Our constructs were designed specifically for the pDB.His.x series plasmids and the pET-28 derived plasmid pTHST, which contains the SUMO protein tag. We chose to use each of these plasmids because they are IPTG inducible, since they have lacI, the lac repressor gene. Once transformed into E. coli, expression can be induced by adding IPTG. After pelleting and lysing cells, it is possible to purify our protein. Before cleavage, our protein has a 6xHis and GST, MBP, or SUMO tag. These tags can be removed using TEV protease, which cleaves between the protein expression tags and our bacteriocins.Using the plasmids above, we designed tagged and untagged constructs. Our tagged constructs were cut using NdeI and XhoI, so our inserts are between the TEV cleavage site and a 6xHis tag that is not expressed. We designed untagged inserts, cut with BglII and XhoI, which removed the protein expression tag from our construct, but these constructs were difficult to work with and we abandoned them. In order to make our inserts BioBrick compatible, we used primers with the BioBrick prefix and suffix and PCRed our inserts.
|BBa_K2196003||Lacticin Z-Aureocin A53 Hybrid|
|BBa_K2196004||Lacticin Z-Epidermicin NI01 Hybrid|
|BBa_K2196005||Lacticin Z-Lacticin Q Hybrid|