(a) piGEM2019-01 digested by XbaⅠ+ VspⅠ (lane 1), piGEM2019-01 digested by SalⅠ+ VspⅠ (lane 2);
(b) piGEM2019-02 digested by SalⅠ+ KpnⅠ (lane 1), piGEM2019-01 digested by Hind Ⅲ + SacⅠ (lane 2);
(c) piGEM2019-03 digested by NdeⅠ+ BamHⅠ (lane 1).
qnrS1 — CIP resistance gene
E.coli DH5α carrying piGEM2019-01 was used to detect ciprofloxacin and produce AHL to activate piGEM2019-02. In order to achieve the better detection function, our detection bacteria must survive in a relatively high concentration of CIP. So, we added a ciprofloxacin resistance gene — qnrS1. We tested qnrS1 by adding gradient concentrations of CIP and measuring the growth curve (Fig. 2).
Moreover, we defined the Relative Bacterial Density to represent the resistance ability of qnrS1. The higher the Relative Bacterial Density is, the stronger the resistance will be. When the value was less than 1, it meant that the growth was suppressed. And we chose the experimental data of 1 mg/L CIP to analyze the Relative Bacterial Density defined above (Fig. 3). It could be seen intuitively from Fig. 3 that E.coli DH5α carrying piGEM2019-01 grew normally. Meanwhile, the values of control were all less than 1. It could be concluded that qnrS1 enhanced the viability of E.coli DH5α carrying piGEM2019-01 in CIP.
PtisAB — CIP responding promoterWhether piGEM2019-01 actually has detection function depends on whether PtisAB responds to CIP. The promoter activity of PtisAB can be detected by green fluorescence intensity . Fig.4 shows fluorescence intensity at different CIP concentrations. It was observed that the fluorescence intensity in E.coli DH5α carrying piGEM2019-01 was significantly stronger than control, implying that PtisAB successfully made response to CIP. We also found that 1 mg/L is the most appropriate concentration for PtisAB.
Detection of CIP by HPLC-UVConsidering the sensitivity and accuracy of High Performance Liquid Chromatography (HPLC) tandem UV detector, we chose HPLC to monitor the degradation of CIP by CrpP. A standard curve of ciprofloxacin was established by HPLC-UV according to the procedure described in literature . Within the range of 50–300 μg/L, the CIP concentration and the peak area showed a good linear relationship (Fig. 6).
Degradation of CIP by CrpP (HPLC-UV detection)After co-transforming piGEM2019-01 and piGEM2019-02 into E.coli DH5α, we added 1 mg/L CIP to induce the expression of CrpP at 37℃ for 12h. Next we used the cell extracts to try to degrade CIP. Although HPLC data gave no significant changes in peak shape of CIP after the treatment, T-test analysis revealed that there was a slight difference between the experimental group (CrpP+) and the control group (CrpP-) (Fig. 7).
CrpP -: E.coli DH5α (piGEM2019-01, without crpP); CrpP +: E.coli DH5α (piGEM2019-01 + piGEM2019-02, expressing CrpP enzyme)
Expression verification of CrpPIn order to find the reason why CrpP had the low enzymatic activity, we conducted SDS-PAGE on the cell extracts. It was surprisingly found that TagRFP was successfully expressed at about 25kD, however, we couldn't distinguish the band of CrpP we expected (Fig. 8a). The poor expression should be responsible for the low enzymatic activity we measured above. In order to make CrpP express better, we inserted corresponding sequences into an IPTG induced vector (pEASY) , where E.coli BL21 (DE3) was used as host cell. The induction of 0.5 mM IPTG gave rise to the appearance of one band at 17 kDa and no similar bands were detected in control group (Fig. 8b). The results indicated that CrpP was successfully expressed in E.coli BL21 (DE3) induced by 0.5 mM IPTG.
CrpP activity by coupled enzymatic assayAfter CrpP was expressed in E.coli BL21 (DE3) (pEASY-crpP), we used a coupled enzymatic assay involving NADH oxidation to measure the activity of cell disruption solution on CIP, as described by Víctor M. Chávez-Jacobo . Fig. 9 presented that NADH oxidation rate of the experimental group (0.5 mM IPTG+) , the control group (0.5 mM IPTG-) and the blank group (PBS buffer). The initial values of the experimental group and the control group were the amount of NAD+ relative to the blank group at 0.5 min. When compared with the control and the blank, NADH oxidation rate was significantly increased following the induction of IPTG, indicating that CrpP expressed here can indeed degrade CIP.
Quorum sensing systemNext, we used E.coli DH5α co-transformed with piGEM2019-01 and piGEM2019-02 to verify quorum sensing . If it can work, the addition of CIP will induce the expression of TagRFP. As we expected, the bacterial liquid in experimental group turned red (seen from Fig. 10). No similar result was observed in control group. This proves that our quorum sensing system can work normally.
Immobilization of bacteriaIn addition, we mixed the E.coli bacteria solution carrying piGEM2019-02 with sodium alginate solution, and dropped the mixture into CaCl2 through the needle (Fig. 11) . We successfully immobilized the bacteria.
Improve our ciprofloxacin disposal system1. Enhance the enzyme activity of CrpP
According to the references, the Km of CrpP is relatively high, indicating the affinity is weak. So, we want to enhance the enzyme activity by site-directed mutation or other methods. As CrpP is a new discovered enzyme, there is a lot of space for improvement. And we can also use our degradation optimization model to find optimal external factors to increase degradation rate.
2. Optimize the expression system
As we want more CrpP enzyme to express, we can enhance PtisAB's response to CIP or just choose a stronger quorum sensing system to enable the expression quantity.
Extend the future market of our hardwareOur device is working normally at present, and we want it to be useful at more situations. In future, we can change PtisAB and CrpP to other specific promoters or genes, so that it can be used to degrade other kinds of antibiotics, and this will help a lot in dealing with the abuse of antibiotics. Besides, it can be better used in larger scenarios, such as sewage plant, laboratory wastewater treatment or pharmaceutical wastewater treatment and so on.
Perfect our safety systemCause our project is based on dealing antibiotics, safety is the prime objective. Although our safety system is working normally, we have to make further improvements to ensure safety. We will continue to work on the blue-light sensitive promoter, using blue light and this promoter to confine our engineered bacteria to our trash can. In addition, we will import a lysin protein with better cell lysis effect to ensure the death of our engineered bacteria cooperating with ultraviolet. In this case, we will do our best to ensure our biosecurity.
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