Genomic Insertion Protocol

• Warning: This protocol needs to be modified an updated based on the results obtained in my first attempt at genomic integration. Please see my lab notebook results until then.***

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 the oligos below to PCR amplify the phi80 locus for confirmation of integration and sequencing.

attPhi80-1: CTGCTTGTGGTGGTGAAT

attPhi80-2: ACTTAACGGCTGACATGG

attPhi80-3: ACGAGTATCGAGATGGCA

attPhi80-4: TAAGGCAAGACGATCAGG

sequence Temp (°C)	No integrant with 1 and 4	Single integrant with 1 and 2, 3 and 4	Multiple integrant with 1 and 2, 3 and 2, 3 and 4
63	546	409, 732	409, 595, 732