Part:BBa_K3606028
cold triggered MazF/MazE Kill Switch
This composite part is a cold triggered kill switch to deprive of survivability of engineered bacteria in the environment when excreted from the intestine. It consists of a MazF/MazE toxin/antitoxin module and an RNA thermometer NoChill-06 to regulate it.
The antitoxin MazE is liable and expressed at a relatively high level. The MazF toxin is constitutively co-expressed with the antitoxin under the control of an RNA thermometer No-Chill 06. Under the body temperature (37℃)，No-Chill 06 unfolds and exposes its ribosome binding site (RBS) to express. MazE and MazF neutralize each other by protein-protein interaction and form a stable complexity in a one-to-two ratio. When the bacteria encounter a cold shock(30℃), MazE is degraded rapidly by an ATP-dependent serine protease ClpAP and releases MazF. The toxin MazF acts as a site-specific endoribonuclease to almost all cellular mRNAs, therefore resulting in cell growth arrest and finally cell death[1].
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Biology
MazF/MazE is one of the mostly used type II toxin/antitoxin systems. The type II toxin/antitoxin system is a mature and stable system that is extensively applied both inside and outside the iGEM competition. In the type II toxin/antitoxin systems, the antitoxin is liable but expressed at a relative high level. And the toxin is constitutively co-expressed with the antitoxin form a stable complexity. When the bacteria encounter special growth conditions, the antitoxin is degraded rapidly by an ATP-dependent serine protease Lon and releases the toxin, therefore resulting cell growth arrest.
In the context of cold triggered MazF/MazE Kill Switch, the toxin MazF is site-specific and single-strand-specific endoribonuclease to cleave mRNA at ACA sequences and tRNA at UUU. MazE is the antagonist of MazF that can form a stable complexity in a two-to-four ratio with MazF. MazE neutralizes the toxicity of MazF by inserting its C-terminal helical segment into the RNA-binding channel of the MazF-MazE complex, preventing MazF from binding RNA[2].
RNA thermometers are short RNA with special hairpin structure that can response to the temperature change. The hairpin structure occludes the ribosome binding site (RBS) and inhibit the small ribosomal subunit from assembly therefore inhibit the translation. Because of a small number of mismatched base pairs in the complimentary sequence, RNA thermometers unfold easily when encounter a temperature increase then exposes its RBS facilitating protein expression. NoChill-06 is a modified RNA thermometer designed by team 19Rice using NUPACK and VSAlgorithm. They mutated the nucleotides by the side of RBS with RBS unchanged obtaining a series of NoChill RNA thermometers that response to temperature change between 25 °C and 37 °C efficiently. NoChill-06 reacts very sensitively when temperature increase from30℃ to 37 °C, so it is appropriate for regulation in human body.
Protocol for characterization
Growth Curve Measurement
1.Plasmids construction and transformation: Insert DNA fragments of BBa_K3606028 in to pSB1C3. Transform the two kinds of constructed plasmids into DH5α strain as experimental groups and empty pSB1C plasmids as control group. Culture three groups in 60mL LB medium (with 50 ng/µl ampicillin) at 37℃ overnight.
2.Cold treatment: Divide each group into two test tubes for 30℃-culture groups and 37℃-culture groups. (3 for each temperature).
3.Measure growth situation: Extract 5μl bacteria solution from each test tube every 1h. Diluted each bacteria solution to 10^7 times and culture them on three LB plate (with 50 ng/µl ampicillin) at 37℃ for 24h. Count the number of colonies in 5 cm^2 per plate after cultured for 24h at 37℃.
4.Draw the growth curve.
References
- ↑ Yamaguchi, Y., Inouye, M. Regulation of growth and death in Escherichia coli by toxin–antitoxin systems. Nat Rev Microbiol 9, 779–790 (2011). https://doi.org/10.1038/nrmicro2651
- ↑ Schifano JM, Cruz JW, Vvedenskaya IO, Edifor R, Ouyang M, Husson RN, Nickels BE, Woychik NA. tRNA is a new target for cleavage by a MazF toxin. Nucleic Acids Res. 2016 Feb 18;44(3):1256-70. doi: 10.1093/nar/gkv1370. Epub 2016 Jan 5. PMID: 26740583; PMCID: PMC4756823.
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