Part:BBa_K2616002
Temperature Kill Switch
This is a kill switch based on temperature: it enables bacteria to survive at human body temperature (37°C) but die at lower temperatures (under 22°C).
The kill-switch we use is based on a toxin/antitoxin combination, CcdB/CcdA. CcdB is a lethal toxin for E. coli and its production is placed under the regulation of a temperature-sensitive promoter. In permissive conditions, i.e. in human body, the expression of the toxin is repressed and the antitoxin is expressed at a constitutive low level in order to counteract any leaky expression of the toxin. This is done thanks to the modification of the Plac promoter, which is explained precisely in the KILL-SWITCH part of this wiki. When the temperature goes lower, the repression is lifted and toxin expression increases. The constitutive low level of antitoxin is no longer sufficient to counter the effects of the toxin, and the bacteria die.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 62
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 430
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1845
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1831
We gene synthesized the genetic construct of our kill-switch commercially. Once we received the sequence, called Seq9, in a commercial plasmid, we transformed competent bacteria E. coli DH5α. After bacterial culture and plasmid DNA extraction, we digested our DNA with restriction enzymes, extracted the inserts from the gel, and ligated it into linearized pSB1C3 for iGEM submission and expression in BL21(DE3).
We proved that our vector contained the insert by DNA electrophoresis (Figure 1).
Figure 1: Agarose gel after electrophoresis of digested pSB1C3 containing Seq9 (Bba_K2616002) in columns 6 to 11. Colonies 2 and 6 have the correct plasmid.
Alignment of sequencing results confirmed that pSB1C3 contained Bba_K2616002
Figure 2: Alignment of sequencing results for BBa_K2616002. Sequencing perform in pSB1C3 and two primers were designed (FOR1 and FOR2) to cover the whole sequence. Image from Geneious. Pairwise Identity: 100%.
The construction was successfully assembled. In Figure 20, we show that we used two different primers, allowing us to cover the whole sequence without mistakes. As visible, the mismatches are only present at the extremities of each primer sequencing. The final basepair identity is 100%.
Kill-switch characterization
To test the efficiency of our kill-switch, we decided to cultivate transformed BL21(DE3) pLysS E. coli at several temperatures (15°C, 20°C, 25°C and 37°C). We used BL21(DE3) pLysS E. coli transformed with the empty pSB1C3 plasmid as the negative control. The bacteria growth was followed by measuring the optical density at 600 nm every 30 minutes for 6 hours, followed by two additional points at 18 hours and at 72 hours. Each experiment was done in triplicate and the standard deviation was calculated for every point. We showed that bacteria transformed with the kill-switch presented no measurable growth at 15°C and at 20°C during the 72 hours of the experiment, whereas the control population grew normally (Figure 3).
At 25°C, the kill-switch population grew more slowly than the control for the first 18 hours, but the growth eventually started to reach normal values at 72 hours.
Finally, at 37°C there was no difference in the growth of the kill-switch population compared to the control bacteria.
Figure 3 Effect of different temperatures on the growth of Cryodeath kill-switch transformed E. coli BL21(DE3) pLysS
Reference: F. Stirling et al., "Rational Design of Evolutionarily Stable Microbial Kill Switches," Mol. Cell, vol. 68, no. 4, p. 686-697.e3, Nov. 2017.
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