Vector

To run our project in the best manner, we have chosen a high-copy plasmid, pUC19 plasmid, which is compatible with RFC10 standard.

Inserts

The next thing was creating our inserts. First of all we had to keep in mind that we work with the RFC10 standard. To ensure the correct codon usage we got rid of various restriction sides. We created two inserts for each of our two strains. One insert is coding for the toxin and the second insert is coding for the antitoxin.

Our antitoxin has a very low half-life and for this reason we used a constitutive promoter. The same was used for our chromo protein, which should give us the possibility to detect the growth of the right bacteria without other measuring methods.

The second insert, carrying the toxin, has to be inducible to make sure our E.coli cells have enough time to produce the antitoxin before being killed by the toxin. Furthermore, the Dam-Methylase, which is responsible for a higher mutation rate should not be expressed from the beginning. So we decided to use the tac promoter which is inducible by IPTG. Unfortunately, we were not aware of the leakiness of tac at that time.

Assembly

Concerning assembly of the parts, we decided that the two inserts should work in the opposite direction. Hence we could use one bidirectional double-terminator in the middle of the two inserts. This design ensured in case the terminator does not work only the antitoxin is expressed by the constitutive promoter.