Subcloning and Sequencing of the Autotransporter Antigen 43 from Escherichia coli K-12 as an Initial Step Toward Understanding the Relationship Between Ag43 and Capsule in Biofilm Formation

The autotransporter Antigen 43 (Ag43) is present in numerous Escherichia coli strains and functions in autoaggregation and adhesion leading to biofilm formation. However, biofilm formation and regulation is not well understood. We hypothesized that Ag43-mediated biofilm formation is prevented by capsule expression. In this study, we describe the construction of plasmid vector encoding the autotransporter protein Ag43 designed to test our hypothesis. Polymerase chain reaction was used to amplify Ag43 from the E. coli K-12 genome. The resulting PCR product was subcloned into pCR2.1-TOPO Cloning Vector and transformed into Escherichia coli strain OneShot TOPO. Clones were selected and isolated plasmids were screened by restriction enzyme digestion followed by agarose gel electrophoresis. The plasmid containing a putative insert, pRCS715, was sequenced and shown to contain the Ag43 sequence.