the fact that it is propagated by phage may increase the mutation frequency. Nonetheless, it will still be contained within a phage specific to and only expressed in <i>S. aureus</i>. On the other hand, if DspB loses its function, then the phage will just have to work alone, but there will not be catastrophic consequences. The probability of this happening is moderate based on the rate of mutation in phages and the numbers of phages produced per infection.<br />
<br/><br/>If the quorum-sensing promoters that control the expression of the phage and DspB mutate to become constitutive or incorrectly activated without the presence of a biofilm, then the phage will simply lyse its host prematurely. If the promoters mutate to become inactivated, then the system will cease to function, but once again there will not be catastrophic consequences. The probability of this happening is moderate as before.<br />
<br/><br/>If we had to imagine the worst case scenario ever... if the phage manages to target various bacteria AND DspB also degrades various biofilms AND the quorum-sensing promoter becomes appropriate to various bacteria, resulting in widespread degradation of all types of biofilms without control, THEN this would have some environmental ramifications since biofilms are found on most natural surfaces. But the probability of this happening is extremely low considering that most bacteria don't even recognize each other's promoters and have internal guard mechanisms to shut down expression of DNA from foreign species.<br />
<br/><br/>If we had to break through the bounds of imagination to imagine the absolute worst case apocalyptic scenario ever, maybe our phage will mutate into a human-specific virus AND DspB will become able to degrade various polymers in humans AND the quorum-sensing promoter will become a constitutive promoter so that our phage will wipe out the human race. But the probability of this happening is as low as that of our team producing a flying pig in time for this year's iGEM Jamboree.<br />
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<h3>2. Do the new BioBrick parts that you made this year raise safety issues? </h3></center><br />
<p><br />
Our new BioBrick part, DspB is a biofilm matrix-degrading enzyme and does not raise any significant safety issues. It has been sequenced and assayed for its enzymatic activity and found to be reliable. In the circumstance that a safety incident occurs, users will be able to contact us and we will update the Registry with their report.</p><br></br><br />
<center><img src="https://static.igem.org/mediawiki/2010/8/84/Ubcs2.jpg"></src><br />
<h3>3. Is there a local biosafety group at your institution?</h3></center><br />
<p><br />
The local biosafety group for our project is the Department of Health Safety and Environment (HSE) of UBC. Our laboratory space and equipment meets all safety requirements as per Canadian regulations and the regulations of the HSE. All members of our team have also taken the required Laboratory health safety course from our local biosafety group. Presently, our team has not embarked on research using pathogenic bacterial strains or phages. Our research also does not involve the transference of toxins or drug resistance that could compromise the use of the drug to control disease agents in humans, veterinary medicine, or agriculture.</p><br></br><br />
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<h3>4. Do you have ideas to deal with safety issues that could be useful for future iGEM competitions? </h3></center><br />
<p><b>4.1. Biosafety engineering and design of a safer chassis</b><br />
<br/><br />
Current safety engineering tools such as Event Tree Analysis (ETA) and Fault Tree Analysis (FTA) have yet to be extensively incorporated into synthetic biology models. One large obstacle is the fact that organisms are complex and not everything is known about their inner processes and community interactions. So while synthetic biologists are able to make logical predictions regarding their designed part or system, the sphere of knowledge of the chassis is greatly limited. This reason also motivates the current search for a safer chassis-one that is understood inside and out. In order to engineer safety into our synthetic biology parts or systems, it is necessary to attain a good understanding of what it does in its natural setting, design safety elements based on this knowledge and its extrapolation, and then collect experimental data on its behavior and mutability in synthetic settings. Just as conventional safety engineering utilizes real engineering data and designs, biosafety engineering has to become a whole and unique field unto itself. Research in controlled system-destruction is growing, and there will probably be research in the mutability of different types of synthetic biological circuits. One field of research that is instrumental to biosafety engineering is that of the intelligent synthesis of whole genomes from scratch. This will no doubt provide valuable insights as to the workings of the organism in study and also lead to the production of synthetic chassis and systems that are much more manipulable, controllable and predictable. <br />
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<b>4.2. Public perception of risks and safety issues</b><br />
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Through our human practices project, we are exploring different perspectives of synthetic biology by asking members of the public, as well as iGEM participants, to create art in the form of visual arts or stories conveying their perception of synthetic biology and its potential impact on the world. We hope that by stimulating the public to learn more about synthetic biology and hopefully engaging in a meaningful exchange of ideas, the public will gain a deeper and sounder understanding of what synthetic biology is and how synthetic biologists also seek to install the necessary safety infrastructure. By asking for public opinion, synthetic biologists also have the opportunity to address public concerns and lay a firmer foundation for future synthetic biology ventures and applications in the real world. Since the benefits and consequences of synthetic biology research are shared by both researchers and the public, the two must actively seek to listen, inform and negotiate. Scientific risk assessment may produce quantitative measures of potential damage, but this is only a model of what may happen in reality. In order to validate the assumptions, applicability and foresight of these risk assessment estimates, we need to receive the input of the public, who are representative of real-life data. As the term “human practices” suggests, synthetic biologists who endeavour to develop the human practices aspect of their research must consider the same things that society considers. Will the fruits of our research be accessible to the poor? Are people in charge trustworthy? Will there be potential consequences for future generations? Are there ways for us to control the applications of our research? In other words, our synthetic biology parts and systems are like children born into the world. Scientific risk assessment is careful planning. Engaging with society is the actual raising of synthetic biology children. There has to be an increase in public awareness of what synthetic biology is and what it can do. There also has to be an increase in dialogue (whether through public forums or through art and entertainment) not just within the scientific community but also with the public.<br />
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<p>Biosafety deals with the containment principles, technologies and practices that are intended to prevent exposure to pathogens and toxins, and their accidental release. <br />
</p><br />
<a href="https://2010.igem.org/Safety">2010 iGEM Safety Questions</a><br></br><br />
<a href="http://www.who.int/csr/delibepidemics/WHO_CDS_CSR_LYO_2004_11/en/">WHO Biosafety Manual</a><br></br><br />
<a href="http://oba.od.nih.gov/rdna_ibc/ibc.html">NIH Institutional Biosafety Committees</a><br></br><br />
<a href="http://www.cdc.gov/biosafety/">CDC Office of Health and Safety</a><br></br><br />
<a href="http://www.hse.ubc.ca/welcome.html">Health Safety and Environment, UBC</a><br></br><br />
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<h3>Risk Perception Quick Links</h3><br />
<p>Risk perception is the subjective judgment that people make about the characteristics and severity of a risk.</p><br />
<a href="http://www.synbiosafe.eu/">Synbiosafe</a><br></br><br />
<a href="http://www.markusschmidt.eu/pdf/Intro_risk_perception_Schmidt.pdf">An Introduction</a><br></br><br />
<a href="http://www.cepis.ops-oms.org/tutorial6/i/pdf/topic_04.pdf">Risk Perception</a><br></br><br />
<a href="http://www.markusschmidt.eu/pdf/slovic_risk-perception.pdf">Placing Risks in Perspective</a><br></br><br />
<a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759424/pdf/11693_2009_Article_9031.pdf">Communicating Synthetic Biology</a><br></br><br />
<a href="http://syntheticbiology.org/Press.html">Synthetic biology Press</a><br></br><br />
<a href="http://ec.europa.eu/european_group_ethics/publications/docs/round_table_ethical_aspects_of_synthetic_biology.pdf">Ethical Aspects of Synthetic Biology</a><br></br><br />
<a href="http://www.springerlink.com/content/h81458455710n37n/fulltext.html">Synthetic Biology and Society</a><br></br><br />
<a href="http://papers.ssrn.com/sol3/papers.cfm?abstract_id=1264804">Culture and Synthetic Biology</a><br></br><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/Project_PhageTeam:British Columbia/Project Phage2010-10-27T15:51:10Z<p>Ayjchan: </p>
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<h3>Introduction</h3><br />
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<p>The goal of the Phage sub-team was to develop the new phage standard for submission to the BioBrick registry and to characterize the phage that would be used for our project.</p><br/><br />
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<p>The <b>phage standard</b> presented itself once we made the decision to use a phage as a vector to attain our project goals. The standard is necessary for three reasons. Firstly, lysogenic phages are natural vectors that have evolved to integrate and propagate their DNA through specific bacterial strains. Secondly, it is impossible to work with phages using existing BioBrick standards due to the illegal cut sites that occur in every lysogenic phage. Lastly, lysogenic phage genomes are too large to be manipulated using normal BioBrick plasmids. Based on these reasons, our phage standard is an important addition to the BioBricks registry.</p><br/><br />
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<p> The objectives of our phage standard include negating the issues of genome size, exploiting phage characteristics for use as a vector, and developing a BioBrick compatible standard applicable to all lysogenic phages.</p><br/><br />
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<h3>Background</h3><br />
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<p>Lysogenic phages have evolved to insert their DNA into the genomes of specific strains of bacteria. Sometimes this insertion is done at random in the case of a non-specific integration site (INSERT A REFERENCE HERE) or it is inserted only at a very specific location in the genome (INSERT REFERENCE HERE). This specificity allows the integration sites of different lysogenic phages to be used as <b>insertion vectors</b>. These insertion vectors (Fig. 1) will be low-copy BioBrick plasmids containing the integration site of a given phage, flanked by chosen restriction sites.</p><br/><br />
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<h3>The Details</h3><br />
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<p>The phage standard describes the process of adding a given Biobrick part, which we will call <b>source DNA</b> into the genome of a lysogenic phage, referred to as <b>host DNA</b>. This will require secondary DNA sequences including the phage genome <b>integration site</b>, some <b>garbage DNA</b> (flanked by essential restriction enzyme cut sites) and the low-copy number <b>BioBrick plasmid</b>.</p><br/><br />
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<p>The first step is to choose restriction enzymes using <a href="http://bio.lundberg.gu.se/cutter2/">webcutter 2.0</a> that <b>appear only once</b>, closest to the region of the phage genome that is going to be modified (Fig. 1).</p><br/><center><img width="600" src="http://i.imgur.com/UVJZQ.jpg"></center><br />
<div align="center"><p><i><b>Figure 1.</b> Phage genome showing region of interest to be modified by <b>source DNA</b> and nearby unique restriction enzyme site</i></p></div><br/><br />
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<p>Next, find a cut site that is absent in (i) the host DNA, (ii) the BioBrick plasmids and (iii) the source DNA. Using PCR, get the integration site off the bacterial genome and flank it with standard BioBrick E, X, S and P in addition to the absent cut site (Fig. 2; A1 refers to the absent restriction enzyme site).</p><br/><center><img width="600" src="http://i.imgur.com/TWVy0.jpg"></center><br />
<div align="center"><p><i><b>Figure 2.</b> Phage integration site flanked by BioBrick restriction enzyme sites and the absent site.</i></p></div><br/><br />
<br />
<p>Using standard BioBrick assembly methods, ligate the integration site onto a low-copy BioBrick plasmid (Fig. 3). Then transform the plasmid into the host bacterial strain and expose the bacteria to the phage. This will allow the phage to infect the bacterial cells as well as the integration site on the low-copy BioBrick plasmid. From this culture, miniprep the DNA - very carefully since it is now a 50 kilo base pair plus plasmid.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/5Ghik.jpg"></center><br />
<div align="center"><p><i><b>Figure 3.</b> Current state of the low copy BioBrick plasmid containing the full phage genome.</i></p></div><br/><br />
<br />
<p>Prepare a high copy BioBrick plasmid with the A1 and Unique site by PCRing these sites onto some garbage DNA and cutting/ligating the DNA onto the plasmid. The garbage DNA doesn’t need to be useless. It is recommended to use GFP or RFP for visual confirmation of a successful cloning procedure.</p><br/><br />
<br />
<p>The next step is to cut at A1 and the Unique site and ligate this DNA onto the higher copy garbage (with cut sites) plasmid. Ideally, this plasmid will be on the order of 4-10 kilo base pairs in total, allowing standard cloning techniques to be used with ease. This plasmid is the sub host DNA.</p><br/><br />
<br />
<p>At this stage it is necessary to create restriction enzyme cut sites in the locations needed. This will involve performing two site-directed mutagenesis (SDM) reactions to insert cut sites flanking the specific region that is to be modified.</p><br/><br />
<br />
<p>For the next step to proceed, it is necessary to modify the source DNA, the BioBrick part, by adding cut sites using PCR. These cut sites should be in on the inside of E, X, S, and P as before (similar to the garbage DNA) and should match the restriction sites that were SDM’ed into the sub host DNA.</p><br/><br />
<br />
<p>Finally, insert the modified source DNA into the sub host DNA.</p><br/><br />
<br />
<p>Cut and ligate it all back together and viola, a modified phage genome is at your disposal.</p><br/><br />
<br />
<h3>Summary of Essentials</h3><br />
<br />
<p>Use PCR to add restriction sites to:<br />
<br />
a) Integration site<br />
b) Source DNA<br />
</p><br/><br />
<br />
<p>Prepare a BioBrick plasmid with the A1 and Unique sites on the inside of the normal BioBrick cut sites (Fig. 4).</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/cBT2d.jpg"></center><br />
<div align="center"><p><i><b>Figure 4.</b> Restriction Enzyme Sites of Integration Site, Source DNA and "Garbage" Plasmid</i></p></div><br/><br />
<br />
<p>Cut and ligate the integration site onto a low copy BioBrick plasmid using standard cut sites.</p><br/><br />
<br />
<p>Allow the phage to insert its genome into the integration site (Fig. 5). <b>IMPORTANT</b> – the genome housing this low-copy integration site plasmid <b>MUST</b> be the bacterial strain normally targeted by the phage.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/rOwEK.jpg"></center><br />
<div align="center"><p><i><b>Figure 5.</b> Phage in integration site plasmid, sub host plasmid before ligation with portion of phage DNA.</i></p></div><br/><br />
<br />
<p>Cut the phage genome at the unique site and the absent site. Ligate this portion of the phage genome onto the prepared BioBrick plasmid.</p><br/><br />
<br />
<p>Use SDM to add cut sites (Fig. 6). Cut/ligate in the source DNA.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/TFM5J.jpg"></center><br />
<div align="center"><p><i><b>Figure 6.</b> Relative Locations of SDM sites.</i></p></div><br/><br />
<br />
<p>Use A1 and the Unique site to reinsert the sub host DNA into the phage (Fig. 7).</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/AejhW.jpg"></center><br />
<div align="center"><p><i><b>Figure 7.</b> Relative Locations of A1 and Unique sites.</i></p></div><br/><br />
<br />
<br />
<p>Enjoy the fruits of your labor – a modified phage genome.</p><br/><br />
<br />
<h3>Conclusions</h3><br />
<br />
<p>Our phage standard solves the problem of phage genomes being too large, negate the problem of phage genomes containing multiple illegal cut sites and allows any lysogenic phage to be used as part of the BioBrick registry.<br />
</p></br><br />
<br />
<h3>The Wet-Lab Phage</h3><br />
<p>Since our wet lab experiments were focusing on <i>S. Aureus</i> biofilms we had a finite list of phages to choose from. We originally chose to work with phage &phi;MR11 since information on it's genome was readily available and it appeared to be the subject of current research. As the summer proceeded and requests for the phage did not materialize we moved on to work on a different phage. We chose &phi;11, a prophage found in <i>S. Aureus</i> strain 8325 along with 2 other prophages. Plans proceeded with developing the phage standard as we attempted acquire both the original phage &phi;MR11 and <i>S. Aureus</i> strain 8325 containing phage &phi;11.</p><br/><br />
<br />
<br />
</div> <!-- end SubWrapper --><br />
<br />
<div id="news" style="height:5500px;"> <br />
<br/><center><h3>References</h3><br />
<br />
<br />
</div> <!-- end news --><br />
</div> <!-- end Super_main_wrapper --><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/Project_PhageTeam:British Columbia/Project Phage2010-10-27T15:50:35Z<p>Ayjchan: </p>
<hr />
<div>__NOTOC__<br />
<br />
{{Template_HD_4}}<br />
<html><body id="project" onload="setPageSize()"><br />
<br />
<div id="super_main_wrapper"><br />
<div id="SubWrapper"> <br />
<br/><br />
<br />
<br />
<h3>Introduction</h3><br />
<br />
<p>The goal of the Phage sub-team was to develop the new phage standard for submission to the BioBrick registry and to characterize the phage that would be used for our project.</p><br/><br />
<br />
<p>The <b>phage standard</b> presented itself once we made the decision to use a phage as a vector to attain our project goals. The standard is necessary for three reasons. Firstly, lysogenic phages are natural vectors that have evolved to integrate and propagate their DNA through specific bacterial strains. Secondly, it is impossible to work with phages using existing BioBrick standards due to the illegal cut sites that occur in every lysogenic phage. Lastly, lysogenic phage genomes are too large to be manipulated using normal BioBrick plasmids. Based on these reasons, our phage standard is an important addition to the BioBricks registry.</p><br/><br />
<br />
<p> The objectives of our phage standard include negating the issues of genome size, exploiting phage characteristics for use as a vector, and developing a BioBrick compatible standard applicable to all lysogenic phages.</p><br/><br />
<br />
<h3>Background</h3><br />
<br />
<p>Lysogenic phages have evolved to insert their DNA into the genomes of specific strains of bacteria. Sometimes this insertion is done at random in the case of a non-specific integration site (INSERT A REFERENCE HERE) or it is inserted only at a very specific location in the genome (INSERT REFERENCE HERE). This specificity allows the integration sites of different lysogenic phages to be used as <b>insertion vectors</b>. These insertion vectors (Fig. 1) will be low-copy BioBrick plasmids containing the integration site of a given phage, flanked by chosen restriction sites.</p><br/><br />
<br />
<h3>The Details</h3><br />
<br />
<p>The phage standard describes the process of adding a given Biobrick part, which we will call <b>source DNA</b> into the genome of a lysogenic phage, referred to as <b>host DNA</b>. This will require secondary DNA sequences including the phage genome <b>integration site</b>, some <b>garbage DNA</b> (flanked by essential restriction enzyme cut sites) and the low-copy number <b>BioBrick plasmid</b>.</p><br/><br />
<br />
<p>The first step is to choose restriction enzymes using <a href="http://bio.lundberg.gu.se/cutter2/">webcutter 2.0</a> that <b>appear only once</b>, closest to the region of the phage genome that is going to be modified (Fig. 1).</p><br/><center><img width="600" src="http://i.imgur.com/UVJZQ.jpg"></center><br />
<div align="center"><p><i><b>Figure 1.</b> Phage genome showing region of interest to be modified by <b>source DNA</b> and nearby unique restriction enzyme site</i></p></div><br/><br />
<br />
<p>Next, find a cut site that is absent in (i) the host DNA, (ii) the BioBrick plasmids and (iii) the source DNA. Using PCR, get the integration site off the bacterial genome and flank it with standard BioBrick E, X, S and P in addition to the absent cut site (Fig. 2; A1 refers to the absent restriction enzyme site).</p><br/><center><img width="600" src="http://i.imgur.com/TWVy0.jpg"></center><br />
<div align="center"><p><i><b>Figure 2.</b> Phage integration site flanked by BioBrick restriction enzyme sites and the absent site.</i></p></div><br/><br />
<br />
<p>Using standard BioBrick assembly methods, ligate the integration site onto a low-copy BioBrick plasmid (Fig. 3). Then transform the plasmid into the host bacterial strain and expose the bacteria to the phage. This will allow the phage to infect the bacterial cells as well as the integration site on the low-copy BioBrick plasmid. From this culture, miniprep the DNA - very carefully since it is now a 50 kilo base pair plus plasmid.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/5Ghik.jpg"></center><br />
<div align="center"><p><i><b>Figure 3.</b> Current state of the low copy BioBrick plasmid containing the full phage genome.</i></p></div><br/><br />
<br />
<p>Prepare a high copy BioBrick plasmid with the A1 and Unique site by PCRing these sites onto some garbage DNA and cutting/ligating the DNA onto the plasmid. The garbage DNA doesn’t need to be useless. It is recommended to use GFP or RFP for visual confirmation of a successful cloning procedure.</p><br/><br />
<br />
<p>The next step is to cut at A1 and the Unique site and ligate this DNA onto the higher copy garbage (with cut sites) plasmid. Ideally, this plasmid will be on the order of 4-10 kilo base pairs in total, allowing standard cloning techniques to be used with ease. This plasmid is the sub host DNA.</p><br/><br />
<br />
<p>At this stage it is necessary to create restriction enzyme cut sites in the locations needed. This will involve performing two site-directed mutagenesis (SDM) reactions to insert cut sites flanking the specific region that is to be modified.</p><br/><br />
<br />
<p>For the next step to proceed, it is necessary to modify the source DNA, the BioBrick part, by adding cut sites using PCR. These cut sites should be in on the inside of E, X, S, and P as before (similar to the garbage DNA) and should match the restriction sites that were SDM’ed into the sub host DNA.</p><br/><br />
<br />
<p>Finally, insert the modified source DNA into the sub host DNA.</p><br/><br />
<br />
<p>Cut and ligate it all back together and viola, a modified phage genome is at your disposal.</p><br/><br />
<br />
<h3>Summary of Essentials</h3><br />
<br />
<p>Use PCR to add restriction sites to:<br />
<br />
a) Integration site<br />
b) Source DNA<br />
</p><br/><br />
<br />
<p>Prepare a BioBrick plasmid with the A1 and Unique sites on the inside of the normal BioBrick cut sites (Fig. 4).</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/cBT2d.jpg"></center><br />
<div align="center"><p><i><b>Figure 4.</b> Restriction Enzyme Sites of Integration Site, Source DNA and "Garbage" Plasmid</i></p></div><br/><br />
<br />
<p>Cut and ligate the integration site onto a low copy BioBrick plasmid using standard cut sites.</p><br/><br />
<br />
<p>Allow the phage to insert its genome into the integration site (Fig. 5). <b>IMPORTANT</b> – the genome housing this low-copy integration site plasmid <b>MUST</b> be the bacterial strain normally targeted by the phage.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/rOwEK.jpg"></center><br />
<div align="center"><p><i><b>Figure 5.</b> Phage in integration site plasmid, sub host plasmid before ligation with portion of phage DNA.</i></p></div><br/><br />
<br />
<p>Cut the phage genome at the unique site and the absent site. Ligate this portion of the phage genome onto the prepared BioBrick plasmid.</p><br/><br />
<br />
<p>Use SDM to add cut sites (Fig. 6). Cut/ligate in the source DNA.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/TFM5J.jpg"></center><br />
<div align="center"><p><i><b>Figure 6.</b> Relative Locations of SDM sites.</i></p></div><br/><br />
<br />
<p>Use A1 and the Unique site to reinsert the sub host DNA into the phage (Fig. 7).</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/AejhW.jpg"></center><br />
<div align="center"><p><i><b>Figure 7.</b> Relative Locations of A1 and Unique sites.</i></p></div><br/><br />
<br />
<br />
<p>Enjoy the fruits of your labor – a modified phage genome.</p><br/><br />
<br />
<h3>Conclusions</h3><br />
<br />
<p>Our phage standard solves the problem of phage genomes being too large, negate the problem of phage genomes containing multiple illegal cut sites and allows any lysogenic phage to be used as part of the BioBrick registry.<br />
</p></br><br />
<br />
<h3>The Wet-Lab Phage</h3><br />
<p>Since our wet lab experiments were focusing on <i>S. Aureus</i> biofilms we had a finite list of phages to choose from. We originally chose to work with phage &phi;MR11 since information on it's genome was readily available and it appeared to be the subject of current research. As the summer proceeded and requests for the phage did not materialize we moved on to work on a different phage. We chose &phi;11, a prophage found in <i>S. Aureus</i> strain 8325 along with 2 other prophages. Plans proceeded with developing the phage standard as we attempted acquire both the original phage &phi;MR11 and <i>S. Aureus</i> strain 8325 containing phage &phi;11.</p></br><br />
<br />
<br />
</div> <!-- end SubWrapper --><br />
<br />
<div id="news" style="height:5000px;"> <br />
<br/><center><h3>References</h3><br />
<br />
<br />
</div> <!-- end news --><br />
</div> <!-- end Super_main_wrapper --><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/Project_PhageTeam:British Columbia/Project Phage2010-10-27T15:37:20Z<p>Ayjchan: </p>
<hr />
<div>__NOTOC__<br />
<br />
{{Template_HD_4}}<br />
<html><body id="project" onload="setPageSize()"><br />
<br />
<div id="super_main_wrapper"><br />
<div id="SubWrapper"> <br />
<br/><br />
<br />
<br />
<h3>Introduction</h3><br />
<br />
<p>The goal of the Phage sub-team was to develop the new phage standard for submission to the BioBrick registry and to characterize the phage that would be used for our project.</p><br/><br />
<br />
<p>The <b>phage standard</b> presented itself once we made the decision to use a phage as a vector to attain our project goals. The standard is necessary for three reasons. Firstly, lysogenic phages are natural vectors that have evolved to integrate and propagate their DNA through specific bacterial strains. Secondly, it is impossible to work with phages using existing BioBrick standards due to the illegal cut sites that occur in every lysogenic phage. Lastly, lysogenic phage genomes are too large to be manipulated using normal BioBrick plasmids. Based on these reasons, our phage standard is an important addition to the BioBricks registry.</p><br/><br />
<br />
<p> The objectives of our phage standard include negating the issues of genome size, exploiting phage characteristics for use as a vector, and developing a BioBrick compatible standard applicable to all lysogenic phages.</p><br/><br />
<br />
<h3>Background</h3><br />
<br />
<p>Lysogenic phages have evolved to insert their DNA into the genomes of specific strains of bacteria. Sometimes this insertion is done at random in the case of a non-specific integration site (INSERT A REFERENCE HERE) or it is inserted only at a very specific location in the genome (INSERT REFERENCE HERE). This specificity allows the integration sites of different lysogenic phages to be used as <b>insertion vectors</b>. These insertion vectors (Fig. 1) will be low-copy BioBrick plasmids containing the integration site of a given phage, flanked by chosen restriction sites.</p><br/><br />
<br />
<h3>The Details</h3><br />
<br />
<p>The phage standard describes the process of adding a given Biobrick part, which we will call <b>source DNA</b> into the genome of a lysogenic phage, referred to as <b>host DNA</b>. This will require secondary DNA sequences including the phage genome <b>integration site</b>, some <b>garbage DNA</b> (flanked by essential restriction enzyme cut sites) and the low-copy number <b>BioBrick plasmid</b>.</p><br/><br />
<br />
<p>The first step is to choose restriction enzymes using <a href="http://bio.lundberg.gu.se/cutter2/">webcutter 2.0</a> that <b>appear only once</b>, closest to the region of the phage genome that is going to be modified (Fig. 1).</p><br/><center><img width="600" src="http://i.imgur.com/UVJZQ.jpg"></center><br />
<div align="center"><p><i><b>Figure 1.</b> Phage genome showing region of interest to be modified by <b>source DNA</b> and nearby unique restriction enzyme site</i></p></div><br/><br />
<br />
<p>Next, find a cut site that is absent in (i) the host DNA, (ii) the BioBrick plasmids and (iii) the source DNA. Using PCR, get the integration site off the bacterial genome and flank it with standard BioBrick E, X, S and P in addition to the absent cut site (Fig. 2; A1 refers to the absent restriction enzyme site).</p><br/><center><img width="600" src="http://i.imgur.com/TWVy0.jpg"></center><br />
<div align="center"><p><i><b>Figure 2.</b> Phage integration site flanked by BioBrick restriction enzyme sites and the absent site.</i></p></div><br/><br />
<br />
<p>Using standard BioBrick assembly methods, ligate the integration site onto a low-copy BioBrick plasmid (Fig. 3). Then transform the plasmid into the host bacterial strain and expose the bacteria to the phage. This will allow the phage to infect the bacterial cells as well as the integration site on the low-copy BioBrick plasmid. From this culture, miniprep the DNA - very carefully since it is now a 50 kilo base pair plus plasmid.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/5Ghik.jpg"></center><br />
<div align="center"><p><i><b>Figure 3.</b> Current state of the low copy BioBrick plasmid containing the full phage genome.</i></p></div><br/><br />
<br />
<p>Prepare a high copy BioBrick plasmid with the A1 and Unique site by PCRing these sites onto some garbage DNA and cutting/ligating the DNA onto the plasmid. The garbage DNA doesn’t need to be useless. It is recommended to use GFP or RFP for visual confirmation of a successful cloning procedure.</p><br/><br />
<br />
<p>The next step is to cut at A1 and the Unique site and ligate this DNA onto the higher copy garbage (with cut sites) plasmid. Ideally, this plasmid will be on the order of 4-10 kilo base pairs in total, allowing standard cloning techniques to be used with ease. This plasmid is the sub host DNA.</p><br/><br />
<br />
<p>At this stage it is necessary to create restriction enzyme cut sites in the locations needed. This will involve performing two site-directed mutagenesis (SDM) reactions to insert cut sites flanking the specific region that is to be modified.</p><br/><br />
<br />
<p>For the next step to proceed, it is necessary to modify the source DNA, the BioBrick part, by adding cut sites using PCR. These cut sites should be in on the inside of E, X, S, and P as before (similar to the garbage DNA) and should match the restriction sites that were SDM’ed into the sub host DNA.</p><br/><br />
<br />
<p>Finally, insert the modified source DNA into the sub host DNA.</p><br/><br />
<br />
<p>Cut and ligate it all back together and viola, a modified phage genome is at your disposal.</p><br/><br />
<br />
<h3>Summary of Essentials</h3><br />
<br />
<p>Use PCR to add restriction sites to:<br />
<br />
a) Integration site<br />
b) Source DNA<br />
</p><br/><br />
<br />
<p>Prepare a BioBrick plasmid with the A1 and Unique sites on the inside of the normal BioBrick cut sites (Fig. 4).</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/cBT2d.jpg"></center><br />
<div align="center"><p><i><b>Figure 4.</b> Restriction Enzyme Sites of Integration Site, Source DNA and "Garbage" Plasmid</i></p></div><br/><br />
<br />
<p>Cut and ligate the integration site onto a low copy BioBrick plasmid using standard cut sites.</p><br/><br />
<br />
<p>Allow the phage to insert its genome into the integration site (Fig. 5). <b>IMPORTANT</b> – the genome housing this low-copy integration site plasmid <b>MUST</b> be the bacterial strain normally targeted by the phage.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/rOwEK.jpg"></center><br />
<div align="center"><p><i><b>Figure 5.</b> Phage in integration site plasmid, sub host plasmid before ligation with portion of phage DNA.</i></p></div><br/><br />
<br />
<p>Cut the phage genome at the unique site and the absent site. Ligate this portion of the phage genome onto the prepared BioBrick plasmid.</p><br/><br />
<br />
<p>Use SDM to add cut sites (Fig. 6). Cut/ligate in the source DNA.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/TFM5J.jpg"></center><br />
<div align="center"><p><i><b>Figure 6.</b> Relative Locations of SDM sites.</i></p></div><br/><br />
<br />
<p>Use A1 and the Unique site to reinsert the sub host DNA into the phage (Fig. 7).</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/AejhW.jpg"></center><br />
<div align="center"><p><i><b>Figure 7.</b> Relative Locations of A1 and Unique sites.</i></p></div><br/><br />
<br />
<br />
<p>Enjoy the fruits of your labor – a modified phage genome.</p><br/><br />
<br />
<h3>Conclusions</h3><br />
<br />
<p>Our phage standard solves the problem of phage genomes being too large, negate the problem of phage genomes containing multiple illegal cut sites and allows any lysogenic phage to be used as part of the BioBrick registry.<br />
</p></br><br />
<br />
<h3>The Wet-Lab Phage</h3><br />
<p>Since our wet lab experiments were focusing on <i>S. Aureus</i> biofilms we had a finite list of phages to choose from. We originally chose to work with phage &phi;MR11 since information on it's genome was readily available and it appeared to be the subject of current research. As the summer proceeded and requests for the phage did not materialize we moved on to work on a different phage. We chose &phi;11, a prophage found in <i>S. Aureus</i> strain 8325 along with 2 other prophages. Plans proceeded with developing the phage standard as we attempted acquire both the original phage &phi;MR11 and <i>S. Aureus</i> strain 8325 containing phage &phi;11.</p></br><br />
<br />
<br />
</div> <!-- end SubWrapper --><br />
<br />
<div id="news" style="height:4000px;"> <br />
<br/><center><h3>References</h3><br />
<br />
<br />
</div> <!-- end news --><br />
</div> <!-- end Super_main_wrapper --><br />
<script type="text/javascript"><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/Project_PhageTeam:British Columbia/Project Phage2010-10-27T15:35:34Z<p>Ayjchan: </p>
<hr />
<div>__NOTOC__<br />
<br />
{{Template_HD_4}}<br />
<html><body id="project" onload="setPageSize()"><br />
<br />
<div id="super_main_wrapper"><br />
<div id="SubWrapper"> <br />
<br/><br />
<br />
<br />
<h3>Introduction</h3><br />
<br />
<p>The goal of the Phage sub-team was to develop the new phage standard for submission to the BioBrick registry and to characterize the phage that would be used for our project.</p><br/><br />
<br />
<p>The <b>phage standard</b> presented itself once we made the decision to use a phage as a vector to attain our project goals. The standard is necessary for three reasons. Firstly, lysogenic phages are natural vectors that have evolved to integrate and propagate their DNA through specific bacterial strains. Secondly, it is impossible to work with phages using existing BioBrick standards due to the illegal cut sites that occur in every lysogenic phage. Lastly, lysogenic phage genomes are too large to be manipulated using normal BioBrick plasmids. Based on these reasons, our phage standard is an important addition to the BioBricks registry.</p><br/><br />
<br />
<p> The objectives of our phage standard include negating the issues of genome size, exploiting phage characteristics for use as a vector, and developing a BioBrick compatible standard applicable to all lysogenic phages.</p><br/><br />
<br />
<h3>Background</h3><br />
<br />
<p>Lysogenic phages have evolved to insert their DNA into the genomes of specific strains of bacteria. Sometimes this insertion is done at random in the case of a non-specific integration site (INSERT A REFERENCE HERE) or it is inserted only at a very specific location in the genome (INSERT REFERENCE HERE). This specificity allows the integration sites of different lysogenic phages to be used as <b>insertion vectors</b>. These insertion vectors (Fig. 1) will be low-copy BioBrick plasmids containing the integration site of a given phage, flanked by chosen restriction sites.</p><br/><br />
<br />
<h3>The Details</h3><br />
<br />
<p>The phage standard describes the process of adding a given Biobrick part, which we will call <b>source DNA</b> into the genome of a lysogenic phage, referred to as <b>host DNA</b>. This will require secondary DNA sequences including the phage genome <b>integration site</b>, some <b>garbage DNA</b> (flanked by essential restriction enzyme cut sites) and the low-copy number <b>BioBrick plasmid</b>.</p><br/><br />
<br />
<p>The first step is to choose restriction enzymes using <a href="http://bio.lundberg.gu.se/cutter2/">webcutter 2.0</a> that <b>appear only once</b>, closest to the region of the phage genome that is going to be modified (Fig. 1).</p><br/><center><img width="600" src="http://i.imgur.com/UVJZQ.jpg"></center><br />
<div align="center"><p><i><b>Figure 1.</b> Phage genome showing region of interest to be modified by <b>source DNA</b> and nearby unique restriction enzyme site</i></p></div><br/><br />
<br />
<p>Next, find a cut site that is absent in (i) the host DNA, (ii) the BioBrick plasmids and (iii) the source DNA. Using PCR, get the integration site off the bacterial genome and flank it with standard BioBrick E, X, S and P in addition to the absent cut site (Fig. 2; A1 refers to the absent restriction enzyme site).</p><br/><center><img width="600" src="http://i.imgur.com/TWVy0.jpg"></center><br />
<div align="center"><p><i><b>Figure 2.</b> Phage integration site flanked by BioBrick restriction enzyme sites and the absent site.</i></p></div><br/><br />
<br />
<p>Using standard BioBrick assembly methods, ligate the integration site onto a low-copy BioBrick plasmid (Fig. 3). Then transform the plasmid into the host bacterial strain and expose the bacteria to the phage. This will allow the phage to infect the bacterial cells as well as the integration site on the low-copy BioBrick plasmid. From this culture, miniprep the DNA - very carefully since it is now a 50 kilo base pair plus plasmid.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/5Ghik.jpg"></center><br />
<div align="center"><p><i><b>Figure 3.</b> Current state of the low copy BioBrick plasmid containing the full phage genome.</i></p></div><br/><br />
<br />
<p>Prepare a high copy BioBrick plasmid with the A1 and Unique site by PCRing these sites onto some garbage DNA and cutting/ligating the DNA onto the plasmid. The garbage DNA doesn’t need to be useless. It is recommended to use GFP or RFP for visual confirmation of a successful cloning procedure.</p><br/><br />
<br />
<p>The next step is to cut at A1 and the Unique site and ligate this DNA onto the higher copy garbage (with cut sites) plasmid. Ideally, this plasmid will be on the order of 4-10 kilo base pairs in total, allowing standard cloning techniques to be used with ease. This plasmid is the sub host DNA.</p><br/><br />
<br />
<p>At this stage it is necessary to create restriction enzyme cut sites in the locations needed. This will involve performing two site-directed mutagenesis (SDM) reactions to insert cut sites flanking the specific region that is to be modified.</p><br/><br />
<br />
<p>For the next step to proceed, it is necessary to modify the source DNA, the BioBrick part, by adding cut sites using PCR. These cut sites should be in on the inside of E, X, S, and P as before (similar to the garbage DNA) and should match the restriction sites that were SDM’ed into the sub host DNA.</p><br/><br />
<br />
<p>Finally, insert the modified source DNA into the sub host DNA.</p><br/><br />
<br />
<p>Cut and ligate it all back together and viola, a modified phage genome is at your disposal.</p><br/><br />
<br />
<h3>Summary of Essentials</h3><br />
<br />
<p>Use PCR to add restriction sites to:<br />
<br />
a) Integration site<br />
b) Source DNA<br />
</p><br/><br />
<br />
<p>Prepare a BioBrick plasmid with the A1 and Unique sites on the inside of the normal BioBrick cut sites (Fig. 4).</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/cBT2d.jpg"></center><br />
<div align="center"><p><i><b>Figure 4.</b> Restriction Enzyme Sites of Integration Site, Source DNA and "Garbage" Plasmid</i></p></div><br/><br />
<br />
<p>Cut and ligate the integration site onto a low copy BioBrick plasmid using standard cut sites.</p><br/><br />
<br />
<p>Allow the phage to insert its genome into the integration site (Fig. 5). <b>IMPORTANT</b> – the genome housing this low-copy integration site plasmid <b>MUST</b> be the bacterial strain normally targeted by the phage.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/rOwEK.jpg"></center><br />
<div align="center"><p><i><b>Figure 5.</b> Phage in integration site plasmid, sub host plasmid before ligation with portion of phage DNA.</i></p></div><br/><br />
<br />
<p>Cut the phage genome at the unique site and the absent site. Ligate this portion of the phage genome onto the prepared BioBrick plasmid.</p><br/><br />
<br />
<p>Use SDM to add cut sites (Fig. 6). Cut/ligate in the source DNA.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/TFM5J.jpg"></center><br />
<div align="center"><p><i><b>Figure 6.</b> Relative Locations of SDM sites.</i></p></div><br/><br />
<br />
<p>Use A1 and the Unique site to reinsert the sub host DNA into the phage (Fig. 7).</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/AejhW.jpg"></center><br />
<div align="center"><p><i><b>Figure 7.</b> Relative Locations of A1 and Unique sites.</i></p></div><br/><br />
<br />
<br />
<p>Enjoy the fruits of your labor – a modified phage genome.</p><br/><br />
<br />
<h3>Conclusions</h3><br />
<br />
<p>Our phage standard solves the problem of phage genomes being too large, negate the problem of phage genomes containing multiple illegal cut sites and allows any lysogenic phage to be used as part of the BioBrick registry.<br />
</p></br><br />
<br />
<h3>The Wet-Lab Phage</h3><br />
<p>Since our wet lab experiments were focusing on <i>S. Aureus</i> biofilms we had a finite list of phages to choose from. We originally chose to work with phage &phi;MR11 since information on it's genome was readily available and it appeared to be the subject of current research. As the summer proceeded and requests for the phage did not materialize we moved on to work on a different phage. We chose &phi;11, a prophage found in <i>S. Aureus</i> strain 8325 along with 2 other prophages. Plans proceeded with developing the phage standard as we attempted acquire both the original phage &phi;MR11 and <i>S. Aureus</i> strain 8325 containing phage &phi;11.</p></br><br />
<br />
<br />
</div> <!-- end SubWrapper --><br />
<br />
<div id="news" style="height:3000px;"> <br />
<br/><center><h3>References</h3><br />
<br />
<br />
</div> <!-- end news --><br />
</div> <!-- end Super_main_wrapper --><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/Project_PhageTeam:British Columbia/Project Phage2010-10-27T15:34:04Z<p>Ayjchan: </p>
<hr />
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<br />
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<br />
<div id="super_main_wrapper"><br />
<div id="SubWrapper"> <br />
<br/><br />
<br />
<br />
<h3>Introduction</h3><br />
<br />
<p>The goal of the Phage sub-team was to develop the new phage standard for submission to the BioBrick registry and to characterize the phage that would be used for our project.</p><br/><br />
<br />
<p>The <b>phage standard</b> presented itself once we made the decision to use a phage as a vector to attain our project goals. The standard is necessary for three reasons. Firstly, lysogenic phages are natural vectors that have evolved to integrate and propagate their DNA through specific bacterial strains. Secondly, it is impossible to work with phages using existing BioBrick standards due to the illegal cut sites that occur in every lysogenic phage. Lastly, lysogenic phage genomes are too large to be manipulated using normal BioBrick plasmids. Based on these reasons, our phage standard is an important addition to the BioBricks registry.</p><br/><br />
<br />
<p> The objectives of our phage standard include negating the issues of genome size, exploiting phage characteristics for use as a vector, and developing a BioBrick compatible standard applicable to all lysogenic phages.</p><br/><br />
<br />
<h3>Background</h3><br />
<br />
<p>Lysogenic phages have evolved to insert their DNA into the genomes of specific strains of bacteria. Sometimes this insertion is done at random in the case of a non-specific integration site (INSERT A REFERENCE HERE) or it is inserted only at a very specific location in the genome (INSERT REFERENCE HERE). This specificity allows the integration sites of different lysogenic phages to be used as <b>insertion vectors</b>. These insertion vectors (Fig. 1) will be low-copy BioBrick plasmids containing the integration site of a given phage, flanked by chosen restriction sites.</p><br/><br />
<br />
<h3>The Details</h3><br />
<br />
<p>The phage standard describes the process of adding a given Biobrick part, which we will call <b>source DNA</b> into the genome of a lysogenic phage, referred to as <b>host DNA</b>. This will require secondary DNA sequences including the phage genome <b>integration site</b>, some <b>garbage DNA</b> (flanked by essential restriction enzyme cut sites) and the low-copy number <b>BioBrick plasmid</b>.</p><br/><br />
<br />
<p>The first step is to choose restriction enzymes using <a href="http://bio.lundberg.gu.se/cutter2/">webcutter 2.0</a> that <b>appear only once</b>, closest to the region of the phage genome that is going to be modified (Fig. 1).</p><br/><center><img width="600" src="http://i.imgur.com/UVJZQ.jpg"></center><br />
<div align="center"><p><i><b>Figure 1.</b> Phage genome showing region of interest to be modified by <b>source DNA</b> and nearby unique restriction enzyme site</i></p></div><br/><br />
<br />
<p>Next, find a cut site that is absent in (i) the host DNA, (ii) the BioBrick plasmids and (iii) the source DNA. Using PCR, get the integration site off the bacterial genome and flank it with standard BioBrick E, X, S and P in addition to the absent cut site (Fig. 2; A1 refers to the absent restriction enzyme site).</p><br/><center><img width="600" src="http://i.imgur.com/TWVy0.jpg"></center><br />
<div align="center"><p><i><b>Figure 2.</b> Phage integration site flanked by BioBrick restriction enzyme sites and the absent site.</i></p></div><br/><br />
<br />
<p>Using standard BioBrick assembly methods, ligate the integration site onto a low-copy BioBrick plasmid (Fig. 3). Then transform the plasmid into the host bacterial strain and expose the bacteria to the phage. This will allow the phage to infect the bacterial cells as well as the integration site on the low-copy BioBrick plasmid. From this culture, miniprep the DNA - very carefully since it is now a 50 kilo base pair plus plasmid.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/5Ghik.jpg"></center><br />
<div align="center"><p><i><b>Figure 3.</b> Current state of the low copy BioBrick plasmid containing the full phage genome.</i></p></div><br/><br />
<br />
<p>Prepare a high copy BioBrick plasmid with the A1 and Unique site by PCRing these sites onto some garbage DNA and cutting/ligating the DNA onto the plasmid. The garbage DNA doesn’t need to be useless. It is recommended to use GFP or RFP for visual confirmation of a successful cloning procedure.</p><br/><br />
<br />
<p>The next step is to cut at A1 and the Unique site and ligate this DNA onto the higher copy garbage (with cut sites) plasmid. Ideally, this plasmid will be on the order of 4-10 kilo base pairs in total, allowing standard cloning techniques to be used with ease. This plasmid is the sub host DNA.</p><br/><br />
<br />
<p>At this stage it is necessary to create restriction enzyme cut sites in the locations needed. This will involve performing two site-directed mutagenesis (SDM) reactions to insert cut sites flanking the specific region that is to be modified.</p><br/><br />
<br />
<p>For the next step to proceed, it is necessary to modify the source DNA, the BioBrick part, by adding cut sites using PCR. These cut sites should be in on the inside of E, X, S, and P as before (similar to the garbage DNA) and should match the restriction sites that were SDM’ed into the sub host DNA.</p><br/><br />
<br />
<p>Finally, insert the modified source DNA into the sub host DNA.</p><br/><br />
<br />
<p>Cut and ligate it all back together and viola, a modified phage genome is at your disposal.</p><br/><br />
<br />
<h3>Summary of Essentials</h3><br />
<br />
<p>Use PCR to add restriction sites to:<br />
<br />
a) Integration site<br />
b) Source DNA<br />
</p><br/><br />
<br />
<p>Prepare a BioBrick plasmid with the A1 and Unique sites on the inside of the normal BioBrick cut sites (Fig. 4).</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/cBT2d.jpg"></center><br />
<div align="center"><p><i><b>Figure 4.</b> Restriction Enzyme Sites of Integration Site, Source DNA and "Garbage" Plasmid</i></p></div><br/><br />
<br />
<p>Cut and ligate the integration site onto a low copy BioBrick plasmid using standard cut sites.</p><br/><br />
<br />
<p>Allow the phage to insert its genome into the integration site (Fig. 5). <b>IMPORTANT</b> – the genome housing this low-copy integration site plasmid <b>MUST</b> be the bacterial strain normally targeted by the phage.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/rOwEK.jpg"></center><br />
<div align="center"><p><i><b>Figure 5.</b> Phage in integration site plasmid, sub host plasmid before ligation with portion of phage DNA.</i></p></div><br/><br />
<br />
<p>Cut the phage genome at the unique site and the absent site. Ligate this portion of the phage genome onto the prepared BioBrick plasmid.</p><br/><br />
<br />
<p>Use SDM to add cut sites (Fig. 6). Cut/ligate in the source DNA.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/TFM5J.jpg"></center><br />
<div align="center"><p><i><b>Figure 6.</b> Relative Locations of SDM sites.</i></p></div><br/><br />
<br />
<p>Use A1 and the Unique site to reinsert the sub host DNA into the phage (Fig. 7).</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/AejhW.jpg"></center><br />
<div align="center"><p><i><b>Figure 7.</b> Relative Locations of A1 and Unique sites.</i></p></div><br/><br />
<br />
<br />
<p>Enjoy the fruits of your labor – a modified phage genome.</p><br/><br />
<br />
<h3>Conclusions</h3><br />
<br />
<p>Our phage standard solves the problem of phage genomes being too large, negate the problem of phage genomes containing multiple illegal cut sites and allows any lysogenic phage to be used as part of the BioBrick registry.<br />
</p></br><br />
<br />
<h3>The Wet-Lab Phage</h3><br />
<p>Since our wet lab experiments were focusing on <i>S. Aureus</i> biofilms we had a finite list of phages to choose from. We originally chose to work with phage &phi;MR11 since information on it's genome was readily available and it appeared to be the subject of current research. As the summer proceeded and requests for the phage did not materialize we moved on to work on a different phage. We chose &phi;11, a prophage found in <i>S. Aureus</i> strain 8325 along with 2 other prophages. Plans proceeded with developing the phage standard as we attempted acquire both the original phage &phi;MR11 and <i>S. Aureus</i> strain 8325 containing phage &phi;11.</p></br><br />
<br />
<br />
</div> <!-- end SubWrapper --><br />
<br />
<div id="news" style="height:1700px;"> <br />
<br/><center><h3>References</h3><br />
<br />
<br />
</div> <!-- end news --><br />
</div> <!-- end Super_main_wrapper --><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/Project_PhageTeam:British Columbia/Project Phage2010-10-27T15:33:29Z<p>Ayjchan: </p>
<hr />
<div>__NOTOC__<br />
<br />
{{Template_HD_4}}<br />
<html><body id="project" onload="setPageSize()"><br />
<br />
<div id="super_main_wrapper"><br />
<div id="SubWrapper"> <br />
<br/><br />
<br />
<br />
<h3>Introduction</h3><br />
<br />
<p>The goal of the Phage sub-team was to develop the new phage standard for submission to the BioBrick registry and to characterize the phage that would be used for our project.</p><br/><br />
<br />
<p>The <b>phage standard</b> presented itself once we made the decision to use a phage as a vector to attain our project goals. The standard is necessary for three reasons. Firstly, lysogenic phages are natural vectors that have evolved to integrate and propagate their DNA through specific bacterial strains. Secondly, it is impossible to work with phages using existing BioBrick standards due to the illegal cut sites that occur in every lysogenic phage. Lastly, lysogenic phage genomes are too large to be manipulated using normal BioBrick plasmids. Based on these reasons, our phage standard is an important addition to the BioBricks registry.</p><br/><br />
<br />
<p> The objectives of our phage standard include negating the issues of genome size, exploiting phage characteristics for use as a vector, and developing a BioBrick compatible standard applicable to all lysogenic phages.</p><br/><br />
<br />
<h3>Background</h3><br />
<br />
<p>Lysogenic phages have evolved to insert their DNA into the genomes of specific strains of bacteria. Sometimes this insertion is done at random in the case of a non-specific integration site (INSERT A REFERENCE HERE) or it is inserted only at a very specific location in the genome (INSERT REFERENCE HERE). This specificity allows the integration sites of different lysogenic phages to be used as <b>insertion vectors</b>. These insertion vectors (Fig. 1) will be low-copy BioBrick plasmids containing the integration site of a given phage, flanked by chosen restriction sites.</p><br/><br />
<br />
<h3>The Details</h3><br />
<br />
<p>The phage standard describes the process of adding a given Biobrick part, which we will call <b>source DNA</b> into the genome of a lysogenic phage, referred to as <b>host DNA</b>. This will require secondary DNA sequences including the phage genome <b>integration site</b>, some <b>garbage DNA</b> (flanked by essential restriction enzyme cut sites) and the low-copy number <b>BioBrick plasmid</b>.</p><br/><br />
<br />
<p>The first step is to choose restriction enzymes using <a href="http://bio.lundberg.gu.se/cutter2/">webcutter 2.0</a> that <b>appear only once</b>, closest to the region of the phage genome that is going to be modified (Fig. 1).</p><br/><center><img width="600" src="http://i.imgur.com/UVJZQ.jpg"></center><br />
<div align="center"><p><i><b>Figure 1.</b> Phage genome showing region of interest to be modified by <b>source DNA</b> and nearby unique restriction enzyme site</i></p></div><br/><br />
<br />
<p>Next, find a cut site that is absent in (i) the host DNA, (ii) the BioBrick plasmids and (iii) the source DNA. Using PCR, get the integration site off the bacterial genome and flank it with standard BioBrick E, X, S and P in addition to the absent cut site (Fig. 2; A1 refers to the absent restriction enzyme site).</p><br/><center><img width="600" src="http://i.imgur.com/TWVy0.jpg"></center><br />
<div align="center"><p><i><b>Figure 2.</b> Phage integration site flanked by BioBrick restriction enzyme sites and the absent site.</i></p></div><br/><br />
<br />
<p>Using standard BioBrick assembly methods, ligate the integration site onto a low-copy BioBrick plasmid (Fig. 3). Then transform the plasmid into the host bacterial strain and expose the bacteria to the phage. This will allow the phage to infect the bacterial cells as well as the integration site on the low-copy BioBrick plasmid. From this culture, miniprep the DNA - very carefully since it is now a 50 kilo base pair plus plasmid.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/5Ghik.jpg"></center><br />
<div align="center"><p><i><b>Figure 3.</b> Current state of the low copy BioBrick plasmid containing the full phage genome.</i></p></div><br/><br />
<br />
<p>Prepare a high copy BioBrick plasmid with the A1 and Unique site by PCRing these sites onto some garbage DNA and cutting/ligating the DNA onto the plasmid. The garbage DNA doesn’t need to be useless. It is recommended to use GFP or RFP for visual confirmation of a successful cloning procedure.</p><br/><br />
<br />
<p>The next step is to cut at A1 and the Unique site and ligate this DNA onto the higher copy garbage (with cut sites) plasmid. Ideally, this plasmid will be on the order of 4-10 kilo base pairs in total, allowing standard cloning techniques to be used with ease. This plasmid is the sub host DNA.</p><br/><br />
<br />
<p>At this stage it is necessary to create restriction enzyme cut sites in the locations needed. This will involve performing two site-directed mutagenesis (SDM) reactions to insert cut sites flanking the specific region that is to be modified.</p><br/><br />
<br />
<p>For the next step to proceed, it is necessary to modify the source DNA, the BioBrick part, by adding cut sites using PCR. These cut sites should be in on the inside of E, X, S, and P as before (similar to the garbage DNA) and should match the restriction sites that were SDM’ed into the sub host DNA.</p><br/><br />
<br />
<p>Finally, insert the modified source DNA into the sub host DNA.</p><br/><br />
<br />
<p>Cut and ligate it all back together and viola, a modified phage genome is at your disposal.</p><br/><br />
<br />
<h3>Summary of Essentials</h3><br />
<br />
<p>Use PCR to add restriction sites to:<br />
<br />
a) Integration site<br />
b) Source DNA<br />
</p><br/><br />
<br />
<p>Prepare a BioBrick plasmid with the A1 and Unique sites on the inside of the normal BioBrick cut sites (Fig. 4).</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/cBT2d.jpg"></center><br />
<div align="center"><p><i><b>Figure 4.</b> Restriction Enzyme Sites of Integration Site, Source DNA and "Garbage" Plasmid</i></p></div><br/><br />
<br />
<p>Cut and ligate the integration site onto a low copy BioBrick plasmid using standard cut sites.</p><br/><br />
<br />
<p>Allow the phage to insert its genome into the integration site (Fig. 5). <b>IMPORTANT</b> – the genome housing this low-copy integration site plasmid <b>MUST</b> be the bacterial strain normally targeted by the phage.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/rOwEK.jpg"></center><br />
<div align="center"><p><i><b>Figure 5.</b> Phage in integration site plasmid, sub host plasmid before ligation with portion of phage DNA.</i></p></div><br/><br />
<br />
<p>Cut the phage genome at the unique site and the absent site. Ligate this portion of the phage genome onto the prepared BioBrick plasmid.</p><br/><br />
<br />
<p>Use SDM to add cut sites (Fig. 6). Cut/ligate in the source DNA.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/TFM5J.jpg"></center><br />
<div align="center"><p><i><b>Figure 6.</b> Relative Locations of SDM sites.</i></p></div><br/><br />
<br />
<p>Use A1 and the Unique site to reinsert the sub host DNA into the phage (Fig. 7).</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/AejhW.jpg"></center><br />
<div align="center"><p><i><b>Figure 7.</b> Relative Locations of A1 and Unique sites.</i></p></div><br/><br />
<br />
<br />
<p>Enjoy the fruits of your labor – a modified phage genome.</p><br/><br />
<br />
<h3>Conclusions</h3><br />
<br />
<p>Our phage standard solves the problem of phage genomes being too large, negate the problem of phage genomes containing multiple illegal cut sites and allows any lysogenic phage to be used as part of the BioBrick registry.<br />
</p></br><br />
<br />
<h3>The Wet-Lab Phage</h3><br />
<p>Since our wet lab experiments were focusing on <i>S. Aureus</i> biofilms we had a finite list of phages to choose from. We originally chose to work with phage &phi;MR11 since information on it's genome was readily available and it appeared to be the subject of current research. As the summer proceeded and requests for the phage did not materialize we moved on to work on a different phage. We chose &phi;11, a prophage found in <i>S. Aureus</i> strain 8325 along with 2 other prophages. Plans proceeded with developing the phage standard as we attempted acquire both the original phage &phi;MR11 and <i>S. Aureus</i> strain 8325 containing phage &phi;11.</p></br><br />
<br />
<br />
</div> <!-- end SubWrapper --><br />
<br />
<div id="news" style="height:1600px;"> <br />
<br/><h3>References</h3><br />
<br />
<br />
</div> <!-- end news --><br />
</div> <!-- end Super_main_wrapper --><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/Project_PhageTeam:British Columbia/Project Phage2010-10-27T15:32:00Z<p>Ayjchan: </p>
<hr />
<div>__NOTOC__<br />
<br />
{{Template_HD_4}}<br />
<html><body id="project" onload="setPageSize()"><br />
<br />
<div id="super_main_wrapper"><br />
<div id="SubWrapper"> <br />
<br/><br />
<br />
<br />
<h3>Introduction</h3><br />
<br />
<p>The goal of the Phage sub-team was to develop the new phage standard for submission to the BioBrick registry and to characterize the phage that would be used for our project.</p><br/><br />
<br />
<p>The <b>phage standard</b> presented itself once we made the decision to use a phage as a vector to attain our project goals. The standard is necessary for three reasons. Firstly, lysogenic phages are natural vectors that have evolved to integrate and propagate their DNA through specific bacterial strains. Secondly, it is impossible to work with phages using existing BioBrick standards due to the illegal cut sites that occur in every lysogenic phage. Lastly, lysogenic phage genomes are too large to be manipulated using normal BioBrick plasmids. Based on these reasons, our phage standard is an important addition to the BioBricks registry.</p><br/><br />
<br />
<p> The objectives of our phage standard include negating the issues of genome size, exploiting phage characteristics for use as a vector, and developing a BioBrick compatible standard applicable to all lysogenic phages.</p><br/><br />
<br />
<h3>Background</h3><br />
<br />
<p>Lysogenic phages have evolved to insert their DNA into the genomes of specific strains of bacteria. Sometimes this insertion is done at random in the case of a non-specific integration site (INSERT A REFERENCE HERE) or it is inserted only at a very specific location in the genome (INSERT REFERENCE HERE). This specificity allows the integration sites of different lysogenic phages to be used as <b>insertion vectors</b>. These insertion vectors (Fig. 1) will be low-copy BioBrick plasmids containing the integration site of a given phage, flanked by chosen restriction sites.</p><br/><br />
<br />
<h3>The Details</h3><br />
<br />
<p>The phage standard describes the process of adding a given Biobrick part, which we will call <b>source DNA</b> into the genome of a lysogenic phage, referred to as <b>host DNA</b>. This will require secondary DNA sequences including the phage genome <b>integration site</b>, some <b>garbage DNA</b> (flanked by essential restriction enzyme cut sites) and the low-copy number <b>BioBrick plasmid</b>.</p><br/><br />
<br />
<p>The first step is to choose restriction enzymes using <a href="http://bio.lundberg.gu.se/cutter2/">webcutter 2.0</a> that <b>appear only once</b>, closest to the region of the phage genome that is going to be modified (Fig. 1).</p><br/><center><img width="600" src="http://i.imgur.com/UVJZQ.jpg"></center><br />
<div align="center"><p><i><b>Figure 1.</b> Phage genome showing region of interest to be modified by <b>source DNA</b> and nearby unique restriction enzyme site</i></p></div><br/><br />
<br />
<p>Next, find a cut site that is absent in (i) the host DNA, (ii) the BioBrick plasmids and (iii) the source DNA. Using PCR, get the integration site off the bacterial genome and flank it with standard BioBrick E, X, S and P in addition to the absent cut site (Fig. 2; A1 refers to the absent restriction enzyme site).</p></br><center><img width="600" src="http://i.imgur.com/TWVy0.jpg"></center><br />
<div align="center"><p><i><b>Figure 2.</b> Phage integration site flanked by BioBrick restriction enzyme sites and the absent site.</i></p></div><br/><br />
<br />
<p>Using standard BioBrick assembly methods, ligate the integration site onto a low-copy BioBrick plasmid (Fig. 3). Then transform the plasmid into the host bacterial strain and expose the bacteria to the phage. This will allow the phage to infect the bacterial cells as well as the integration site on the low-copy BioBrick plasmid. From this culture, miniprep the DNA - very carefully since it is now a 50 kilo base pair plus plasmid.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/5Ghik.jpg"></center><br />
<div align="center"><p><i><b>Figure 3.</b> Current state of the low copy BioBrick plasmid containing the full phage genome.</i></p></div><br/><br />
<br />
<p>Prepare a high copy BioBrick plasmid with the A1 and Unique site by PCRing these sites onto some garbage DNA and cutting/ligating the DNA onto the plasmid. The garbage DNA doesn’t need to be useless. It is recommended to use GFP or RFP for visual confirmation of a successful cloning procedure.</p><br/><br />
<br />
<p>The next step is to cut at A1 and the Unique site and ligate this DNA onto the higher copy garbage (with cut sites) plasmid. Ideally, this plasmid will be on the order of 4-10 kilo base pairs in total, allowing standard cloning techniques to be used with ease. This plasmid is the sub host DNA.</p><br/><br />
<br />
<p>At this stage it is necessary to create restriction enzyme cut sites in the locations needed. This will involve performing two site-directed mutagenesis (SDM) reactions to insert cut sites flanking the specific region that is to be modified.</p><br/><br />
<br />
<p>For the next step to proceed, it is necessary to modify the source DNA, the BioBrick part, by adding cut sites using PCR. These cut sites should be in on the inside of E, X, S, and P as before (similar to the garbage DNA) and should match the restriction sites that were SDM’ed into the sub host DNA.</p><br/><br />
<br />
<p>Finally, insert the modified source DNA into the sub host DNA.</p><br/><br />
<br />
<p>Cut and ligate it all back together and viola, a modified phage genome is at your disposal.</p><br/><br />
<br />
<h3>Summary of Essentials</h3><br />
<br />
<p>Use PCR to add restriction sites to:<br />
<br />
a) Integration site<br />
b) Source DNA<br />
</p><br/><br />
<br />
<p>Prepare a BioBrick plasmid with the A1 and Unique sites on the inside of the normal BioBrick cut sites (Fig. 4).</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/cBT2d.jpg"></center><br />
<div align="center"><p><i><b>Figure 4.</b> Restriction Enzyme Sites of Integration Site, Source DNA and "Garbage" Plasmid</i></p></div><br/><br />
<br />
<p>Cut and ligate the integration site onto a low copy BioBrick plasmid using standard cut sites.</p><br/><br />
<br />
<p>Allow the phage to insert its genome into the integration site (Fig. 5). <b>IMPORTANT</b> – the genome housing this low-copy integration site plasmid <b>MUST</b> be the bacterial strain normally targeted by the phage.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/rOwEK.jpg"></center><br />
<div align="center"><p><i><b>Figure 5.</b> Phage in integration site plasmid, sub host plasmid before ligation with portion of phage DNA.</i></p></div><br/><br />
<br />
<p>Cut the phage genome at the unique site and the absent site. Ligate this portion of the phage genome onto the prepared BioBrick plasmid.</p><br/><br />
<br />
<p>Use SDM to add cut sites (Fig. 6). Cut/ligate in the source DNA.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/TFM5J.jpg"></center><br />
<div align="center"><p><i><b>Figure 6.</b> Relative Locations of SDM sites.</i></p></div><br/><br />
<br />
<p>Use A1 and the Unique site to reinsert the sub host DNA into the phage (Fig. 7).</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/AejhW.jpg"></center><br />
<div align="center"><p><i><b>Figure 7</b> Relative Locations of A1 and Unique sites.</i></p></div><br/><br />
<br />
<br />
<p>Enjoy the fruits of your labor – a modified phage genome.</p><br/><br />
<br />
<h3>Conclusions</h3><br />
<br />
<p>Our phage standard solves the problem of phage genomes being too large, negate the problem of phage genomes containing multiple illegal cut sites and allows any lysogenic phage to be used as part of the BioBrick registry.<br />
</p></br><br />
<br />
<h3>The Wet-Lab Phage</h3><br />
<p>Since our wet lab experiments were focusing on <i>S. Aureus</i> biofilms we had a finite list of phages to choose from. We originally chose to work with phage &phi;MR11 since information on it's genome was readily available and it appeared to be the subject of current research. As the summer proceeded and requests for the phage did not materialize we moved on to work on a different phage. We chose &phi;11, a prophage found in <i>S. Aureus</i> strain 8325 along with 2 other prophages. Plans proceeded with developing the phage standard as we attempted acquire both the original phage &phi;MR11 and <i>S. Aureus</i> strain 8325 containing phage &phi;11.</p></br><br />
<br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/Project_PhageTeam:British Columbia/Project Phage2010-10-27T15:26:18Z<p>Ayjchan: </p>
<hr />
<div>__NOTOC__<br />
<br />
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<br/><br />
<br />
<br />
<h3>Introduction</h3><br />
<br />
<p>The goal of the Phage sub-team was to develop the new phage standard for submission to the BioBrick registry and to characterize the phage that would be used for our project.</p><br/><br />
<br />
<p>The <b>phage standard</b> presented itself once we made the decision to use a phage as a vector to attain our project goals. The standard is necessary for three reasons. Firstly, lysogenic phages are natural vectors that have evolved to integrate and propagate their DNA through specific bacterial strains. Secondly, it is impossible to work with phages using existing BioBrick standards due to the illegal cut sites that occur in every lysogenic phage. Lastly, lysogenic phage genomes are too large to be manipulated using normal BioBrick plasmids. Based on these reasons, our phage standard is an important addition to the BioBricks registry.</p><br/><br />
<br />
<p> The objectives of our phage standard include negating the issues of genome size, exploiting phage characteristics for use as a vector, and developing a BioBrick compatible standard applicable to all lysogenic phages.</p><br/><br />
<br />
<h3>Background</h3><br />
<br />
<p>Lysogenic phages have evolved to insert their DNA into the genomes of specific strains of bacteria. Sometimes this insertion is done at random in the case of a non-specific integration site (INSERT A REFERENCE HERE) or it is inserted only at a very specific location in the genome (INSERT REFERENCE HERE). This specificity allows the integration sites of different lysogenic phages to be used as <b>insertion vectors</b>. These insertion vectors (Fig. 1) will be low-copy BioBrick plasmids containing the integration site of a given phage, flanked by chosen restriction sites.</p><br/><br />
<br />
<h3>The Details</h3><br />
<br />
<p>The phage standard describes the process of adding a given Biobrick part, which we will call <b>source DNA</b> into the genome of a lysogenic phage, referred to as <b>host DNA</b>. This will require secondary DNA sequences including the phage genome <b>integration site</b>, some <b>garbage DNA</b> (flanked by essential restriction enzyme cut sites) and the low-copy number <b>BioBrick plasmid</b>.</p><br/><br />
<br />
<p>The first step is to choose restriction enzymes using <a href="http://bio.lundberg.gu.se/cutter2/">webcutter 2.0</a> that <b>appear only once</b>, closest to the region of the phage genome that is going to be modified (Fig. 1).</p><br/><center><img width="600" src="http://i.imgur.com/UVJZQ.jpg"></center><br />
<div align="center"><p><i>Figure 1: Phage genome showing region of interest to be modified by <b>source DNA</b> and nearby unique restriction enzyme site</i></p></div><br/><br />
<br />
<p>Next, a cut site is found which is absent in (i) the host DNA, (ii) the BioBrick plasmids and (iii) the source DNA. Using PCR, get the integration site off the bacterial genome and flank it with standard BioBrick E, X, S and P in addition to the absent cut site. Please see Figure 2 for more details, note that A1 refers to the absent restriction enzyme site.</p></br><center><img width="600" src="http://i.imgur.com/TWVy0.jpg"></center><br />
<div align="center"><p><i>Figure 2: Phage integration site flanked by BioBrick restriction enzyme sites and the absent site.</i></p></div><br/><br />
<br />
<p>Using standard BioBrick assembly methods, ligate the integration site onto a low-copy BioBrick plasmid. Then transform the plasmid into the host bacterial strain and expose the bacteria to the phage. This will allow the phage to infect the bacterial cells as well as the integration site on the low-copy BioBrick plasmid. From this culture, miniprep the DNA - very carefully since it is now a 50 kilo base pair plus plasmid. The state of the plasmid is shown below in Figure 3.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/5Ghik.jpg"></center><br />
<div align="center"><p><i>Figure 3: Current state of the low copy BioBrick plasmid containing the full phage genome.</i></p></div><br/><br />
<br />
<p>Prepare a high copy BioBrick plasmid with the A1 and Unique site by PCRing these sites onto some garbage DNA and cutting/ligating the DNA onto the plasmid. The garbage DNA doesn’t need to be useless, it is recommended to use GFP or RFP for visual confirmation of a successful cloning procedure.</p><br/><br />
<br />
<p>The next step is to cut at A1 and the Unique site and ligate this DNA onto the higher copy garbage (with cut sites) plasmid. Ideally, this plasmid will be on the order of 4-10 kilo base pairs in total, allowing standard cloning techniques to be used with ease. This plasmid is the sub host DNA.</p><br/><br />
<br />
<p>At this stage it is necessary to create restriction enzyme cut sites in the locations needed. This will involve performing two site-directed mutagenesis (SDM) reactions to insert cut sites flanking the specific region that is to be modified.</p><br/><br />
<br />
<p>For the next step to proceed, it is necessary to modify the source DNA, the BioBrick part, by adding cut sites using PCR. These cut sites should be in on the inside of E, X, S, and P as before (similar to the garbage DNA) and should match the restriction sites that were SDM’ed into the sub host DNA.</p><br/><br />
<br />
<p>Finally, insert the modified source DNA into the sub host DNA.</p><br/><br />
<br />
<p>Cut and ligate it all back together and viola, a modified phage genome is at your disposal.</p><br/><br />
<br />
<h3>Summary of Essentials</h3><br />
<br />
<p>Use PCR to add restriction sites to:<br />
<br />
a) Integration site<br />
b) Source DNA<br />
</p><br/><br />
<br />
<p>Prepare a BioBrick plasmid with the A1 and Unique sites on the inside of the normal BioBrick cut sites (Figure 4).</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/cBT2d.jpg"></center><br />
<div align="center"><p><i>Figure 4: Restriction Enzyme Sites of Integration Site, Source DNA and "Garbage" Plasmid</i></p></div><br/><br />
<br />
<p>Cut and ligate the integration site onto a low copy BioBrick plasmid using standard cut sites.</p><br/><br />
<br />
<p>Allow the phage to insert its genome into the integration site (Figure 5). <b>IMPORTANT</b> – the genome housing this low-copy integration site plasmid <b>MUST</b> be the bacterial strain normally targeted by the phage.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/rOwEK.jpg"></center><br />
<div align="center"><p><i>Figure 5: Phage in integration site plasmid, sub host plasmid before ligation with portion of phage DNA.</i></p></div><br/><br />
<br />
<p>Cut the phage genome at the unique site and the absent site. Ligate this portion of the phage genome onto the prepared BioBrick plasmid.</p><br/><br />
<br />
<p>Use SDM to add cut sites (Figure 6). Cut/ligate in the source DNA.</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/TFM5J.jpg"></center><br />
<div align="center"><p><i>Figure 6: Relative Locations of SDM sites.</i></p></div><br/><br />
<br />
<p>Use A1 and the Unique site to reinsert the sub host DNA into the phage (Figure 7).</p><br/><br />
<br />
<center><img width="600" src="http://i.imgur.com/AejhW.jpg"></center><br />
<div align="center"><p><i>Figure 7: Relative Locations of A1 and Unique sites.</i></p></div><br/><br />
<br />
<br />
<p>Enjoy the fruits of your labor – a modified phage genome.</p><br/><br />
<br />
<h3>Conclusions</h3><br />
<br />
<p>Our phage standard solves the problem of phage genomes being too large, negate the problem of phage genomes containing multiple illegal cut sites and allows any lysogenic phage to be used as part of the BioBrick registry.<br />
</p></br><br />
<br />
<h3>The Wet-Lab Phage</h3><br />
<p>Since our wet lab experiments were focusing on <i>S. Aureus</i> biofilms we had a finite list of phages to choose from. We originally chose to work with phage &phi;MR11 since information on it's genome was readily available and it appeared to be the subject of current research. As the summer proceeded and requests for the phage did not materialize we moved on to work on a different phage. We chose &phi;11, a prophage found in <i>S. Aureus</i> strain 8325 along with 2 other prophages. Plans proceeded with developing the phage standard as we attempted acquire both the original phage &phi;MR11 and <i>S. Aureus</i> strain 8325 containing phage &phi;11.</p></br><br />
<br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/NotebookTeam:British Columbia/Notebook2010-10-27T15:23:36Z<p>Ayjchan: </p>
<hr />
<div>__NOTOC__<br />
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<h3>Notebook: Need to Know</h3></center><br />
<p>Welcome to our wiki notebook! We have organized our notebook according to sub-teams. Each page will provide you with a link to our actual notebook on <a href="http://openwetware.org/wiki/Main_Page">OpenWetWare</a>. To deliver the essentials here on the wiki (so you don't have to read through 6 months of experiments to get our message), we discuss the protocols, experimental outline, troubleshooting and optimization, and potential implications for iGEM.</p><br/><br />
<center><a href="https://2010.igem.org/Team:British_Columbia/Notebook_Biofilm"><img src="https://static.igem.org/mediawiki/2010/7/74/Bfnotebk.jpg"></a><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia/Notebook_QS"><img src="https://static.igem.org/mediawiki/2010/b/b5/Qsnotebk.jpg"></a><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia/Notebook_DspB"><img src="https://static.igem.org/mediawiki/2010/a/a4/Dspbnote.jpg"></a><br />
<br />
<a href="https://2010.igem.org/Team:British_Columbia/Notebook_Phage"><img src="https://static.igem.org/mediawiki/2010/6/69/Phagenotebk.jpg"></a><br />
<br></br><br/><br />
<h3>Standard Operating Protocols (SOPS)</h3><br />
<br />
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<br/><center><a href="http://openwetware.org/wiki/Main_Page"><img src="https://static.igem.org/mediawiki/2010/2/21/OWW_Sticker.jpg"></a></center><br/><br />
<p><a href="http://openwetware.org/wiki/Main_Page">OpenWetWare</a> (OWW) is an effort to promote the sharing of information, know-how, and wisdom among researchers and groups who are working in biology & biological engineering. OWW hosts lab/research wikis, course wikis, protocol wikis and wiki blogs.<br></br><br/><br />
See our <a href="http://openwetware.org/wiki/IGEM:UBC/2009/Notebook/UBC_iGEM_2010">UBC OWW notebook</a>.</p><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Template:Template_HD_3Template:Template HD 32010-10-27T15:22:45Z<p>Ayjchan: </p>
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/Notebook_QSTeam:British Columbia/Notebook QS2010-10-27T15:21:52Z<p>Ayjchan: </p>
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<div>__NOTOC__<br />
<br />
{{Template_HD_4}}<br />
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<p>Click <a href="http://openwetware.org/wiki/IGEM:UBC/2009/Notebook/UBC_iGEM_2010">here</a> to view our lab notebook for more details of our experiments and protocols. Listed below are protocols specifically used for the Quorum Sensing Track. Scroll down to see what we learned this summer!</p><br />
<br />
<p><p><p><h3>Cell growth and preparation of cells for electroporation</h3><br />
<ol><li>An overnight culture grown in B2 broth (Table 1) with constant aeration at 37C was diluted 1/25 in 25mL of fresh b2 broth in a 300mL flask.</li><br />
<li>The cells were grown with constant aeration at 37C until they reached an OD600 of about 0.4 and were then harvested by centrifugation.</li><br />
<li>Upon harvesting the cells from the B2 broth, the cells were washed three times in an equal volume of deionized water, followed by second washes with 10% glycerol solution.</li><br />
<li>Following resuspension of the cells in the second 10% glycerol wash solution, the cell suspension was incubated for 15 min., centrifuged and the cell pellet resuspended in 800 ul of a l0% glycerol solution.</li><br />
<li>The final cell concentrations obtained were between 1 and 3 x 10^10 cell per mL.</li><br />
<li>All wash solutions and incubation were at 20C. All centrifugation were at 4000 rpm, 5 min, 20oC. The electrocompetent cells were used directly after preparation.</li><br />
<li>Alternatively, 70 ul aliquots of electrocompetent cells were frozen in microfuge tube at -80oC immediately after preparation.</li></p></p></p><br />
<br />
<h3>Electroporation protocol for <i>S. aureus</i> RN4220</h3><br />
<ol><li>Remove competent cells (70uL aliquots) from -80C and thaw on ice for 30 minutes</li><br />
<li>Add ligation mix (1ug of DNA)</li><br />
<li>Incubate on ice for 30 minutes</li><br />
<li>Transfer 60uL of cell suspension-DNA mixture to 0.1cm gap electroporation cuvette. Make sure the cuvettes are on ice prior to this.</li><br />
<li>Cells and DNA electroporated at 20C, 100ohm resistance, 25uF capacitance (optimum time constant = 2.5ms), and 2.3kV in a Gene Pulser apparatus with pulse controller.</li><br />
<li>Place cells on ice and immediately resuspend in 940uL of B2 broth.</li><br />
<li>Transfer cells, DNA, and broth to eppendorf microcentrifuge tube. Make sure microcentrifuge tubes were previously on ice.</li><br />
<li>Incubate for at least 2 hours at 37C.</li><br />
<li>Plate on Tryptic Soy Agar (TSA) or NYE agar with appropriate antibiotic. In this case, erthrymycin.</li><br />
<li>Incubate plates at 37C for 48 hours.</li></p><br />
<br />
<div><table align="center" width="65%" cellspacing="0" border="1" cellpadding="1"><caption> Table 1</caption><br />
<tr><td>Media<td>Ingredients<td>References/Sources</td></tr><br />
<tr><td>B2<td><ul><li>1.0 % casein hydrolysate</li><li>2.5 % yeast extract</li><li>0.1% K2HPO4</li><li>0.5% glucose</li><li>2.5% NaCl</li><li>adjust pH to 7.5</li></ul><td>Schenk and Laddaga (1992)</td></tr><br />
<tr><td>NYE<td><ul><li>1.0 % casein hydrolysate</li><li>0.5% yeast extract</li><li>0.5% NaCl</li><li>adjust pH to 7.2</li></ul><td>Schenk and Laddaga (1992)</td></tr></table></div><br/><br />
<br />
<p>The above protocols (table included) are from the following paper: Schenk S, Laddaga RA. Improved method for electroporation of Staphylococcus aureus. FEMS Microbiol Lett. 1992 Jul 1;73(1-2):133–138</p><br />
<h3>Lessons Learned</h3><br />
<br />
<p>1. The 3-A method usually works. However, some parts seem harder to join to other parts. In the case of P2 (BBa_I764104), it took 4-5 times as long as expected to join it to 2 different GFP constructs. We haven't found a fix to this problem, even after varying several variables: insert:vector ratio, ligation volume, and changing ligase buffer. </p><br />
<p>2. PCR is a very good technique for verifying inserts. It is not so good for verifying small inserts (e.g. RBS) since the band would barely change. Sometimes, even when the PCR does not show correct bands, further restriction digests and sequencing yields the correct insert. </p><br />
<p>3. It is possible to clone genes directly from non-purified bacterial DNA. Simply pick a colony of cells containing the desired gene in the genome. This is of course harder to do than plasmid DNA. We've found that addition of DMSO (5-10%) helps the PCR (by presumably allowing primers and reagents to better reach the desired gene). This is true in the case of agrAC, where PCR was unsuccessful without the addition of DMSO.</p><br />
<p>4. Don't try to measure PCR product concentration using spectrophotometry right after a PCR. The concentration before and after reaction is basically the same because dNTP's also absorb similarly to strands of DNA. After purification, do measure DNA concentration.</p><br />
<p>5. Don't be afraid of going to the lab and start working. The best way to learn is through practice and troubleshooting. It also helps build up a good work flow.</p><br />
<p>6. Use polymerases with error checking ability (e.g. Phusion) when cloning genes. </p><br />
<p>7. Check antibiotics before using them. </p> <br />
<p>8. DNA is incredibly stable. DNA stocks will last a long time when stored properly, even in water.</p><br />
<p>9. Don't be afraid of asking for help or asking to use other lab groups' equipment.</p><br />
<p>10. DNA purification kits don't always work very well. Do check with a nanodrop or other method. Pay close attention to contaminants absorbing at other wavelengths (large peak), as this can fool the machine into thinking there is DNA. </p><br />
<p>11. Always sequence constructs if possible, even if simply joining 2 registry parts together. It is the only way to be sure the right parts have joined and reduces panic when parts do not work as expected.</p><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/Notebook_BiofilmTeam:British Columbia/Notebook Biofilm2010-10-27T15:21:32Z<p>Ayjchan: </p>
<hr />
<div>__NOTOC__<br />
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<p>Click <a href="http://openwetware.org/wiki/IGEM:UBC/2009/Notebook/UBC_iGEM_2010">here</a> to view our lab notebook for more details of our experiments and protocols. Listed below are protocols specifically used for the Biofilm Track. Scroll down to see what we learned this summer!</p><br />
<h3>Biofilm Quantification Protocol</h3><br />
<p><b>Day 1</b></p> <br />
<p>1. PIck an isolated colony from a Trypticase Soy Agar (TSA) plate and incubate in 5 mL of Trypticase Soy Broth (TSB) without shaking overnight</p><br />
<p><b>Day 2</b></p><br />
<p>1. Vortex the overnight culture and dilute 30 uL of culture by 1:100 in a 1% glucose solution diluted with TSB</p><br />
<p>2. Vortex to mix bacteria sample</p><br />
<p>3. Innoculate 200 uL of dilute sample into pre-determined wells of a microtiter plate</p><br />
<p>4. Use 1% glucose solution diluted with TSB, but without bacterial sample, as control and test samples in triplicate</p><br />
<p>5. Place lid onto microtiter plate and place in a non-shaking incubator for 24 hours at 37C</p><br />
<p><b>Day 3</b></p><br />
<p>1. Take microtiter plate out of the incubator and decant all liquid from the wells into a biohazard container</p> <br />
<p>2. Wash each well with 3 x 300 uL of Phosphate Buffered Saline (PBS) at room temperature</p><br />
<p>3. Heat fix remaining bacteria through exposure for 1 hour to 60C air using a heat block</p><br />
<p>4. Leave plate inverted overnight at room temperature</p> <br />
<p><b>Day 4</b></p><br />
<p>1. Add 150 uL of 0.1% crystal violet dye diluted with deionized water to each well</p><br />
<p>2. Allow staining to occur for 15 minutes</p><br />
<p>3. Decant crystal violet dye from each well into a biohazard container</p> <br />
<p>4. Wash wells with deionized water until crystal violet is no longer present in the water</p><br />
<p>5. Air dry plate</p><br />
<p>6. Take OD readings at an absorbance of 550 nm on a plate reader</p> <br />
<br />
<h3>Optimization</h3><br />
<p>Initial experiments were conducted to determine the conditions that were suitable for optimal biofilm growth. The original protocol called for incubation of bacteria in media with a glucose concentration ranging from 0.25%-2%. Through our experiments which tested the OD readings of various glucose concentrations, we concluded that 1% glucose media provided the best environment for biofilm growth of <i>S. aureus</i> strains RN4220 and 8325-4. As well, we determined using identical samples of bacteria and media solution that the outer edges of the Greiner 96-well flat bottom microtiter plate showed abnormal growth possibly due to increased exposure to oxygen when compared to the center wells. Therefore, we decided to use only the center wells from C3 to E10 for our experiments as to avoid extraneous variables. Day 3 of the original protocol was altered as it called for the aspiration of liquid from the wells with a micropipette, whereas we simply inverted the plate into a biohazard container as it was decided that the original method often resulted in biofilm disturbance.</p><br />
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<hr />
<div>__NOTOC__<br />
<br />
{{Template_HD_4}}<br />
<html><body id="notebook" onload="setPageSize()"><br />
<br />
<div id="super_main_wrapper"><br />
<div id="SubWrapper"> <br />
<br/><p>Click <a href="http://openwetware.org/wiki/IGEM:UBC/2009/Notebook/UBC_iGEM_2010">here</a> to view our lab notebook for more details of our experiments and protocols. Listed below are protocols specifically used for the DspB Track. Scroll down to see what we learned this summer!</p><br />
<br />
<h3>Sonication Protocol (with sonicator)</h3><br />
<ol><br />
<li>Add 100uL of sdH2O to 5mg of lysozyme</li><br />
<li>Pipet this into the cell pellet and resuspend</li><br />
<li>Add 50uL of 0.5M EDTA pH 8.0 to a final concentration of 25uM</li><br />
<li>Add 850uL of TE or EB buffer (fill up to 1mL)</li><br />
<li>Sonicate (ear protectors on!)</li><br />
<ol type="i"><br />
<li>Turn sonicator on</li><br />
<li>Wipe down probe with kimwipe wetted with ethanol</li><br />
<li>Press start</li><br />
<li>Turn knob up to desired level (in this case, 5)</li><br />
<li>Place mirocentrifuge tube under the probe and move it up and down for 15 seconds</li><br />
<li>Take the tube out and place on ice</li><br />
<li>Repeat 5 more times</li><br />
<li>Turn off sonicator</li></ol></ol><br />
<br />
<h3>Sonication Protocol (Lysozyme method)</h3><br />
<ol><li>Take the overnight culture out of 37C incubator</li><br />
<li>Transfer the culture into a microcentrifuge tube and spin it down into a pellet</li><br />
<li>Resuspend the culture in approximately 800uL of TE/EB buffer</li><br />
<li>Weigh out lysozyme in microcentrifuge tube (10mg)</li><br />
<li>Dissolve the lysozyme in 100uL of sdH2O</li><br />
<li>Add the 100uL of dissolved lysozyme to the 800uL of culture</li><br />
<li>Place the tube in the 37C incubator for at least 2 hours</li><br />
<li>Centrifuge/spin it down into a pellet</li><br />
<li>Take the supernatant and transfer to another microcentrige tube</li><br />
<li>Use for crude cell assay</li></ol><br />
<br />
<h3>Substrate Assay Protocol</h3><br />
<ol><li>Take a 96-well plate and assign wells for 6 well replicates for 4 conditions, for a total of 24 wells</li><br />
<li>For the first six wells, add 100uL of phosphate buffer only</li><br />
<li>For the next six wells, add 70uL of phosphate buffer and 30uL of substrate</li><br />
<li>For the next six wells, add 60uL of phosphate buffer, 30uL of substrate, and 10uL of DspB lysate</li><br />
<li>For the final six wells, add 60uL of phosphate buffer, 30uL of substrate, 10uL of control lysate.</li><br />
<li>Take measurements at absorbance wavelength = 405nm using a plate reader. In this case, Tecan M200 Plate Reader is used.</li></ol><br />
<br />
<h3>Lessons Learned</h3><br />
<br />
<ol><li>Make your own aliquots of reagents, especially sdH2O.</li><br />
<li>There's no need to aliquot buffers from kits into a falcon tube before using.</li><br />
<li>You don't need three people to load a gel.</li><br />
<li>You don't need to gel extract your PCR product.</li><br />
<br />
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<p><a href="http://openwetware.org/wiki/Main_Page">OpenWetWare</a> (OWW) is an effort to promote the sharing of information, know-how, and wisdom among researchers and groups who are working in biology & biological engineering. OWW hosts lab/research wikis, course wikis, protocol wikis and wiki blogs.<br></br><br/><br />
See our <a href="http://openwetware.org/wiki/IGEM:UBC/2009/Notebook/UBC_iGEM_2010">UBC OWW notebook</a>.</p><br />
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</body></html></div>Ayjchanhttp://2010.igem.org/Team:British_Columbia/Notebook_DspBTeam:British Columbia/Notebook DspB2010-10-27T15:19:26Z<p>Ayjchan: </p>
<hr />
<div>__NOTOC__<br />
<br />
{{Template_HD_4}}<br />
<html><body id="notebook" onload="setPageSize()"><br />
<br />
<div id="super_main_wrapper"><br />
<div id="SubWrapper"> <br />
<br/><p>Click <a href="http://openwetware.org/wiki/IGEM:UBC/2009/Notebook/UBC_iGEM_2010">here</a> to view our lab notebook for more details of our experiments and protocols. Listed below are protocols specifically used for the DspB Track. Scroll down to see what we learned this summer!</p><br />
<br />
<h3>Sonication Protocol (with sonicator)</h3><br />
<ol><br />
<li>Add 100uL of sdH2O to 5mg of lysozyme</li><br />
<li>Pipet this into the cell pellet and resuspend</li><br />
<li>Add 50uL of 0.5M EDTA pH 8.0 to a final concentration of 25uM</li><br />
<li>Add 850uL of TE or EB buffer (fill up to 1mL)</li><br />
<li>Sonicate (ear protectors on!)</li><br />
<ol type="i"><br />
<li>Turn sonicator on</li><br />
<li>Wipe down probe with kimwipe wetted with ethanol</li><br />
<li>Press start</li><br />
<li>Turn knob up to desired level (in this case, 5)</li><br />
<li>Place mirocentrifuge tube under the probe and move it up and down for 15 seconds</li><br />
<li>Take the tube out and place on ice</li><br />
<li>Repeat 5 more times</li><br />
<li>Turn off sonicator</li></ol></ol><br />
<br />
<h3>Sonication Protocol (Lysozyme method)</h3><br />
<ol><li>Take the overnight culture out of 37C incubator</li><br />
<li>Transfer the culture into a microcentrifuge tube and spin it down into a pellet</li><br />
<li>Resuspend the culture in approximately 800uL of TE/EB buffer</li><br />
<li>Weigh out lysozyme in microcentrifuge tube (10mg)</li><br />
<li>Dissolve the lysozyme in 100uL of sdH2O</li><br />
<li>Add the 100uL of dissolved lysozyme to the 800uL of culture</li><br />
<li>Place the tube in the 37C incubator for at least 2 hours</li><br />
<li>Centrifuge/spin it down into a pellet</li><br />
<li>Take the supernatant and transfer to another microcentrige tube</li><br />
<li>Use for crude cell assay</li></ol><br />
<br />
<h3>Substrate Assay Protocol</h3><br />
<ol><li>Take a 96-well plate and assign wells for 6 well replicates for 4 conditions, for a total of 24 wells</li><br />
<li>For the first six wells, add 100uL of phosphate buffer only</li><br />
<li>For the next six wells, add 70uL of phosphate buffer and 30uL of substrate</li><br />
<li>For the next six wells, add 60uL of phosphate buffer, 30uL of substrate, and 10uL of DspB lysate</li><br />
<li>For the final six wells, add 60uL of phosphate buffer, 30uL of substrate, 10uL of control lysate.</li><br />
<li>Take measurements at absorbance wavelength = 405nm using a plate reader. In this case, Tecan M200 Plate Reader is used.</li></ol><br />
<br />
<h3>Lessons Learned</h3><br />
<br />
<ol><li>Make your own aliquots of reagents, especially sdH2O.</li><br />
<li>There's no need to aliquot buffers from kits into a falcon tube before using.</li><br />
<li>You don't need three people to load a gel.</li><br />
<li>You don't need to gel extract your PCR product.</li><br />
<li><br />
<br />
<br />
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<br/><center><a href="http://openwetware.org/wiki/Main_Page"><img src="https://static.igem.org/mediawiki/2010/2/21/OWW_Sticker.jpg"></a></center><br/><br />
<p><a href="http://openwetware.org/wiki/Main_Page">OpenWetWare</a> (OWW) is an effort to promote the sharing of information, know-how, and wisdom among researchers and groups who are working in biology & biological engineering. OWW hosts lab/research wikis, course wikis, protocol wikis and wiki blogs.<br></br><br/><br />
See our <a href="http://openwetware.org/wiki/IGEM:UBC/2009/Notebook/UBC_iGEM_2010">UBC OWW notebook</a>.</p><br />
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