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 Table1.
| No. | Vector | E.coli resistance | Description |
|---|---|---|---|
| 1 | piGEM2019-01 | Amp | PtisAB+LuxI+GFP+AmpR+ori+J23119+qnrS1 |
| 2 | piGEM2019-02 | Kan | PLuxR+PelB-5D+CrpP+6×His+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 above constructed vectors 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 HindⅢ+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 HindⅢ+SacⅠ(lane 2);
(c) piGEM2019-03 digested by NdeⅠ+BamHⅠ(lane 1).
Ciprofloxacin detection
QnrS1 enhances the CIP resistance of E.coli
QnrS1 prevents E.coli from death
QnrS1 prevents E.coli from death
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 Negative Control(a) and E.coli DH5α carrying piGEM2019-01(b) at 0, 0.3, 1, 10, 50mg/L of CIP.
From the results, we determined the MIC value of E.coli DH5α carrying piGEM2019-01 was between 10-50mg/L while the MIC value of negative control was between 0.3-1mg/L.
Moreover, we defined the Relative Bacterial Density to represent the resistance ability of qnrS1. The higher the Relative Bacterial Density, the stronger the resistance. 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 negative 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, the stronger the resistance. 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 negative 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 and Negative Control at 1mg/L CIP. Relative Bacterial Density = The OD600 corresponding to 1mg/L CIP / The OD600 corresponding to 0mg/L CIP(0.19)
PtisAB responds to CIP successfully
Whether piGEM2019-01 actually has detection function depends on whether PtisAB 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 negative control.
Whether piGEM2019-01 actually has detection function depends on whether PtisAB 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 negative control.
Fig. 4. Fold of Fluorescence Intensity (FI) in unit OD for negative control(a) and E.coli DH5α carrying piGEM2019-01(b). (Negative control carries a plasmid which only have AmpR. Fold induction is GFP unit fluorescence after 2/4/6 h of exposure normalized to initial unit fluorescence. Unit fluorescence=green fluorescence of 1.5ml bacteria/OD600 of 0.2ml bacteria).
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).
Fig. 5. Fold of Fluorescence Intensity (FI) in unit OD for E.coli DH5α carrying piGEM2019-01 and negative control. (Negative control carries a plasmid which has AmpR and GFP, and GFP is initiated by the IPTG-induced promoter. Fold means GFP unit fluorescence after 2h of exposure normalized to initial unit fluorescence. Unit fluorescence=green fluorescence of 1.5ml bacteria/OD600 of 0.2ml bacteria).
For E.coli DH5α carrying piGEM2019-01, we could see PtisAB responds differently to CIP at different concentrations, among them, 1mg/L is the most appropriate response concentration for PtisAB.
Besides, we could infer the concentration of ciprofloxacin based on the green fluorescence intensity. At a concentration of 0-1mg/L of ciprofloxacin, its relationship with green fluorescence intensity is basically in accordance with y = 0.3698x + 0.5477. R2 = 0.9721. Where y is the green fluorescence intensity and x is the ciprofloxacin concentration.
Besides, we could infer the concentration of ciprofloxacin based on the green fluorescence intensity. At a concentration of 0-1mg/L of ciprofloxacin, its relationship with green fluorescence intensity is basically in accordance with y = 0.3698x + 0.5477. R2 = 0.9721. Where y is the green fluorescence intensity and x is the ciprofloxacin concentration.
Ciprofloxacin Degradation
Construct CIP concentration standard curve by HPLC
E.coli BL21(DE3) carrying piGEM2019-02 was used to degrade ciprofloxacin. CrpP is the most important enzyme to realize this function in the pathway which can validated by monitoring the degradation of ciprofloxacin. Therefore, we first need to establish a ciprofloxacin standard curve for the follow-up verification of the function of CrpP enzyme.
A standard curve of 50-300ug/L concentration of ciprofloxacin was established by HPLC (Fig. 6)[2]. As shown in Table.2, we successfully used the standard curve to test for ciprofloxacin in two common drugs. Between them, the content of ciprofloxacin detected by us in the ciprofloxacin hydrochloride suppository is almost completely consistent with that in the drug specification. The results show that our method of detecting ciprofloxacin is applicable and accurate.
E.coli BL21(DE3) carrying piGEM2019-02 was used to degrade ciprofloxacin. CrpP is the most important enzyme to realize this function in the pathway which can validated by monitoring the degradation of ciprofloxacin. Therefore, we first need to establish a ciprofloxacin standard curve for the follow-up verification of the function of CrpP enzyme.
A standard curve of 50-300ug/L concentration of ciprofloxacin was established by HPLC (Fig. 6)[2]. As shown in Table.2, we successfully used the standard curve to test for ciprofloxacin in two common drugs. Between them, the content of ciprofloxacin detected by us in the ciprofloxacin hydrochloride suppository is almost completely consistent with that in the drug specification. The results show that our method of detecting ciprofloxacin is applicable and accurate.
Fig. 6. The standard curve of CIP concentration for HPLC peak area.
| Drug Name | Detection |
|---|---|
| Ciprofloxacin hydrochloride eye drops | 1.57g/L |
| Ciprofloxacin hydrochloride suppository | 0.24g in 1.785g |
CIP is degraded by CrpP
In order to detect CrpP’s function, High Performance Liquid Chromatography (HPLC) was chosen. Although HPLC has a relatively higher sensitivity, the concentration of CIP inferred from the Km and Vmax given by Víctor M. Chávez-Jacobo et al.(2018) [3] is beyond HPLC’s limit of detection(LOD). So, we had to low down the CIP’s concentration. However, 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(＜1mg/L).
In order to detect CrpP’s function, High Performance Liquid Chromatography (HPLC) was chosen. Although HPLC has a relatively higher sensitivity, the concentration of CIP inferred from the Km and Vmax given by Víctor M. Chávez-Jacobo et al.(2018) [3] is beyond HPLC’s limit of detection(LOD). So, we had to low down the CIP’s concentration. However, 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(＜1mg/L).
Fig. 7. Degradation of CIP by CrpP.
(CrpP-: E.coli without crpP gene;CrpP+: E.coli express CrpP)
Statistics methods were introduced to help to solve this problem. Same concentration of CIP was incubated in a 1.9mL mix including 2 mM ATP, with or without cell extract that contains CrpP, at 37℃ for 30 minutes. Our experimental group(CrpP+) has 30 samples and control group(CrpP-) has 6 samples. Reaction was terminated by adding 2μL 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 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.
Varify the expression of CrpP-His
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 first. We co-transformed piGEM2019-01 and piGEM2019-02 into E.coli DH5α, and SDS-PAGE was selected to monitor CrpP’s expression. 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. In order to make CrpP better expression so as to be able to be detected, we inserted pelB-5D, CrpP, Histag and TagRFP into an IPTG induced vector(pEASY) and transformed it into E.coli BL21(DE3). More CrpP protein would also obtain more 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).
Varify the expression of CrpP-His
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 first. We co-transformed piGEM2019-01 and piGEM2019-02 into E.coli DH5α, and SDS-PAGE was selected to monitor CrpP’s expression. 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. In order to make CrpP better expression so as to be able to be detected, we inserted pelB-5D, CrpP, Histag and TagRFP into an IPTG induced vector(pEASY) and transformed it into E.coli BL21(DE3). More CrpP protein would also obtain more 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).
Fig. 8. SDS-PAGE analysis of TagRFP and CrpP from E.coli DH5α co-transformed piGEM2019-01 and piGEM2019-02(a) and E.coli BL21(DE3) transformed pEASY with pelB-5D, CrpP, Histag and TagRFP(b). In (a), 3,4 were the IPTG induction group, 1,2 were the control group. In (b), 1,2 were the IPTG induction group, 3,4 were the control group. The band at 25kDa is TagRFP, and the band at 17kDa is CrpP with Histag.
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.9). Based on this result, we can verify that AHL produced by plasmid piGEM2019-01 can normally activate the expression of plasmid piGEM2019-02. This proves that our quorum sensing system can work normally.
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.9). Based on this result, we can verify that AHL produced by plasmid piGEM2019-01 can normally activate the expression of plasmid piGEM2019-02. This proves that our quorum sensing system can work normally.
Fig. 9. The validation of quorum sensing. Left one is control group(without CIP-induced), and right one is exprimental group(with CIP-induced).
Immobilization of bacteria
In addition, we mixed the E.coli bacteria solution carrying plasmid piGEM2019-02 with sodium alginate solution, and dropped the mixture into CaCl2 through the needle (Fig. 10)[4]. We successfully embedded the bacteria that could degrade CIP and immobilized the bacteria.
In addition, we mixed the E.coli bacteria solution carrying plasmid piGEM2019-02 with sodium alginate solution, and dropped the mixture into CaCl2 through the needle (Fig. 10)[4]. We successfully embedded the bacteria that could degrade CIP and immobilized the bacteria.
Fig. 10. Immobilization of bacteria. The left one is sodium alginate pellets embedded E.coli DH5α carrying piGEM2019-02. The right one is sodium alginate solution.
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 0.5mM)[5]. Transformed it into E.coli BL21(DE3). And we measured the growth curve to represent intracellular cleavage effect (Fig. 11). As Fig. 10 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. 11. Growth curve of E.coli BL21 carrying Lysep3-D8 protein with IPTG-induction and without IPTG-induction.
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. 12).
Fig. 12. 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. 13). 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. 13. 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. 14). It shown our device has excellent performance.
Fig. 14. 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 CrpP
According to the references, the Km of CrpP is pretty 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, it’s easy to remold it.
2. Improve 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.
3. Further application
Our system is working normally at present, and we want it to be useful at more situations. We can change PtisAB and CrpP to other specific promoters and genes. And our system can be used to degrade other kinds of antibiotics, and this will help a lot in dealing with the abuse of antibiotics.
Improve our hardware
At present, our hardware is only a small device, and it works perfectly. In future, we can make some improvements to it so that it can be better used in larger scenarios, such as sewage plant, laboratory wastewater treatment or pharmaceutical wastewater treatment and so on.
Improve our safety
As for further experiments, it’s important for us to improve the function of blue-sensitive promoter and lysin protein. Match the darkroom and ultraviolet light in the device to prevent the escape of engineered bacteria, providing a double guarantee for the biosafety of our project.
1. Enhance the enzyme activity of CrpP
According to the references, the Km of CrpP is pretty 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, it’s easy to remold it.
2. Improve 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.
3. Further application
Our system is working normally at present, and we want it to be useful at more situations. We can change PtisAB and CrpP to other specific promoters and genes. And our system can be used to degrade other kinds of antibiotics, and this will help a lot in dealing with the abuse of antibiotics.
Improve our hardware
At present, our hardware is only a small device, and it works perfectly. In future, we can make some improvements to it so that it can be better used in larger scenarios, such as sewage plant, laboratory wastewater treatment or pharmaceutical wastewater treatment and so on.
Improve our safety
As for further experiments, it’s important for us to improve the function of blue-sensitive promoter and lysin protein. Match the darkroom and ultraviolet light in the device to prevent the escape of engineered bacteria, providing a double guarantee for the biosafety of our project.
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]National pharmacopoeia commission. Pharmacopoeia of the People's Republic of China [M]. Part ii. Beijing: China medical science and technology press, 2015: 580
[3]Chávez-Jacobo, V. M., Hernández-Ramírez, K. C., Romo-Rodríguez, P., Pérez-Gallardo, R. V., Campos-García, J., Gutiérrez-Corona, J. F., ... & Ramírez-Díaz, M. I. (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., Zhang, W., Liu, J., Wu, Y., ... & Cheng, F. (2018). A light-controlled cell lysis system in bacteria. Journal of industrial microbiology & biotechnology, 45(6), 429-432.
[2]National pharmacopoeia commission. Pharmacopoeia of the People's Republic of China [M]. Part ii. Beijing: China medical science and technology press, 2015: 580
[3]Chávez-Jacobo, V. M., Hernández-Ramírez, K. C., Romo-Rodríguez, P., Pérez-Gallardo, R. V., Campos-García, J., Gutiérrez-Corona, J. F., ... & Ramírez-Díaz, M. I. (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., Zhang, W., Liu, J., Wu, Y., ... & Cheng, F. (2018). A light-controlled cell lysis system in bacteria. Journal of industrial microbiology & biotechnology, 45(6), 429-432.