Difference between revisions of "Part:BBa K3482004"

Revision as of 13:05, 22 October 2020

IPTG-inducible miniColicin toxin

This part allows for IPTG inducible expression of the DNAase miniColicin in E. coli. The degradation of DNA , which leads to cell death, can therefore be induced by IPTG.

Biology

Colicin E2 is an endonuclease that cut on both single and double-stranded DNA. It has no specific cutting site. Colicin toxin are toxins that can be produced by E.coli and closely related bacteria.

Usage

We used that part to test if the expression of the toxin work in our E.coli nissle 1917 strain and if it was sufficient do provoc cell death

Characterization

Functional killswitch assay with IPTG and aTc gradients on agar plate

E.coli Nissle 1917 were transformed with a plasmid containing the part BBa K3482004 and the part BBa_K3482014 (IM2 antitoxin part) and plated with a gradient of aTc and IPTG on agar plate. The plate shows strong stimulation/activity of the IM2 antitoxin with aTc induction, also IPTG induction shows induction of the MiniColicin E2 toxin, and a number of surving cells (probable mutants) proportional to the dilution of the plated culture.

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
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 124
1000
COMPATIBLE WITH RFC[1000]

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 [[File:BBa K3482004 kill-switch plate.jpeg|200px|]]
-E.coli Nissle 1917 were transformed with a plasmid containing the part BBa K3482004 and the part <html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K3482014"> BBa_K3482014 </a></html> (IM2 antitoxin part) and plated with a gradient of aTc and IPTG on agar plate
+E.coli Nissle 1917 were transformed with a plasmid containing the part BBa K3482004 and the part <html><a style="padding: 0px; margin: 0px;" href="https://parts.igem.org/Part:BBa_K3482014"> BBa_K3482014 </a></html> (IM2 antitoxin part) and plated with a gradient of aTc and IPTG on agar plate.
 The plate shows strong stimulation/activity of the IM2 antitoxin with aTc induction, also IPTG induction shows induction of the MiniColicin E2 toxin, and a number of surving cells (probable mutants) proportional to the dilution of the plated culture.