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	=Thessaly 2021 Literature Characterization=		=Thessaly 2021 Literature Characterization=
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		+	[https://2021.igem.org/Team:Thessaly Thessaly 2021]contributed to the characterization of this part by adding new documentation learned from the literature highlighting why CymR is a better repressor for the cumate kill-switch.
		+
		+	<p>We chose to characterize CymR because it is a repressor with no documented data in iGEM registry and it is suitable for our kill-switch.
		+	Our kill-switch is based on the Cumate gene-switch. Cumate gene-switch consists of a repressor, CymR, a Cumate Operator, CuO, and the inducer, cumate.In presence of cumate CymR loses its affinity for CuO. This system is dose dependent, non-toxic and inexpensive, while cumate as an inducer is water-soluble and easily penetrates the cells(Horbal, L. et al.(2014)).
		+	CymR repressor belongs to the widespread and poorly characterized Rrf2 family of regulators. The rrf2-type HTH domain is a DNA-binding, winged helix-turn-helix (wHTH) domain of about 130 residues present in transcription regulators of the rrf2 family.("InterPro", 2021) The global regulator CymR represses the transcription of a large set of genes, involved in cysteine metabolism in Bacillus subtilis and Staphylococcus aureus. (Shepard, W.(2011)). </p>
		+	<p>So, we chose CymR for our kill-switch because :
		+	it is characterized by small leakage
		+	it is sensitive while it is induced by small amounts of cumate
		+	it has high affinity to its cumate operator as there is no other inducer
		+	its inducer is non toxic and it is a cheap compound
		+	It is reversible because in the absence of cumate it binds again in its CuO operator
		+	it is orthogonal as its inducer is not naturally found in the gut
		+	Mutant strains are not required as CymR is not native in E. coli</p>
		+
		+
		+
		+
		+	<p>Kill switches are safety mechanisms needed to secure the desirable function of a biological system.There are many biological kill-switches used daily in synthetic systems in order to ensure the safety of environmental, human and animal health. </p>
		+
		+	<p>IIn figure 1, we see the cumate gene switch. It consists of a repressor, the repressor CymR, the inducer cumate, an operator site, CuO, and a target gene. In the absence of cumate, CymR binds to the CuO operator and blocks the production of the target gene, a toxin in our case. On the other hand when cumate is present, it binds to the binding domain of CymR, losing its affinity for CuO, and so the target gene-toxin is expressed, killing the bacteria.This system allows for tight control of gene expression over a linear range in a cumate concentration-dependent manner, which is particularly important for the expression of toxic proteins(Seo SO, Schmidt-Dannert C.(2019))./p>
		+
		+	[[File:T--Thessaly--cumate.png|700px|thumb|none|<i><b>Fig.2:</b>Figure 1. The mechanism of cumate gene switch, represented in a schematic diagram, developed in E.coli strains.(Seo SO, Schmidt-Dannert C.2019).</i>]]
		+
		+	<p>In figure 2, the literature shows the characterization of the functionality of the cumate-inducible system using as reporter gene, a fluorescent protein like GFP. The plate on the left indicates the repression of the GFP gene, while on the right plate we see that the addition of cumate activates the expression of GFP.</p>
		+
		+	[[File:T--Thessaly--MIT2.png|700px|thumb|none|<i><b>Fig.2:</b> Figure 2. Testing the cumate gene switch in E.coli bacteria.Choi, Y. J.(2010)</i>]]
		+
		+	<p>In figure 3, we observe on the Y axis the levels of fluorescence and on the X axis the concentration of cumate that induces the system. As we see, small concentrations of cumate provoke a response of the system indicating the high sensitivity of our system and also we can see the exact concentration of cumate, in which, fluorescence reaches a plateau. </p>
		+
		+	[[File:T--Thessaly--MIT3.png|700px|thumb|none|<i><b>Fig.2:</b>Figure 3. Diagram for the response of the system in different concentrations of cumate.(Choi, Y. J.(2010)</i>]]
		+
		+	<p>In figure 4, we see on the Y axis the relative fluorescent units and on the X axis the time in which fluorescence appears. Experts indicate the difference between the Cumate gene-switch and the IPTG gene-switch. The green line shows the signal induction in the cumate-inducible system, while the blue line indicate the signal induction in the  IPTG-inducible system. As we observe, the cumate system has a quicker and more intense response than the IPTG system.</p>
		+
		+	[[File:T--Thessaly--MIT4.png|700px|thumb|none|<i><b>Fig.2:</b>Figure 4. Comparison of cumate gene switch and IPTG gene switch.Choi, Y. J.(2010)</i>]]
		+
		+	=References=
		+	*Choi, Y. J., Morel, L., Le François, T., Bourque, D., Bourget, L., Groleau, D., Massie, B., & Míguez, C. B. (2010). Novel, versatile, and tightly regulated expression system for Escherichia coli strains. Applied and environmental microbiology, 76(15), 5058–5066. https://doi.org/10.1128/AEM.00413-10
		+	*InterPro. (2021). Retrieved 6 October 2021, from https://www.ebi.ac.uk/interpro/entry/InterPro/IPR030489/
		+	*Kaczmarczyk A, Vorholt JA, Francez-Charlot A. Cumate-inducible gene expression system for sphingomonads and other Alphaproteobacteria. Appl Environ Microbiol. 2013 Nov;79(21):6795-802. doi: 10.1128/AEM.02296-13. Epub 2013 Aug 30. PMID: 23995928; PMCID: PMC3811519.
		+	*Seo SO, Schmidt-Dannert C. Development of a synthetic cumate-inducible gene expression system for Bacillus. Appl Microbiol Biotechnol. 2019 Jan;103(1):303-313. doi: 10.1007/s00253-018-9485-4. Epub 2018 Nov 3. PMID: 30392122.
		+	*Shepard, W., Soutourina, O., Courtois, E., England, P., Haouz, A. and Martin-Verstraete, I. (2011), Insights into the Rrf2 repressor family – the structure of CymR, the global cysteine regulator of Bacillus subtilis. The FEBS Journal, 278: 2689-2701. https://doi.org/10.1111/j.1742-4658.2011.08195.
		+	*Horbal, L., Fedorenko, V. & Luzhetskyy, A. Novel and tightly regulated resorcinol and cumate-inducible expression systems for Streptomyces and other actinobacteria. Appl Microbiol Biotechnol 98, 8641–8655 (2014). https://doi.org/10.1007/s00253-014-5918-x
		+

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Latest revision as of 10:11, 13 October 2021

cymR Repressor Protein

The cymR repressor tightly and efficiently binds the cmt operator. In conjunction with part BBa_K415200, this repressor may be used to create an inverter or similar logical circuitry. cymR changes conformation upon induction with cumate (isopropylbenozate), and may be used in a manner similar to the lacI/IPTG system.

The cymR repressor appears to bind the complementary cmt operator more efficiently than lac repressor binds the lac operator, according to Choi, et al. in a recent (as of 2010) paper titled, "A novel, versatile, and tightly regulated expression system for E. Coli strains."

Thessaly 2021 Literature Characterization

Thessaly 2021contributed to the characterization of this part by adding new documentation learned from the literature highlighting why CymR is a better repressor for the cumate kill-switch.

We chose to characterize CymR because it is a repressor with no documented data in iGEM registry and it is suitable for our kill-switch. Our kill-switch is based on the Cumate gene-switch. Cumate gene-switch consists of a repressor, CymR, a Cumate Operator, CuO, and the inducer, cumate.In presence of cumate CymR loses its affinity for CuO. This system is dose dependent, non-toxic and inexpensive, while cumate as an inducer is water-soluble and easily penetrates the cells(Horbal, L. et al.(2014)). CymR repressor belongs to the widespread and poorly characterized Rrf2 family of regulators. The rrf2-type HTH domain is a DNA-binding, winged helix-turn-helix (wHTH) domain of about 130 residues present in transcription regulators of the rrf2 family.("InterPro", 2021) The global regulator CymR represses the transcription of a large set of genes, involved in cysteine metabolism in Bacillus subtilis and Staphylococcus aureus. (Shepard, W.(2011)).

So, we chose CymR for our kill-switch because : it is characterized by small leakage it is sensitive while it is induced by small amounts of cumate it has high affinity to its cumate operator as there is no other inducer its inducer is non toxic and it is a cheap compound It is reversible because in the absence of cumate it binds again in its CuO operator it is orthogonal as its inducer is not naturally found in the gut Mutant strains are not required as CymR is not native in E. coli

Kill switches are safety mechanisms needed to secure the desirable function of a biological system.There are many biological kill-switches used daily in synthetic systems in order to ensure the safety of environmental, human and animal health.

IIn figure 1, we see the cumate gene switch. It consists of a repressor, the repressor CymR, the inducer cumate, an operator site, CuO, and a target gene. In the absence of cumate, CymR binds to the CuO operator and blocks the production of the target gene, a toxin in our case. On the other hand when cumate is present, it binds to the binding domain of CymR, losing its affinity for CuO, and so the target gene-toxin is expressed, killing the bacteria.This system allows for tight control of gene expression over a linear range in a cumate concentration-dependent manner, which is particularly important for the expression of toxic proteins(Seo SO, Schmidt-Dannert C.(2019))./p>

<p>In figure 2, the literature shows the characterization of the functionality of the cumate-inducible system using as reporter gene, a fluorescent protein like GFP. The plate on the left indicates the repression of the GFP gene, while on the right plate we see that the addition of cumate activates the expression of GFP.

In figure 3, we observe on the Y axis the levels of fluorescence and on the X axis the concentration of cumate that induces the system. As we see, small concentrations of cumate provoke a response of the system indicating the high sensitivity of our system and also we can see the exact concentration of cumate, in which, fluorescence reaches a plateau.

In figure 4, we see on the Y axis the relative fluorescent units and on the X axis the time in which fluorescence appears. Experts indicate the difference between the Cumate gene-switch and the IPTG gene-switch. The green line shows the signal induction in the cumate-inducible system, while the blue line indicate the signal induction in the IPTG-inducible system. As we observe, the cumate system has a quicker and more intense response than the IPTG system.

References

• Choi, Y. J., Morel, L., Le François, T., Bourque, D., Bourget, L., Groleau, D., Massie, B., & Míguez, C. B. (2010). Novel, versatile, and tightly regulated expression system for Escherichia coli strains. Applied and environmental microbiology, 76(15), 5058–5066. https://doi.org/10.1128/AEM.00413-10
• InterPro. (2021). Retrieved 6 October 2021, from https://www.ebi.ac.uk/interpro/entry/InterPro/IPR030489/
• Kaczmarczyk A, Vorholt JA, Francez-Charlot A. Cumate-inducible gene expression system for sphingomonads and other Alphaproteobacteria. Appl Environ Microbiol. 2013 Nov;79(21):6795-802. doi: 10.1128/AEM.02296-13. Epub 2013 Aug 30. PMID: 23995928; PMCID: PMC3811519.
• Seo SO, Schmidt-Dannert C. Development of a synthetic cumate-inducible gene expression system for Bacillus. Appl Microbiol Biotechnol. 2019 Jan;103(1):303-313. doi: 10.1007/s00253-018-9485-4. Epub 2018 Nov 3. PMID: 30392122.
• Shepard, W., Soutourina, O., Courtois, E., England, P., Haouz, A. and Martin-Verstraete, I. (2011), Insights into the Rrf2 repressor family – the structure of CymR, the global cysteine regulator of Bacillus subtilis. The FEBS Journal, 278: 2689-2701. https://doi.org/10.1111/j.1742-4658.2011.08195.
• Horbal, L., Fedorenko, V. & Luzhetskyy, A. Novel and tightly regulated resorcinol and cumate-inducible expression systems for Streptomyces and other actinobacteria. Appl Microbiol Biotechnol 98, 8641–8655 (2014). https://doi.org/10.1007/s00253-014-5918-x

Sequence and Features