Pathway construction

For efficient expression of multiple enzymes in E.coli, codon optimization of all target genes were performed before DNA synthesis. The obtained genes were subsequently cloned into different vectors by using Gibson Assembly and Golden Gate strategies. The resulting vectors piGEM2019-01, piGEM2019-02, piGEM2019-03 are listed in Table 1.

Illustration of three constructed vectors

No. Vector E.coli resistance Description
1 piGEM2019-01 Amp PtisAB+LuxI+GFP+AmpR+ori+J23119+QnrS
2 piGEM2019-02 Kan PLuxR+PelB-5D+CrpP+TagRFP+J23100+LuxR+KanR+p15Aori
3 piGEM2019-03 Amp J23100+YF1+FixJ+Ter+PFixK2+Lysep3-D8+Ter+AmpR+ori
Before DNA sequencing, those vectors were verified by restriction enzyme digestion. After electrophoresis analysis, the samples which contained the desired bands were selected and sent for sequencing. The sequencing results showed that all the vectors constructed above were successful (Fig. 1).
Fig. 1. Double restriction enzyme digestion of three constructed vectors analyzed by using agarose gel electrophoresis.
(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).

Ciprofloxacin detection

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).

Fig. 2. Growth curve of control (qnrS1-, a) and E.coli DH5α carrying piGEM2019-01 (qnrS1+, b) in LB containing 0, 0.3, 1, 10, 50 mg/L of CIP.
From the results, we determined the MIC value of E.coli DH5α carrying piGEM2019-01 was between 10 – 50 mg/L while the MIC value of control was between 0.3 – 1 mg/L.

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.
Fig. 3. Relative Bacterial Density (OD600) of E.coli DH5α carrying piGEM2019-01 (qnrS1+) and Control (qnrS1-) at 1 mg/L CIP. Relative Bacterial Density is defined as the OD600 corresponding to 1 mg/L CIP devided by the OD600 corresponding to 0 mg/L CIP. The OD600 corresponding to 0 mg/L CIP is 0.19.

PtisAB — CIP responding promoter

Whether 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 [1]. 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.
Fig. 4. Fold of Fluorescence Intensity per OD for control (PtisAB-, a) and E.coli DH5α carrying piGEM2019-01 (PtisAB+, b). (Fold means GFP unit fluorescence after 2/4/6 h of exposure to CIP normalized to initial unit fluorescence. Unit fluorescence is fluorescence intensity per OD).
Next, we narrowed the range of CIP concentration and found that there was a linear relationship between green fluorescence intensity and CIP concentration (Fig. 5). At a concentration of 0 – 1 mg/L of ciprofloxacin, it followed the formula y = 0.3698x + 0.5477. R2 = 0.9721 (y: the green fluorescence intensity; x: the ciprofloxacin concentration). No similar phenomenon was observed for the control group.
Fig. 5. Fold of Fluorescence Intensity per OD for E.coli DH5α carrying piGEM2019-01 (PtisAB+) and control (PtisAB-). (Fold means GFP unit fluorescence after 2h of exposure to CIP normalized to initial unit fluorescence. Unit fluorescence is fluorescence intensity per OD).

Ciprofloxacin Degradation

Detection of CIP by HPLC-UV

Considering 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 [2]. Within the range of 50–300 μg/L, the CIP concentration and the peak area showed a good linear relationship (Fig. 6).
Fig. 6. The standard curve of CIP concentration detected by HPLC-UV.

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).
Fig. 7. Degradation of CIP using cell extracts.
CrpP -: E.coli DH5α (piGEM2019-01, without crpP); CrpP +: E.coli DH5α (piGEM2019-01 + piGEM2019-02, expressing CrpP enzyme)

Expression verification of CrpP

In 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.
Fig. 8. SDS-PAGE analysis of cell extracts of engineered E.coli. In panel (a), lane 1,2: control group (E.coli DH5α (piGEM2019-01)); lane 3,4: experimental group(E.coli DH5α (piGEM2019-01 + piGEM2019-02)). In panel (b), lane 1,2: experimental group E.coli BL21 (DE3) (pEASY-crpP) (0.5 mM IPTG +); lane 3,4: control group E.coli BL21 (DE3) (pEASY-crpP) (0.5 mM IPTG -). The bands of TagRFP and CrpP (with Histag) are shown as arrows.

CrpP activity by coupled enzymatic assay

After 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 [3]. 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.
Fig. 9. NADH oxidation rate comparisons between the experimental group with CrpP expression (IPTG+), the control group without CrpP expression (IPTG-) and the blank group (PBS). Data are means from three independent duplicate assays, with standard errors.

Quorum sensing system

Next, 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.
Fig. 10. The effect of CIP on the engineered E.coli DH5α (piGEM2019-01 + piGEM2019-02) growing in fluid medium. Left: control group (without CIP induction); Right: exprimental group (with CIP induction).

Immobilization of bacteria

In 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) [4]. We successfully immobilized the bacteria.
Fig. 11. Immobilization of engineered bacteria. Left: the mixture of sodium alginate solution and E.coli DH5α carrying piGEM2019-02; Right: the embedded E.coli DH5α.

Kill switch

In order to check if piGEM2019-03 is working, we investigated the growth situation of engineered E.coli under the irradiation of blue light. Unfortunately, the engineered E.coli could still survive in the darkness (data not shown). Next, we tested whether lysin gene lysep3-D8 can work independently by inserting corresponding sequences into an IPTG-inducing vector pEASY [5, 6]. It was found from Fig. 12 that the growth of engineered E.coli BL21 (DE3) was suppressed after the induction of 0.5 mM IPTG, and the inhibition rate was about 30.7%. Although the efficiency was not high, we could conclude that lysep3-D8 gene was working. We also checked the function of the promoter, but it hasn't worked yet.
Fig. 12. Growth curve of E.coli BL21 carrying lysep3-D8 gene. Orange line: IPTG-; Grey line: IPTG+.
From the above results, the Lysep3-D8 protein induced by IPTG has the effect of inhibiting growth. Combined with the second safety mechanism-UV sterilization mechanism, we have minimized the risk of leakage of engineered bacteria and made the biosafety of the hardware a strong guarantee.
In order to detect ciprofloxacin in our environment using E.coli DH5α carrying piGEM2019-01, we designed and manufactured a lightweight fluorescence detection device. The entire device is modular in design and most modules can be made using 3D printing technology (Fig. 13).
Fig. 13. Detection device.
To verify the function of our detection device, we compared it with a microplate reader (Thermo Scientific Varioskan LUX). And a standard curve of fluorescence intensity was measured. As we could see from Fig. 14, the standard curve of our detection device has the same trend with the microplate reader, indicating our hardware has the ability to detect the fluorescence on the cellular level.
Fig. 14. The comparison of our fluorescence detection device and a microplate device.
We used our detection device to measure fluorescence intensity of E.coli DH5α carrying piGEM2019-01 at different CIP concentrations (Fig. 15). It showed that the collected voltage was linearly dependent on the concentration of CIP. We can roughly conclude that our device was suitable for quantitative analysis of CIP. In the near future, we aim to optimize and develop this device to monitor CIP in the environment.
Fig. 15. The connection between voltage of the device and the concentration of CIP.
We also manufactured one CIP-degradation device, where the immobilized engineered bacteria were injected into the pipeline. We have also set up the circuit to operate the device smoothly. It will be feasible to combine the detection and degradation device together to achieve more function.
Fig. 17. CIP-degradation device
Work going on

Improve our ciprofloxacin disposal system

1. 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 hardware

Our 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 system

Cause 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.
[1]Dörr, T., Vulić, M., & Lewis, K. (2010). Ciprofloxacin causes persister formation by inducing the TisB toxin in Escherichia coli. PLoS biology, 8(2), e1000317.
[2]China Medical Science and Technology Press. (2015). Pharmacopoeia of the People's Republic of China, Volume 2. Beijing.
[3]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. Antimicrobial agents and chemotherapy, 62(6), e02629-17.
[4]Cho, E., Jang, G., Kim, D., & Lee, T. S. (2017). Fabrication of hollow-centered sodium-alginate-based hydrogels embedded with various particles. Molecular Crystals and Liquid Crystals, 659(1), 71-76.
[5]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.
[6]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.
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