Part:BBa_K4645022

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).

Figure 1.Circuit design

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 1.0mM 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 3.. 
Figure 2. Bacterial culture plates.

Figure 3. Bacterial OD₆₀₀ over time.

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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 856
  Illegal NheI site found at 1561
  Illegal NheI site found at 1584
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]