"
Team:INSA-Lyon/Project/Stage1/Strategy
From 2010.igem.org
Strategy
Total plasmid synthesis
In 1988 Slater et al (ref) developed the plasmid pSB20. Then the construction of a total DNA bank of Alcaligenes eutrophus permit to identified two restriction sites which surround the three genes and their promoter: KnpI and EcoRI. With the help of a logiciel serial cloner and the sequence of Ralstonia eutropha, a 5800 pb length sequence was isolated. It contains the promoter and the phaCAB genes. This sequence had been inserted into a plasmide 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.
Two new strategies
A phaC gene had been design in order to be synthesized.
(design part phaC)
In one hand we try to get phaA and phaB by PCR
(design des primers)
and to ligate the PCR product together and then with the synthesized phaC
In the other hand we try a ligation between two registry parts:BBa_K125501 and BBa_K125502.They correspond to a phaA and a phaB gene. This product will be ligated with the synthesized phaC
