Difference between revisions of "Team:Cambridge-JIC/Collaborations"
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− | <h1 style="line-height:1.295em"> Collaborations </h1> | + | <center><h1 style="line-height:1.295em"> Collaborations </h1></center> |
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− | <h2>William and Mary Team</h2> | + | <center><h2>William and Mary Team</h2></center> |
− | <p>As part of their project, the William and Mary iGEM Team were imaging cells expressing a variety of fluorescent | + | <h4><center><i>Transforming & Fluorescence</i></center></h4> |
+ | <p>As part of their project, the William and Mary (W&M) iGEM Team were imaging cells expressing a variety of fluorescent proteins, including RFP, CFP, GFP, and YFP. Their constructs are being used for the Interlab Measurement study. As part of the measurement track the W&M team assembled fluorescent constructs, and measured and reported their fluorescence. The samples were sent to us to confirm the correct assembly of the parts by validating their fluorescence. Their interest in seeing how their constructs performed (in terms of fluorescent output) perfectly matched our own need to test OpenScope on cell samples rather than fluorescent beads. In addition, optical components were obtained to extend our imaging capabilities to both GFP and RFP. In particular, RFP imaging has not been tested previously. </p> | ||
<p>The W&M team were kind enough to send us dried DNA samples containing constructs for GFP and RFP expression in <i>E. coli</i>. Details on the protocols used to resuspend the DNA and to transform the cells can be found below. </p> | <p>The W&M team were kind enough to send us dried DNA samples containing constructs for GFP and RFP expression in <i>E. coli</i>. Details on the protocols used to resuspend the DNA and to transform the cells can be found below. </p> | ||
<br> | <br> | ||
− | <h3>Transformation Protocol | + | <h3>Transformation Protocol</h3> |
<br> | <br> | ||
<h4> Context </h4> | <h4> Context </h4> | ||
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</ol> | </ol> | ||
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− | < | + | <h4> Imaging:</h4> |
− | <p> | + | <p>Fluorescent samples were then visualised with OpenScope using two set-ups:</p> |
+ | <ol> | ||
+ | <li><p><b>Standard set-up</b>, using single LED illumination (470 nm, 100mW) and GFP epi-cube</li></p> | ||
+ | <li><p><b>Non-standard set-up</b>, using high brightness LED (470nm, 3W) and GFP epi-cube</li></p> | ||
+ | </ol> | ||
+ | |||
+ | <br> | ||
+ | <h4> Results:</h4> | ||
<p>Earlier testing using the standard set-up (single LED illumination, GFP epi-cube) indicated that visualising fluorescent beads was possible with OpenScope (Fig. 1). </p> | <p>Earlier testing using the standard set-up (single LED illumination, GFP epi-cube) indicated that visualising fluorescent beads was possible with OpenScope (Fig. 1). </p> | ||
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− | < | + | <p><center><img src="https://static.igem.org/mediawiki/2015/6/67/CamJIC-FBead1.png" style="height:200px;margin:5px"><img src="https://static.igem.org/mediawiki/2015/e/ee/CamJIC-FBead2.png" style="height:200px;margin:5px"></center></p> |
− | + | <p><center><i><b>Fig. 1: </b>GFP-coated fluorescent beads visualised using OpenScope under standard set-up. Images were captured on 14.09.15 using the Webshell, and are unedited.</i></center></p> | |
− | < | + | <br style="clear: both"> |
+ | |||
+ | <p>Testing using the commercial fluorescence microscope confirmed that the samples had phenotypes as reported by William and Mary. Both constructs resulted in GFP expression in E. coli. J23106+I13504 demonstrated increased fluorescence intensity relative to J23117+I13504.</p> | ||
+ | <p> A control slide (sample taken from the agar of the control plate, untransformed cells plated on Amp) was first tested to establish a fluorescence-free baseline (Fig. 2a). The duplicate J23106+I13504 samples were then tested using the standard set-up and non-standard set-up. Fluorescence was detected (Fig. 2b and 2c).</p> | ||
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+ | <p><center><img src="https://static.igem.org/mediawiki/2015/9/94/CamJIC-W%26MControl.png" style="height:200px;margin:5px"><img src="https://static.igem.org/mediawiki/2015/3/33/CamJIC-W%26M100.png" style="height:200px;margin:5px"><img src="https://static.igem.org/mediawiki/2015/4/48/CamJIC-W%26M3%281%29.png" style="height:200px;margin:5px"></center></p> | ||
+ | <p><center><i><b>Fig. 2:</b> <b>a</b> Control slide (left) showing no fluorescence <b>b</b> J23106+I13504 samples imaged using standard set-up showing fluorescence under 470nm excitation (centre) <b>c</b> J23106+I13504 samples imaged using non-standard set-up (right). All images were captured using the Webshell on 15.09.15 and are unedited.</i></center></p> | ||
+ | <br style="clear: both"> | ||
<p>Duplicate J23117+I13504 samples were then tested using standard set-up and the non-standard set-up, but the brightness was insufficient to image the fluorescence. This confirms the reduced fluorescence intensity of the J23117+I13504 samples compared to the J23106+I13504 samples. </p> | <p>Duplicate J23117+I13504 samples were then tested using standard set-up and the non-standard set-up, but the brightness was insufficient to image the fluorescence. This confirms the reduced fluorescence intensity of the J23117+I13504 samples compared to the J23106+I13504 samples. </p> | ||
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− | <h3> Conclusions:</h3> | + | |
+ | </div></div></section> | ||
+ | <section style="background-color:#fff"> | ||
+ | <div class="slide" style="min-height:0px"> | ||
+ | <div style="width: 90%; margin: 30px 50px;color:#000"> | ||
+ | <p><center><a href="//2015.igem.org/Team:Glasgow"><img src="https://static.igem.org/mediawiki/2015/a/ae/2015-Glasgow-sticker.png"style="height: 75px"></a> | ||
+ | <h2>Glasgow Team</h2></center> | ||
+ | <h4><center><i>We can see how you fluoresce</i></center></h4></p> | ||
+ | <p>Collaboration with the Glasgow iGEM Team was also based on fluorescence imaging: as part of their project, the Glasgow team are characterising different fluorescence constructs expressing RFP and GFP. It was therefore beneficial for them to have an independent group analyse the bacterial expression of their constructs, and for us to have some living fluorescent samples to image. Specifically, the bacterial transformants they supplied were reported to have the following phenotypes: </p> | ||
+ | <ul class="RFPlist"> | ||
+ | <li><p>DH5α - No antibiotic resistance, no fluorescence (control)</p></li> | ||
+ | <li><p>DH5α - Kanamycin resistance, GFP and RFP fluorescence (in the same cell)</p></li> | ||
+ | <li><p>DH5α - Kanamycin and Chloramphenicol resistance, RFP fluorescence</p></li> | ||
+ | <li><p>DH5α - Kanamycin and Chloramphenicol resistance, GFP fluorescence</p></li> | ||
+ | <li><p>DH5α - Kanamycin and Chloramphenicol resistance, mostly GFP fluorescence with a small proportion of cells on the plate expressing RFP</p></li> | ||
+ | </ul> | ||
+ | <p>Our objective was therefore twofold: firstly to test the capabilities of OpenScope in analysing bacterial samples for GFP and RFP fluorescence, and secondly to report back to Glasgow with our phenotypic analysis of their bacterial strains.</p> | ||
+ | <br> | ||
+ | <h4> Results:</h4> | ||
+ | <p>After preliminary testing using the RFP epi-cube, it was decided that imaging of RFP at this stage would not be possible. Hence only bacterial strains expressing GFP (as confirmed earlier) were tested against the control.</p> | ||
+ | <p>Testing using the commercial fluorescence microscope confirmed that the samples had phenotypes as reported by Glasgow. From tube 5, a small proportion of the cells were expressing RFP and the majority expressed GFP as predicted.</p> | ||
+ | <p>Results from preliminary testing of DH5α cells with p126.1 and p56.1 (confirmed: GFP expression only) using the standard set-up indicated illumination brightness was insufficient to detect GFP. The non-standard set-up (with a brighter LED) was used, and GFP expression was confirmed in p126.1, p126.+p56.1 and p126.1+p80.1 cells as expected (Fig. 3a-d). The images display an artefact of the square shape of the LED used, as there is an area of increased fluorescence in the outline of a square at the centre of the images (Fig 3c and d). </p> | ||
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+ | <div style="float:left; margin-right: 20px"><img src="https://static.igem.org/mediawiki/2015/f/f0/CamJIC-GlasgowControl.png" style="height: 175px"></div> | ||
+ | <div style="float:left; margin-right: 20px"><img src="https://static.igem.org/mediawiki/2015/7/72/CamJIC-Glasgowp126.1.png" style="height: 175px"></div> | ||
+ | <div style="float:left; margin-right: 20px"><img src="https://static.igem.org/mediawiki/2015/4/4f/CamJIC-Glasgowp126.1p80.1.png" style="height: 175px"></div> | ||
+ | <div style="float:left; margin-right: 20px"><img src="https://static.igem.org/mediawiki/2015/a/a9/CamJIC-Glasgowp126.1p56.1.png" style="height: 175px"></div> | ||
+ | <div style="float:left; margin-right: 20px"><p><i><b>Fig. 3:</b> <b>a</b> Control slide (left) showing no fluorescence <b>b</b> p126.1 samples imaged using non-standard set-up under 470nm excitation (centre left) <b>c</b> p126.1+p80.1 samples imaged using non-standard set-up showing fluorescence (centre right) <b>d</b> p126.1+p56.1 samples imaged using non-standard set-up (right). All images were captured using the Webshell on 15.09.15 and are unedited.</i></p></div> | ||
+ | <br style="clear: both"> | ||
+ | </div></div></section> | ||
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+ | <section style="background-color:#fff"> | ||
+ | <div class="slide" style="min-height:0px"> | ||
+ | <div style="width: 90%; margin: 30px 50px;color:#000"> | ||
+ | <center><h3> Conclusions:</h3></center> | ||
<p>The first objective of the collaboration was to confirm the phenotypes of the bacterial strains and the expression of fluorescent proteins. Due to the technical limitations of OpenScope (see below), RFP could not be visualised. Hence a commercially available microscope was used to confirm RFP expression. In addition, GFP was reliably confirmed using the same microscope. Across the board, expression of the fluorescent proteins was as reported by Glasgow and W&M iGEM teams. This confirms the functionality of the plasmids. </p> | <p>The first objective of the collaboration was to confirm the phenotypes of the bacterial strains and the expression of fluorescent proteins. Due to the technical limitations of OpenScope (see below), RFP could not be visualised. Hence a commercially available microscope was used to confirm RFP expression. In addition, GFP was reliably confirmed using the same microscope. Across the board, expression of the fluorescent proteins was as reported by Glasgow and W&M iGEM teams. This confirms the functionality of the plasmids. </p> | ||
− | <p>After successful visualisation of fluorescent beads labeled with GFP (Fig. 1), it was expected that OpenScope would enable visualisation of E. coli expressing GFP. However, results indicate that reliable detection was not possible using the standard set-up (single LED illumination, GFP epi-cube). Possible | + | <p>After successful visualisation of fluorescent beads labeled with GFP (Fig. 1), it was expected that OpenScope would enable visualisation of E. coli expressing GFP. However, results indicate that reliable detection was not possible using the standard set-up (single LED illumination, GFP epi-cube). Possible reasons are:</p> |
<ol> | <ol> | ||
<li><p>The fluorescent beads used are far brighter than biological samples, and therefore can be visualised with low-brightness LEDs</li></p> | <li><p>The fluorescent beads used are far brighter than biological samples, and therefore can be visualised with low-brightness LEDs</li></p> | ||
− | <li><p> | + | <li><p>Bacterial cells are widely dispersed on a microscope slide when prepared from a liquid culture. Fluorescence can only be detected in clustered bacterial cells and colonies, therefore OpenScope cannot resolve individual fluorescent bacterial cells</li></p> |
</ol> | </ol> | ||
− | <p>Replacement of the 100mW LED with the 3W LED allowed visualisation of samples with reduced fluorescence intensity in the case of the p126.1, p126.+p56.1 and p126.1+p80.1 cells. The 100mW LED was sufficient only to image the J23106+I13504 samples. Overall, the results suggest that in order to reliably detect fluorescence the 3W LED is more appropriate. However, this is still not sufficiently reliable to make OpenScope useful for fluorescence screening at this stage. In addition, the | + | <p>Replacement of the 100mW LED with the 3W LED allowed visualisation of samples with reduced fluorescence intensity in the case of the p126.1, p126.+p56.1 and p126.1+p80.1 cells. The 100mW LED was sufficient only to image the J23106+I13504 samples. Overall, the results suggest that in order to reliably detect fluorescence the 3W high-power surface-mount LED is more appropriate. However, this is still not sufficiently reliable to make OpenScope useful for fluorescence screening at this stage. In addition, the artefact (image of the LED itself) seen when using the 3W LED means that the uniformity of illumination must be improved.</p> |
− | <p>RFP is more challenging to image, as it has a narrow gap between the excitation (584nm) and emission (607nm). | + | <p>RFP is more challenging to image, as it has a narrow gap between the excitation (584nm) and emission (607nm). For more information, see the <a href="//2015.igem.org/Team:Cambridge-JIC/Modeling" class="blue">Modeling Page</a>. |
</div></div></section> | </div></div></section> | ||
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<section style="background-color:#fff"> | <section style="background-color:#fff"> | ||
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− | <h2>UK Teams Meetup (Organised by Westminster Team)</h2> | + | <center><img src="https://static.igem.org/mediawiki/2015/2/25/Team_Westminster_Logo.png" style="height:40px;margin:5px"><h2>UK Teams Meetup (Organised by Westminster Team)</h2></center> |
<h4><center><i>Bringing microscopy to iGEMmers</i></center></h4> | <h4><center><i>Bringing microscopy to iGEMmers</i></center></h4> | ||
− | < | + | <p><center><img src="https://static.igem.org/mediawiki/2015/b/b1/CamJIC-UKmeet_general.JPG" style="width:250px;margin:20px"> |
− | + | <img src="https://static.igem.org/mediawiki/2015/5/5e/CamJIC-UKmeet_team.JPG" style="width:250px;margin:20px"></center></p> | |
− | <p>In order for UK iGEM teams to meet, socialise and discuss their projects ahead of the Jamboree, the Westminster iGEM team organised a two day meeting in London. As the Hardware track was new to this year’s competition, and being the only team following this track in the UK, we felt this would be a fantastic opportunity to introduce other teams to | + | <p>In order for UK iGEM teams to meet, socialise and discuss their projects ahead of the Jamboree, the Westminster iGEM team organised a two day meeting in London. As the Hardware track was new to this year’s competition, and being the only team following this track in the UK, we felt this would be a fantastic opportunity to introduce other teams to the track and highlight the important role of hardware in synthetic biology. |
<br>Our workshop took the form of three stations, each covering a slightly different aspect of our project and each with a different discussion focus:</p> | <br>Our workshop took the form of three stations, each covering a slightly different aspect of our project and each with a different discussion focus:</p> | ||
<div class="factcol-container"> | <div class="factcol-container"> | ||
− | <div class="factcol" style="background-color:# | + | <div class="factcol" style="background-color:#b1e7ad;color:#1b4f18"> |
<h4><center>Optics Bench</center></h4> | <h4><center>Optics Bench</center></h4> | ||
<p>A working set-up of our first optics bench, and demonstration 3D printed material. Discussion points:</p> | <p>A working set-up of our first optics bench, and demonstration 3D printed material. Discussion points:</p> | ||
− | <ul | + | <ul> |
<li><p>3D printing and rapid prototyping</p></li> | <li><p>3D printing and rapid prototyping</p></li> | ||
<li><p>Principles of open-source hardware</p></li> | <li><p>Principles of open-source hardware</p></li> | ||
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</ul> | </ul> | ||
</div> | </div> | ||
− | <div class="factcol" style="background-color:# | + | <div class="factcol" style="background-color:#c6b2e4;color:#4c366e"> |
<h4><center>Microscopy Awaits</center></h4> | <h4><center>Microscopy Awaits</center></h4> | ||
<p>A working set-up of our latest microscope prototype, brightfield and fluorescence modes, fully motorised and video streaming to the Webshell. Discussion points:</p> | <p>A working set-up of our latest microscope prototype, brightfield and fluorescence modes, fully motorised and video streaming to the Webshell. Discussion points:</p> | ||
<ul> | <ul> | ||
− | <li><p>Importance of | + | <li><p>Importance of, and our difficulty with, illumination</li></p> |
<li><p>Remote control over Webshell</li></p> | <li><p>Remote control over Webshell</li></p> | ||
<li><p>Epi-cube explanation</li></p> | <li><p>Epi-cube explanation</li></p> | ||
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</ul> | </ul> | ||
</div> | </div> | ||
− | <div class="factcol" style="background-color:# | + | <div class="factcol" style="background-color:#bde9fe;color:#123a68"> |
<h4><center>Build Your Own</center></h4> | <h4><center>Build Your Own</center></h4> | ||
<p>An interactive demonstration on building our microscope from scratch. Discussion points:</p> | <p>An interactive demonstration on building our microscope from scratch. Discussion points:</p> | ||
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<br style="clear: both" /> | <br style="clear: both" /> | ||
</div> | </div> | ||
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− | + | <p><center><img src="https://static.igem.org/mediawiki/2015/4/49/CamJIC-UKmeet_action.jpg" style="width:320px;margin:20px"><img src="https://static.igem.org/mediawiki/2015/1/1b/CamJIC-UKmeet_webshell.JPG" style="width:320px;margin:20px"><img src="https://static.igem.org/mediawiki/2015/b/b9/CamJIC-UKmeet_openscope.JPG" style="width:320px;margin:20px"></center></p> | |
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<p>The entire team was involved, and this allowed members of other teams to chat and ask questions at all three stations while the equipment was being run. | <p>The entire team was involved, and this allowed members of other teams to chat and ask questions at all three stations while the equipment was being run. |
Latest revision as of 16:40, 16 September 2015