To visualize RbmA during biofilm growth, the wild type copy of
rbmA was exchanged by a FLAG-tagged
rbmA (16 (link)) (with the
octapeptide DYKDDDDK) by mating the plasmid pNUT462 into the strain KDV148,
resulting in V. cholerae strain KDV829. Successful FLAG-tagging
of RbmA was confirmed by PCR and sequencing. The final strain KDV835 was
generated by conjugating the fluorescence protein expression plasmid pNUT542
into strain KDV829. For RbmA visualization in flow chambers, biofilms were grown
in M9 medium containing 1 μg/mL of FLAG tag monoclonal antibody (L5)
conjugated to AlexaFluor 555 (Thermo Scientific) and 1 mg/mL of
filter-sterilized bovine serum albumin (BSA).
rbmA was exchanged by a FLAG-tagged
rbmA (16 (link)) (with the
octapeptide DYKDDDDK) by mating the plasmid pNUT462 into the strain KDV148,
resulting in V. cholerae strain KDV829. Successful FLAG-tagging
of RbmA was confirmed by PCR and sequencing. The final strain KDV835 was
generated by conjugating the fluorescence protein expression plasmid pNUT542
into strain KDV829. For RbmA visualization in flow chambers, biofilms were grown
in M9 medium containing 1 μg/mL of FLAG tag monoclonal antibody (L5)
conjugated to AlexaFluor 555 (Thermo Scientific) and 1 mg/mL of
filter-sterilized bovine serum albumin (BSA).