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Image Search Results
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:
Techniques: Plasmid Preparation, Transformation Assay, Selection, Standard Deviation