Pt18mobsacb

Mycobacterium neoaurum HGMS2, a non-pathogenic industrial Mycobacterium, was maintained in our laboratory and deposited at China Center for Type Culture Collection (CCTCC No: M2012522) [55 (link)] and its genome sequence was deposited in GenBank (CP031414.1). The plasmids p2NIL (Cat. 20188) [56 (link)] and pT18mobsacB (Cat. 72648) were purchased from AddGene (Watertown, MA, USA).
The genomic DNAs of Mycobacterium sp. HGMS2 and its mutants were extracted using a Bacterial Genomic DNA Extraction Kit from Tiangen (Beijing, China). Plasmids were purified using a Plasmid Purification Kit from Tiangen (Beijing, China). PCR fragments were purified using an agarose gel purification kit (Qiagen, USA).

The previously described ΔgacA::pK18mobsacB suicide vector [17 ] was conjugated into DC3000 by tri-parental mating using an E. coli helper strain carrying pRK600 [43 ]. Merodiploid colonies were passaged 1–3 days in KB broth with 50 μg/mL rifampicin prior to counter-selection on KB agar with 15% sucrose. Sucrose-resistant colonies were plated on KB agar with 50 μg/mL rifampicin supplemented with or without selective antibiotics. PCR reactions with primers gacA-F and gacA-R (S2 Table) were used to screen genomic DNA isolated from kanamycin-sensitive colonies for deletion of gacA. To generate a ΔgacA deletion in AC811 (Kan^R), the ΔgacA deletion cassette from pK18mobsacB was sub-cloned into pT18mobsacB (Addgene #72648, Tc^R) using the Gibson assembly method. Primers pT18gacA-F and pT18gacA-R (S2 Table) were used to PCR amplify the ΔgacA deletion cassette from pK18mobsacB. Primers pT18-F and pT18-R (S2 Table) were used to PCR amplify the entire backbone of pT18mobsacB. PCR products were assembled into an intact plasmid using NEBuilder HiFi mix (NEB). Plasmids were transformed into E. coli DH5a competent cells (NEB) by heat shock. Clones were screened for the presence of the ΔgacA deletion cassette by colony PCR with primers M13F and M13R, and constructs were confirmed by Sanger sequencing.