Pneb193 vector

The E. coli–mycobacterial shuttle integrative vector, plasmid pJH223.HIVA, was previously constructed in our laboratory. The aph gene, conferring kanamycin resistance, was removed from pJH223.HIVA plasmid by SpeI digestion. Following aph gene excision, the glyA gene cassette was ligated into the plasmid and subsequently transformed into the E. coli M15ΔglyA strain. Briefly, this cassette included the following elements: the α fragment, containing the weak constitutive P3 promoter; the β fragment containing the glyA gene, which encodes the enzyme serine hydroxymethyl transferase; and its own terminator sequence T1, a string of termination codons. In E. coli, the synthesis of intracellular glycine is mainly performed by the serine hydroxymethyl transferase enzyme. The αβT1 DNA fragment was amplified by PCR using the pQEαβT1FucA plasmid DNA as a template and provided by Prof. Pau Ferrer. The primers were designed to incorporate SpeI and SmaI sites at both 5′ and 3′ termini of the amplified DNA fragment to be subcloned into pNEB193 vector (NEB, Ipswich, MA). Finally, the pNEB193.GlyA plasmid DNA was digested by SpeI, and the released αβT1 DNA fragment was cloned into pJH223.HIVA vector (lacking the kanamycin resistance gene after SpeI digestion), to generate the p2auxo.HIVA^int plasmid DNA.

The E. coli–mycobacterial shuttle vector, plasmid pJH222.HIVA, was previously constructed in our laboratory.^{5 (link)} The aph gene, conferring kanamycin resistance, was removed from pJH222.HIVA plasmid by SpeI digestion. Following aph gene excision, the glyA gene cassette was ligated into this plasmid and subsequently transformed into E. coli M15ΔglyA strain. Briefly, this cassette included the following elements: the α fragment, containing the weak constitutive P3 promoter; the β fragment containing the glyA gene, which encodes the enzyme serine hydroxymethyl transferase; and its own terminator sequence T1, a string of termination codons. In E. coli, the synthesis of intracellular glycine is carried out mainly by serine hydroxymethyl transferase enzyme. The αβT1 DNA fragment was amplified by PCR using the pQEαβT1FucA plasmid DNA as template.^{44 (link)} The primers were designed to incorporate SpeI and SmaI sites at both 5′ and 3′ termini of the amplified DNA fragment to be subcloned into pNEB193 vector (NEB, Ipswich, MA). Finally, the pNEB193.GlyA plasmid DNA was digested by SpeI, and the released αβT1 DNA fragment was cloned into pJH222.HIVA vector (lacking the kanamycin resistance gene after SpeI digestion), to generate the p2auxo.HIVA plasmid DNA.