Difference between revisions of "Part:BBa K3606027"

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cold triggered RelE/RelB 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 RelE/RelB toxin/antitoxin module and an RNA thermometer NoChill-06 to regulate it.

The antitoxin RelB is liable and expressed at a relatively high level. The RelE 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. RelE and RelB neutralize each other by protein-protein interaction and form a stable complexity. When the bacteria encounter a cold shock(30℃), RelB is degraded rapidly by an ATP-dependent serine protease Lon and releases RelE. The toxin RelE acts as a global translational inhibitor to cleave mRNAs under translating at the ribosomal A site, therefore resulting in cell growth arrest and finally cell death^[1].

design of cold triggered RelE/RelB Kill Switch

Sequence and Features

Biology

RelE/RelB is one of the best characterized 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 RelE/RelB Kill Switch, the toxin RelE is a global translational inhibitor that cleaves mRNA after the second nucleotide in the empty ribosomal A sites. RelE does not exhibit endoribonuclease activity by itself but act as a ribosome-associated factor tightly interacting with 16S rRNA to executive cleavage function. And RelE shows a modest sequence preference for select codons (CAG, UCG and the stop codon UAG)^[2].

RelB is the antagonist of RelE that can form a stable complexity in a two-to-two or one-to-two ratio with RelE^[3].RelB can be degraded rapidly by an ATP-dependent serine protease Lon and releases RelE. 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_K3606027 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.

Attention for RelE/RelB

1. Potential pathogenicity of RelE Although RelE/RelB module poses minimized adverse effects on hosts as it expanding in microbial communities [2], it has several other effects on the human intestine. Firstly, RelE is a very rare toxin that has activity against both prokaryotic and eukaryotic organisms in a similar mechanism. It has been observed that RelE can induce apoptosis in cultured human cell lines. Secondly, RelE/RelB module tends to have unexpected enrichment in bacteria that is not observed for other toxin/antitoxin modules such as MazE/MazF and ParD/ParE. Such enrichment may be associated with the formation of the persistence of microbes in the gut. Thirdly, the divisions of bacteria carrying homologs of the RelE toxin involved in comprising the human gut microbiota.

All this effect has only been testified in artificial mammalian gene expression systems, so whether it has tangible impacts on the human gut microbiome need further study. iGEM teams that focus on intestinal probiotics and have special safety requirements ought to pay special attention to these. 2. Chromosomally encoded RelE/RelB in gut microbiota What’s more, RelE/RelB and other toxin/antitoxin modules have homology searches on bacterial chromosomes. RelB-RelE complex can inhibit the promotor of chromosomally encoded RelE/RelB [3]. 3. Homonymic “Relb”

1. ↑ Unterholzner, Simon J et al. “Toxin-antitoxin systems: Biology, identification, and application.” Mobile genetic elements vol. 3,5 (2013): e26219. doi:10.4161/mge.26219
2. ↑ Hwang, Jae-Yeon, and Allen R Buskirk. “A ribosome profiling study of mRNA cleavage by the endonuclease RelE.” Nucleic acids research vol. 45,1 (2017): 327-336. doi:10.1093/nar/gkw944
3. ↑ Hwang, Jae-Yeon, and Allen R Buskirk. “A ribosome profiling study of mRNA cleavage by the endonuclease RelE.” Nucleic acids research vol. 45,1 (2017): 327-336. doi:10.1093/nar/gkw944