Construction of pAN71-ltak63-lysA Vectors

The pAN71-ltak63 vector containing pAN promoter and driving low expression of the LTAK63 adjuvant was previously constructed by our group (5 (link)). A PCR-amplified lysA gene (containing a Shine-Dalgarno, SD), or a lysA expression cassette digested from pJH152 were used. The pAN71-ltak63 vector was digested with NotI/PvuII (New England Biolabs) and the lysA fragment inserted in tandem with ltak63 under the same pAN promoter, generating pAN71-ltak63-lysA(t) (Figure 2A). In this construct, the kanamycin resistance marker was removed by digestion with NsiI (New England Biolabs) and self-ligated. The pAN71-ltak63 vector was also digested with NotI/ClaI (New England Biolabs) to clone the lysA expression cassette, generating pAN71-ltak63-lysA(c) (Figure 2B). The kanamycin resistance marker was removed by digestion with ClaI (New England Biolabs) and self-ligated. Both vectors were electroporated into M. smegmatis mc² 1493 (lysine auxotroph) (19 (link)) and transformants plated onto MB7H10. Transformants were then grown in MB7H9 with and without kanamycin to confirm construction of the unmarked pAN71-ltak63-lysA vectors. Plasmid extraction from M. smegmatis was performed using Wizard Plus SV Minipreps DNA Purification kit (Promega, Madison, WI, USA) and the extracted plasmids were used to transform the lysine auxotrophic BCG (BCGΔlysA).