Export of type 3 secretion (T3S) substrates is traditionally evaluated using trichloroacetic acid (TCA) precipitation of cultured cell supernatants followed by western blot analysis of the secreted substrates. In our lab, we have developed β-lactamase (Bla), lacking its Sec secretion signal, as a reporter for the export of flagellar proteins into the periplasm via the flagellar T3S system. Bla is normally exported into the periplasm through the SecYEG translocon. Bla must be secreted into the periplasm in order to fold into an active conformation, where it acts to cleave β-lactams (such as ampicillin) to confer ampicillin resistance (ApR) to the cell. The use of Bla as a reporter for flagellar T3S allows the relative comparison of translocation efficiency of a particular fusion protein in different genetic backgrounds. In addition, it can also be used as a positive selection for secretion.Graphical overviewUtilization of β-lactamase (Bla) lacking its Sec secretion signal and fused to flagellar proteins to assay the secretion of exported flagellar substrates, into the periplasm, through the flagellar T3S system.A. Bla is normally transported into the periplasm space through the Sec secretion pathway, where it folds into an active conformation and allows resistance to ampicillin (ApR). B. Bla, lacking its Sec secretion signal, is fused to flagellar proteins to assay the secretion of exported flagellar proteins into the periplasm through the flagellar T3S system.