Team:UESTC-China/Design
Overview
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 quorum sensing system to enhance the expression of CrpP by E.coli. Moreover, to prevent bacterias containing CrpP gene from escaping and causing more biosecurity and environmental problems, we have designed an anti-escape light-controlled kill switch--piGEM2019-03.
The ciprofloxacin disposal system
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.
Detection of ciprofloxacin
In piGEM2019-01 (Fig.1), we have chosen the promoter PtisAB 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 PtisAB [1] (Fig.2).
When PtisAB 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].
When PtisAB 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].
Fig. 1. Schematic map of piGEM2019-01. PtisAB: a promoter sensitive to CIP; RBS: ribosome binding site; qnrS: an anti-CIP protein. LuxI: a protein converts SAM into AHL. GFP: green fluorescent protein used as reporter protein. 2Ter: double terminator consists of rrnB T1 terminator and rrnB T2 terminator. AmpR: ampicillin resistance protein.
Fig. 2. Schematic diagram of machenism of promoter PtisAB. PtisAB: a promoter sensitive to CIP; RBS: ribosome binding site; qnrS: an anti-CIP protein. LuxI: a protein converts SAM into AHL. LexA: repressor inhibits PtisAB from initiating.
Degradation of ciprofloxacin
In order to quickly produce a large number of degrading enzymes after CIP is detected, we add quorum sensing system to piGEM2019-02 (Fig.3). 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 (PLuxR) [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.4. Under natural conditions, 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. Therefore, we speculate that it has certain value [5] [6].
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.
CrpP is a novel ciprofloxacin-modifying enzyme which can phosphorylate and degrade CIP [4]. The degradation pathway is shown in Fig.4. Under natural conditions, 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. Therefore, we speculate that it has certain value [5] [6].
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.
Fig. 3. Schematic map of piGEM2019-02. PelB-5D: signal peptide. CrpP: a novel CIP-modifying enzyme found in 2018.
TagRFP: a kind of red fluorescent protein used as reporter protein. RBS: ribosome binding site. 2Ter: double terminator consists of rrnB T1 terminator and T7Te terminator. J23100: a constitutive promoter. LuxR: a protein that can combine with AHL, then the complex can activate PLuxR. KanR: kanamycin resistance protein.
Fig. 4. Reaction mechanism of CIP inactivation and biodegradation of phosphorylated CIP.
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].
Kill Switch
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.5).
Fig. 5. Schematic map of piGEM2019-03. YF1: encodes a kinase; FixJ: is phosphorylated by YF1 in darkness and unphosphorylated in light; PFixK2: a promoter that can be activated by phosphorylated FixJ; Lysep3-D8: a kind of protein that lyses cells; Ter: a terminator; RBS: ribosome binding site; AmpR: ampicillin resistance gene.
References
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[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.
[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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[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.