Deletion of bcsABC operon. Non-fluorescent deletion candidates for bcsABC were isolated using the same workflow as outlined for ldh and screened for deletions or wild-type (wt) alleles by PCR amplification. (A) The location of primers used to distinguish between the wt and the deletion genotype is indicated by arrows. (B,C) Agarose gel electrophoresis of products from PCR amplification of five non-fluorescent candidate colonies. (B) Amplification using primers P 22 and P 23 yielded a DNA fragment of 1.4 kb for the deletion of bcsABC (three total), while wt cells are expected to produce an amplicon of ∼10 kb, which was poorly amplified under our reaction conditions. (C) To verify the genotype of wt strains, a second PCR experiment using primers P21 and P22 was included. In this case, wt strains are expected to yield an amplicon of 0.7 kb while the deletion strain should not produce a band. Similar results were obtained from six biological replicates of this workflow. (D) To test the phenotype of our mutant, we compared flocculation of the wt (left) and deletion strain (right) after growth for 15 h in glucose minimal media under aerobic conditions. Flocs are indicated by the arrow. (E) Growth curve of wt and mutant strains grown in glucose minimal media under anaerobic conditions.
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