Difference between revisions of "Part:BBa K3482004"

Latest revision as of 20:16, 27 October 2020

IPTG-inducible miniColicin toxin

This part allows for IPTG inducible expression of the DNAase miniColicin E2 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 cuts on both single and double-stranded DNA. It has no specific cutting site. Colicins 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 kill switch 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 activity of the IM2 antitoxin with aTc induction, whereas IPTG induction promotes production of the MiniColicin E2 toxin, resulting in a number of surviving cells (probable mutants) proportional to the dilution of the plated culture.

We also tested our pKC1 plasmid encoding for the miniColicin E2 toxin and IM2 antitoxin in E. coli Nissle 1917 ΔclbA. Again, we used different concentrations of IPTG and aTc to study the effect of differential expression of the toxin and antitoxin on the growth of our strain. We compared this to the strain transformed with the empty vector pAND

Dose-response growth curve of E. coli Nissle 1917 ΔclbA harboring kill switch plasmid pKC1 at 37°C. E. coli Nissle with pAND (red line), E. coli Nissle with pKC1 (green line). The lines and shade represent the mean ± standard error.

With E. coli Nissle 1917 with pKA1, we observed desired growth inhibition of the strain with pKC1 at high IPTG and low aTc concentrations, while the pAND strain showed no alteration in growth in any of the tested conditions. Contrastingly to E. coli Nissle 1917 transformed with pKA1, the induction of aTc was clearly able to rescue cell growth. For increasing concentrations of IPTG, also increasing concentrations of aTc were necessary to rescue cell growth. Similar to E. coli Nissle 1917 pKA1, cell growth could be completely inhibited for 6-8 h (IPTG: 100 µM, aTc: 0 ng/mL) before observing rapid growth. Also similarly to pKA1, the reduction in IPTG concentration gave a lesser but more constant growth inhibition, which also persisted to at least 10 h. Overall, we demonstrated that the miniColicin E2/IM2 encoding pKC1 plasmid could efficiently inhibit the growth of E. coli Nissle 1917 at 100 µM IPTG for up to 6 h and provided even longer growth inhibition at 5 µM IPTG.

Sequence and Features