Genomic Insertion Protocol

Note that you'll need a pir strain for replication of pG80 plasmids. You can drop your biobrick into the Eco/pst sites of pG80ko, transform the pir116 cells. Grow up, miniprep, and map a single colony. Make competent cells of pInt80-649 in your target strain (plate them on Amp). It is temperature sensitive, so do all growth manipulations at 30 degrees. Transform in your pG80 derivative, plate on 15ug/mL gentamicin plates at 37 degrees. Grow a single colony to saturation at 37 in LB+15ug/mL gentamicin, then restreak on a gentamicin plate at 43 degrees. You can use oligos ca603F and R (see below) to pcr amplify the phi80 locus for confirmation of integration and sequencing.

If you want to do the "refactored" markerless version of the CRIMs, all the relevant parts for making such things in BBa have been made and are in the Registry distribution except a phi80 part. You could easily make such a part from pG80ko.

ca603F PCR of Phi80 att locus CTGCTTGTGGTGGTGAAT

ca603R PCR of Phi80 att locus TAAGGCAAGACGATCAGG