From 2007.igem.org

Back

Making Shewanella Competent and Transforming Plasmid

Zymo is a company that sells a package of wash buffer, competent buffer and dilution buffer. Using the protocol listed on this website, shewanella was made competent, meaning able to take in a plasmid.

The plasmid pJQ200 was transformed into shewanella. Since this plasmid has a gentamycin resistance gene, the shewanella was plated on gentamycin plates. After overnight incubation the plates were filled with bacteria, proving that the bacteria had taken in the plasmid and gained resistance due to it. To confirm that the plasmid was definitely pJQ200, a portion of bacteria was plated on a 10% sucrose and gentamycin plate. No cells grew after incubation, as expected if transformation worked correctly due to pJQ200's sacB gene, which acts as a suicide vector in the presence of 10% sucrose, killing the bacteria housing it.

While pJQ200 was originally chosen due to its usefulness for conjugation, and with zymo successful conjugation with E. coli may no longer be necessary, the plan is to continue using pJQ200, firstly because it is a low copy plasmid, meaning its replication is slower and it will not overreplicate mutations, and secondly because its natural sacB gene allows for double selection.