Part:BBa_K4342009

acrB Upstream

Usage and Biology

This gel demonstrates that the tdk/kan cassette replaced the acrB gene and then was knocked out to produce a scarless deletion of acrB

The acrB gene participates in the efflux of β-lactam antibiotics such as ampicillin and carbenicillin. The efflux process makes ADP1 more resistant to these antibiotics. Deleting the acrB gene increases ADP1's susceptibility to ampicillin, carbenicillin, and other β-lactams. Additionally, acrB gene is non-essential for ADP1’s survival, thus this gene serves as an ideal location for inserting genetic constructs.

Design

The acrB upstream homology part comprises the 1000 base pair region directly upstream of the acrB gene in ADP1. This part has bsaI and bsmbI restriction sites attached to the 3’ end which are designed to delete the acrB gene through a two-step process involving selection and counterselection.

BsaI Restriction Site

The bsaI site is designed to ligate to the 5’ end of the tdk/kan cassette (BBa_K4342000) creating the acrB tdk/kan cassette composite part (BBa). This composite part permits the selection of transformants through kanamycin resistance.

Bsmbi Restriction Site

The bsmbI site is designed to ligate to the 5’ end of the acrB downstream homology (BBa_K4342010) creating the acrB rescue cassette homologies composite part (BBa). This composite part permits the counterselection of transformants when plating on Azidothymidine (AZT).