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]