Strain construction To construct strain NF33, a 400 bp DNA fragment from the region upstream of B. cereus lysK was amplified by PCR using primers NF36F and NF36R, cut with EcoRI and BamHI, and ligated into
similarly restricted pDG268  to produce the plasmid pBCJ307. pBCJ307 was inserted into the amyE locus of the B. subtilis lysine auxotroph strain 1A765 by double crossover to produce strain NF33. In order to analyze the effect of a reduction of the cellular level of charged tRNALys on expression of a P lysK(T box) lacZ fusion, strain BCJ367 was constructed. Plasmid pBCJ307 was integrated into the B. subtilis chromosome AZD1480 clinical trial by a double crossover event at the amyE locus to produce strain BCJ363. To place the endogenous lysS gene of B. subtilis under IPTG inducible control, plasmid pMUTIN4  was digested with SalI and BsiWI and eluted from an agarose
gel Omipalisib purchase to remove the 2 kb lacZ gene. The ends of the plasmid molecule were blunt ended using Klenow polymerase and religated, resulting in plasmid pMUTINXZ. A 670 bp DNA fragment encoding the end of the yacF gene was amplified with oligonucleotides NF2F and NF2R using B. subtiliis strain 168 chromosomal DNA as a template. This fragment was digested with EcoRI and inserted into the EcoRI site of pMUTINXZ, resulting in plasmid pXZ2. Plasmid pXZ2 was then integrated onto the chromosome of strain BCJ363 by a Campbell enough type event check details generating strain BCJ366 thereby placing expression of the lysS gene under the control of the IPTG inducible Pspac promoter. To effect tight control of the Pspac promoter, replicating plasmid pMap65  that encodes a lacI gene, was transformed into BCJ366 to produce strain BCJ367. Strain NF54 was made to assess whether a B. subtilis strain expressing a T-box regulated lysK gene was viable. A 1.95 kb fragment of the B. cereus chromosome encoding the lysK promoter, leader region and structural gene was
generated by PCR using oligonucleotides NF36F and NF9R. This fragment was digested with EcoRI and cloned into the EcoRI site of plasmid pBCJ102 that has transcriptional terminators flanking the multiple cloning site, to generate plasmid pNF30 . A 2567 bp fragment encoding the lysK promoter, T box element and structural gene flanked by transcriptional terminator sequences was amplified using the pBluescript T7 and M13 reverse primers and plasmid pBCJ102 as template. The ends of this fragment were phosphoryalted using T4 polynucleotide kinase (Promega) and it was then cloned into the EcoRV site of plasmid pDG1730  to produce the plasmid pNF48. Plasmid pNF48 was integrated at the amyE locus of the B. subtilis chromosome by a double crossover event to produce strain NF52.