Part:BBa_K4645015
The pH-antitoxin system
The pH-antitoxin system contains two gene pathways: pH-sensitive pathway, and toxin-antitoxin pathway. The pH-sensitive circuit can detect pH changes when engineered bacteria enter a cat's digestive tract and take effect, expressing CI protein to inhibit the expression of antitoxin protein MntA. At the same time, it expresses the two-component system RstAB. RstB can sense the decrease in pH to phosphorylate RstA, and RstA can increase the activity of the acid-induced promoter Pasr, thus quickly increasing the expression of CI protein. The toxin protein HepT and antitoxin protein MntA form a toxin-antitoxin pathway, where the expression of MntA protein is inhibited by CI protein.The circuit design is shown in the diagram below.
Basic Elements
Pasr is an acid sensitive promoter, in acid condition of PH5-6 , it could be activated .
CI protein is the regulatory protein of λ phage lysogenic cleavage pathway .
RstAB is a two-component system, RstB can sense the decrease in pH to phosphorylate RstA, and RstA can increase the activity of the acid-induced promoter Pasr.
HepT is a toxin, strongly inhibiting cell growth in S.oneidensis and Escherichia coli.
Basic Elements Functional validation
Pasr
P asr is a promoter that is activated in an acidic environment. It is the promoter of the acid shock protein ASP in E. coli, and its sequence contains a RstA box that can be bound by phosphorylated RstA to enhance its promoter activity in a dose-dependent manner[1]. The team obtained the corresponding promoter fragment from the E. coli MG1655 genome by PCR using a high-fidelity DNA polymerase.
Test Method Design
Charaterization of Pasr with fluorescence intensity measurement
To validate the obtained promoter activity and its activation range in response to pH, we connected Amcyan protein after this promoter and introduced it into E. coli BL21(DE3). When E. coli grew to OD600=0.6, we used HCl aqueous solution and NaOH aqueous solution to adjust the pH and built a gradient from pH 8 to pH 3. Fluorescence intensity (Exλ:453nm Emλ:486nm) was measured every 30 minutes for 5 hours while OD600 was measured simultaneously. The final fluorescence intensity divided by OD600 gave the relative fluorescence intensity.
Test Protocol
1) Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli BL21. Then spread it onto an LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37 ℃ in an incubator.
2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated on two identical media with 5mL LB medium containing 50 μg/mL kanamycin and cultured at temperatures(36 ℃) respectively while shaking at 200 rpm .
3) Measure the OD₆₀₀ value of the resuspending culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader)until the OD₆₀₀ is in the range of 0.4 and 0.6.
4) Use HCl aqueous solution and NaOH aqueous solution to adjust the pH of the medium, and construct a gradient from pH 8 to pH 3.
5) Samples were grouped and incubated continuously at 37 ° C while shaking at 200 rpm for four hours.
6) Fluorescence intensity (Ex.lamda.: 453 nm, Em.lamda.: 486 nm) and OD₆₀₀ were measured continuously using a an automatic microplate reader (Synergy H1 hybrid multimodal reader). At the end of the detection, the measured fluorescence intensity was divided by OD₆₀₀ to obtain the relative fluorescence intensity, and the results were analyzed by plotting.
Result
Analysis of the experimental results
As shown in Figure 3, Pasr has almost no expression under pH 7-8 conditions, but begins low-dose expression at pH 6 and gradually increases as the pH decreases, reaching a peak at pH = 5, then maintaining a relatively low expression level as the pH continues to decline. As shown in Figure 4,We selected the final values for differential analysis, which showed extremely significant differences between pH 6, pH 5 and the control group. This experiment validated that this promoter has almost no expression under pH 7-8 conditions, consistent with our project design expectations. Pasr can be used to activate the suicide circuit in the gastric environment of cats to cause engineered bacteria death, thus avoiding potential hazards from leakage. This promoter can be used to respond to changes in environmental pH and has some reference value for projects with corresponding environmental condition changes.
HepT Toxin Protein
Test Design
We referred to the method of verifying Hept by iGEM21_HZAU-China BBa_K3733010. To verify the cytotoxicity of HepT, we connected the HepT toxin protein after the lactose promoter and transferred it to E. coli BL21(DE3) using pet28a. E. coli strains were cultured to OD600 = 0.4-0.6 and induced with 0.5mM, 0.75mM IPTG, then allowed to grow continuously for 5 hours at 37°C. In 96-well plates, cytotoxicity was measured by comparing OD600 between the experimental group and control group using Synergy H1 enzyme-labeled detector.
Test Protocol
Toxin protein HepT OD600 detection
1) Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli BL21. Then spread it onto LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37 °C in an incubator.
2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated in two identical media with 5 ml LB medium containing 50 μg/mL kanamycin and cultured at temperature (37 °C) respectively while shaking at 200 rpm.
3) Measure the OD600 value of the resuspension culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader) until the OD600 of the bacteria solution reaches 0.4-0.6.
4) Add IPTG to the bacteria solution of the experimental group to induce expression of the toxin proteins.
5) Plot the OD600 value curves of the resuspension culture media over time in an automatic microplate reader (SynergyH1 hybrid multimodal reader). Incubate the cultures for 4 hours at 37°C while shaking at 200 rpm. Take samples at intervals or continuously measure the OD600 data of the bacteria solution with a UV spectrophotometer. And then convert the raw data into OD600. Compare the data of experimental groups and control group and compare curves in two schemes with each other.
Result
Analysis of the experimental results
As shown in Figure 5, the toxin protein HepT has a very obvious bactericidal effect, and this effect is dose-dependent, correlating with the induction concentration of IPTG within a certain range.
Composite Elements Functional validation
The pH-antitoxin system
We constructed a circuit to validate the function of the toxin-antitoxin system (HepT/MntA) and the CI-PR system.We constructed the CI protein downstream of the LacI promoter, and constitutively expressed HepT, and connected the antitoxin protein MntA downstream of a promoter repressed by the CI protein, using the pet28a plasmid transformed into E. coli BL21(DE3).
Since the toxin protein HepT is highly toxic, and a strong promoter J23119 and strong RBS B0034 were used, if MntA cannot function, the bacteria transformed with this plasmid should grow slowly or even not survive. But as shown in Figure 6., the bacteria grew well on the plates.
We also cultured the E. coli strain to OD600 = 0.4-0.6, induced with 1.0mM IPTG, and allowed continuous growth for 4 hours at 37°C. In 96-well plates, by comparing the OD600 between the experimental group and control group using the Synergy H1 enzyme-labeled tester, it displayed the function of the antitoxin protein MntA. The experimental results are shown in Figure 7..
Test Protocol
Anti-Toxin protein MntA OD600 detection
1) Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli BL21. Then spread it onto LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37 °C in an incubator.
2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated in two identical media with 5 ml LB medium containing 50 μg/mL kanamycin and cultured at temperature (37 °C) respectively while shaking at 200 rpm.
3) Measure the OD600 value of the resuspension culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader) until the OD600 of the bacteria solution reaches 0.4-0.6.
4) Add IPTG to the bacteria solution of the experimental group to induce expression of the toxin proteins.
5) Plot the OD600 value curves of the resuspension culture media over time in an automatic microplate reader (SynergyH1 hybrid multimodal reader). Incubate the cultures for 4 hours at 37°C while shaking at 200 rpm. Take samples at intervals or continuously measure the OD600 data of the bacteria solution with a UV spectrophotometer. And then convert the raw data into OD600. Compare the data of experimental groups and control group and compare curves in two schemes with each other.
Analysis of the experimental results
Through analysis of the experimental results, we can find that MntA plays an important role in maintaining the survival of bacteria with constitutive expression of HepT. We also successfully verified the availability of the CI-PR (PCI) system. We also found the bacteria in the control group grew slightly slower than normal bacteria, which may be due to low-dose leakage from the LacI promoter.
Reference
[1]Ogasawara H, Hasegawa A, Kanda E, Miki T, Yamamoto K, Ishihama A. Genomic SELEX search for target promoters under the control of the PhoQP-RstBA signal relay cascade. J Bacteriol. 2007 Jul;189(13):4791-9.
[2] Stirling F, Naydich A, Bramante J, Barocio R, Certo M, Wellington H, Redfield E, O'Keefe S, Gao S, Cusolito A, Way J, Silver P. Synthetic Cassettes for pH-Mediated Sensing, Counting, and Containment. Cell Rep. 2020 Mar 3;30(9):3139-3148.e4.
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