Difference between revisions of "Part:BBa K4645022"
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<partinfo>BBa_K4645022 short</partinfo> | <partinfo>BBa_K4645022 short</partinfo> | ||
==The pH-antitoxin system== | ==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 | + | We constructed a circuit to validate the function of the <b>toxin-antitoxin system (HepT/MntA) and the CI-PR system</b>.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 <i>E. coli</i> BL21(DE3). |
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<center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/lacimnta2.jpg" style="width:80%; "></center> | <center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/lacimnta2.jpg" style="width:80%; "></center> | ||
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| − | <center><b>Figure 1.Circuit design</b> </center> | + | <center><b>Figure 1.Circuit design.</b> </center> |
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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 <b>Figure 2.</b>, the bacteria grew well on the plates. | 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 <b>Figure 2.</b>, the bacteria grew well on the plates. | ||
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| − | We also cultured the E. coli strain to OD<sub>600</sub> = 0.4-0.6, induced with 1. | + | We also cultured the <i>E. coli</i> strain to OD<sub>600</sub> = 0.4-0.6, induced with 1.0 mM IPTG, and allowed continuous growth for 4 hours at 37°C. In 96-well plates, by comparing the OD<sub>600</sub> between the experimental group and control group using the <b>Synergy</b><sup>TM</sup> H1 enzyme-labeled tester, it displayed the function of the antitoxin protein MntA. The experimental results are shown in <b>Figure 3.</b>. |
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| − | <center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/cimnta.jpg" style="width: | + | <center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/cimnta.jpg" style="width:55%; "></center> |
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<center><b>Figure 2. Bacterial culture plates.</b> </center> | <center><b>Figure 2. Bacterial culture plates.</b> </center> | ||
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===Test Protocol=== | ===Test Protocol=== | ||
| − | <b>Anti-Toxin protein MntA | + | <b>Anti-Toxin protein MntA OD<sub>600</sub> detection</b> |
| − | <p> 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 | + | <p> 1) Methods of molecular cloning and transformation are described above. Transform this plasmid into <i>E. coli</i> BL21. Then spread it onto LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37°C in an incubator. </p> |
| − | <p> 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 ( | + | <p> 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. </p> |
| − | <p> 3) Measure the | + | <p> 3) Measure the OD<sub>600</sub> value of the resuspension culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader) until the OD<sub>600</sub> of the bacteria solution reaches 0.4-0.6. </p> |
<p> 4) Add IPTG to the bacteria solution of the experimental group to induce expression of the toxin proteins.</p> | <p> 4) Add IPTG to the bacteria solution of the experimental group to induce expression of the toxin proteins.</p> | ||
| − | <p> 5) Plot the | + | <p> 5) Plot the OD<sub>600</sub> value curves of the resuspension culture media over time in an automatic microplate reader (<b>Synergy</b><sup>TM</sup> H1 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 OD<sub>600</sub> data of the bacteria solution with a UV spectrophotometer. And then convert the raw data into OD<sub>600</sub>. Compare the data of experimental groups and control group and compare curves in two schemes with each other.</p> |
===Analysis of the experimental results=== | ===Analysis of the experimental results=== | ||
Latest revision as of 15:16, 12 October 2023
HepT-MntA toxin-antitoxin--CI-PR System
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 2., the bacteria grew well on the plates.
We also cultured the E. coli strain to OD600 = 0.4-0.6, induced with 1.0 mM 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 SynergyTM H1 enzyme-labeled tester, it displayed the function of the antitoxin protein MntA. The experimental results are shown in Figure 3..
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 (SynergyTM H1 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 856
Illegal NheI site found at 1561
Illegal NheI site found at 1584 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]