Team:UESTC-China/Demonstrate
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 expression vectors by using Gibson Assembly and Golden Gate strategies. The resulting vectors piGEM2019-01, piGEM2019-02, piGEM2019-03 are listed in Table 1.
| 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).
(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.
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 expression of PtisAB can be detected by green fluorescence intensity. Fluorescence intensity was tested at different CIP concentrations(Fig. 4) [1]. The results showed that the fluorescence intensity in E.coli DH5α carrying piGEM2019-01 was significantly stronger than control, implying that PtisAB responded to CIP.
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).
We narrowed the range of concentration gradients to find the most appropriate concentration of ciprofloxacin and the linear relationship between green fluorescence intensity and CIP concentration(Fig. 5). In this experiment, we choose Wild-type Ⅱ as the control (with an arabinose-inducible promoter), since arabinose-inducible promoter won’t be induced CIP.
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).
For E.coli DH5α carrying piGEM2019-01, we could see PtisAB responds differently to CIP at different concentrations, comparison shows that 1 mg/L is the most appropriate response concentration for PtisAB.
Besides, we could infer the concentration of ciprofloxacin from the green fluorescence intensity. 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).
Besides, we could infer the concentration of ciprofloxacin from the green fluorescence intensity. 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).
Ciprofloxacin Degradation
Detection of CIP by HPLC-UV
Considering the sensitivity and accuracy of High Performance Liquid Chromatography (HPLC) tandem UV detector, we chose it to monitor the degradation of CIP by CrpP. However, during our experiment, we found that the concentration of CIP inferred from the Km and Vmax given by Víctor M. Chávez-Jacobo., et al. (2018) [2] (≈ 150 mg/L) is beyond the response range of our UV detector, so we had to reduce the concentration of CIP. Therefore, we first need to establish a standard curve of ciprofloxacin concentration for the follow-up verification of the CIP-degrading function of CrpP enzyme.A standard curve of 50-300 μg/L ciprofloxacin was established by HPLC-UV (Fig. 6) as a protocol described [3]. Within this range, the CIP concentration and the peak area shows a good linear relationship.
Fig. 6. The standard curve of CIP concentration detected by HPLC-UV.
Degradation of CIP by CrpP (HPLC-UV detection)
Then we co-transformed piGEM2019-01 and piGEM2019-02 into E.coli DH5α, and transformants were selected on LB agar plates using ampicillin and kanamycin. Overnight cultures of E. coli DH5α (piGEM2019-01 + piGEM2019-02) were diluted into 25 mL of fresh LB medium and were cultured at 37°C. When the culture was shaked for 1.5 hours, we added 1 mg/L CIP to induce the expression of CrpP, and the culture was incubated for additional 12 hours. Cells were harvested by centrifugation, and the pellets were suspended in 5mL PBS Buffer (pH = 7.4). Then the mix was treated with sonication. We used the cell extracts to degrade CIP, but it was difficult to tell the difference between experimental group (CrpP+) and control group (CrpP-) directly through peak shape in such a low CIP concentration.In order to solve the problem mentioned above, statistics methods were introduced. Same concentration of CIP was incubated in both groups in a 1.9mL mix including 2 mM ATP, with or without cell extracts that contain CrpP, at 37℃ for 30 minutes. Reaction was terminated by adjusting pH to about 2 (by adding Concentrated Hydrochloric Acid). It is reasonable to reckon that the average concentration of CIP of both groups has no difference in the beginning, but we can clearly notice that the experimental group's average concentration is lower than that of control group (Fig. 7). Moreover, T-test for comparison of pooled data mean was introduced to analyze our results. The results (P = 0.036) indicate that in the case of a probability of error of less than 5%, there is a significant difference between the experimental group (CrpP+) and the control group (CrpP-). Thus, we can come to the conclusion that CrpP has the capability of degrading CIP.
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)
CrpP -: E.coli DH5α (piGEM2019-01, without crpP); CrpP +: E.coli DH5α (piGEM2019-01 + piGEM2019-02, expressing CrpP enzyme)
Expression verification of CrpP
Although we used statistical methods to test the function of CrpP, we did not get the desired effect directly, so we decided to verify the expression of CrpP. We treated E.coli DH5α (piGEM2019-01 + piGEM2019-02) according to the procedure mentioned in section Degradation of CIP by CrpP (HPLC-UV). As shown in Fig. 8a, we observed one band at 17 kDa corresponding to TagRFP, indicating that the expression system was working. Unfortunately, we couldn’t distinguish the band of CrpP we expected (CrpP expression quantity is low). In order to make CrpP better express, we inserted corresponding sequences into an IPTG induced vector (pEASY) and 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. 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), 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 [2]. 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). 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 embedded the bacteria that could degrade CIP and immobilized the bacteria.
Fig. 11. Immobilization of bacteria. Left: sodium alginate solution. Right: sodium alginate pellets embedded E.coli DH5α carrying piGEM2019-02.
Kill switch
In order to ensure the biosecurity of our project, we used piGEM2019-03 to accomplish our purpose. First of all, we tested the lysin gene lysep3-D8 separately, by inserting it into an expression vector induced by IPTG (final concentration is 0.5 mM) [5,6]. Transformed it into E.coli BL21 (DE3). And we measured the growth curve to represent intracellular cleavage effect (Fig. 12). As Fig. 12 shown, the Lysep3-D8 protein inhibited the growth of E.coli BL21, and the inhibition rate was about 30.7%. The Lysep3-D8 protein can only inhibit the growth but can’t kill it.
Fig. 12. Growth curve of E.coli BL21 carrying lysep3-D8 gene. Orange line: IPTG-; Grey line: IPTG+.
Then, we tested the blue-sensitive promoter switch, but it hasn’t worked yet. We will continue to test its function in future experiments.
From the above results, the Lysep3-D8 protein induced by IPTG strong inducer 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.
From the above results, the Lysep3-D8 protein induced by IPTG strong inducer 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.
Hardware
In order to detect ciprofloxacin in our environment using E.coli DH5α carrying piGEM2019-01, we designed and manufactured a lightweight fluorescence detection device that we use to detect green fluorescence. The entire device is modular in design and most 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 (Fig. 14). As we could see from the result, the standard curve of our detection device has the same trend with the microplate reader, indicating our hardware has the ability to detect the CIP concentrations in environment.
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 shown our device has excellent performance.
Fig. 15. The connection between voltage of the device and the concentration of CIP.
Work going on
Improve our ciprofloxacin disposal system
1. Enhance the enzyme activity of CrpPAccording 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.
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.
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. Nucleic acids research, 36(19), 6249-6259.
[2] 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.
[3] China Medical Science and Technology Press. (2015). Pharmacopoeia of the People's Republic of China, Volume 2. Beijing.
[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.
[2] 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.
[3] China Medical Science and Technology Press. (2015). Pharmacopoeia of the People's Republic of China, Volume 2. Beijing.
[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.