The 2012 UCSF iGEM Team used this part to determine if toxins and antitoxins could be used as a form of communication between two different strains of E. coli to tune population ratios.
We tried several different experiments to determine if the toxin an antitoxin pair could move between cells to cause an effect on growth. The final experiment we performed utilized protein induction at 30C which we believe helped maintain the stability of these small proteins.
- A flask with 150ml LB+Spec was inoculated with either – Toxin strain, Antitoxin Strain, or BL21 (empty vector) control cells.
- Each flask was started at an initial OD600nm of 0.05 and was grown for 2 hours at 37C before being induced with 0.5 ul/ml of 1M IPTG.
- After the addition of IPTG, the flasks were transferred to 30C to promote stable protein production. These cultures were grown overnight, ~16 hours.
- The next morning the cultures were centrifuged and the supernatant was filtered over a 0.2um filter to remove any cell debris, but retain any small proteins or molecules in the supernatant.
- The filtered supernatant were then diluted (25ml supernatant + 75ml fresh LB) to provide a fresh supply of nutrients.
- Then toxin, antitoxin, and control cells were inoculated into these filtered, diluted supernatents and induced with IPTG after 2 hours of growth at 37C.
- The effect of supernatents potentially containing toxin or antitoxin proteins was observed in the growth of each strain and is shown in the graphs below.
The graphs shown below demonstrate growth of toxin (YoeB), antitoxin (YefM), and control cells in the media produced after step 5 above.
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