Genomic cassettes were inserted
into the chromosome of E. coli DH5α using
lambda red recombineering.59 (link) Selected PAB
variants were transferred to the pKIKOarsBKM integration
vector,60 (link) and then the integration cassette
was amplified with primers AB 39/40 and cleaned up with a Qiagen PCR
purification column. E. coli DH5α was
transformed with the pSIM18 vector, grown to an OD600 of
∼0.3 and heat-shocked at 42 °C for 15 min to induce expression
of the λ Red recombinase proteins. The cells were washed 5 times
in ice-cold sterile water then electroporated with 300 ng of PCR product
and the transformants selected on LB plates supplemented with kanamycin
at 37 °C. Confirmation of cassette insertion at the correct locus
was confirmed by colony PCR with primers AB 34/61. To cure the strains
of pSIM18, clones were subcultured overnight on LB plus kanamycin
at 42 °C and restreaked onto LB plates containing kanamycin (growth)
or hygromycin (no growth) to confirm loss of pSIM18.
into the chromosome of E. coli DH5α using
lambda red recombineering.59 (link) Selected PAB
variants were transferred to the pKIKOarsBKM integration
vector,60 (link) and then the integration cassette
was amplified with primers AB 39/40 and cleaned up with a Qiagen PCR
purification column. E. coli DH5α was
transformed with the pSIM18 vector, grown to an OD600 of
∼0.3 and heat-shocked at 42 °C for 15 min to induce expression
of the λ Red recombinase proteins. The cells were washed 5 times
in ice-cold sterile water then electroporated with 300 ng of PCR product
and the transformants selected on LB plates supplemented with kanamycin
at 37 °C. Confirmation of cassette insertion at the correct locus
was confirmed by colony PCR with primers AB 34/61. To cure the strains
of pSIM18, clones were subcultured overnight on LB plus kanamycin
at 42 °C and restreaked onto LB plates containing kanamycin (growth)
or hygromycin (no growth) to confirm loss of pSIM18.
