e coli s2060  (Addgene inc)

Journal: Cell

Article Title: Phage-assisted evolution and protein engineering yield compact, efficient prime editors.

doi: 10.1016/j.cell.2023.07.039

Figure Lengend Snippet: Figure 2. Development and validation of a prime editing PACE selection (A) Schematic of PE-PACE selection circuit. Upon infection of E. coli by selection phage (blue), the NpuN intein and NpuC intein (pink) mediate reconstitution of the PE2 prime editor (purple and pink), which engages a pegRNA (dark green) and corrects a frameshift in T7 RNAP (orange) via PE. Functional T7 RNAP then transcribes gIII (light green), which enables SP propagation. (B) Phage replication levels from overnight propagation of empty phage (red), NpuC-PE2-RT phage (purple), and T7-RNAP phage (green) in PE-PACE host cells before pegRNA optimization.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and virus strains One Shot Mach1 T1 Phage-Resistant Chemically Competent E. coli Thermo Fisher Scientific Cat#C862003 E. coli S2060 Addgene #105064 Chemicals, peptides, and recombinant proteins BsaI-HFv2 New England BioLabs Cat#R3733S LguI (SapI) Thermo Fisher Scientific Cat#ER1932 T4 DNA Ligase New England BioLabs Cat#M0202S NEBuilder HiFi DNA assembly master mix New England BioLabs Cat#E2621S Dimethyl sulfoxide Sigma-Aldrich Cat#D8418-50ML Poly(ethylene glycol) 3350 Sigma-Aldrich Cat#P4338-500G DNaseI (Rnase-free) New England BioLabs Cat#M0303 Magnesium chloride solution Sigma-Aldrich Cat#M1028-10X1ML Carbenicillin Gold Biotechnology Cat#C-103 Chloramphenicol Gold Biotechnology Cat#C-105 Tetracycline Gold Biotechnology Cat#T-101 Streptomycin Gold Biotechnology Cat#S-150 L-arabinose Gold Biotechnology Cat#A-300 Glucose Sigma-Aldrich Cat#G7021 Bluo-gal Gold Biotechnology Cat#B-673-10 dNTPs New England BioLabs Cat#N0447S Lipofectamine 2000 Thermo Fisher Scientific Cat#11668019 TrypLE Thermo Fisher Scientific Cat#12605010 Proteinase K, recombinant, PCR grade Thermo Fisher Scientific Cat#11668019 SDS (10% wt/vol) Thermo Fisher Scientific Cat#15553027 DNAdvance Kit Beckman Coulter Cat#A48705 AMPure XP Beckman Coulter Cat#B23318 CleanCap Reagent AG TriLink BioTechnologies Cat#N-7113 N1 -Methylpseudouridine50 -Triphosphate TriLink BioTechnologies Cat#N-1081 LiCl Precipitation Solution (7.5 M) Thermo Fisher Scientific Cat#AM9480 DMEM, high glucose, GlutaMAX supplement Thermo Fisher Scientific Cat#10566016 Fetal bovine serum Thermo Fisher Scientific Cat#16000044 L-Glutamine Corning Cat#25-005-Cl Penicillin-Streptomycin Thermo Fisher Scientific Cat#15070063 GlutaMAX supplement Thermo Fisher Scientific Cat#35050061 N-acetyl-L-cysteine Sigma-Aldrich Cat#A7250-100G Human AB Serum Valley Biomedical Cat#HP1022HI Recombinant Human IL-2 Peprotech Cat#200-02 Lymphoprep density gradient medium STEMCELL Technologies Cat#07801 (Continued on next page) Cell 186, 3983–4002.e1–e13, August 31, 2023 e1

Techniques: Biomarker Discovery, Selection, Infection, Functional Assay

Journal: eLife

Article Title: Measuring the tolerance of the genetic code to altered codon size

doi: 10.7554/elife.76941

Figure Lengend Snippet: Figure 2. Engineering qtRNAs with codon patterns. (A) We measured quadruplet tRNAs (qtRNAs) that might arise through point insertions in the anticodon loop. Each qtRNA is based upon a tRNA from the Escherichia coli genome that serves as a ‘scaffold’ (Supplementary file 1). We tested two quadruplet codon patterns: a tRNA decoding the triplet codon ‘XYZ’ to a qtRNA decoding the quadruplet codon ‘XYZZ’ or ‘XYYZ’. In instances in which XYZZ and XYYZ are the same, the qtRNA is depicted on the XYZZ graph. We use ‘qtRNA’-‘three letter scaffold’-‘four letter codon’ nomenclature to refer to qtRNAs; for example, a serine qtRNA bearing a 5’-UCUA-3’ anticodon that recognizes 5’-UAGA-3’ in mRNA transcripts is referred to as qtRNASer TAGA. (B) We measured qtRNAs using a luciferase readthrough assay. Measurements are taken kinetically and normalized to culture density, and efficiency is reported relative to luminescence produced by a wildtype (WT), all triplet luciferase transcript. qtRNAs that are statistically >0 are annotated with their one-sample t-test p-value: 0.033(*), 0.0021(**), 0.0002(***), < 0.001(****). fMet qtRNAs are measured with a luciferase reporter bearing a quadruplet codon at residue 1; all others are measured with a quadruplet codon at residue 357 of luxAB. (C) Expression of qtRNAs can be toxic. Here, we report the fractional OD600 density difference between cultures where qtRNA expression had been induced versus suppressed. Data in (B and C) represent the mean and standard deviation of three to eight technical replicates in one biological replicate. For raw data, see Figure 2—source data 1.

Article Snippet: Standard phage cloning Competent E. coli S2060 cells were prepared containing pJC175e (Addgene #79219), a plasmid expressing pIII under control of the phage shock promoter (Badran et al., 2016), which enables propagation of ΔpIII M13 bacteriophage through complementation.

Techniques: Luciferase, Produced, Residue, Expressing, Standard Deviation