Figure 1. Validation of conditional dysregulation of DnaA.

(A) Two sgRNAs were designed to knock down borrelial dnaA transcription by CRISPRi. One sgRNA targeted the template strand directly upstream of the ORF (dnaA_T1), while the other targeted the template strand within the ORF (dnaA_NT1). (B) The dnaA CRISPRi bacteria, CRISPRi empty vector bacteria, and the strain with the dnaA overexpression plasmid grew at the same rate as the parental e2. Adding IPTG to these strains resulted in appropriate knockdown or overexpression of DnaA protein (C) and transcript (D), as assessed by immunoblot and qRT-PCR. The overexpression shuttle vector encodes a DnaA with an N-terminal 3xFLAG moiety and thus migrates above the native protein. Error bars in (B) and (D) represent the standard error of the mean (SEM).