Journal: Nucleic Acids Research
Article Title: Dual expression of CCA-adding enzyme and RNase T in Escherichia coli generates a distinct cca growth phenotype with diverse applications
Figure Lengend Snippet: Dual expression of RNase T and tRNA nucleotidyltransferase in E. coli Δ cca enforces selective growth phenotype. ( A ) Schematic presentation of the established in vivo system based on the dual expression of RNase T (orange) and tRNA nucleotidyltransferase (blue) from a pETDuet vector construct. In E. coli , both activities compete in the turnover of tRNA 3′ ends. ( B ) in vivo complementation requires efficient A-adding activity. Streak outs display E. coli JM109(DE3) Δ cca (top) or wild type (bottom) transformed with the indicated pETDuet construct plated onto LB-amp. Eco CCA, E. coli CCA-adding enzyme; DxD, wild type enzyme with catalytically active carboxylates; AxA, inactive enzyme variant with catalytically active carboxylates replaced by alanine residues; DxA, inactive enzyme with catalytically active aspartate at position 23 replaced by alanine; Bha A, B. halodurans A-adding enzyme; Δ cca, cca gene disruption; wt, wild type. ( C ) Radiolabeled yeast tRNA Phe(GAA) (top) or tRNA Phe(GAA) -CC (bottom), respectively, was incubated in the presence of 20 % (v/v) crude extract from different E. coli JM109(DE3) samples containing the indicated recombinantly expressed enzymes. Reaction products were resolved on a 10 % denaturing PAGE. Δ cca , extract prepared from the strain carrying cca gene disruption; wt, extract from wild type strain; C, control incubation without extract, Eco CCA, extract from E. coli expressing recombinant CCA-adding enzyme; Bha A, E. coli extract expressing B. halodurans A-adding enzyme. ( D ) Utilization of dual expression as a selection system. A vector library with a semi-randomized GNN sequence at codon position 23 in the open reading frame for E. coli CCA-adding enzyme was generated. Introduction into E. coli JM109(DE3) Δ cca resulted in colonies carrying vectors with GAT codons.
Article Snippet: Following DpnI digest, E. coli NEB 5-alpha cells (NEB) were transformed with the DNA sample.
Techniques: Expressing, In Vivo, Plasmid Preparation, Construct, Activity Assay, Transformation Assay, Variant Assay, Incubation, Polyacrylamide Gel Electrophoresis, Recombinant, Selection, Sequencing, Generated