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. In addition, we added a Lux quorum sensing system to enhance the expression of CrpP by E.coli. Moreover, we have designed an anti-escape light-controlled kill switch piGEM2019-03 to prevent bacteria containing crpP gene from escaping and causing more biosecurity and environmental problems.
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].
When PtisAB senses ciprofloxacin, green fluorescent protein (GFP) and LuxI will be expressed. By measuring the fluorescence intensity, 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].
PtisAB
LuxI
Fig. 1. Schematic map of piGEM2019-01. PtisAB : a promoter sensitive to CIP;
RBS: ribosome binding site; qnrS : gene encoding an anti-CIP protein; LuxI : gene coding for a protein which can convert SAM into AHL; gfp : gene encoding green fluorescent protein used as reporter protein; 2Ter : double terminator consists of rrnB T1 terminator and rrnB T2 terminator; AmpR : gene encoding ampicillin resistance protein.
Degradation of ciprofloxacin
In order to quickly produce a large number of degrading enzymes after CIP is detected, we added Lux quorum sensing system (Fig. 2). LuxR is a constitutively expressed protein that can bind AHL. When bound to AHL (produced by piGEM2019-01), LuxR can activate the right hand Lux promoter (PLuxR) [2] , and then initiate the 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 reported that 1, 4-dihydroquinoline can be used as carriers for specific brain delivery on mice [5,6], indicating that it is valuable.
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, a red fluorescent protein, is used as a reporter protein to response if CrpP is expressed or not.
LuxR
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 reported that 1, 4-dihydroquinoline can be used as carriers for specific brain delivery on mice [5,6], indicating that it is valuable.
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, a red fluorescent protein, is used as a reporter protein to response if CrpP is expressed or not.
Fig. 2. Schematic map of piGEM2019-02. PelB-5D : gene encoding signal peptide; CrpP : gene coding for a novel CIP-modifying enzyme found in 2018; tagrfp : gene encoding 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 : gene encoding a protein that can combine with AHL, then the complex can activate PLuxR; KanR : gene encoding kanamycin resistance protein.
Fig. 3. Reaction mechanism of CIP degradation catalyzed by CrpP originally from the literature [4] with some modifications.
In order to keep our engineered bacteria living longer in the drug solution, we used sodium alginate (SA) as entrapping agent to immobilize the engineered E.coli carrying piGEM2019-02. This immobilization method has 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 survive only when it is exposed to blue light [9,10] (Fig. 4).
Fig. 4. Schematic map of piGEM2019-03. YF1 : gene encodeing a kinase; FixJ : is phosphorylated by YF1 in darkness and unphosphorylated in light; PFixK2 : encodes a promoter that can be activated by phosphorylated FixJ; Lysep3-D8 : encodes a kind of protein that lyses cells; Ter : a terminator; RBS : ribosome binding site; AmpR : encodes ampicillin resistance gene.
References
[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.
[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.