TABLE 1.
Bacterial strains and plasmids used in this study
| Strain or plasmid | Relevant characteristic(s) | Source or reference |
|---|---|---|
| Strains | ||
| Escherichia coli strain E. cloni 10G | F−mcrA Δ(mrr-hsdRMS-mcrBC) endA1 recA1 φ80dlacZΔM15 ΔlacX74 araD139 Δ(ara leu)7697 galU galK rpsL nupG λ−tonA | Lucigen |
| E. coli Rosetta 2 | F−ompT hsdSB(rB− mB−) gal dcm pRARE2 (Camr and tRNA genes argU, argW, argX, ileX, glyT, leuW, proL, metT, thrT, tyrU, and thrU) | Novagen |
| Mycobacterium smegmatis mc2155 | Mycobacterial expression host with high transformation efficiency | ATCC 700084 |
| Plasmids | ||
| pQE80 | Commercially available Escherichia coli expression vector | Qiagen |
| pVV16 | Shuttle vector and mycobacterial expression vector controlled by constitutive Hsp60 promoter | 23 |
| pMYC | Derived from PVV16 to express target genes with a C-terminal myc tag | This work |
| pQE80-Rv3230c | rv3230c in pQE80 | 6 |
| pVV16-DesA3 | desA3 in pVV16 | 6 |
| pVV16-DesA3-His6 | DesA3-His6 in pVV16 | This work |
| pMYC-DesA3-myc | DesA3-myc expressed from pMYC | This work |
| pMYC-DesA3-DKD | DesA3 with the last three amino acid changed from LAA to DKD expressed from pMYC | This work |
| pMYC-DesA3-LEA | DesA3 with the last three amino acid changed from LAA to LEA expressed from pMYC | This work |
| pUV15TetORs | Cloning shuttle vector and mycobacterial expression vector controlled by tetracycline-inducible promoter | 11 |
| pTet | Modified from pUV15TetORs to replace the original GFP gene with a useful multicloning site for further cloning work | This work |
| PGFPStop | Enhanced GFP gene with a stop codon in the multicloning site of pTet. | This work |
| Pgfp | Enhanced GFP gene without a stop codon in the multicloning site of pTet. This vector was used to make the constructs with variations in DesA3 C terminus described below. | This work |
| pGFP-DesA3-LAA | Expression of the GFP with a C-terminal fusion to native DesA3-LAA | This work |
| pGFP-DesA3-LAK | Expression of the GFP with a C-terminal fusion to native DesA3-LAK | This work |
| pGFP-DesA3-LAD | Expression of the GFP with a C-terminal fusion to native DesA3-LAD | This work |
| pGFP-DesA3-LKA | Expression of the GFP with a C-terminal fusion to native DesA3-LKA | This work |
| pGFP-DesA3-LDA | Expression of the GFP with a C-terminal fusion to native DesA3-LDA | This work |
| pGFP-DesA3-KAA | Expression of the GFP with a C-terminal fusion to native DesA3-KAA | This work |
| pGFP-DesA3-DAA | Expression of the GFP with a C-terminal fusion to native DesA3-DAA | This work |