Screening strategy for subsurface OspA:mRFP1 fusions. A random mutagenesis oligo was synthesized to change mRFP1 codons E4 and D5 in OspA20:mRFP1 to any amino acid, with a bias against stop codons (except for amber UAG, see text). The oligo was converted to a double-stranded linker and ligated with a shuttle vector carrying the 5' and 3' portions of the OspA20:mRFP1 fusion gene. The resulting library was amplified in E. coli and used to transform B. burgdorferi. A presorted population of red fluorescent spirochetes was incubated with proteinase K, washed, and sorted again for red fluorescence. Clones grown from individual colonies were grown in 96-well plates and subjected to a confirmatory in situ proteolysis assay. PCR and DNA sequence analysis revealed the mutant genotypes. Numbered arrows indicate specific oligonucleotides used (Table 1). For details, see the Materials and Methods section.