Difference between revisions of "Part:BBa K1659100"
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We are interested in the antibacterial activity of MccS against antibiotic-resistant strains of bacteria found in urinary tract infections, namely ''E. coli'' and ''P. aeruginosa''. However, in the interest of lab usage safety, for our wet lab work we will only test the antibacterial potency of MccS against Biosafety Level 1 laboratory strains of ''E. coli'' and ''P. putida''.
 
We are interested in the antibacterial activity of MccS against antibiotic-resistant strains of bacteria found in urinary tract infections, namely ''E. coli'' and ''P. aeruginosa''. However, in the interest of lab usage safety, for our wet lab work we will only test the antibacterial potency of MccS against Biosafety Level 1 laboratory strains of ''E. coli'' and ''P. putida''.
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===Characterization===
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To characterize this part, we moved the MccS coding sequence into the commercial expression vector [https://www.thermofisher.com/order/catalog/product/V43001 pBAD/HisB] by adding a BspHI restriction site to the 5' site of the coding sequence using PCR and performing digestion-ligation at BspHI(insert)-NcoI(plasmid) and PstI, making the expression of the MccS coding gene inducible by L-arabinose. This MccS[pBAD] plasmid is then cloned into ''E. coli'' MG1655 and RP437 ∆FliC.
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'''Toxicity Test'''
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The literature describing MccS as a narrow-spectrum antibacterial protein that is selectively antibacterial towards bacterial strains that are closely related to the original microcin-producing strain. As we did not know how genotypically-alike were our expression host strains MG1655 and RP437 to the original microcin-producing strain G3/10, we were unable to determine beforehand whether MccS would be toxic towards our host strains and hence we conducted a toxicity test to find out whether expression of MccS would kill our host strains.
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 +
We induced the production of MccS using 0.2%, 0.02%, and 0.002% L-arabinose in both strains and measuring cell density as a function of time.
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<html>
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    <figure>
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        <img src="https://static.igem.org/mediawiki/parts/0/06/Oxford15-Mccs_curve_30.png" width="350" height="300" style="float:center">
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        <figcaption>
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        <p style="font-size:11px">
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Expression host cell cultures were grown in a 96-well plate at 30°C with 200 rpm shaking.
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        </p>
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        </figcaption>
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    </figure>
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    <figure>
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        <img src="https://static.igem.org/mediawiki/parts/2/2d/Oxford15-Mccs_curve_37.png" width="640" height="300" style="float:center">
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        <figcaption>
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        <p style="font-size:11px">
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Expression host cell cultures were grown in a 96-well plate at 37°C with 200 rpm shaking.
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        </p>
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        </figcaption>
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    </figure>
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</html>
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From the data we can clearly draw 2 conclusions:
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1. MccS is more toxic to RP437 than MG1655, to the extent where even low induction (0.002% arabinose) inhibits cell growth in RP437 almost entirely.
 +
2. MccS appears more toxic at 30°C than 37°C.
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Revision as of 23:24, 18 September 2015

Microcin S, a Class II microcin produced by probiotic E. coli G3/10


This part contains the sequence for the antimicrobial protein microcin S (MccS).


Biology

Microcins are a subclass of antibacterial proteins known as bacteriocins, which have have in recent years garnered a significant amount of interest as antibiotic candidates due to their high antibacterial potency against drug-resistant bacterial as well as their narrow-spectrum antibacterial activity being potentially less harmful to our commensal microbiota [1]. Microcins are small, enterobacteria-produced bacteriocins that exert antibacterial activity against closely-related species, and microcin S is produced by Escherichia coli G3/10 which is present in the probiotic drug Symbioflor 2 that has been shown to successfully treat gastrointestinal disorders [2].

Zschüttig et al., the team which originally discovered and characterized MccS, discovered the gene encoding for the protein as well as its corresponding resistance gene in the E. coli G3/10 megaplasmid pSYM1. Enteropathogenic E. coli strains that were cloned with the MccS-coding gene was shown to be significantly less able to adhere to human intestinal epithelial cell lines upon the induction of MccS production [2].

In 2012, Hwang et al. more conclusively proved the antibacterial potency of MccS. They fused the protein YebF, which is able to facilitate the export of passenger proteins via the Sec and Omp pathways, to the N-terminal of MccS, and cloned the gene encoding for this fusion protein along with the MccS resistance gene into E. coli TOP10 and was able to use this engineered strain of E. coli to effectively kill the pathogenic Pseudomonas aeruginosa strain PAO1 [3][4].


Usage

We are interested in the antibacterial activity of MccS against antibiotic-resistant strains of bacteria found in urinary tract infections, namely E. coli and P. aeruginosa. However, in the interest of lab usage safety, for our wet lab work we will only test the antibacterial potency of MccS against Biosafety Level 1 laboratory strains of E. coli and P. putida.


Characterization

To characterize this part, we moved the MccS coding sequence into the commercial expression vector pBAD/HisB by adding a BspHI restriction site to the 5' site of the coding sequence using PCR and performing digestion-ligation at BspHI(insert)-NcoI(plasmid) and PstI, making the expression of the MccS coding gene inducible by L-arabinose. This MccS[pBAD] plasmid is then cloned into E. coli MG1655 and RP437 ∆FliC.

Toxicity Test

The literature describing MccS as a narrow-spectrum antibacterial protein that is selectively antibacterial towards bacterial strains that are closely related to the original microcin-producing strain. As we did not know how genotypically-alike were our expression host strains MG1655 and RP437 to the original microcin-producing strain G3/10, we were unable to determine beforehand whether MccS would be toxic towards our host strains and hence we conducted a toxicity test to find out whether expression of MccS would kill our host strains.

We induced the production of MccS using 0.2%, 0.02%, and 0.002% L-arabinose in both strains and measuring cell density as a function of time.

Expression host cell cultures were grown in a 96-well plate at 30°C with 200 rpm shaking.

Expression host cell cultures were grown in a 96-well plate at 37°C with 200 rpm shaking.

From the data we can clearly draw 2 conclusions: 1. MccS is more toxic to RP437 than MG1655, to the extent where even low induction (0.002% arabinose) inhibits cell growth in RP437 almost entirely. 2. MccS appears more toxic at 30°C than 37°C.



References

[1] Cotter, P.D., Ross, R.P. & Hill, C., 2013. Bacteriocins - a viable alternative to antibiotics? Nature reviews. Microbiology, 11(2), pp.95–105. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23268227.

[2] Zschüttig, A. et al., 2012. Identification and characterization of microcin S, a new antibacterial peptide produced by probiotic Escherichia coli G3/10. PLoS ONE, 7(3), pp.1–9.

[3] Prehna, G. et al., 2012. A protein export pathway involving Escherichia coli porins. Structure, 20(7), pp.1154–1166. Available at: http://dx.doi.org/10.1016/j.str.2012.04.014.

[4] Hwang, I.Y. et al., 2014. Reprogramming microbes to be pathogen-Seeking killers. ACS Synthetic Biology, 3(4), pp.228–237.