Team:INSA-Lyon/Project/Stage1/Strategy

In 1988 Slater et al (ref) developed the plasmid pSB20. Then the construction of a total DNA bank of Alcaligenes eutrophus permits to identified two restriction sites which surround the three genes and their promoter: KnpI and EcoRI. With the help of a software (serial cloner) and the sequence of Ralstonia eutropha, a 5800 pb length sequence Had been isolated. It contains the promoter and the phaCAB genes. This sequence had been inserted into a plasmid pUC18. A site HindIII had been added before the promoter and also a unique restriction site XhoI between the promoter and the operon (so we could change the promoter). This is the plasmid pILI1.

Map of the plasmid PiliI

We send this sequence to GeneCust to be synthesized. The product of this synthesis had been transformed in competent bacteria NM522.

However this plasmid doesn’t match with the iGEM criteria. Indeed some forbidden restriction sites was present in the sequence.

PhaABC Operon synthesis: two strategies

To construct the phaCAB operon we tried two strategies. In one hand we designed a phaC gene in BioBrick standard from the pILI1 sequence: as there were two Pst1 and one Not1 sites inside the gene, we looked for silent mutations in those sites and modified the sequence to make the sites disappear. Then we added the sequence of iGEM prefix:

at the beginning and the suffix one

at the end in order to have the sequence in the BioBrick standard. For this first strategy we also designed primers to get phaA and phaB from the pILI1 sequence (they didn’t have the wrong restriction sites in their original sequence): we designed them using a software to make them the most stable and the most specific that we could, adding the prefix sequence at the beginning of the forward primer and the suffix sequence at the end of the reverse primer. Then we wanted to ligate the three genes in the original order.

In the other hand, we got the three genes directly from the iGEM registry, and we tried to ligate them. The BioBricks we used are : BBa_K156012 for phaA, BBa_K156013 for phaB and BBa_K156014 for phaC. Unfortunately, the phaC gene we found had an Xba1 missing site so we couldn’t use our new operon. Our two final strategies were to ligate phaC with either PCR product either iGEM phaA and phaB. With more time we could have designed a primer with the right restriction sites but we were not sure the phaC gene had just this problem.