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+eGFP+AmpR+ori+J23119+qnrS |
| 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
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 Cell Density to represent the resistance ability of qnrS1. The higher the Relative Cell 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 Cell Density to represent the resistance ability of qnrS1. The higher the Relative Cell 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 Cell Density (OD600) of E.coli DH5α carrying piGEM2019-01 and Negative Control at 1mg/L CIP. Relative Cell Density = The OD600 corresponding to 1mg/L CIP / The OD600 corresponding to 0mg/L CIP(0.19)
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
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.
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 |
In order to verify the expression of CrpP in E.coli BL21, SDS-PAGE was selected for detection. The SDS-PAGE result of E.coli BL21 carrying piGEM2019-01 and piGEM2019-02 together was not ideal, which had so many bands. And we couldn’t distinguish which one was CrpP. So, we amplified pelB-5D, CrpP, Histag and TagRFP, and inserted them into an expression vector induced by IPTG (pEASY). More CrpP protein would also obtain more by IPTG induction. As shown in Fig. 7, the 1 and 2 tubes were the IPTG induction group, while the others were the control group. It can be seen that there were obvious bands around 17kDa in the induction group. The results indicated that CrpP was successfully expressed in E.coli BL21(DE3) with piGEM2019-02.
Fig. 7 SDS-PAGE of His-CrpP in E.coli BL21(DE3).
(Line 1 and 2 are IPTG-induced groups, line 3 and 4 are negative groups)
Next, since CrpP expression is regulated by quorum sensing, we need to verify whether quorum sensing works properly. We co-transferred the plasmid piGEM2019-01 and piGEM2019-02 to E.coli DH5α. Ciprofloxacin was added in the experimental group with a final concentration of 1mg/L, but not in the control group. After the same time of culture, the bacterial liquid in the experimental group was red but the bacterial liquid in the experimental group was not red (Fig. 8). That indicated that TagRFP in the experimental group was normally expressed, while no TagRFP was expressed in the control group.
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.
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. 9)[3]. 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. 9)[3]. We successfully embedded the bacteria that could degrade CIP and immobilized the bacteria.
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)[4]. Transformed it into E.coli BL21(DE3). And we measured the growth curve to represent intracellular cleavage effect (Fig. 10). 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. 10. 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. 11).
Fig. 11. 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. 12). 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. 12. The comparison of our fluorescence detection device and a microplate device.
Work going on
Improve the blue-sensitive promoter switch
Although we have not yet verified the promoter's function, we hope to make some improvements to make it work properly. Then this can better limit the engineering bacteria inside our device. 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.
Import a stronger lysin gene
At present, the intracellular cleavage effect of Lysep3-D8 is not ideal. So, we can change it to a more efficient lysin gene which kills the bacteria instead of inhibiting its growth.
Improve the enzyme activity of CrpP
As for further experiments, it’s important for us to improve the function of CrpP. We can use a stronger quorum sensing system or genetically modify it to enhance the degradation ability.
Although we have not yet verified the promoter's function, we hope to make some improvements to make it work properly. Then this can better limit the engineering bacteria inside our device. 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.
Import a stronger lysin gene
At present, the intracellular cleavage effect of Lysep3-D8 is not ideal. So, we can change it to a more efficient lysin gene which kills the bacteria instead of inhibiting its growth.
Improve the enzyme activity of CrpP
As for further experiments, it’s important for us to improve the function of CrpP. We can use a stronger quorum sensing system or genetically modify it to enhance the degradation ability.
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.
[4] 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.
[4] 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.