Figure 6: Excision of selection cassette using FLAEM restores expression of tmeB when targeting tmeA and does not disrupt downstream genes.

(A) Relative mRNA level of C. trachomatis tmeA and tmeB mutant strains. The presence of transcripts downstream of tmeA was determined by reverse transcriptase (RT) quantitative PCR. The region immediately downstream of the tmeA/B operon encodes ct696. Total RNA was isolated at 24 hpi from McCoy cells infected at an MOI of 1 with WT, L2 tmeA, L2 tmeB, or L2^Rif tmeA-lx. Transcripts for tmeA, tmeB, and ct696 were detected by qRT-PCR. Signals are presented after normalization to rpoD. ND = none detected. (B) Western blot for presence of TmeA and TmeB in C. trachomatis mutant strains. Equal quantities of whole-culture material from 24 hpi cultures infected with equal inclusion forming units of WT, L2 tmeA, L2^Rif tmeA-lx, or L2^Rif tmeA-lx ptmeA were probed in immunoblots for TmeA and TmeB. Hsp60 was used as a loading control, and proteins were visualized by chemiluminescence. Figure has been modified from Keb et al.¹².