Revision as of 01:49, 5 October 2022

acrB Downstream

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, the AcrB gene is non-essential for ADP1’s survival, thus this gene serves as an ideal location for inserting genetic constructs.

Design

The acrB downstream homology part comprises the 1011 base pair region directly downstream of the acrB gene in ADP1. This part has bsaI and bsmbI restriction sites attached to the 5’ end. These restriction sites 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 3’ end of the tdk/kan cassette (BBa) 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 3’ end of the AcrB Upstream homology (BBa) creating the ΔAcrB homologies composite part (BBa). This composite part permits the counterselection of transformants when plating on Azidothymidine (AZT).

Characterization

References