Difference between revisions of "Team:UESTC-China/Design"

We designed two modules in the present work to integrate the two related pathways of ciprofloxacin (CIP) detection and degradation. The first module piGEM2019-01 was designed to achieve the automatic detection of ciprofloxacin. We want to initiate the degradation of ciprofloxacin after detecting ciprofloxacin in the water source, so the other module piGEM2019-02 was designed to produce a novel ciprofloxacin-modifying enzyme--CrpP and perform its function. In addition, we added a Lux quorum sensing system to enhance the expression of CrpP by E.coli. Moreover, to prevent bacteria containing CrpP gene from escaping and causing more biosecurity and environmental problems, we have designed an anti-escape light-controlled kill switch--piGEM2019-03.

There are two modules synthesized in our ciprofloxacin disposal system, including detection and degradation of ciprofloxacin. Prior to the DNA synthesis, we carried out the codon optimization for the purpose to gain better expression for the target genes in E.coli.

In piGEM2019-01 (Fig. 1), we have chosen the promoter P_tisAB which is sensitive to CIP. Fluoroquinolones such as ciprofloxacin induce the SOS response by blocking the ligase activity of DNA gyrase and topoisomerase, converting them into endonucleases, which up-regulates DNA repair functions. The repair function can activate RecA gene, triggering induction of P_tisAB [1]. When P_tisAB senses ciprofloxacin, green fluorescent protein (GFP) and LuxI will express. By measuring the fluorescence intensity through our standard curve prepared in advance, the concentration of CIP can be estimated. LuxI is a synthase that converts S-adenosylmethionine (SAM) into a small molecule called acyl-homoserine lactone (AHL) which can diffuse across cell membranes and “activate” the other module [2]. Ciprofloxacin resistance gene controlled by the constitutive promoter J23119, qnrS1, codes for pentapeptide repeat proteins which reduce susceptibility to quinolones by protecting the complex of DNA and DNA gyrase enzyme from the inhibitory effect of quinolones. Therefore, qnrS1 can reduce the damage of ciprofloxacin to our E.coli [3].

In order to quickly produce a large number of degrading enzymes after CIP is detected, based on the priciple of quorum sensing, we constructed piGEM2019-02 (Fig. 2). LuxR is a constitutively expressed protein that can bind AHL. When bound to AHL (produced by piGEM2019-01), it can activate the right hand Lux promoter (P_LuxR) [2] , then start transcription.
CrpP is a novel ciprofloxacin-modifying enzyme which can phosphorylate and degrade CIP [4]. The degradation pathway is shown in Fig. 3. Under natural conditions, phosphorylated CIP will be gradually degraded and the final product--1, 4-dihydroquinoline is obtained. It has been pointed out in some papers that 1, 4-dihydroquinoline can be used as carriers for central acting agents and tested on mice [5,6]. Therefore, we speculate that it has certain value.

Besides, an extra signal peptide pelB followed by five aspartate repeats were introduced to facilitate the extracellular expression of CrpP in E.coli [7]. TagRFP is a fluorescent protein used as a reporter protein. We can judge whether the CrpP is correctly transcribed according to the red of bacteria liquid.

In order to keep our engineered bacteria living longer in the drug solution, sodium alginate (SA) was selected as entrapping agent to immobilize the engineered E.coli carrying piGEM2019-02 due to its good mechanical strength, internal porous structure and small toxicity [8].

Because our engineered bacteria have certain antibiotic resistance, it is especially important to prevent them from escaping. To this end, we designed an anti-escape light-controlled kill switch (piGEM2019-03) that allows the engineered bacteria to live only with blue light [9,10] (Fig. 4).

[1] Weel-Sneve, R., Bjørås, M., & Kristiansen, K. I. (2008). Overexpression of the LexA-regulated tisAB RNA in E.coli inhibits SOS functions; implications for regulation of the SOS response.

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(19), 6249-6259.
[2]http://parts.igem.org/Lux?tdsourcetag=s_pctim_aiomsg
[3] Strahilevitz, J., Jacoby, G. A., Hooper, D. C., & Robicsek, A. (2009). Plasmid-mediated quinolone resistance: a multifaceted threat.

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[4] Chávez-Jacobo, V. M., Hernández-Ramírez, K. C., Romo-Rodríguez, P., et al. (2018). CrpP is a novel ciprofloxacin-modifying enzyme encoded by the Pseudomonas aeruginosa pUM505 plasmid.

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[5] Foucout, L., Gourand, F., Dhilly, M., et al. (2009). Synthesis, radiosynthesis and biological evaluation of 1, 4-dihydroquinoline derivatives as new carriers for specific brain delivery.

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[6] Gourand, F., Tintas, M. L., Henry, A., et al. (2017). Delivering FLT to the central nervous system by means of a promising targeting System: Synthesis,[11C] radiosynthesis, and in vivo evaluation.

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(11), 2457-2467.
[7] Kim SK, Park YC, Lee HH, Jeon ST, Min WK & Seo JH. 2015. Simple amino acid tags improve both expression and secretion of Candida antarctica lipase B in recombinant Escherichia coli.

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, 112: 346-355.
[8] Orive, G., Santos, E., Poncelet, D., et al. (2015). Cell encapsulation: technical and clinical advances.

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[9] Wang, G., Lu, X., Zhu, Y., et al. (2018). A light-controlled cell lysis system in bacteria. Journal of industrial microbiology & biotechnology, 45(6), 429-432.
[10] Wang, S., Gu, J., Lv, M. et al. (2017). The antibacterial activity of E. coli bacteriophage lysin lysep3 is enhanced by fusing the Bacillus amyloliquefaciens bacteriophage endolysin binding domain D8 to the C-terminal region. Journal of Microbiology, 55: 403.