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| | <p class="title"><font color="#002B9B"> | | <p class="title"><font color="#002B9B"> |
| - | An introduction to MightyColi
| + | <font color="#CCD6EA">'Mighty Coli, an</font> $Introduction$ <font color="#CCD6EA">to Mighty Coli'</font> |
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| - | <h1>Purpose and definitions</h1>
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| - | $\Ogo$ur $\MyColi$ project aims at improving the yield and the quality of protein production in bioreactors.</p>
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| - | Indeed, production of recombinant proteins by microorganisms such as bacteria ($\small Escherichia$ $\small coli$) or yeasts ($\small Saccharomyces$ $\small cerevisiae$, $\small Pichia$ $\small pastoris$) is a key process in pharmacy (vaccines, insulin) and biotechnology (enzymes, antibodies). The tank in which the protein-producing microorganisms are grown is called a <i>bioreactor</i>. One can define it as a controlled environment where a chemical reaction (in our case, protein synthesis) is catalyzed by micro-organisms.
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| - | <h3>Heterogeneity in bioreactors</h3>
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| - | Micro-organisms used to produce recombinant proteins in bioreactors are often seen as a homogeneous population. However, stressed subpopulations may appear, resulting in reduced quantity and quality of the production. Indeed, stressed cells consume nutrients and space but have a reduced productivity (Davey et al., 1996). Several factors may induce heterogeneity in a population : desynchronisation in cell cycle phases, emergence of mutants or local variations in physico-chemical conditions within the reactor (Muller et al., 2010). Getting rid of these stressed subpopulations might be an effective way to increase both quality and quantity of production in bioreactors. </p>
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| - | <h3>The Mighty Coli solution</h3>
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| - | <p>We figured that the underlying problem of population heterogeneity is that the micro-organism do not have any advantage in the production of the protein of interest (PI), it's a unnecessary energy charge for the cell. They do not gain in fitness if they produce, and do not lose in fitness if they stop producing. If anything ... it would rather be the opposite!</p>
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| - | We thus decided to give microorganisms some incentive to overproduce the PI. That would be done by killing the non-productive bacteria, or that would enter a stressed physiological state.</p>
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| - | We used <i>two genetic compounds</i> to apply this principle and design $\MyColi$: the TA system and the 2A peptide.
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| - | <br/><font size="1"><b>Figure 0 </b>:
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| - | <h1>Toxin-Antitoxin Systems</h1>
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| - | <p>$\small Toxin$-$\small Antitoxin$ (TA) systems are
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| - | two linked genes encoding respectively for a stable toxic protein and an unstable inhibitor of this toxin.
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| - | Thus, to ensure the survival of bacteria expressing one of these toxins, the corresponding antitoxin must be continuously expressed in order to compensate its unstability.
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| - | TA systems naturally occur in mobile genetic elements such as plasmids, and are used by plasmids to maintain themselves in a microbial population. When a bacterium possessing such a plasmid divides, the generated daughter cells might not inherit a copy of the plasmid due to a stochastic partition. In this case, the antitoxin, unstable, is quickly degraded, allowing the toxin to perform its function and kill the daugther cell in a process known as $\small Post$-$\small Segregational$ $\small Killing$ (PSK) [<b>Fig. 1</b>]. This system allows a plasmid to be selected and maintained in a bacterial population even if it does not confer any advantage for the host. Therefore, TA systems can be seen as selfish entities, virtually making bacteria addicted to them (Hayes & Van Melderen, 2011).</p>
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| - | <center><img src="https://static.igem.org/mediawiki/2014/a/ad/PSK.png">
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| - | <br/><font size="1"><b>Figure 1 </b>$:\hspace{0.16cm}$ PSK and plasmid addiction. Cells that inherit a plasmid encoding a TA system can grow normally. As there is no system to part plasmids equivalently in daughter cells, some cells might not receive a plasmid during division. Such cells would still have toxins and antitoxins in their cytosol but these will not be renewed due to the loss of TA genes. Antitoxins are often unstable and quickly degraded by a protease under these conditions, leaving the toxins free and able to kill the cell, effectively eliminating cells losing plasmids that encode TA systems.</font>
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| - | <p>TA systems can be divided into three groups, depending on the nature and mode of action of its components (Hayes & Van Melderen, 2011). We will mainly use type II TAs in which both components are proteins and the antitoxin binds to the toxin, preventing it from performing its function [<b>Fig. 2</b>]. Toxin functions and structures in type II TA systems are diversified, allowing us to chose how cells will die when stressed. Two systems will be used to illustrate our project : $\small CcdBA$ and $\small Kid/Kis$.</p>
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| - | <center><img src="https://static.igem.org/mediawiki/2014/5/57/TIITA.png">
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| - | <br/><font size="1"><b>Figure 2 </b>: Type II TA systems. The toxin is a protein that inhibits a vital function of the cell (translation, replication, peptidoglycan synthesis, etc.) and the antitoxin is another protein that binds to this toxin, preventing it from causing harm to the cell. This antitoxin is often unstable and subject to a quick degradation. Should the transcription of the TA operon stops, the antitoxin would swiftly be degraded, allowing the toxin to kill or damage the cell. Type II TA operons are negatively regulated.</font>
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| - | <h3>CcdBA and the DNA gyrase</h3>
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| - | CcdBA is the most studied type II TA system where CcdB is the toxin and CcdA the antitoxin. CcdB is an inhibitor of the DNA gyrase, an enzyme that supercoils circular DNA, such as bacterial chromosomes and plasmids, making it more compact. DNA gyrase complexes must bind to DNA to perform negative supercoiling during replication. CcdB binds the DNA gyrase subunit that binds to DNA and inhibits its activity when it is bound to DNA, resulting in DNA double strand breaks due to the progession of the DNA polymerase. It cause the activation of emergency signals and possibly death of the cell <b>[Fig. 3]</b> (Dao-Thi et al., 2005).
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| - | <center><img src="https://static.igem.org/mediawiki/2014/7/79/Ccdbgyrase.png">
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| - | <br/><font size="1"><b>Figure 3 </b>$:\hspace{0.16cm}$ DNA gyrase mechanism & CcdB poisoning. $\hspace{0.2cm}a)$ The DNA gyrase is a tetramer made of two GyrA and two GyrB subunits. $\hspace{0.2cm}b)$ An undefined DNAsegment (G-segment) can bind between two GyrB subunits. $\hspace{0.2cm}c)$ Another segment (T-segment) binds, situated between both GyrA subunits of the complex. $\hspace{0.2cm}d)$ The gyrase catalyses a double strand break in the G-segment. $\hspace{0.2cm}e)$ The T-segment is translocated from GyrA to GyrB subunits, above the G-segment. $\hspace{0.2cm}f)$ Both ends of the G-segment are joined and supercoiled DNA is released from the complex. $\hspace{0.2cm}g)$ CcdB binds to GyrA subunits. $\hspace{0.2cm}h)$ CcdB prevents the gyrase from joining G-segment ends, resulting in DNA double strand breaks.</font>
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| - | <p> We want to build a biological system selecting only highly productive cells. By coupling the production of a $\small Protein$ $\small of$ $\small Interest$ (PI) with an antitoxin (A-Tox) in a strain expressing its corresponding toxin, we hope that stressed cells experiencing a drop in PI productivity will be killed by the ensuing drop in A-Tox production. There are many ways to couple the production of two proteins, f.e. polycistronic mRNAs in procaryotes or Internal Ribosome Entry Sites. We chose to use a 2A peptide to pair the PI and the A-Tox.</p>
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| - | <h3>Stability of Mighty Coli</h3>
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| - | The regulation of natural TA system can be complex, and is not always well understood. Since we want our system to be as simple, as safe and as productive as possible, we decided to use well characterized promoters for our system. Using different promoters for our toxin and our antitoxin would also allow us to build a safety device directly into our system (cf. biosafety page). We thus decided to synthetize first the toxin and the antitoxin from different plasmids, each under the control of a different promoter and each bearing a different antibiotic resistance gene. The TA system is thus used to boost protein production and the plasmid stability is assured by antibiotic resistance.<p>
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| - | <center><img src="https://static.igem.org/mediawiki/2014/7/7f/ULB-Brussels_Intro-Plasmids%2BTA.png">
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| - | </center>
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| - | <br/><font size="1"><b>Figure 4 </b>:
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| - | Distinction between Toxin and Antitoxin Vectors: the plasmid on the left containing the toxin gene (when not added in the bacterial chromosome) and the second plasmid (regulated by the T7 constitutive promoter) containing the protein of interest (RFP/GFP marker) and the antitoxin gene.
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| - | <h1>The 2A Peptide</h1>
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| - | <p>The 2A peptide sequence (18 amino-acids) allows the post-transcriptional cleavage of 1 ARN sequence into 2 amino-acid sequences: one upstream and one downstream of 2A peptide. This cleavage is done by ribosome skipping between the last amino-acid of the 2A peptide and the first amino-acid of the downstream protein, if this first amino-acid is a proline. It results in the termination of the translation of the upstream mRNA sequence. The ribosome can then continue to translate the downstream sequence into a second, separated protein. Since the downstream protein does not necessarily begins by a prolin, we added the DNA sequence for the prolin at the end of the gene for the 2A peptide. This ensures that the 2A peptide will always be followed by a proline undependantely of the nature of the downstream protein. It is thus this construction of 2A peptide + proline that is referred to as "2A peptide" in the other sections of our project. Hence, the C-terminal extremity of the upstream protein is fused with the 2A peptide, and N-terminal extremity of the downstream protein is fused with the proline we added to the 2A peptide. <p>
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| - | A great advantage of the use of the 2A peptide over other methods is that it allows a powerful quality control: the antitoxin will be produced only and only if the upstream protein is correctly translated (or punctually muted, which is very rare). Any premature stop codon or “frame-shift” will be detected.</p>
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| - | There are several 2A-like sequences, but a 2A peptide that worked within E.coli could not be found in the literature. However, the 2A peptide P2A was found to function properly in S.cerevisiae. We thus decided to separate our wetlab in two separate projects: on the first hand, we would try to find a 2A peptide that works in procaryotes (Escherichia coli); on the other hand, we will build the Mighty Coli system in eukaryotes (Saccharomyces cerevisiae) using the P2A peptide. We will thus use two different T-A systems: ccdB-ccdA for E.coli, and Kid-Kis for S.cerevisiae (respectively Toxin-Antitoxin)
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| - | <h1>Project overview</h1>
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| - | The aim of our project is thus double. On the one hand, we will test several 2A peptide sequences to find one that would work efficiently in E.coli. On the other hand we would try to build Mighty Coli into two different chassis (E.coli and S.cerevisiae), and characterize the quantity and quality of their protein production.</p>
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| - | </section>
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| - | <!-- A REECRIRE/COMPLETER : (C'est l'intro de Quentin)
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| - | We have written a lot about ccdBA, a little about the promotors and plasmids, and no evidence with p2A.
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| - | <p style=”text-align: justify;”> The ULB-Brussels team aims to work out a system which fights against the appearance of less or none productive microbial sub-populations in bioreactors by repressing :</p>
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| - | <li style=”text-align: left;”>cells that have lost their transforming vector;</li>
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| - | <li style=”text-align: left;”>cells that have turned off the expression of the target gene;</li>
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| - | <li style=”text-align: left;”>cells producing muted target protein;</li>
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| - | <li style=”text-align: left;”>cells stressed by aging or starvation.</li>
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| - |
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| - | <p style=”text-align: justify;”> Our focus will be on <i>E.Coli</i> and <i>S.Cerevisiae</i>. We will maintain the target gene in cells by a mechanism wildly used by the plasmids themselves [4].
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| - | Plasmids are closed collections of DNA sequences often find in bacteria and added to the bacterial chromosome. There are actually broad elements for the cell, like virus and transposons, and they have their own existence. They aim to be maximally amplified. They so carry on the exact inverse strategy of the virus: they bring genes that are not essential for the survival of the micro-organism but that give clear evolutionary advantages such as gene of resistance to antibiotics. Despite the metabolic overhead that they represent, the cell can thus have interest to let them breed and to keep them. However some plasmids are greedy and they want to pull the evolutionary phenomenon to their own advantage. When the cell is split, it is important that the plasmid an its copies are present in the both daughter cells but the replication of the plasmids is independent of that of the bacterial chromosome and can't thus use the microtubules. Some plasmids bear genes for a toxin and its antitoxin, the antitoxin being less stable than the toxin, so that the daughter cell which doesn’t inherit at least one copy of the plasmid is sentenced to death: the cell won’t be able to renew its supply of toxin and antitoxin whereas the inherited antitoxin will be quickly degraded, freeing the action of the inherited toxin. That is how some plasmids manipulate the laws of the natural selection…</p>
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| - | <p style=”text-align: justify;”> The fact is that the plasmids are the privileged transforming vectors for bacteria and the yeasts. It is rather easy to insert the target gene on a plasmid and then integrate it inside the micro-organisms. To be ensured that the plasmid, and then the target gene, is maintained into the microbial strains, we could transfer the toxin-antitoxin strategy wildly adopted by numerous plasmids into the process. Moreover we suggest the coupling of the production of the target protein to that of the antitoxin while the toxin is produced independently. So the cells that would have turned off the target gene and thus the antitoxin gene will die. We will actually build what is called a bicistronic gene: the target protein and the antitoxin are set on the same order form (mRNA) for the protein factory (ribosome). A special DNA sequence has to be put between these two genes if one wants that the ribosome produce two distinct proteins with only one mRNA. We will use the gene of the 2A peptide (18 aa.), in both the yeast ($\SCere$) and bacteria model ($\EColi$). There is nowadays few literature about the use of 2A peptide in prokaryotes and an important part of our work will aim to improve our knowledge. This peptide is a trick used by some virus to condensate their genome. When the ribosome translates the last amino acid of the 2A peptide, the nascent polypeptide chain is trapped because of steric obstruction inside the ribosomal complex. The translation is momentarily paused. The congestion can be relieved by the hydrolysis of the ester link between the tRNA (linked to the mRNA into the P site of the ribosome) and the last amino acid, which allows the release of the nascent chain, formed by the first target protein in fusion with the 2A peptide. If the second target protein begins by a prolyl residue, the translation can restart. A great advantage of the use of the 2A peptide, unlike other methods, is that allows carrying on a mighty quality control: the antitoxin will be produced only and only if the upstream protein is correctly translated (or punctually muted, which is very rare). Any premature stop codon or “frame-shift” will be detected.</p>
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| - | <p style=”text-align: justify;”> At last, the group of stressed cells because of aging or starvation would be repressed thanks to the exploitation of another wild function of the toxin-antitoxin systems. They are also responsible for the sacrifice of part of the population during an important stress in order to maximize the survival of the remaining cells [6].
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| - | The system we will tune will be rather low sensitive so that the micro-organisms won’t be killed for any stress that is inherent to the process (change in the set points, sub-optimal settings, micro-spatial variations, etc.).</p>
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| - | <p> The cues we suggest to fight against the appearance of less or none productive sub-populations are universal and would be found in any process using $\EColi$ and $\SCere$.
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| - | Here is a great strength of our project: it's a quite simple method that could be easily transferred into many processes!
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| - | </p>
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| - | <h3>Bibliography</h3>
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| - | <ul>
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| - | <li>H.M. Davey & D.B. Kell, (1996). Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses. Microbiological reviews, 60(4), 641-696.</li>
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| - | <li>F. Hayes & L. Van Melderen, (2011). Toxins-antitoxins: diversity, evolution and function. Critical Reviews in Biochemistry and Molecular Biology, 46(5), 386-408.</li>
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| - | <li>S. Müller, H. Harms & T. Bley, (2010). Origin and analysis of microbial population heterogeneity in bioprocesses. Current opinion in biotechnology, 21(1), 100-113.</li>
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| - | <li>M.H. Dao-Thi, L. Van Melderen, E. De Genst, H. Afif, L. Buts, L. Wyns & R. Loris, (2005). Molecular basis of gyrase poisoning by the addiction toxin CcdB. Journal of molecular biology, 348(5), 1091-1102.</li>
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| - | </ul>
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| | <!-- | | <!-- |
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| | -- Previous and Next pages -- | | -- Previous and Next pages -- |
| - | <tr style="background-color:rgb(245,245,245);"><td width="50%"><section style="text-align: left"><b></b><br/><br/><br/></section> </td><td><section style="text-align: right"> | + | <tr style="background-color:rgb(245,245,245);"><td width="50%"><section style="text-align: left"> |
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| | + | <br/><br/><br/></section> </td><td><section style="text-align: right"> |
| | <a href="https://2014.igem.org/Team:ULB-Brussels/Project/WetLab"><b> WetLab & Methods > </b></a> | | <a href="https://2014.igem.org/Team:ULB-Brussels/Project/WetLab"><b> WetLab & Methods > </b></a> |
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| | <a href="https://2014.igem.org/Team:ULB-Brussels/Project/WetLab"> WetLab & Methods > </a> | | <a href="https://2014.igem.org/Team:ULB-Brussels/Project/WetLab"> WetLab & Methods > </a> |
| | </section> | | </section> |
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$~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
\newcommand{\MyColi}{{\small Mighty\hspace{0.12cm}Coli}}
\newcommand{\Stabi}{\small Stabi}$
$\newcommand{\EColi}{\small E.coli}
\newcommand{\SCere}{\small S.cerevisae}\\[0cm]
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
\newcommand{\PI}{\small PI}$
$\newcommand{\Igo}{\Large\mathcal{I}}
\newcommand{\Tgo}{\Large\mathcal{T}}
\newcommand{\Ogo}{\Large\mathcal{O}}
~$
"
- Université Libre de Bruxelles -
