In strain NF54, 373 amino acids of LysRS are deleted leaving only the C-terminal 126 amino acids). Importantly, in this strain the P lysK (Tbox) lysK construct is flanked by transcriptional terminators so that lysK expression is solely dependent on the P lysK (Tbox) promoter. To insert the P lysK (Tbox) lacZ reporter fusion into the chromosome of B. subtilis strain NF54, learn more plasmid pBCJ307 was integrated at the amyE locus, thereby generating strain NF206. To construct B.
subtilis strain NF113, that has expression of the endogenous lysS gene under the control of the lysK promoter and T box element, a 423 bp DNA fragment encoding the B. cereus lysK promoter and T box element (generated using oligonucleotides NF36F and NF15R) was fused to a 672 bp fragment of the lysS gene (generated using oligonucleotides OSI-027 manufacturer NF15F and NF3R/2) by overlapping PCR (using the outside
primers NF36F and NF3R/2). This DNA fragment was then digested with EcoRI and BamHI and cloned into EcoRI digested pBCJ102  to generate the plasmid pNF112: the P lysK (Tbox) lysS insert is flanked by transcriptional terminators in this plasmid. Plasmid pNF112 was then integrated into the B. subtilis chromosome at the lysS locus by a Campbell-type event to produce the strain NF113. To introduce the P lysK (Tbox) lacZ reporter fusion into strain NF113, it was transformed with chromosomal DNA from strain NF204 that contains the P lysK (Tbox) lacZ reporter fusion at the amyE locus, thereby generating strain NF205. Strain NF204 was constructed by transformation of strain 1A717  with pBCJ307. selleck chemicals llc To construct B. subtilis strain
NF60 in which expression of the endogenous asnS gene is placed under the control of the IPTG-dependent PSpac promoter and containing the P lysK(T box) lacZ fusion, a 516 bp DNA fragment encoding the asnS promoter region was amplified using oligonucleotides NF16F and NF16R, digested with HindIII and cloned into HindIII digested pMutinXZ to produce plasmid pNF40. Plasmid pNF40 was transformed into B. subtilis strain BCJ363 by a Campbell-type event to produce strain NF58. Plasmid pMAP65 (encoding the lacI gene) was then established in strain NF58 to ensure strict IPTG-dependent asnS expression, thereby Protein kinase N1 generating strain NF60. Measurement of tRNA charging by Northern analysis Establishing the level of charged tRNALys was carried out as previously described . B. subtilis tRNALys was detected with an oligonucleotide probe complementary to nucleotides 26-51 that was labeled either with DIG oligonucleotide Tailing Kit (Roche, East Sussex, UK) or with biotin (New England Biolabs, USA). Detection used either the DIG labeling kit (Roche, East Sussex, UK) or the NEB blot phototope kit (New England Biolabs, USA) according to the manufacturer’s instructions. Determination of β-galactosidase activity Measurement of β-galactosidase activity was as previously described .