Journal: bioRxiv

Article Title: Preventing escape and malfunction of recoded cells due to tRNA base changes

doi: 10.1101/2024.07.18.604179

Figure Lengend Snippet: a , Wild-type beta-lactamase gene in plasmid pUC57, conferring ampicillin/carbenicillin resistance. Black arrows locate all wild-type serine codons, and blue arrows highlight TCA/TCG (TCR) codons. b , Top: Schematic representation of the transformation and selection process performed with all plasmids to study the escape rate of Ec_Syn61Δ3. Bottom left: Engineered genetic code of Ec_Syn61Δ3. Missing codons, anticodons, and genes are marked with dashed lines. Remaining codons, anticodons, and genes are marked with bold lines. Bottom right: Schema of central dogma of biology including our observation. c , Relative escape rate [(cfu/mL)/(cfu/mL)] defined as the number of colonies formed by Ec_Syn61Δ3 with (+) and without (-) the wild-type plasmid pUC57 relative to those of parallel experiments with pUC57-dTCR upon selection onto carbenicillin-containing agar plates ( n = 4 ). Escapees of Ec_Syn61Δ3 without the pUC57 plasmid were not detected (ND) onto carbenicillin-containing agar plates with up to 1 mL of OD 600 plated (Methods). The dashed bar length represents the ratio of 10 -9 cfu/mL and measured number of cells of Ec_Syn61Δ3 with pUC57-dTCR ( n = 4 ). d , Number of colonies formed by Ec_Syn61Δ3 escapees with selected plasmids including TCR-containing antibiotic resistance genes upon selection onto antibiotic-containing agar plates (mean ± standard deviation (SD), n = 2-4 ). The table below describes the number of TCR codons, plasmid copy number, presence of a his-tag on the antibiotic resistance protein, and antibiotic resistance for each plasmid. High or low plasmid copy number is denoted by + or -. Presence or absence of a His-tag is denoted by + or -. Antibiotic resistance acronyms: ChlR, chloramphenicol; KanR, kanamycin; AmpR/CarbR, ampicillin/carbenicillin; SpecR, spectinomycin; and GentR, gentamicin. Designs tagged as “max” or “min” contain TCR codons in serine positions with the highest or lowest probability for serine, respectively, based on the protein language model ESM-1b (Supplementary Fig. 3). Antibiotic acronyms: A, chloramphenicol; B, kanamycin; C, carbenicillin; D, spectinomycin; and E, gentamicin.

Article Snippet: We worked with the following antibiotics and final concentrations: Chloramphenicol (C0316, Teknova) at 25 µg/mL, Kanamycin (K2199, Teknova) at 50 µg/mL, Carbenicillin (E0096, Teknova) at 100 µg/mL, Spectinomycin (S9525, Teknova) at 75 µg/mL, and Gentamicin (15750060, Thermo Fisher Scientific, Life Technologies) at 15 µg/mL.

Techniques: Plasmid Preparation, Transformation Assay, Selection, Standard Deviation

Journal: bioRxiv

Article Title: Preventing escape and malfunction of recoded cells due to tRNA base changes

doi: 10.1101/2024.07.18.604179

Figure Lengend Snippet: a , Schematic representation of data collected to identify the mechanism of escape in Ec_Syn61Δ3. b , Mass spectrum of the beta-lactamase enzyme (encoded in a gene containing 3 TCR codons, pCmin design) as detected in escapees of Ec_Syn61Δ3. We detected serine incorporated into the third TCR codon with the peptide QIAEIGASLIK (see Supplementary Figures 7-11 for complementary data). c , Differential, untargeted proteomics analysis of Ec_Syn61Δ3 escapees with the pUC57 plasmid relative to Ec_Syn61Δ3 ( n = 3 ). We highlight the overexpressed lysU . d , Detection of mutation in the anticodon of the tRNA Ser serW/X G34U. e , In vitro aminoacylation of the wild-type tRNA Ser serW/X and the mutants of serW/X with anticodons UGA and CGA show similar aminoacylation levels. f , Fluorescence per OD 600 over time in Ec_Syn61Δ3 carbenicillin escapees with our genetic-code-sensitive reporter (light green), compared to fluorescence of this reporter in Ec_Syn61 with tRNA Ser genes serT and serU (dark green) and in Ec_Syn61Δ3 when it is part of a plasmid with a recoded carbenicillin resistance gene (gray) (mean ± SD; n = 5 ).

Article Snippet: We worked with the following antibiotics and final concentrations: Chloramphenicol (C0316, Teknova) at 25 µg/mL, Kanamycin (K2199, Teknova) at 50 µg/mL, Carbenicillin (E0096, Teknova) at 100 µg/mL, Spectinomycin (S9525, Teknova) at 75 µg/mL, and Gentamicin (15750060, Thermo Fisher Scientific, Life Technologies) at 15 µg/mL.

Techniques: Plasmid Preparation, Mutagenesis, In Vitro, Fluorescence