|
|
| (149 intermediate revisions not shown) |
| Line 2: |
Line 2: |
| | | | |
| | <html> | | <html> |
| | + | <!-- NB: ''Our wiki uses active compiling. Please use a recent Web browser (ok on Firefox & Chrome)'' |
| | + | <br/> |
| | + | <__ Next endpages Hypertxt <tr style="background-color:#ebebeb> --> |
| | | | |
| - | <!-- Next pages Hypertxt <tr style="background-color:#ebebeb> -->
| |
| - | <!--requirements section -->
| |
| | <table style="background-color:#CCD6EA; box-shadow: 1px 1px 10px #555; " width="90%" align="center"> | | <table style="background-color:#CCD6EA; box-shadow: 1px 1px 10px #555; " width="90%" align="center"> |
| | <tr style="background-color:#CCD6EA; "><td colspan="2"> | | <tr style="background-color:#CCD6EA; "><td colspan="2"> |
| | <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> |
| | </font></p> | | </font></p> |
| | </td></tr> | | </td></tr> |
| Line 23: |
Line 24: |
| | </section> | | </section> |
| | --> | | --> |
| - | | + | <section style="margin: -40px"></section> |
| - | $\Ogo$ur $\MyColi$ project is based on several biological concepts, like production mechanisms of proteins & toxin-antitoxin systems, and bioengineering topics, like bioreactor optimization & effect of an additional peptide.
| + | |
| - | | + | |
| | <section style="text-align: justify; margin: 50px"> | | <section style="text-align: justify; margin: 50px"> |
| - | <h1>Recombinant Proteins</h1>
| |
| | | | |
| - | <p>Production of recombinant proteins by microorganisms such as bacteria ($\small Escherichia$ $\small Coli$) or yeasts ($\small Saccharomyces$ $\SCere$, $\small Pichia$ $\small Pastoris$) is a key process in pharmacy (vaccines, insulin) and biotechnology (enzymes, antibodies). On industrial scale, proteins and other biological molecules are produced in bioreactors.
| |
| - | <h3>Heterogeneity in bioreactors</h3>
| |
| - | Microorganisms used to produce recombinant proteins in bioreactors are often seen as a homogeneous population, yet stressed subpopulations may arise in these reactors, resulting in reduced quantity and quality of production, as stressed cells consume nutrients and space but have an altered productivity (Davey et al., 1996). Several factors may induce heterogeneity in a population : desynchronisation in cell cycle phases, emergence of mutants and new genotypes or variation 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>
| |
| - | <h3>The Mighty Coli solution</h3>
| |
| - | <p>To reduce inefficient production due to population heterogeneity in bioreactors, we designed $\MyColi$, a synthetic $\EColi$ that suicides when it enters a stressed physiological state. </p>
| |
| - | <!-- or when its production goes under a threshold. ...</p> What threshold? do we obtain an experimental result like this? -->
| |
| - | <br/>
| |
| - | <!--
| |
| - | Why this image as the first in this page?
| |
| - | <center><img src="https://static.igem.org/mediawiki/2014/6/66/Mighty.png">
| |
| - | </center>
| |
| - | <br/><font size="1"><b>Figure 0 </b>:
| |
| - | A nouveau, il y a du texte soukigné de vagues rouges : à enlever
| |
| - | </font>
| |
| - | -->
| |
| - |
| |
| - | <br/>
| |
| - | <br/>
| |
| - |
| |
| - | </section>
| |
| - | <section style="margin: -60px"></section>
| |
| - |
| |
| - | <section style="text-align: justify; margin: 50px">
| |
| - | <h1>Toxin-Antitoxin Systems</h1>
| |
| - |
| |
| - | <p>$\small Toxin$-$\small Antitoxin$ (TA) systems are
| |
| - | couples of linked genes encoding for a toxic protein and an inhibitor of this toxin.
| |
| - | To ensure the survival of bacteria expressing one of these toxins, the corresponding antitoxin must also be expressed.
| |
| - | TA systems can be contained on mobile genetic elements such as plasmids. When a bacteria possessing such a plasmid divides, the generated daughter cells might not inherit this plasmid due to a stochastic partition. In this case, the antitoxin, which is often 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 doesn't confer any advantage. Therefore, TA systems can be seen as selfish entities, virtually making bacteria addicted to them (Hayes & Van Melderen, 2011).</p>
| |
| - | <br/>
| |
| - | <center><img src="https://static.igem.org/mediawiki/2014/a/ad/PSK.png">
| |
| - | </center>
| |
| - | <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 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>
| |
| - |
| |
| - | <br/>
| |
| - | <br/>
| |
| - |
| |
| - | <p>TA systems can be divided into five groups, depending on the molecular nature of its components (Hayes & Van Melderen, 2011). We will mainly use 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 : CcdBA and Kid/Kis.</p>
| |
| - |
| |
| - | <br/>
| |
| - | <center><img src="https://static.igem.org/mediawiki/2014/5/57/TIITA.png">
| |
| - | </center>
| |
| - | <br/><font size="1"><b>Figure 2 </b>: Type II TA systems. In such systems, toxins and antitoxins are translated from the same polycistronic mRNA. The toxin is a protein that inhibits a vital function (translation, replication, peptidoglycan synthesis, etc.) and the antitoxin is another protein that binds this toxin, preventing it from causing harm to the cell. This antitoxin is often unstable and subject to proteolytic 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 their respective toxin-antitoxin complexes.</font>
| |
| - | <br/>
| |
| - | <br/>
| |
| - |
| |
| - | </section>
| |
| - | <section style=" margin: -30px"></section>
| |
| - |
| |
| - |
| |
| - | <section style="text-align: justify; margin: 50px">
| |
| - | <h3>CcdBA and the DNA gyrase</h3>
| |
| - |
| |
| - | 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 supercoiling. CcdB binds this subunit when bound to DNA, inhibiting its activity, resulting in DNA double strand breaks, activation of emergency signals and possibly death <b>[Fig. 3]</b> (Dao-Thi et al., 2005).
| |
| - | </p>
| |
| - |
| |
| - | <br/>
| |
| - | <center><img src="https://static.igem.org/mediawiki/2014/7/79/Ccdbgyrase.png">
| |
| - | </center>
| |
| - | <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>
| |
| - | <br/>
| |
| - | <br/>
| |
| - | <p> We want to construct 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>
| |
| - |
| |
| - | </section>
| |
| - | <section style=" margin: 10px"></section>
| |
| - |
| |
| - |
| |
| - | <section style="text-align: justify; margin: 50px">
| |
| - | <h1>The 2A Peptide</h1>
| |
| - |
| |
| - | <p>New text about p2A in <a href="https://2014.igem.org/Team:ULB-Brussels/Project/WetLab"> this </a> page.</p>
| |
| - | </section>
| |
| - |
| |
| - | <!-- A REECRIRE/COMPLETER : (C'est l'intro de Quentin)
| |
| - | We have written a lot about ccdBA, a little about the promotors and plasmids, and no evidence with p2A.
| |
| - |
| |
| - | <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>
| |
| - | <li style=”text-align: left;”>cells that have lost their transforming vector;</li>
| |
| - | <li style=”text-align: left;”>cells that have turned off the expression of the target gene;</li>
| |
| - | <li style=”text-align: left;”>cells producing muted target protein;</li>
| |
| - | <li style=”text-align: left;”>cells stressed by aging or starvation.</li>
| |
| - |
| |
| - | <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].
| |
| - | 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>
| |
| - |
| |
| - | <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>
| |
| - | <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].
| |
| - | 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>
| |
| - | <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 $\SCere$.
| |
| - | Here is a great strength of our project: it's a quite simple method that could be easily transferred into many processes!
| |
| - | </p>
| |
| - | -->
| |
| - | </section>
| |
| - |
| |
| - | <section style="text-align: justify; margin: 50px">
| |
| - | <h3>Bibliography</h3>
| |
| - | <ul>
| |
| - | <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>
| |
| - | <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>
| |
| - | <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>
| |
| - | <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>
| |
| - | </ul>
| |
| - | </section>
| |
| - |
| |
| - | </td>
| |
| | <!-- | | <!-- |
| | </tr> | | </tr> |
| | -- 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"> |
| | + | <b></b> |
| | + | <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> |
| | <br/><br/><br/></section></td></tr> --> | | <br/><br/><br/></section></td></tr> --> |
| Line 146: |
Line 40: |
| | <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> |
| - | </tr> | + | </tr> |
| - | | + | |
| | <tr><td><br/><br/></td></tr> | | <tr><td><br/><br/></td></tr> |
| - | </table></th></tr>
| + | </th></tr> |
| | | | |
| | </div> | | </div> |
$~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
\newcommand{\MyColi}{{\small Mighty\hspace{0.12cm}Coli}}
\newcommand{\Stabi}{\small Stabi}$
$\newcommand{\EColi}{\small E.coli}
\newcommand{\SCere}{\small S.cerevisae}\\[0cm]
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
\newcommand{\PI}{\small PI}$
$\newcommand{\Igo}{\Large\mathcal{I}}
\newcommand{\Tgo}{\Large\mathcal{T}}
\newcommand{\Ogo}{\Large\mathcal{O}}
~$
"
- Université Libre de Bruxelles -
