Mutant-specific amino acid sequences are listed in single letter code on the × axis. n indicates the number of times a particular mutant was isolated from the unsorted (pre) and sorted (post) population. Unanalyzed mutants are listed in Additional File 1-Table S1. (B) Boxplots of surface percentage values of the unsorted (pre) and sorted (post) populations. For each dataset, the box outlines the first and third quartiles, the horizontal red line indicates the median,
and the vertical lines extend to the minimum and maximum values. A total of 172 random clones from the pRJS1016-derived ABT-737 ic50 library were analyzed by DNA sequencing. 38 clones were from a population sampled prior to proteolytic shaving and sorting (unsorted), and 134 clones were from a population sampled after proteolytic shaving and sorting (sorted). 63 mutants
were identified, 8 being unique to the unsorted population, 40 unique to the sorted population, and 15 common to both populations. Within the sorted population, the majority of the mutants (40 out of 55, i.e. 73%) were recovered repeatedly, e.g. 11 times for Ser-Gly (Figure 3A and Additional File 1-Table S1). This suggested that we were approaching saturation in this experimental setting. As predicted, sorting for fluorescent cells significantly selected against the presence of non-expressing cells: the incidence of “”amber”" stops within the two mutated find more codons was reduced 18-fold, from 5 clones in the unsorted to 1 in the sorted population. We randomly chose 93 clones from the sorted population for further analysis. This cohort covered 43 individual mutants, 11 of which PI3K Inhibitor Library were also identified in the presorted population (Figure 3A as well as Additional File 1-Table S1). The mutants were assessed for (i) protein levels and (ii) protein localization within the spirochetal cell envelope by in situ proteolysis and membrane fractionation. The observed protein levels provided a measure of fusion protein stability in vivo, as expression of all mutant proteins was driven
by an identical promoter. Furthermore, there was no correlation between the genomic frequency of the introduced codons and protein levels; correlation coefficients were -0.06 and -0.30 for Methisazone the first and second codon, respectively. All experiments were done in triplicate. Mutant phenotypes are summarized in Figure 3A and Additional File 1-Table S1. Figure 4 shows a representative raw dataset of mutants discussed in more detail below, while raw data for all 43 mutants can be found in the Additional Files (Additional File 2-Figures S1 and S2). OspA28:mRFP1 and OspA20:mRFP1 (labeled as ED in all figures and tables) were included as controls. Surface localization of the OspA:mRFP1 mutants was assessed by proteolytic shaving with proteinase K followed by Western immunoblotting of whole cell lysates (Figure 4A and Additional File 2-Figure S1).