| | + | content.text= "Our system of light inducible protein degradation can be utilized to degrade any specific protein and is therefore usable to realize a light induced kill-switch system. Therefore a connection between the degradation system and an toxin-antitoxin module like MazEF or ccdA/ccdB is needed. You could either light inducible degrade the toxin or the antitoxin by adding an ssrA-tag to its encoding gene, that is detected by our degradation system: <ul><li><b>Using the degradation of the toxin:</b> For that purpose the insertion of a plasmid containing the ssrA-tagged toxin encoding gene is needed. As the predominance of the toxin leads into a cell death pathway in bacteria, a bacterium containing a module that allows light inducible degradation of the toxin would only relive, when the toxin is light induced degraded. When light turns off, the overexpression of the toxin is no longer compensated and the toxin leads the bacterium into apoptosis. Apart from the use in lab security such a kill-switch system would also be useful in environmental applications of bacteria, as you can control the time those bacteria are living and you avoid that they may live in areas where no light is. If you e.g. want to use bacteria in a lake to improve its ecological stability you could be sure, that after one night all genetically modified bacteria are dead.</li><li><b>Using the degradation of the antitoxin:</b> Two plasmids are needed: The first one needs to express the toxin and the second one the ssra-tagged antitoxin, so that the amounts of the toxin and the antitoxin are in equilibrium. If light induces the degradation system, the antitoxin is degraded and the predominant toxin will kill the bacterium. </li></ul> Regarding our idea to improve lab security by inserting a kill-switch system, both described ways seem possible. Using the first one, you need to cultivate and work with the bacteria steadily under blue light, as darkness would kill them. Realizing the second one, you must avoid any blue light in the lab. If bacteria get into touch with daylight our any blue light, they will be killed. As we suppose our light inducible degradation system to be activated via daylight, using the degradation of the toxin for lab security would be unlikely. Bacteria that escape from the lab could go on living simply by getting into touch with daylight. So finally we focused on the second system (via the degradation of the antitoxin).</br> We described the realization of a light inducible kill-switch system via the insertion of plasmids into bacteria, but we also consider a final kill-switch system to be realized in the genomic DNA, as it would raise the security of such a system. Plasmids in bacteria can get lost, for instance via cell division, whereas a genomic DNA mutation is less probable.</br>Finally we have to add that we consider a MazEF or ccdA/ccdB kill-switch system to be a part of a much larger system in bacteria, that raises lab security. This system should contain much more than one kill-switch system to compensate errors of single kill-switch systems."; |
| - | content.text= "Our system of light inducible protein degradation can be utilized to degrade any specific protein and is therefore usable to realize a light induced kill-switch system. A connection of protein degradation to a cell death pathway is represented by the stress-induced toxin-antitoxin module <i>mazE</i>F in Escherichia coli. <i>mazE</i>F is located on a chromosome in E.coli that is associated with programmed cell death. The toxin-antitoxin system is composed by an upstream gene <i>mazE</i>, encoding a labile antitoxin, and a downstream gene mazF, that encodes a stable toxin. </br>The product of <i>mazF</i> cleaves mRNAs and tmRNAs at a specific site, which leads to an inhibition of translation. MazF shows a specific cleaving mechanism, which is not well understood yet, but explains that there exists also protein synthesis that is not affected by MazF. These proteins are supposed to be part of a cell death pathway. </br>The action of <i>mazF</i> is hindered by the Product of <i>mazE</i> which is degraded by the Protease ClpAP in bacteria. The follow of stressful conditions is a lowered expression of the chromosomally <i>mazE</i>F module and finally an imbalance between the products of <i>mazF</i> and <i>mazE</i>: Whereas the stable toxin of <i>mazF</i> still exists, the labile antitoxin of <i>mazE</i> is degraded and can no longer compensate the action of <i>mazF</i>. </br><i>mazE</i>F-mediated cell death is supposed to be caused by:<ul><li>extreme amino acid starvation<sup><a href = '#1'>[17.1]</a></sup></li><li> inhibition of transcription and/or translation by antibiotics such as rifampin, chloramphenicol, and spectinomycin under specific growth conditions<sup><a href = '#1'>[17.1]</a></sup></li><li>inhibition of translation by the Doc protein of prophage P1<sup><a href = '#1'>[17.1]</a></sup></li><li>DNA damage caused by thymine starvation as well as by mitomycin C, nalidixic acid, and UV irradiation<sup><a href = '#1'>[17.1]</a></sup></li><li>oxidative stress (H2O2)<sup><a href = '#1'>[17.1]</a></sup></li></ul>Amitai et al. tested in 2004 the Hypothesis of Pedersen et al.<sup><a href = '#2'>[17.2]</a></sup>, that chromosomal toxin-antitoxin systems may rather cause a state of reversible bacteriostasis than programmed cell death<sup><a href = '#1'>[17.1]</a></sup>.Therefore E.coli strain MC4100 Δ<i>mazE</i>F relA1 lacIq was cotransformated with:<ul><li>pBad-<i>mazF</i></li><li>pQE-Δhis-<i>mazE</i></li></ul><i>mazF</i>-expression can be induced by the addition of Arabinose via the pBad promoter of the first plasmid. The transformation of the second plasmid results firstly in the repression of <i>mazE</i> expression, whereas when IPTG is added <i>mazE</i> production is induced.</br><div class='content-image' align='center' height=501 width=410><a href='https://static.igem.org/mediawiki/2013/a/ac/Team_Bonn_MazF_1.png'><img src='https://static.igem.org/mediawiki/2013/a/ac/Team_Bonn_MazF_1.png' height=491 width=400></a></br><i>Ability of E. coli cells that had been ectopically overexpressing MazF in liquid medium to form colonies when ectopically overexpressing MazE on plates. The cultures were grown in LB medium (A) or M9 minimal medium with 0.5% glycerol (B) at 37°C to midlogarithmic phase (OD600, 0.5)<sup><a href = '#1'>[17.1]</a></sup>.</i></div>Using these tools, Amitai et al. tested the effect of MazE overproduction on MazF-overproducing bacteria during growth in liquid medium.</br>The E.coli strain was incubated in LB medium. After <i>mazF</i> expression was induced by adding arabinose at several time points two samples were taken. To repress <i>mazF</i> expression to both of them glucose was added. One portion got also IPTG to induce <i>mazE</i> expression and was compared to the other one via the level of protein synthesis and OD600.</br>Finally Amitai et al. confirmed the assumption that the overproduction of MazE could resuscitate E.coli cells overproducing MazF during a period of 6h in LB medium (Fig. 1A)<sup><a href = '#1'>[17.1]</a></sup>, but the longer MazF was induced the minor cells could be resuscitated by MazE.</br>Whereas MazE overproduction can reverse the inhibitory effect of MazF on translation, it cannot reverse the effect of MazF on colony formation, which is shown in figure 2. Only 1h after the induction of MazE expression, the rate of translation was restored to around 100% (Fig.2 Aa, Ab, Ac) but the bacteriocidic effect could not be reversed (Fig.2 Ba, Bb, Bc).</br>Additionally, Amtai et al. found out, that in M9 medium MazE was less able to reverse the effects of MazF overexpression than in LB medium (Fig.1B vs. 1A). They concluded that there is a point of no return, when MazE is inable to resuscitate a MazF damaged cell, which occurs earlier in a M9 medium than in a LB medium.</br>Based on their results they build a model of the MazEF mechanism: A <i>mazF</i>-mediated cascade leads into a cell death pathway, but can nevertheless be stopped at several intermediary steps by e.g. <i>mazE</i>. When a point of no return is reached, the cascade cannot be stopped.<div class='content-image' align='center' height=827 width=784><a href='https://static.igem.org/mediawiki/2013/9/9e/Team_Bonn_MazF_2.png'><img src='https://static.igem.org/mediawiki/2013/9/9e/Team_Bonn_MazF_2.png' height=817 width=764></a></br><i>Effect of MazE overproduction during growth in liquid medium on the ability of MazF-overproducing E. coli cells to synthesize proteins and to form colonies.To induce <i>mazE</i> expression, IPTG was added to the bacterial culture at 1 h (Aa and Ba), 4 h (Ab and Bb), and 6 h (Ac and Bc) after <i>mazF</i> induction at time zero. The effects of the ectopic overexpression of MazE were measured at 1 and 3 h after the induction of <i>mazE</i> expression.<sup><a href = '#1'>[17.1]</a></sup>.</i></div></br>Back to our project and to the idea of a light inducible kill-switch system:</br>As we described in general for both of our kill-switch systems, considering the MazEF module, you could either degrade MazF or MazE:<ul><li>To use the degradation of MazF, you need to insert an additional plasmid containing a constantly active promoter and an ssra-tagged <i>mazF</i> gene. As the predominance of MazF leads into a cell death pathway in bacteria, a bacterium containing this plasmid would only relive, when the degradation tool is light induced.</li><li>Using light inducible degradation of MazE, two additional plasmids need to be inserted into bacteria. The first one needs to express MazF and the second one an ssra-tagged MazE, so that the amounts of MazF and MazE are in equilibrium. If light induces the degradation system, MazE is degraded and the predominant MazF toxin will kill the bacterium.</li></ul>As we explained in the general kill-switch system text we finally focused on the second system (via the degradation of MazE).</br>Designing a MazEF kill-switch system you have to consider the possibility to resuscitate bacteria in the way Amitai et al. showed. A predominant MazF could kill a bacterium in LB within about two hours, but it needs to be predominant over a long period (>7h) to be sure, that it dies with more than 50% probability and cannot be resuscitated by an again active <i>mazE</i> expression (Fig.1A).</br>Surely, these facts seem to be unfavourable for the realization of a kill switch system via MazEF, but fortunately our system of heterodimerization (Lungu et al.) allows a pretty long off-time hours <sup><a href = '#3'>[17.3]</a></sup>, which means that perhaps a short exposure time could as well be enough to kill a bacterium. Additionally, Amitai et al. showed that the less nutrition is available for a bacterium, the earlier the point of no return appears. If a bacterium leaves the lab, it is supposed to get less nutrition than in a LB medium. It might reach the point of no return earlier.</br>We described the realization of a light inducible MazEF kill-switch system via the insertion of plasmids into bacteria, but we also consider a final kill-switch system to be realized in the genomic DNA, as it would raise the security of such a system. Plasmids in bacteria can get lost, for instance via cell division, whereas a genomic DNA mutation is less probable.</br>Finally we have to add that we consider the MazEF kill-switch system to be a part of a much larger system in bacteria, that raises lab security. This system should countain much more than one kill-switch system to compensate errors of single kill-switch systems.</br></br>References</br><a name = '1'><sup>[17.1]</sup></a><a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC532418/'>MazF-Mediated Cell Death in Escherichia coli: a Point of No Return, Shahar Amitai et al., Journal of Bacteriology Vol. 186, No. 24, 2004, p.8295–8300.</a></br><a name = '2'><sup>[17.2]</sup></a><a href = 'http://www.ncbi.nlm.nih.gov/pubmed/?term=Rapid+induction+and+reversal+of+bacteriostatic+conditions+by+controlled+expression+of+toxins+and+antitoxins'>Rapid induction and reversal of bacteriostatic conditions by controlled expression of toxins and antitoxins, Pedersen et al., Molecular Microbiology 45, 2002, 501–510.</a></br><a name = '3'><sup>[17.3]</sup></a><a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334866/'>Designing Photoswitchable Peptides Using the AsLOV2 Domain, Oana I. Lungu et al., Chem Biol. 2012, 19(4):507-17.</a>";
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