Table 1.
List of oligos used in this study.
Restriction enzyme sites, wherever present, are shown in bold letters.
Fig 1.
Schematic representation of proposed vector with pMA-His as example.
Panel A depicts the PCR-generated amplicons of 6x-His fragment (amplicon 1; green bar) and the kanamycin resistance marker (amplicon 2; yellow bar). Primers used in these PCR reactions are shown as arrows. TAG (stop codon) and relevant restriction enzyme sites are shown. Panel B shows the graphical representation of proposed plasmid with acetamide-inducible promoter system (labelled as acetamidase). oriM and oriE represent the origin of replication for mycobacteria and E. coli, respectively. KanR is the kanamycin resistance marker. The nucleotide sequence of the region between EcoRV and HpaI is shown having the following elements: NdeI–EcoRV–Ser-Gly-Ser linker (italicized sequence)–reporter tags (either 6x-His, GFP or GST)–Ser-Gly-Ser linker (italicized sequence)–HpaI–Stop codon. For presentation purpose, 6x-histidine tag is shown as underlined sequence.
Table 2.
List of plasmids used in this study.
All the plasmids carry kanamycin resistance marker. pMA, pMT, and pMH represent plasmids carrying acetamidase, tetRO, and hsp60 promoter, respectively. 6xHis, hexa-histidine tag; GFP, Green Fluorescent Protein; GST, Glutathione S-transferases tag.
Fig 2.
Schematic representation of subcloning of various tags from pMA tag vectors to heat inducible promoter systems.
Respective tag-containing fragments (6xHis, GFP or GST) were obtained by digestion of pMA-His, pMA-GFP, or pMA-GST vectors by NdeI and SpeI (shown in the vectors) and ligated downstream of hsp60 promoter at the same sites. oriM and oriE represent the origin of replication for mycobacteria and E. coli, respectively. KanR is the kanamycin resistance marker. Similarly, cloning of these tags was carried out under tetRO promoter, thus giving rise to pMT vectors.
Fig 3.
Schematic representation of cloning of lacZ gene in given plasmids.
Panel A depicts site-directed mutagenesis to abolish NdeI restriction enzyme site (shown as strikethrough) within lacZ gene. Panel B depicts subcloning of lacZ gene in pMT-HIS and pMA-HIS vectors, which was carried out using NdeI and SpeI enzymes. Here, tetRO and acetamidase represent the tetracycline- and acetamide-inducible promoters. oriM, oriE, and KanR represent the origin of replication for mycobacteria and E. coli, and the kanamycin resistance marker, respectively.
Fig 4.
Expression of lacZ and other tags under different promoter systems.
The Western blot data presented here depict the production of β-galactosidase (Panel A), GFP (Panel B), and GST (Panel C). Expression in each case is shown under acetamidase, hsp60, and tetRO promoters, in presence (+) and absence (-) of the inducer. In each panel, the first lane depicts the molecular weight marker with few bands marked in kDa. Panel D shows the fluorescence microscopy imaging of M. smegmatis cells expressing GFP from pMA-GFP vector. Cells were also stained with FM4-64 dye and imaged.
Fig 5.
Schematic representation the proposed vector.
A schematic of the vector developed in this study is shown as pMX, where X = inducible promoter system (A, acetamide; H, hsp60; T, tetRO). The plasmid retains one of the promoters (acetamidase, hsp60, or tetRO) depicted in yellow. Downstream to the promoter is the EcoRV and HpaI restriction enzyme sites that are to be used for cloning purpose. These sites sandwich one of the three tags (viz. 6xHis, GFP, or GST) shown in black. oriM, oriE, and KanR represent the origin of replication for mycobacteria and E. coli, and the kanamycin resistance marker, respectively.