Modification of a Flavobacterium johnsoniae Suicide Vector for Rapid Screening in an Allelic Exchange System
Presentation
Overview
Overview
Description
Genetic manipulation, including allelic exchange, has been important in identifying and characterizing genes involved in motility. The cloning steps required for allelic exchange include amplifying the regions upstream and downstream of the site of interest and sequentially cloning these regions into pRR51. Following each cloning step a colony PCR must be performed to confirm insertion of the PCR fragment, a time-consuming process that would benefit from an alternative approach. With blue-white screening, identifying colonies carrying recombinant plasmids is relatively easy as colonies carrying plasmids with inserts that interrupt the lacZα gene exhibit a white hue as opposed to a blue colony color indicative of no insert. pRR51 was modified for blue-white screening through restriction digest removal of its multiple cloning site (MCS) and insertion of a pUC18 lacZα gene and associated MCS to generate pSEE03.
