Part:BBa_K3733010

HepT: A toxin can cleave mRNA

HepT is a toxin, a member of the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) superfamily, strongly inhibiting cell growth in S.oneidensis and Escherichia coli. The HepT toxin (HEPN-domain protein) could function as an RNase with a RX4-6H active motif and cleave mRNA to inhibit cell growth.[1]

HepT/MntA is one kind of Toxin/Antitoxin (TA) systems in which the enzyme antitoxin chemically modifies the toxin to neutralize it. [1]

Usage and Biology

The mechanism of the HepT toxin is common in bacteria and archaea. Cytotoxicity is based on the fact that HepT can bind to mRNA, and induce degradation of the mRNA.

HepT dimerization enables the formation of a deep cleft at the HEPN-domain interface harboring a composite Rx4-6H active site that functions as an RNA-cleaving ribonuclease. [2][3]

Functional Parameters

To verify the cytotoxicity of HepT, we transferred pET-28a(+)-HepT into E.coil BL21(DE3). The E.coil strain was cultured to OD₆₀₀ = 0.4 ~ 0.6, induced with or without 0.5 mM IPTG, and was allowed to grow overnight at 37℃. In the 96-well plates, the Synergy H1 microplate reader was used to measure the cytotoxicity by comparing the OD₆₀₀ between experimental group and control group. The results obtained from triple independent experiments are shown below (Figure 1).

Figure 1. A. Turbidity change of the experimental group (induced by IPTG) and control group (without IPTG). B. The result of OD₆₀₀ (-IPTG and +IPTG) presents the cytotoxicity of HepT. The error bars are obtained from three independent experiments.

Reference

[1] Yao J, Zhen X, Tang K, et al. Novel polyadenylylation-dependent neutralization mechanism of the HEPN/MNT toxin/antitoxin system[J]. Nucleic acids research, 2020, 48(19): 11054-11067.
[2] Yao J, Guo Y, Zeng Z, et al. Identification and characterization of a HEPN‐MNT family type II toxin–antitoxin in S hewanella oneidensis[J]. Microbial biotechnology, 2015, 8(6): 961-973.
[3] Jia X, Yao J, Gao Z, et al. Structure–function analyses reveal the molecular architecture and neutralization mechanism of a bacterial HEPN–MNT toxin–antitoxin system[J]. Journal of Biological Chemistry, 2018, 293(18): 6812-6823.

Experience

This part was used in iGEM21_HZAU-China`s project “P.E.T”. Considering intestinal temperature of dogs is normally higher than room temperature, we decided to enable the bacteria to kill themselves at low temperatures and only survive at intestinal temperature. To achieve such a function, we utilized RNA thermometers and the HepT toxin to build this module. We used HepT as toxin to let the engineered bacteria suicide or lose competitive advantage in the nature environment until they die off.

Sequence and Features

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterized by HZAU_China 2023

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

Figure 1. Bacterial OD600 over time .

Analysis of the experimental results

As shown in Figure 1., 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.