Revision as of 13:36, 18 September 2015

Ferment It Yourself

iGEM Paris-Bettencourt 2O15

In order to gain trust from the population, the technology should belong to everyone. In a way 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.

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 way 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?

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 self-replicative tiny factories, 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.

For the product to be usable, several specifications have to be taken into account:

It must be easily extendable:
Our micro-organism should be a chassis allowing for quick addition of standard cassettes
It must be modular:
The different metabolic pathways should be independant so they can be put together without going through tedious troubleshooting,
It must survive in the real world:
To make our micro-organism more resistant to contamination, we need to design it so our modifications come with a minimal fitness cost.
It must be all-in-one:
For people with limited equipment, having only one strain that does everything is a huge advantage, because only one bioreactor and one production line is needed. This makes it accessible to community labs or NGOs that would want to start producing their own version of our product.

Specification

All in one

make manufacturing simpler only one production line

An extendable chassis

limit R&D expenses

https://static.igem.org/mediawiki/2015/9/98/PB_framework_construction.png

Our design

From the lab to the world

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 all along. Our design must therefore provide strategies to create an durable, usable product. On paper, the plan is simple: the manufacturers grow the micro-organism, distribute it and save a little fraction to start a new culture with. This could in principle last forever, but in reality the universal rules of biology soon kick back in.

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 cycle, the worst seems unavoidable: the micro-organism that will be distributed will not be the right one. Not only this one doesn't produce nutrients, but it might not ferment the rice well or even be pathogenic.

These contamination events bring a lot of hassle for the manufacturer, so our design must provide solutions for making them as rare as possible.

Our approaches is based on two strategies:

Reducing the fitness burden: To make our micro-organism more resistant to contamination, we need to design it so our modifications come with a minimal fitness cost.
Identifying the contamination: If a contamination occurs, it is essential that it does not go unnoticed. Our design must allow the manufacturer to detect contamination, and check that what he is growing is exactly what he wants to grow.

It seems impossible to make a strain that fullfills its nutrient-producing functions while growing as fast as the wild type, so we found a workaround: the cells that people use are not the cells that people grow.
We embedded a differentiation system into our organism, so the vitamin-producing pathways are only expressed after a recombination event. First, the cells that are grown are almost identical to the wild-type cells. Before distribution, the differentiation is induced and the cells start to produce vitamins in high quantity. The battle against contaminants is now a fair fight.

We protected our product against foreign organisms, but one threat remains: our organism's own mutants. If a mutation occurs in the active site of an enzyme, or in the promoter of an operon, the functionality of the organism might be impaired. How can we prevent our organism from mutating?

Fortunately, our friends at the Vanderbilt University iGEM team worked precisely on that problem this summer. We worked hand in hand with them to see what a real-life application of their invention would mean practically. They invented an algorithm to scan the sequences looking for regions that are likely to mutate, and proposed alternative versions of our sequences.
As they worked on this project while we were working on ours, we obviously could not use their optimized sequences for our constructs. However, we relied on gene synthesis for a lot of parts, so it would not have been a problem to use the optimized sequences instead. Their algorithm is therefore a valuable tool for any synthetic biologist willing to create durable products, and we applaud their work.

https://static.igem.org/mediawiki/2015/8/88/PB_growth.png

caption

Our differentiation system is inspired by the Brainbow system, initially developed for tracking the axons of neurons in mammalian's brain. We modified it so it becomes extendable.

This system is randomized on a single-cell level, so each cell produce one, and only one, vitamin pathway. Having one cell expressing only one pathway should theoretically preclude unexpected interactions between different pathways, thus making an extendable framework where every synthesis function is decoupled.

https://static.igem.org/mediawiki/2015/9/98/PB_framework_construction.png

How is it possible?

The chassis

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 followed by a terminator.

Constitutive promoter: Thanks to this promoter, a RNA transcript of the cassette will be produced until the first terminator is reached.

The Lox Array: The original LoxP site comes from the phage P1. When an enzyme called the CRE recombinase is expressed, all the DNA between two Lox sites is deleted. Each lox site is made of one overlap region (in bold) surrounded by two complementary flanking regions. The middle of the sequence can be modified, but two LoxP sites will recombine together only if the sequence is exactly identical for both (Richier 2015). The flanking regions cannot be mutated and determine the specificity for one recombination enzyme.

Here are the four orthogonal Lox sites we used:

LoxP: ATAACTTCGTATAATGTATGCTATACGAAGTTAT
Lox2272: ATAACTTCGTATAAAGTATCCTATACGAAGTTAT
LoxN: ATAACTTCGTATAAGGTATACTATACGAAGTTAT
Lox5171: ATAACTTCGTATAATGTGTACTATACGAAGTTAT

The ID gene: This gene is entirely optional but can be used as a barcode to identify the strain. This allows for quality control of what is inoculated when a new production culture is started.

The landing pad: This part allows for easy integration of new gene cassettes into the system.

CRE-recombinase: The CRE recombinase should be integrated in the chromosome as well, so we do not have to use an antibiotic for maintaining the plasmid. It has to be under the control of an inducible promoter. The expression of CRE will trigger the differentiation.

The Landing Pad

Starting from this chassis, up to four metabolic pathways can be added by using the attB sequence as a landing pad. Like the Lox sites, this sequence comes from a bacteriophage: the PhiC31 phage who uses it to integrate itself in the genome of the host. To insert a new sequence in it, all you need to do is build a plasmid with the matching "attP" site and express the PhiC31 integrase.

attB: GTGCGGGTGCCAGGGCGTGCCCTTGGGCTCCCCGGGCGCGTACTCCA
attP: AGTGCCCCAACTGGGGTAACCTTTGAGTTCTCTCAGTTGGGGGCGT

When inserting something in the landing pad, a new landing pad should be added for subsequent integration. This landing pad should be orthogonal to the first one to avoid multiple successive integrations. The same integrase can be used, the central TT just has to be replaced by CC to make the two sites orthogonal.

quick forking in a lot of parallel projects

For clarity, the selection method has not been depicted. Using CRISPR-based system for selection could be a good idea as it avoids the problem of antibiotic resistance removal.

Division of labour

https://static.igem.org/mediawiki/2015/6/65/PB_brainbow.png

Chemical / Absence of chemical

Heat

Light

Constitutive

leakiness

decoupling the different metabolic pathways

Results

Construction of the system

We succesfully assembled a prototype version of this system in the model bacteria Escherichia coli. The genes involved in vitamin production are replaced with fluorescent proteins, allowing for easy monitoring of their production. Our construct contains mCherry as a reporter gene, and two other fluorescent proteins to mimick pathways operons. It also has a phage PhiC31 integration site for subsequent addition of new genes.

https://static.igem.org/mediawiki/2015/8/8f/PB_colibow_sequence.png

This cassette was constructed by Gibson Assembly and assembled in a self-integrating plasmid vector which integrates in the site of the phage HK022 in E. coli's chromosome. This plasmid was electroporated in the bacteria and the phage HK022 integrase was induced.

Integration in the bacterial cells

To check that the cassette has correctly been integrated in the right locus, we performed an analytical PCR on the whole genome of the transformants, with a set of four primers mixed altogether.

Integrity of the cassette

fluorescent proteins are present

Sequencing of the Lox Array

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.

Function of the promoter

BBa_K1678005

The LoxP array does not theoretically interfere with translation, as in prokaryotes the 30S subunit of the ribosome binds directly to the ribosome binding site even if it is not right at the beginning of the mRNA transcript. It can however interfere with the transcription. During the transcription, the RNA polymerase has to go through the LoxP array, which is made of repetitive sequences that are likely to form a hairpin. We show that this has an impact on the transcription efficiency (Mann-Whitney test, p-value < 10^-6), as the amount of protein is reduced on average by 9%. However, it still allows for strong protein expression and the 91% of RNA polymerases that get through should be more than enough for our design.

We have also sequenced it.

When integrated (Mann-Whitney test, p-value < 10^-6)

suitability for quality control DIlambda

Induction of the differentiation

https://static.igem.org/mediawiki/2015/c/c1/PB_empty.png

Effects on growth

Outlook

link

Attribution

This project was designed and accomplished by Antoine Vigouroux in consultation with Jason Bland and Ihab Boulas. 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 Nivia 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 it so much fun.

@@ Line 13: / Line 13: @@
 For the product to be usable, several specifications have to be taken into account:
 <ul>
-<li><b>It must be easily extendable:</b> Our micro-organism should be a chassis allowing for quick addition of standard cassettes</li>
+<li><b>It must be easily extendable:</b><br/>Our micro-organism should be a chassis allowing for quick addition of standard cassettes</li>
-<li><b>It must be modular:</b> The different metabolic pathways should be independant so they can be put together without going through tedious troubleshooting,</li>
+<li><b>It must be modular:</b><br/> The different metabolic pathways should be independant so they can be put together without going through tedious troubleshooting,</li>
-<li><b>It must survive in the real world:</b> To make our micro-organism more resistant to contamination, we need to design it so our modifications come with a minimal fitness cost.</li>
+<li><b>It must survive in the real world:</b><br/> To make our micro-organism more resistant to contamination, we need to design it so our modifications come with a minimal fitness cost.</li>
-<li><b>It must be all-in-one:</b> For people with limited equipment, having only one strain that does everything is a huge advantage, because only one bioreactor and one production line is needed. This makes it accessible to community labs or NGOs that would want to start producing their own version of our product.</li>
+<li><b>It must be all-in-one:</b><br/> For people with limited equipment, having only one strain that does everything is a huge advantage, because only one bioreactor and one production line is needed. This makes it accessible to community labs or NGOs that would want to start producing their own version of our product.</li>
 </ul>
 </div>
 <div style="clear:both"></div>
 <h1 id="specification">Specification</h1>
@@ Line 35: / Line 37: @@
 <h1 class="date two" id="our-design">Our design</h1>
+<!-- Fitness burden -->
 <h2 id="from-the-lab-to-the-world">From the lab to the world</h2>
@@ Line 52: / Line 56: @@
 <div class="column-right">
-<p>It seems impossible to make a strain that fullfills its nutrient-producing functions while growing as fast as the wild type, so we found a workaround: the cells that people use are not the cells that people grow. We embedded a differentiation system into our organism, so the vitamin-producing pathways are only expressed after a recombination event. The cells that are grown are almost identical to the wild-type cells. The battle against contaminants is now a fair fight.</p>
+<p>It seems impossible to make a strain that fullfills its nutrient-producing functions while growing as fast as the wild type, so we found a workaround: the cells that people use are not the cells that people grow.<br/>
-<p>It is inspired by the Brainbow system, initially developed for tracking the axons of neurons in mammalian's brain. We modified it so it becomes extendable.</p>
-<p>This system is randomized on a single-cell level, so each cell produce one, and only one, vitamin pathway. Having one cell expressing only one pathway should theoretically preclude unexpected interactions between different pathways, thus making an extendable framework where every synthesis function is decoupled.</p>
+We embedded a differentiation system into our organism, so the vitamin-producing pathways are only expressed after a recombination event. First, the cells that are grown are almost identical to the wild-type cells. Before distribution, the differentiation is induced and the cells start to produce vitamins in high quantity. The battle against contaminants is now a fair fight.</p>
+<p>We protected our product against foreign organisms, but one threat remains: our organism's own mutants. If a mutation occurs in the active site of an enzyme, or in the promoter of an operon, the functionality of the organism might be impaired. How can we prevent our organism from mutating?</p>
+<p>Fortunately, our friends at the Vanderbilt University iGEM team worked precisely on that problem this summer. We worked hand in hand with them to see what a real-life application of their invention would mean practically. They invented an algorithm to scan the sequences looking for regions that are likely to mutate, and proposed alternative versions of our sequences.<br/>
+As they worked on this project while we were working on ours, we obviously could not use their optimized sequences for our constructs. However, we relied on gene synthesis for a lot of parts, so it would not have been a problem to use the optimized sequences instead. Their algorithm is therefore a valuable tool for any synthetic biologist willing to create durable products, and we applaud their work.
+</p>
 <img src="https://static.igem.org/mediawiki/2015/8/88/PB_growth.png" title="https://static.igem.org/mediawiki/2015/8/88/PB_growth.png" alt="https://static.igem.org/mediawiki/2015/8/88/PB_growth.png" />
@@ Line 61: / Line 71: @@
 <div style="clear:both"></div>
-<p>How does this all work?</p>
+<!-- Extendability -->
+<div class="column-left">
+<p>Our differentiation system is inspired by the Brainbow system, initially developed for tracking the axons of neurons in mammalian's brain. We modified it so it becomes extendable.</p>
+<p>This system is randomized on a single-cell level, so each cell produce one, and only one, vitamin pathway. Having one cell expressing only one pathway should theoretically preclude unexpected interactions between different pathways, thus making an extendable framework where every synthesis function is decoupled.</p>
+</div>
+<div class="column-right">
+<div class="figure">
+<img src="https://static.igem.org/mediawiki/2015/9/98/PB_framework_construction.png" title="https://static.igem.org/mediawiki/2015/9/98/PB_framework_construction.png" alt="https://static.igem.org/mediawiki/2015/9/98/PB_framework_construction.png" />
+<p class="caption">https://static.igem.org/mediawiki/2015/9/98/PB_framework_construction.png</p>
+</div>
+</div>
+<div style="clear:both"></div>
+<center><p>How is it possible?</p></center>
 <h2 id="the-chassis">The chassis</h2>
+Before addition of any metabolic pathways, this is what our empty chassis would look like. The following cassette is integrated in the chromosome.
 <div class="figure">
-<img src="https://static.igem.org/mediawiki/2015/1/14/PB_chassis.png" title="https://static.igem.org/mediawiki/2015/1/14/PB_chassis.png" alt="https://static.igem.org/mediawiki/2015/1/14/PB_chassis.png" />
+<img src="https://static.igem.org/mediawiki/2015/1/14/PB_chassis.png" style="width:100%" />
-<p class="caption">https://static.igem.org/mediawiki/2015/1/14/PB_chassis.png</p>
 </div>
-<p><img src="https://static.igem.org/mediawiki/2015/e/ee/PB_1.png" title="fig:https://static.igem.org/mediawiki/2015/e/ee/PB_1.png" alt="https://static.igem.org/mediawiki/2015/e/ee/PB_1.png" /> <strong>Constitutive promoter</strong></p>
+All proteins' coding regions are followed by a terminator.
-<p><img src="https://static.igem.org/mediawiki/2015/c/c8/PB_2.png" title="fig:https://static.igem.org/mediawiki/2015/c/c8/PB_2.png" alt="https://static.igem.org/mediawiki/2015/c/c8/PB_2.png" /> <strong>The Lox Array</strong> Each lox site is made of one <em>overlap region</em> surrounded by two complementary <em>flanking regions</em>. The flanking regions cannot be mutated and determine the specificity for one recombination enzyme. The overlap region can be modified, but two LoxP sites will recombine together only if it's identical for both. (richier 2015) LoxP: ATAACTTCGTATAATGTATGCTATACGAAGTTAT Lox2272: ATAACTTCGTATAAAGTATCCTATACGAAGTTAT LoxN: ATAACTTCGTATAAGGTATACTATACGAAGTTAT Lox5171: ATAACTTCGTATAATGTGTACTATACGAAGTTAT</p>
-<p><img src="https://static.igem.org/mediawiki/2015/e/e6/PB_3.png" title="fig:https://static.igem.org/mediawiki/2015/e/e6/PB_3.png" alt="https://static.igem.org/mediawiki/2015/e/e6/PB_3.png" /> <strong>The ID gene</strong></p>
-<p><img src="https://static.igem.org/mediawiki/2015/8/8d/PB_4.png" title="fig:https://static.igem.org/mediawiki/2015/8/8d/PB_4.png" alt="https://static.igem.org/mediawiki/2015/8/8d/PB_4.png" /> <strong>The landing pad</strong> Landing pad A: attB: GTGCGGGTGCCAGGGCGTGCCC<strong>TT</strong>GGGCTCCCCGGGCGCGTACTCCA attP: AGTGCCCCAACTGGGGTAACCT<strong>TT</strong>GAGTTCTCTCAGTTGGGGGCGT</p>
-<p>Landing pad B: attB: GTGCGGGTGCCAGGGCGTGCCC<strong>CC</strong>GGGCTCCCCGGGCGCGTACTCCA attP: AGTGCCCCAACTGGGGTAACCT<strong>CC</strong>GAGTTCTCTCAGTTGGGGGCGT</p>
-<p><img src="https://static.igem.org/mediawiki/2015/9/9d/PB_5.png" title="fig:https://static.igem.org/mediawiki/2015/9/9d/PB_5.png" alt="https://static.igem.org/mediawiki/2015/9/9d/PB_5.png" /> <strong>CRE-recombinase</strong></p>
-<h2 id="division-of-labour">Division of labour</h2>
+<p><img src="https://static.igem.org/mediawiki/2015/e/ee/PB_1.png"/> <strong>Constitutive promoter:</strong> Thanks to this promoter, a RNA transcript of the cassette will be produced until the first terminator is reached.</p>
+<p><img src="https://static.igem.org/mediawiki/2015/c/c8/PB_2.png"/> <strong>The Lox Array:</strong>
+The original LoxP site comes from the phage P1.
+When an enzyme called the <em>CRE recombinase</em> is expressed, all the DNA between two Lox sites is deleted. Each lox site is made of one <em>overlap region</em> (in bold) surrounded by two complementary <em>flanking regions</em>. The middle of the sequence can be modified, but two LoxP sites will recombine together only if the sequence is exactly identical for both (Richier 2015). The flanking regions cannot be mutated and determine the specificity for one recombination enzyme.</p>
+<p>Here are the four orthogonal Lox sites we used:
+<ul>
+    <li><b>LoxP:</b>    ATAACTTCGTATA<strong>ATGTATGC</strong>TATACGAAGTTAT</li>
+    <li><b>Lox2272:</b> ATAACTTCGTATA<strong>AAGTATCC</strong>TATACGAAGTTAT</li>
+    <li><b>LoxN:</b>    ATAACTTCGTATA<strong>AGGTATAC</strong>TATACGAAGTTAT</li>
+    <li><b>Lox5171:</b> ATAACTTCGTATA<strong>ATGTGTAC</strong>TATACGAAGTTAT</li>
+</ul>
+</p>
+<p><img src="https://static.igem.org/mediawiki/2015/e/e6/PB_3.png"/> <strong>The ID gene: </strong>This gene is entirely optional but can be used as a barcode to identify the strain. This allows for quality control of what is inoculated when a new production culture is started.</p>
+<p><img src="https://static.igem.org/mediawiki/2015/8/8d/PB_4.png"/> <strong>The landing pad: </strong>
+This part allows for easy integration of new gene cassettes into the system.
+<p><img src="https://static.igem.org/mediawiki/2015/9/9d/PB_5.png"/> <strong>CRE-recombinase: </strong>The CRE recombinase should be integrated in the chromosome as well, so we do not have to use an antibiotic for maintaining the plasmid. It has to be under the control of an inducible promoter. The expression of CRE will trigger the differentiation.</p>
+<!-- LANDING PAD -->
+<h2 id="landing-pads">The Landing Pad</h2>
+<div class="column-left">
+<p>Starting from this chassis, up to four metabolic pathways can be added by using the attB sequence as a landing pad. Like the Lox sites, this sequence comes from a bacteriophage: the PhiC31 phage who uses it to integrate itself in the genome of the host. To insert a new sequence in it, all you need to do is build a plasmid with the matching "attP" site and express the PhiC31 integrase.
+<ul>
+<li>attB: GTGCGGGTGCCAGGGCGTGCCC<strong>TT</strong>GGGCTCCCCGGGCGCGTACTCCA</li>
+<li>attP: AGTGCCCCAACTGGGGTAACCT<strong>TT</strong>GAGTTCTCTCAGTTGGGGGCGT</li>
+</ul>
+When inserting something in the landing pad, a new landing pad should be added for subsequent integration. This landing pad should be orthogonal to the first one to avoid multiple successive integrations. The same integrase can be used, the central <strong>TT</strong> just has to be replaced by <strong>CC</strong> to make the two sites orthogonal.</p>
+quick forking in a lot of parallel projects</p>
+</div>
+<div class="column-right">
+<img src="https://static.igem.org/mediawiki/2015/7/75/PB_landingpad.png"/>
+<span class="legend">For clarity, the selection method has not been depicted. Using CRISPR-based system for selection could be a good idea as it avoids the problem of antibiotic resistance removal.</span>
+</div>
+<div style="clear:both"></div>
+<!--DIVISION OF LABOUR -->
+<h2 id="division-of-labour">Division of labour</h2>
 <div class="figure">
-<img src="https://static.igem.org/mediawiki/2015/6/65/PB_brainbow.png" title="https://static.igem.org/mediawiki/2015/6/65/PB_brainbow.png" alt="https://static.igem.org/mediawiki/2015/6/65/PB_brainbow.png" />
+<img src="https://static.igem.org/mediawiki/2015/6/65/PB_brainbow.png"/>
 <p class="caption">https://static.igem.org/mediawiki/2015/6/65/PB_brainbow.png</p>
 </div>
@@ Line 86: / Line 147: @@
 <p>leakiness</p>
 <p>decoupling the different metabolic pathways <img src="https://static.igem.org/mediawiki/2015/1/16/PB_prototype.png" title="fig:https://static.igem.org/mediawiki/2015/1/16/PB_prototype.png" alt="https://static.igem.org/mediawiki/2015/1/16/PB_prototype.png" /></p>
-<h2 id="landing-pads">Landing pads</h2>
-<p>quick forking in a lot of parallel projects <img src="https://static.igem.org/mediawiki/2015/7/75/PB_landingpad.png" title="fig:https://static.igem.org/mediawiki/2015/7/75/PB_landingpad.png" alt="https://static.igem.org/mediawiki/2015/7/75/PB_landingpad.png" /></p>
-<h2 id="genetic-stability">Genetic stability</h2>
-<p>Now we protected our product against foreign organisms, but one threat remains: our organism's own mutants. If a mutation occurs in the active site of an enzyme, or in the promoter of an operon, the functionality of the organism might be impaired. Moreover, the ID gene doesn't account for this.</p>
-<p>How can we prevent our organism from mutating?</p>
-<p>Fortunately, our friends at the Vanderbilt University iGEM team worked precisely on that problem this summer. We worked hand in hand with them to see what a real-life application of their invention would mean practically.</p>
 <h1 class="date three" id="results">Results</>
 <h2 id="construction-of-the-system">Construction of the system</h2>
@@ Line 124: / Line 182: @@
 This project was designed and accomplished by Antoine Vigouroux in consultation with Jason Bland and Ihab Boulas.
-Most of the strains (DH5alpha, Top10, NEB turbo, Pir116) were kindly provided by Inserm U1001. Plasmids pFHC2938 and pMEV250 were provided by Didier Mazel's lab at Institut Pasteur. Plasmids pL1F2 and pR6K-shortened were provided by Inserm U1001. The plasmid pIT5-KH was provided by 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 Nivia 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 it so much fun.
 </html>
 {{Paris_Bettencourt/footer}}

Difference between revisions of "Team:Paris Bettencourt/Sustainability/Continuity"