Difference between revisions of "Team:UESTC-China/Demonstrate"

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 Table1.

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).

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).

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 a significant concentration 1mg/L to represent the growth situation (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.

P_tisAB —— CIP responding promoter

Whether piGEM2019-01 actually has detection function depends on whether P_tisAB responds to CIP. Fluorescence intensity was tested at different CIP concentrations every two hours (Fig. 4) [1]. The results showed that the fluorescence intensity in E.coli DH5α carrying piGEM2019-01 was significantly stronger than control.

To determine that our green fluorescence is produced by ciprofloxacin-induced promoter expression, we performed the same experimental procedure on WTII (with an arabinose-inducible promoter), in order to make sure that other promoters won’t be induced by CIP. Besides, 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).

For E.coli DH5α carrying piGEM2019-01, we could see P_tisAB responds differently to CIP at different concentrations, among them, 1mg/L is the most appropriate response concentration for P_tisAB.

Besides, we could infer the concentration of ciprofloxacin from the green fluorescence intensity. At a concentration of 0-1 mg/L of ciprofloxacin, its relationship with green fluorescence intensity is basically in accordance with y = 0.3698x + 0.5477. R² = 0.9721 (y: the green fluorescence intensity; x: the ciprofloxacin concentration).

Detection of CIP by HPLC

Considering the sensitivity and accuracy of High Performance Liquid Chromatography (HPLC), 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 K_m and V_max 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 low down 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 (Fig. 6) as a protocol described [3]. Within this range, the CIP concentration and the peak area shows a good linear relationship.

Degradation of CIP by CrpP

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 at a 1:100 ratio 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+, E.coli express CrpP enzyme) and control group (CrpP-, E.coli without crpP gene) 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 a 1.9mL mix including 2 mM ATP, with or without cell extracts that contain CrpP, at 37℃ for 30 minutes. Our experimental group (CrpP+) has 30 samples and control group (CrpP-) has 6 samples. 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 group 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. T-test 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.

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 above. But from the result (Fig. 8a), we could only see the band of TagRFP, indicating that the expression system was working. But we couldn’t distinguish which one was the band of CrpP (CrpP expression quantity is low). In order to make CrpP better express so as to be able to be detected, we inserted pelB-5D, CrpP and TagRFP into an IPTG induced vector (pEASY) and transformed it into E.coli BL21 (DE3). More CrpP protein would been obtained by IPTG induction, and the concentration of IPTG was 0.5mM. As shown in Fig. 8b, there were obvious bands around 17kDa in the induction group. The results indicated that CrpP was successfully expressed in E.coli BL21 (DE3) induced by IPTG.

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 crude enzyme on CIP, as described by Víctor M. Chávez-Jacobo [2]. NADH oxidation rate of the experimental group (IPTG+) , the control group (IPTG-) and the blank group are shown in Fig. 9. The blank group only uses PBS buffer solution. The experimental (control) group is cell disruption solution induced with (without) 0.5 mM IPTG. Our experimental data illustrates that, in the presence of CrpP, NADH oxidation increases with the reaction time, showing that CrpP can indeed degrade CIP.

Quorum sensing system

Next, we wanted to verify quorum sensing using co-transformed E.coli DH5α. CIP was added to the experimental group, but not to the control group. After the same time, the bacterial liquid in the experimental group turned red, indicating that TagRFP was normally expressed, while the control group did not express TagRFP (Fig. 10). Based on this result, we can verify that AHL produced by piGEM2019-01 can normally activate the expression of piGEM2019-02. This proves that our quorum sensing system can work normally.

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.

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.

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.

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).

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.

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.

Improve our ciprofloxacin disposal system

1. Enhance the enzyme activity of CrpP
According to the references, the K_m 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 P_tisAB'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 can do quantitative detection of fluorescence and degrade CIP at present, and we want it to be useful at more situations. In future, we can make some improvements to it 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 safety system

Cause our project is based on antibiotics dealing, safety is the prime objective.

[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.