Vector Construction for Inducible bosR Expression in B. burgdorferi

To manipulate bosR expression in B. burgdorferi, an IPTG-inducible bosR expression construct was created by using a lac-based inducible expression system (Blevins et al., 2007). First, bosR was amplified from strain 297 via PCR using primers ZM113F and ZM195 (Table 2). Purified PCR product was digested with restriction enzymes NdeI and BglII, and then cloned into pOY99.2 (Ouyang et al., 2011) digested with same enzymes. In the resultant shuttle vector pOY152, bosR expression was directly controlled by the IPTG-inducible T5 promoter (PpQE30) of the protein overexpression vector pQE30 (Qiagen, Valencia, CA).

Oligonucleotide primers used in this study.

Restriction enzymes sites are underlined; mutation nucleotides are in bold.