Targeted and Random Mutagenesis of Ehrlichia chaffeensis for the Identification of Genes Required for In vivo Infection
Figure 5
Clonal isolation of the E. chaffeensis mCherry and GFP mutants.
Host cell-free organisms recovered from nearly 90% infected culture flask were used to make 10-fold serial dilutions and cultured in DH82 cultures. The culture flasks resulting in E. chaffeensis growth from the highest dilution were used to isolate genomic DNA and for assessing the Himar1 insertions by performing insertion-specific PCRs. Lanes 1–8, genomic DNA from mixed populations of mutants prior to cloning by serial dilution was used as the template for PCRs targeting to the 8 Himar genomic insertion sites (as listed in Figure 3); Lanes 1c–8c, as in lanes 1–8, but using DNAs recovered after serial dilution cloning of the organisms; Lanes with the letter N refer to no template DNA controls for each of the PCRs and lane M contained the 1 kb+ DNA molecular weight marker.