Difference between revisions of "Team:USP UNIFESP-Brazil/Proof"

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<p class=black>Our iGEM idea was not only to produce spider silk proteins and antibiotics.
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We also aimed to expand the tools and improve <i>Chlamydomonas reinhardtii</i> as a chassis for synthetic biology!
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We were able to transform through electroporation and successfully express a reporter protein, mCherry, in <i> Chlamydomonas</i> and detect its fluorescence. Our composite part, <a href=http://parts.igem.org/Part:BBa_K2136010>BBa_K2136010</a> will be of great value for any other teams working with microalgae in iGEM’s future!
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Even though this organism offers exciting possibilities, as well as many other photosynthetic chassis, a limited number of parts is available for synthetic work is available in the registry. That motivated us to change this and help the growth of this part of the synbio community!
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One of our goals within iGEM was to develop a “Chlamydomonas molecular toolkit”, with basic parts for everyone’s use. We had a construct, <a href=http://parts.igem.org/Part:BBa_K2136010>BBa_K2136010</a>, synthesized by IDT gBlocks technology which included:
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<li class=black> An enhancer/promoter construct for transcription, <a href=http://parts.igem.org/Part:BBa_K2136013>BBa_K2136013</a></li>
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<li class=black> A resistance gene, selection marker, against the antibiotic zeocin, <a href=http://parts.igem.org/Part:BBa_K2136014>BBa_K2136014</a></li>
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<li class=black> A self cleaving peptide from foot-and-mouth disease virus, <a href=http://parts.igem.org/Part:BBa_K2136017>BBa_K2136017</a></li>
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<li class=black> A signal peptide which enable secretion of the protein of interest, <a href=http://parts.igem.org/Part:BBa_K2136018>BBa_K2136018</a> </li>
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This construct was already assembled through an scarless method with mCherry codon optimized coding sequence  <a href=http://parts.igem.org/Part:BBa_K2136016>BBa_K2136016</a> and a <i>Chlamydomonas</i> terminator,  <a  href=http://parts.igem.org/Part:BBa_K2136015>BBa_K2136015</a>
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The clones were then selected in Petri dishes containing zeocyn and screened in 96 well plates for mCherry fluorescence (excitation at 587nm, emission at 611nm). Even though the vast majority of the cells were productive, there was a variation, in great part due to the random assembly in <i>C. reinhardtii</i>’s genome. This, hopefully, will be surpassed with the development of new expression methods in the future (perhaps even  by iGEM teams!). Here we show the expression profile during growth of the best 5 clones in our first and second screening:
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<img src="https://static.igem.org/mediawiki/parts/7/73/T--USP_UNIFESP-Brazil-result_Screen_1_mCherry.png" width="450px" style="margin-bottom:20px; margin-top:0px;" />
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<p class="fig-label" class=black>Image 1 - mCherry emission/excitation in different cells in our first screen</p>
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<img src="https://static.igem.org/mediawiki/2016/2/22/T--USP_UNIFESP-Brazil--result_Screen_2_mCherry.png" width="450px" style="margin-bottom:20px; margin-top:0px;" />
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<p class="fig-label" class=black>Image 2 - mCherry emission/excitation in different cells in our second screen</p>
  
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<h2> Proof of concept</h2>
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The excitation/emission fluorescence spectrum of the mCherry supernatant (without cell lysis, indicating secretion) was also characterized, not only confirming the detection, but improving characterization of this protein (<a href=http://parts.igem.org/wiki/index.php?title=Part:BBa_J06505>BBa_J06505</a> and <a href=http://parts.igem.org/Part:BBa_K2136016>BBa_K2136016</a> )in the context of iGEM. 
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Revision as of 02:14, 19 October 2016

AlgAranha Team USP_UNIFESP-Brazil

iGEM 2016

Our iGEM idea was not only to produce spider silk proteins and antibiotics. We also aimed to expand the tools and improve Chlamydomonas reinhardtii as a chassis for synthetic biology! We were able to transform through electroporation and successfully express a reporter protein, mCherry, in Chlamydomonas and detect its fluorescence. Our composite part, BBa_K2136010 will be of great value for any other teams working with microalgae in iGEM’s future!

Even though this organism offers exciting possibilities, as well as many other photosynthetic chassis, a limited number of parts is available for synthetic work is available in the registry. That motivated us to change this and help the growth of this part of the synbio community!

One of our goals within iGEM was to develop a “Chlamydomonas molecular toolkit”, with basic parts for everyone’s use. We had a construct, BBa_K2136010, synthesized by IDT gBlocks technology which included:

  • An enhancer/promoter construct for transcription, BBa_K2136013
  • A resistance gene, selection marker, against the antibiotic zeocin, BBa_K2136014
  • A self cleaving peptide from foot-and-mouth disease virus, BBa_K2136017
  • A signal peptide which enable secretion of the protein of interest, BBa_K2136018

This construct was already assembled through an scarless method with mCherry codon optimized coding sequence BBa_K2136016 and a Chlamydomonas terminator, BBa_K2136015

.

The clones were then selected in Petri dishes containing zeocyn and screened in 96 well plates for mCherry fluorescence (excitation at 587nm, emission at 611nm). Even though the vast majority of the cells were productive, there was a variation, in great part due to the random assembly in C. reinhardtii’s genome. This, hopefully, will be surpassed with the development of new expression methods in the future (perhaps even by iGEM teams!). Here we show the expression profile during growth of the best 5 clones in our first and second screening:



Image 1 - mCherry emission/excitation in different cells in our first screen

Image 2 - mCherry emission/excitation in different cells in our second screen

The excitation/emission fluorescence spectrum of the mCherry supernatant (without cell lysis, indicating secretion) was also characterized, not only confirming the detection, but improving characterization of this protein (BBa_J06505 and BBa_K2136016 )in the context of iGEM.