Composite

Part:BBa_K3606027

Designed by: Jingqi Zhang   Group: iGEM20_Fudan   (2020-10-09)
Revision as of 02:55, 25 October 2020 by Zjq11037 (Talk | contribs)


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].


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 164
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



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.
  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
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