Difference between revisions of "Team:Paris Bettencourt/Project/Continuity"
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− | <p>In order to gain trust | + | <p>In order to gain the trust of the public, we believe the technology should belong to everyone. In a manner similar to the open-source software industry, people should be able to improve our project or create their own versions of it. This idea of openness is very common among the community of synthetic biologists, but a lot of pitfalls have to be overcome to make it a sustainable reality.</p> |
− | <p>A parallel could be drawn with electronics in the 1960's, when computer programming was extremely low-level and belonged to the realm of academia. Since then, it has reached a | + | <p>A parallel could be drawn with electronics in the 1960's, when computer programming was extremely low-level and belonged to the realm of academia. Since then, it has reached a far wider population thanks to the creation of frameworks allowing for abstraction of the most technical parts. How could the same principles be applied to synthetic biology, in the context of metabolic engineering and vitamins production?</p> |
<p>Even though a lot of lab strains designed for easier modification have been designed in the past, they usually have a very general purpose and biotechnology remains a matter of specialists where every modification has to be made from scratch. We imagined a repurposed organism made especially for the quick construction of these <em>self-replicative tiny factories</em>, that could be easily used by startups, community labs or just by enthusiasts. In the following section we discuss the constraints associated with it, and what such an organism could look like.</p> | <p>Even though a lot of lab strains designed for easier modification have been designed in the past, they usually have a very general purpose and biotechnology remains a matter of specialists where every modification has to be made from scratch. We imagined a repurposed organism made especially for the quick construction of these <em>self-replicative tiny factories</em>, that could be easily used by startups, community labs or just by enthusiasts. In the following section we discuss the constraints associated with it, and what such an organism could look like.</p> | ||
</div> | </div> | ||
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<div class="column-left"> | <div class="column-left"> | ||
− | <p>For a biological product to leave the benches and actually reach the population, it's essential to foresee its life in the hands of the people who will cultivate it and make sure it stays alive | + | <p>For a biological product to leave the benches and actually reach the population, it's essential to foresee its life in the hands of the people who will cultivate it and make sure it stays alive for the future. Our design must therefore provide strategies to create a durable, usable product. On paper, the plan is simple: the manufacturers grow the micro-organism, distribute it and save a little fraction with which to start a new culture. This could in principle continue ad infinitum, but in reality the universal rules of biology soon kick back in.</p> |
− | <p>Let's consider the following scenario: a wild type organism sneaks into the incubator and starts to replicate along with the engineered organism. Our microbe cannot compete: this contaminant has been selected precisely for its ability to sneak into environments and replicate, during hundreds of years, while our microbe has the burden of producing tons of enzymes to make the precious vitamins. Additionally, unnatural proteins and metabolites can have toxic effects when their production rate is high. After a couple of growth | + | <p>Let's consider the following scenario: a wild type organism sneaks into the incubator and starts to replicate along with the engineered organism. Our microbe cannot compete: this contaminant has been selected precisely for its ability to sneak into environments and replicate, during hundreds of years, while our microbe has the burden of producing tons of enzymes to make the precious vitamins. Additionally, unnatural proteins and metabolites can have toxic effects when their production rate is high. After a couple of growth cycles, the worst seems unavoidable: the microorganism that will be distributed will not be the right one. Not only does it not produce nutrients, but it might not ferment the rice well or even be pathogenic.</p> |
<p>These contamination events bring a lot of hassle for the manufacturer, so our design must provide solutions for making them as rare as possible.</p> | <p>These contamination events bring a lot of hassle for the manufacturer, so our design must provide solutions for making them as rare as possible.</p> | ||
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<img src="https://static.igem.org/mediawiki/2015/8/88/PB_growth.png"/> | <img src="https://static.igem.org/mediawiki/2015/8/88/PB_growth.png"/> | ||
− | <p class="caption"> | + | <p class="caption"><b>Our model for decreasing the fitness burden of our organism during the production phase.</b> The cells do not produce any vitamin during the manufacturing process. The vitamin production is triggered before the distribution.</p> |
</div> | </div> | ||
<div style="clear:both"></div> | <div style="clear:both"></div> | ||
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<h2>The ID gene</h2> | <h2>The ID gene</h2> | ||
− | <div class="column-left"> | + | <div class="column-left" style="width:40%"> |
− | <p>The quality control of our system is possible thanks to an ID gene, which consists | + | <p>The quality control of our system is possible thanks to an ID gene, which consists of the fluorescent protein mCherry expressed at low levels. We chose this protein because its fluorescence colour is easily distinguishable from the media that will be used for growth, hence a better signal.</p> |
<p>The aim of this gene is to provide a quick and reliable way to determine whether the strain that will be distributed is the one intended. After growing the micro-organism in a bioreactor, a sample is taken in order to start a new culture from it. We suggest that, while doing so, the sample has to pass a quality check where its fluorescence is measured.</p> | <p>The aim of this gene is to provide a quick and reliable way to determine whether the strain that will be distributed is the one intended. After growing the micro-organism in a bioreactor, a sample is taken in order to start a new culture from it. We suggest that, while doing so, the sample has to pass a quality check where its fluorescence is measured.</p> | ||
<p>If the sample displays fluorescence, the culture is sent for packaging and a new culture can be launched. If the fluorescence in not sufficient, the sample is discarded and a new blister stock of the original strain is used to start the culture.</p> | <p>If the sample displays fluorescence, the culture is sent for packaging and a new culture can be launched. If the fluorescence in not sufficient, the sample is discarded and a new blister stock of the original strain is used to start the culture.</p> | ||
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This fluorescence measurement is a good example of real-life use for the DIλ spectrophotometer, a low-budget device that is developed by our neighbours at the Openlab. Heavy development is currently ongoing to make it capable of fluorescence measurements. | This fluorescence measurement is a good example of real-life use for the DIλ spectrophotometer, a low-budget device that is developed by our neighbours at the Openlab. Heavy development is currently ongoing to make it capable of fluorescence measurements. | ||
</p> | </p> | ||
− | <a href="https://2015.igem.org/Team:Paris_Bettencourt/ | + | <a href="https://2015.igem.org/Team:Paris_Bettencourt/Practices/DILambda" class="readMore buttonCyan">Click here to learn more about the DIλ spectrophotometer</a> |
</div> | </div> | ||
− | <div class="column-right"> | + | <div class="column-right" style="width:55%"> |
+ | <a href="https://static.igem.org/mediawiki/2015/f/ff/PB_workflow.png"> | ||
<img src="https://static.igem.org/mediawiki/2015/f/ff/PB_workflow.png"/> | <img src="https://static.igem.org/mediawiki/2015/f/ff/PB_workflow.png"/> | ||
+ | </a> | ||
+ | <br/> | ||
+ | <span class="caption"><b>How quality control will be performed.</b> The transfer of the inoculate should be done in sterile conditions in a container that also plays the role of fluorimeter cuvette. This ensures that even low-budget labs will always distribute the right strain to people.</span> | ||
</div> | </div> | ||
<div style="clear:both"></div> | <div style="clear:both"></div> | ||
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<div class="column-right" style="width: 60%"> | <div class="column-right" style="width: 60%"> | ||
<a href="https://static.igem.org/mediawiki/2015/9/98/PB_framework_construction.png"> | <a href="https://static.igem.org/mediawiki/2015/9/98/PB_framework_construction.png"> | ||
− | <img src="https://static.igem.org/mediawiki/2015/9/98/PB_framework_construction.png" style="width: | + | <img src="https://static.igem.org/mediawiki/2015/9/98/PB_framework_construction.png" style="width:96 |
+ | %"/> | ||
</a> | </a> | ||
</div> | </div> | ||
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<h2 id="the-chassis">The chassis</h2> | <h2 id="the-chassis">The chassis</h2> | ||
− | Let us see how it works under the hood.<br/> | + | <p>Let us see how it works under the hood.<br/> |
− | Before addition of any metabolic pathways, this is what our empty chassis would look like. The following cassette is integrated in the chromosome. | + | Before addition of any metabolic pathways, this is what our empty chassis would look like. The following cassette is integrated in the chromosome. All proteins' coding regions are preceded by a RBS (<em>Ribosome Binding Site</em>) and followed by a transcription terminator.</p> |
<br/> | <br/> | ||
<br/> | <br/> | ||
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</a> | </a> | ||
<br/> | <br/> | ||
− | |||
<br/> | <br/> | ||
<br/> | <br/> | ||
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<a href="https://static.igem.org/mediawiki/2015/7/75/PB_landingpad.png"><img src="https://static.igem.org/mediawiki/2015/7/75/PB_landingpad.png" style="width:90%"/></a> | <a href="https://static.igem.org/mediawiki/2015/7/75/PB_landingpad.png"><img src="https://static.igem.org/mediawiki/2015/7/75/PB_landingpad.png" style="width:90%"/></a> | ||
<br/> | <br/> | ||
+ | <span class="caption"><b>The proposed process for addition of new operons to the chassis.</b> A standard cassette is integrated in the PhiC31 locus. It then becomes a new possible outcome for the random differentiation, thanks to the addition of a new LoxP site.</span> | ||
</div> | </div> | ||
<div style="clear:both"></div> | <div style="clear:both"></div> | ||
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<img src="https://static.igem.org/mediawiki/2015/6/65/PB_brainbow.png" style="width:100%" align="middle"/> | <img src="https://static.igem.org/mediawiki/2015/6/65/PB_brainbow.png" style="width:100%" align="middle"/> | ||
</a> | </a> | ||
− | <p class="caption"> | + | <p class="caption"> |
+ | <b>Overview of the differentiation process.</b> For a complete organism with four metabolic operons, there are four possible outcomes, each of them leading to the expression of only one operon. In other words, each cell randomly choses one operon to express. | ||
+ | </p> | ||
</div> | </div> | ||
<div style="clear:both"></div> | <div style="clear:both"></div> | ||
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<h3>How to induce the differentiation?</h3> | <h3>How to induce the differentiation?</h3> | ||
There are different ways the CRE recombinase can be induced. | There are different ways the CRE recombinase can be induced. | ||
− | <h4>Chemical</h4> | + | <h4>Chemical induction</h4> |
A chemical would be one of the most predictable, efficient way to differentiate the cells. However, it requires to have access to this chemical, and to open the reactor which can be impractical for community labs with low resources to maintain sterility. It is nevertheless the solution of choice for funded factories. Carbohydrates such as glucose, arabinose or lactose seem to be the best options since they are not toxic. | A chemical would be one of the most predictable, efficient way to differentiate the cells. However, it requires to have access to this chemical, and to open the reactor which can be impractical for community labs with low resources to maintain sterility. It is nevertheless the solution of choice for funded factories. Carbohydrates such as glucose, arabinose or lactose seem to be the best options since they are not toxic. | ||
<h4>Heat</h4> | <h4>Heat</h4> | ||
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To answer this question, we created a mathematical and computational model of the situation. Given the growth rate of the mother cells and the daughter cells, it is possible to calculate the optimal differentiation rate, and chose the strength of the promoter accordingly. | To answer this question, we created a mathematical and computational model of the situation. Given the growth rate of the mother cells and the daughter cells, it is possible to calculate the optimal differentiation rate, and chose the strength of the promoter accordingly. | ||
</p> | </p> | ||
− | <a href="https://2015.igem.org/Team:Paris_Bettencourt/Modeling" class="readMore buttonCyan">Click here to learn more about the model</a> | + | <a href="https://2015.igem.org/Team:Paris_Bettencourt/Modeling" class="readMore buttonCyan">Click here to learn more about the model</a> |
+ | <br/> | ||
+ | <br/> | ||
+ | <br/> | ||
<h1>Results</> | <h1>Results</> | ||
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<p>In theory, the cells with this cassette integrated in the chromosome are expected to emit a red fluorescence. Upon induction of the CRE-recombinase, they should lose the red fluorescence and start to express either mCerualean (a cyan fluorescent protein) or mVenus (a yellow fluorescent protein). Each cell should express only one of those two proteins at the same time.</p> | <p>In theory, the cells with this cassette integrated in the chromosome are expected to emit a red fluorescence. Upon induction of the CRE-recombinase, they should lose the red fluorescence and start to express either mCerualean (a cyan fluorescent protein) or mVenus (a yellow fluorescent protein). Each cell should express only one of those two proteins at the same time.</p> | ||
− | < | + | <p><img src="https://static.igem.org/mediawiki/2015/8/8f/PB_colibow_sequence.png"/></p> |
+ | <span class="caption"><b>Map of the DNA sequence we constructed.</b> The promoter BBaJ23199 is constitutive. mCherry, mCerulean, mVenus are fluorescent proteins of different colours.</span> | ||
+ | <br/> | ||
+ | <br/> | ||
<div class="column-left"> | <div class="column-left"> | ||
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<div class="column-right"> | <div class="column-right"> | ||
<img src="https://static.igem.org/mediawiki/2015/a/a7/PB_colibow_integrated.png" style="width:80%"/> | <img src="https://static.igem.org/mediawiki/2015/a/a7/PB_colibow_integrated.png" style="width:80%"/> | ||
+ | <br/> | ||
+ | <span class="caption"><b>Gel electrophoresis after PCR for checking the integration.</b> We amplified the junctions between the artificial cassette and <i>E. coli </i>'s chromosome. For every screened clone, the two bands have the expected sizes, which proves that the cassette is integrated in the correct locus.</span> | ||
</div> | </div> | ||
<div style="clear:both"></div> | <div style="clear:both"></div> | ||
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<div class="column-left"> | <div class="column-left"> | ||
<img src="https://static.igem.org/mediawiki/2015/5/5b/PB_colibow_proteins.png" style="width:80%"/> | <img src="https://static.igem.org/mediawiki/2015/5/5b/PB_colibow_proteins.png" style="width:80%"/> | ||
+ | <br/> | ||
+ | <span class="caption"><b>Gel electrophoresis after PCR for checking the presence of the three fluorescent proteins.</b> For the clone shown here, it means that all three proteins ORFs are present on the chromosome, even though only the first one is actually expressed.</span> | ||
</div> | </div> | ||
<div class="column-right"> | <div class="column-right"> | ||
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<h3>Sequencing of the Lox Array</h3> | <h3>Sequencing of the Lox Array</h3> | ||
− | <p>To investigate whether unexpected recombination occured within the LoxP sites due to homologous recombination, we performed sequencing on the first part of the integrated cassette, where the Lox Array is. This way we could make sure that it was still intact and contained no PCR-induced mutations.</p> | + | <p>To investigate whether unexpected recombination occured within the LoxP sites due to homologous recombination, we performed Sanger sequencing on the first part of the integrated cassette, where the Lox Array is. This way we could make sure that it was still intact and contained no PCR-induced mutations.</p> |
</div> | </div> | ||
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<span class="legend"> | <span class="legend"> | ||
+ | <b>Characterization of the promoter followed by the four LoxP sites.</b><br/> | ||
Using standard biobrick assembly, three plasmids were constructed and transformed into <i>E. coli</i>:</span> | Using standard biobrick assembly, three plasmids were constructed and transformed into <i>E. coli</i>:</span> | ||
<ul style="font-size:13px"> | <ul style="font-size:13px"> | ||
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<img src="https://static.igem.org/mediawiki/2015/b/be/PB_colibow_fluorescence.png"/> | <img src="https://static.igem.org/mediawiki/2015/b/be/PB_colibow_fluorescence.png"/> | ||
<span class="legend"> | <span class="legend"> | ||
+ | <b>Expression of the first protein of the cassette in the chromosome.</b><br/> | ||
A "mother cell" with our differentiation system integrated in the chromosome was grown to exponential phase and its fluorescence was measured when OD<sub>600</sub> reached 0.3. As a negative control, a cell without fluorescent underwent the same treatment. | A "mother cell" with our differentiation system integrated in the chromosome was grown to exponential phase and its fluorescence was measured when OD<sub>600</sub> reached 0.3. As a negative control, a cell without fluorescent underwent the same treatment. | ||
Excitation wavelength: 585 nm. | Excitation wavelength: 585 nm. | ||
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<p> | <p> | ||
− | The cells exhibit clear fluorescence (Mann-Whitney test, p-value < 10<sup>-6</sup>), even though it was not visible to the naked eye. | + | The cells exhibit clear fluorescence (Mann-Whitney test, p-value < 10<sup>-6</sup>), even though it was not visible to the naked eye. The mCherry fluorescent protein is therefore a suitable reporter for quality control of the strain. |
</p> | </p> | ||
− | |||
− | |||
</div> | </div> | ||
<div style="clear:both"></div> | <div style="clear:both"></div> | ||
− | <h2 | + | <h2>Induction of the differentiation</h2> |
− | <div class=" | + | <div class="column-left"> |
− | < | + | <p>We then aimed to trigger the differentiation of our newly constructed strain. A strain carrying the plasmid pFHC2938, that allows for expression of the CRE recombinase upon arabinose induction (Nielsen 2006), was aquired.<br/> |
− | <p | + | Unfortunately, all our attempts at transforming our strain with it have been unsuccesful, even when resorting to very efficient techniques such as electroporation. It always lead to either nothing, or a lawn of bacteria that did not seem to carry the antibiotic resistance using for selecting the plasmid. To troubleshoot this transformation, we made three hypothesis: |
+ | <ul> | ||
+ | <li>Is there something in the cell that interferes transformation?</li> | ||
+ | <li>Is the strain not suitable for CRE expression, e.g. there are LoxP sites somewhere that result in deletions in the genome?</li> | ||
+ | <li>Is the plasmid the wrong one?</li> | ||
+ | </ul></p> | ||
+ | |||
+ | <p> | ||
+ | To figure this out, we transformed our strain with the CRE recombinase plasmid along with a standard pSB1C3-mRFP plasmid. The transformation of the CRE-recombinase gave a lawn, while the control plasmid gave clear colonies. We then performed the same 4-primer PCR that was used to check for integration on the bacteria present on the plates. The bacteria transformed with pSB1C3-mRFP still contained the integrated cassette, while the bacteria on the pFHC2938 plate did not display any band, meaning that they were contaminants. This means that the transformation process is not the problem.</p> | ||
+ | |||
+ | <p>We then transformed a non-modified strain without our integrated cassete with the pFHC2938 plasmid. At the same time, the integrative plasmid was transformed into another <i>E. coli</i> strain (STBL). None of the transformations yielded any colonies, meaning that the problem came from the plasmid and not from the strain.</p> | ||
</div> | </div> | ||
− | |||
− | |||
− | |||
− | < | + | <div class="column-right"> |
− | < | + | <img src="https://static.igem.org/mediawiki/2015/d/da/PB_beforeaftercre.png"/> |
− | < | + | <br/> |
− | < | + | <span class="caption"><b>Troubleshooting of the transformation.</b> <strong>A.</strong> Strain carrying the integrated cassette before transformation. <em>B.</em> Cells picked from the lawn after pFHC2938 transformation. <strong>C.</strong> Colony picked after the transformation with a control plasmid (pSB1C3-mRFP).</span> |
− | < | + | |
− | < | + | <img src="https://static.igem.org/mediawiki/2015/c/c1/PB_empty.png" /><br/> |
− | < | + | <p class="caption">Unmodified Top10 lab strain after transformation with pFHC2938. The great nothingness.</p> |
− | </ | + | </div> |
+ | |||
+ | <div style="clear:both"></div> | ||
+ | |||
+ | <h1>Outlook</h1> | ||
+ | <h2>More than just operons</h2> | ||
+ | <p>The system we invented, as presented here, works primarily when the whole vitamin pathway fits in one operon. It was notably the case for our pathway for vitamin A, where all the required enzymes are tied together in one big polycistron.<br/> | ||
+ | It is still possible to implement this differentiation system for a pathway that needs several promoters to function. For this, we can put the different operons under promoters that are activated by another factor, and this factor is put in the differentiation system.<br/> | ||
+ | Two related technologies, <em>CRISPR interference</em> and <em>CRISPR activation</em> (Bikard 2013), appear as an ideal way to do this. It relies on a mutant of Cas9 defective for nuclease activity (dCas9), that can be targeted at about any place in the genome. This can lead to either repression of transcription, or activation by fusing a transcription activation factor to dCas9. It works on both prokaryotes and eukaryotes (Perez-Pinera 2013), and is extremely versatile and programmable. As seen in our phytase project, inactivation of a gene is sometimes useful for nutrient production. | ||
+ | </p> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2015/f/fb/PB_tentacles.png" style="width:96%"> | ||
+ | |||
+ | <p>By expressing dCas9 and replacing the fluorescent proteins in our construct with CRISPR arrays, it could be possible to widely change the expression profile of the micro-organism while keeping the advantages of differentiation.</p> | ||
+ | |||
<h1>Attribution</h1> | <h1>Attribution</h1> | ||
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Most of the strains (DH5alpha, Top10, NEB turbo, Pir116) were kindly provided by Inserm U1001. Plasmids pFHC2938 and pMEV250 were provided by Jason Bland and Aleksandra Nivina at Didier Mazel's lab at Institut Pasteur. Plasmids pL1F2 and pR6K-shortened were provided by Antoine Decrulle and Ihab Boulas at Inserm U1001. The pIT5-KH vector was provided by Lun Cui at David Bikard's lab at Institut Pasteur. | Most of the strains (DH5alpha, Top10, NEB turbo, Pir116) were kindly provided by Inserm U1001. Plasmids pFHC2938 and pMEV250 were provided by Jason Bland and Aleksandra Nivina at Didier Mazel's lab at Institut Pasteur. Plasmids pL1F2 and pR6K-shortened were provided by Antoine Decrulle and Ihab Boulas at Inserm U1001. The pIT5-KH vector was provided by Lun Cui at David Bikard's lab at Institut Pasteur. | ||
− | Special thanks to all the people who gave me an hand during this project, and all the Paris Bettencourt team for making | + | Special thanks to all the people who gave me an hand during this project, and all the Paris Bettencourt team for making this adventure so much fun. |
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{{Paris_Bettencourt/footer}} | {{Paris_Bettencourt/footer}} |
Latest revision as of 16:08, 15 October 2015