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| − | <h1 class="date three" id="design">The polycistron</h1>
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| − | <p>In 2014, Beekwilder & als. assembled the crtE, crtYB and crtI genes into a polycistronic construct where the individual Crt proteins were separated by the T2A sequences of the <i>Thosea asigna</i> virus.<br> Their polycistron is under the regulation of the strong yeast promoter TDH3, and the terminator TEF1.
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| − | They were able to show that the addition of those genes to <i>Saccharomyces cerevisiae</i> was enough to make it produce ß-carotene.<br>
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| − | <br><div align="center"><img src="https://static.igem.org/mediawiki/2015/9/95/ParisBettencourt_polycistron_schema.jpg" width="500px"></img></div>
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| − | <div class="column-right" align="left"><b>2A sequences or cis-acting hydrolase element:</b> <br>
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| − | 2A like sequences are able to force the ribosome to "skip" a codon. The ribosome releases the part that it has already translated and to keep translating the mRNA. It allows transcription of multiple proteins from only 1 mRNA with 1 promoter, like bacterial polycistronic elements, but also with only one kozac sequence (yeast RBS) which ensures that the quantities of all the translated product are the same.<br>
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| − | <h1 class="date one" id="overview">Growth, blabla</h1>
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| − | We used strain with polycistron from blabla.
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| − | <br>cool figures
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| − | <div class="column-right" align="left">Lorem ipsum
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| − | <h1 class="date one" id="overview">Improvement??? (help to find cool title!)</h1></div>
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| − | <b>An optimized polycistron</b>
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| − | <br>The amount of ß-carotene produced by the polycistronic strain is not enough to meet the daily requirement if the idli fermented with the engineered strain is the only source of vitamin A eaten in a day; which is why we aimed to strongly increase the ß-carotene yield of those yeast.
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| − | <br>For this purpose, we designed a construct very similar to theirs, except that we moved the crtE gene to the first place of the polycistron, in order to increase the carotenoid yield. Indeed, it has been shown that the efficiency of translation decreases after every 2A sequence (de Felipe et al. 2006), and that an increase of CrtE may improve the ß-carotene production (Verwaal et al. 2007). We kept the same 2A sequences between the cistrons, as well as the same strong promoter TDH3 and the same terminator TEF1.
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| − | <br>We also codon-optimized the three genes for <i>S. cerevisiae</i> in order to increase the genes expression. The study from Li et al. (2013) had shown that the optimization of 5 codons in the sequence of crtI, and 8 codons in the sequence of crtYB had increased the ß-carotene production in <i>S. cerevisiae</i> by 200%, so we had high hopes that codon-optimizing the whole genes would lead to even better yield.
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| − | <br>The whole construct we designed was synthesized by IDT in 5 gBlocks.
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| − | <br><br><br><div align="center"><img src="https://static.igem.org/mediawiki/2015/5/52/ParisBettencourt_new_polycistron.jpg" width="500px"></img></div>
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| − | <br><br>
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| − | <b>An optimized HMG gene</b>
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| − | <br>Additionally, we codon-optimized for <i>S. cerevisiae</i> the HMG-CoA reductase gene from <i>S. aureus</i> that had been used by Li & al. in 2013. Indeed, their study had shown that <i>S. cerevisiae</i> transformed with this gene had a better ß-carotene yield than the ones transformed by the tHMG1 from <i>S. cerevisiae</i>; it is highly probable than a codon-optimized version of this gene from <i>S. aureus</i> would produce even more ß-carotene.
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| − | <b>Chromosomal integration</b>
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| − | <br>In the strain containing the polycistron designed by Beekwilder, the polycistronic construct is on a plasmid (pUDC082). But since in our final product we would like our yeast to grow on non selective media and to keep the polycistron, we designed a way to integrate the construct in the yeast chromosome. Our plan was to use the HO-Poly-KanMX4-HO plasmid (AddGene plasmid #51662) as a backbone for our construct: it's a yeast plasmid for chromosomal integration into the HO locus, with a selection marker for yeast (KanMX4). This plasmid also has an origin of replication for <i>E. coli</i> and a selection marker for bacteria (Ampicillin).
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| − | <br><br>Map of the HO-Poly-KanMX4-HO plasmid containing our optimized polycistron:
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| − | <div class="container content" ng-init="sample.size=375">
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| − | <filter id="dropshadow" height="120%">
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| − | <feGaussianBlur in="SourceAlpha" stdDeviation="5"/>
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| − | <feOffset dx="2" dy="2" result="offsetblur"/>
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| − | <feFuncA type="linear" slope="0.3"/>
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| − | </feComponentTransfer>
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| − | <feMergeNode in="SourceGraphic"/>
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| − | </feMerge>
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| − | </defs>
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| − | </svg>
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| − | <plasmid sequencelength='12010' plasmidheight="{{sample.size}}" plasmidwidth="{{sample.size}}">
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| − | <plasmidtrack width="5" trackstyle='fill:#ccc;stroke:#999;filter:url(#dropshadow)' style='fill:#FABE44;stroke:#000000'>
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| − | <tracklabel text='HO-E/YB/I-Poly-KanMX4-HO' labelstyle="font-size:13px"></tracklabel>
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| − | <tracklabel labelstyle="font-size:12px;font-weight:400;fill:#999" text="12,010 bp" vadjust="18"></tracklabel>
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| − | <trackmarker start='296' end='1075' markerstyle='stroke:#000;fill:#FF0000;filter:url(#dropshadow)' arrowendlength="7" arrowendwidth="5" wadjust="10" vadjust="-5">
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| − | <markerlabel text='KanMX4' vadjust="40" hadjust="2" valign="outer" style="font-size:14px;font-weight:400;fill:#000000" showline="1" linevadjust="-15" linevadjust="-15" linelabelstyle="stroke:#cc0;stroke-dasharray:2,2;stroke-width:2px;"></markerlabel>
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| − | </trackmarker>
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| − | <trackmarker start='1332' end='1831' markerstyle='stroke:#000;fill:#FF4000;filter:url(#dropshadow)' wadjust="10" vadjust="-5">
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| − | <markerlabel text='HO-R' vadjust="40" hadjust="2" valign="outer" style="font-size:14px;font-weight:400;fill:#000000" showline="1" linevadjust="-15" linevadjust="-15" linelabelstyle="stroke:#cc0;stroke-dasharray:2,2;stroke-width:2px;"></markerlabel>
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| − | </trackmarker>
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| − | <trackmarker start='2897' end='3757' markerstyle='stroke:#000;fill:#FF8000;filter:url(#dropshadow)' arrowendlength="7" arrowendwidth="5" wadjust="10" vadjust="-5">
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| − | <markerlabel text='AmpR' vadjust="40" hadjust="2" valign="outer" style="font-size:14px;font-weight:400;fill:#000000" showline="1" linevadjust="-15" linevadjust="-15" linelabelstyle="stroke:#cc0;stroke-dasharray:2,2;stroke-width:2px;"></markerlabel>
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| − | </trackmarker>
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| − | <trackmarker start='3912' end='4531' markerstyle='stroke:#000;fill:#FACC2E;filter:url(#dropshadow)' arrowendlength="7" arrowendwidth="5" wadjust="10" vadjust="-5">
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| − | <markerlabel text='pBR322' vadjust="40" hadjust="2" valign="outer" style="font-size:14px;font-weight:400;fill:#000000" showline="1" linevadjust="-15" linevadjust="-15" linelabelstyle="stroke:#cc0;stroke-dasharray:2,2;stroke-width:2px;"></markerlabel>
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| − | </trackmarker>
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| − | <trackmarker start='4983' end='5895' markerstyle='stroke:#000;fill:#01DF01;filter:url(#dropshadow)' wadjust="10" vadjust="-5">
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| − | <markerlabel text='HO-L' vadjust="40" hadjust="2" valign="outer" style="font-size:14px;font-weight:400;fill:#000000" showline="1" linevadjust="-15" linevadjust="-15" linelabelstyle="stroke:#cc0;stroke-dasharray:2,2;stroke-width:2px;"></markerlabel>
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| − | </trackmarker>
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| − | <trackmarker start='6017' end='6671' markerstyle='stroke:#000;fill:#01DFA5;filter:url(#dropshadow)' wadjust="10" vadjust="-5">
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| − | <markerlabel text='ADH1 promoter' vadjust="40" hadjust="2" valign="outer" style="font-size:14px;font-weight:400;fill:#000000" showline="1" linevadjust="-15" linevadjust="-15" linelabelstyle="stroke:#cc0;stroke-dasharray:2,2;stroke-width:2px;"></markerlabel>
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| − | </trackmarker>
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| − | <trackmarker start='6690' end='7817' markerstyle='stroke:#000;fill:#01A9DB;filter:url(#dropshadow)' arrowendlength="7" arrowendwidth="5" wadjust="10" vadjust="-5">
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| − | <markerlabel text="crtE" vadjust="40" hadjust="2" valign="outer" style="font-size:14px;font-weight:400;fill:#000000" showline="1" linevadjust="-15" linevadjust="-15" linelabelstyle="stroke:#cc0;stroke-dasharray:2,2;stroke-width:2px;"></markerlabel>
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| − | </trackmarker>
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| − | <trackmarker start='7818' end='7877' markerstyle='stroke:#000;fill:#666' wadjust="20" vadjust="-10">
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| − | <markerlabel text='2A' vadjust="40" hadjust="2" valign="outer" style="font-size:14px;font-weight:400;fill:#000000" showline="1" linevadjust="-15" linevadjust="-15" linelabelstyle="stroke:#cc0;stroke-dasharray:2,2;stroke-width:2px;"></markerlabel>
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| − | </trackmarker>
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| − | <trackmarker start='7878' end='9893' markerstyle='stroke:#000;fill:#0000FF;filter:url(#dropshadow)' arrowendlength="7" arrowendwidth="5" wadjust="10" vadjust="-5">
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| − | <markerlabel text='crtYB' vadjust="40" hadjust="2" valign="outer" style="font-size:14px;font-weight:400;fill:#000000" showline="1" linevadjust="-15" linevadjust="-15" linelabelstyle="stroke:#cc0;stroke-dasharray:2,2;stroke-width:2px;"></markerlabel>
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| − | </trackmarker>
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| − | <trackmarker start='9894' end='9953' markerstyle='stroke:#000;fill:#666' wadjust="20" vadjust="-10">
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| − | <markerlabel text='2A' vadjust="40" hadjust="2" valign="outer" style="font-size:14px;font-weight:400;fill:#000000" showline="1" linevadjust="-15" linevadjust="-15" linelabelstyle="stroke:#cc0;stroke-dasharray:2,2;stroke-width:2px;"></markerlabel>
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| − | </trackmarker>
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| − | <trackmarker start='9954' end='11696' markerstyle='stroke:#000;fill:#8000FF;filter:url(#dropshadow)' arrowendlength="7" arrowendwidth="5" wadjust="10" vadjust="-5">
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| − | <markerlabel text='crtI' vadjust="40" hadjust="2" valign="outer" style="font-size:14px;font-weight:400;fill:#000000" showline="1" linevadjust="-15" linevadjust="-15" linelabelstyle="stroke:#cc0;stroke-dasharray:2,2;stroke-width:2px;"></markerlabel>
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| − | </trackmarker>
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| − | <trackmarker start='11767' end='11980' markerstyle='stroke:#000;fill:#F5A9E1;filter:url(#dropshadow)' wadjust="10" vadjust="-5">
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| − | <markerlabel text='TEF1 terminator' vadjust="40" hadjust="2" valign="outer" style="font-size:14px;font-weight:400;fill:#000000" showline="1" linevadjust="-15" linevadjust="-15" linelabelstyle="stroke:#cc0;stroke-dasharray:2,2;stroke-width:2px;"></markerlabel>
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| − | </trackmarker>
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| − |
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| − | </plasmidtrack>
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| − | </plasmid>
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| − | <br><br>
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| − | <b>Mutation optimization</b>
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| − | <br>Another very different way to improve the sequence of the pathway would be to replace the regions that are the most likely to mutate by alternative, more stable versions. Indeed, our strain is meant to be released in the environment without containment and to be consumed by people, so if mutations were to happen in the genes we have engineered, the yeast's ability to produce ß-carotene could be greatly reduced, or lost. Uncontrolled mutations could also have undesirable consequences which could be dangerous for the environment or for the consumers' health.
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| − | <br>To address this problem, we made a collaboration with the Vanderbilt iGEM Team 2015, who invented an algorithm to scan the sequences looking for regions that are likely to mutate. Thanks to their software they were able to find alternative versions of the crtE, crtI, crtYB and HMG-CoA genes that were more robust and durable.
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| − | </div>
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| − | <br><br><br>
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| − | <h4>Bibliography</h4>
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| − | <ul>
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| − | <li>Li, Q., Sun, Z., Li, J. & Zhang, Y. Enhancing beta-carotene production in Saccharomyces cerevisiae by metabolic engineering. <i>FEMS Microbiology Letters</i> <b>345</b>, 94-101 (2013).</li>
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| − | <li>Beekwilder, J. et al. Polycistronic expression of a ß-carotene biosynthetic pathway in Saccharomyces cerevisiae coupled to ß-ionone production. <i>Journal of Biotechnology</i> (2014).</li>
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| − | <li>Voth, W.P., Richards, J.D., Shaw, J.M. & Stillman, D.J. Yeast vectors for integration at the HO locus. <i>Nucleic acids research</i> <b>29</b>, E59-E59 (2001).</li>
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| − | <li>Gietz, R.D. & Schiestl, R.H. High-efficiency yeast transformation using the LiAc / SS carrier DNA / PEG method. <i>Nature Protocols</i> <b>2</b>, 31-35 (2008).</li>
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| − | </ul>
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