Tetracysteine tag construction and expression. (a) Graphical depiction of F. tularensis LVS ripA locus showing the location of SOE PCR primers used to insert the C terminal TC tag (marked in gray). (b) Nucleotide and amino acid sequence of the C terminal TCtag showing the overlapping sequence of the SOE PCR primers. (c) In gel fluorescence of RipA-TC (black arrow) from dilution series of F. tularensis LVS (plasmid) pKK ripA'-TC and F. tularensis LVS (chromosomal)ripA'-TC using 6000 ng to 60 ng total protein of whole cell lysates. F. tularensis LVS lysates (wt) used as a non TC tagged control displaying three non specific bands (gray arrows) at a higher molecular weight than RipA-TC.