Journal: Nature protocols

Article Title: Phage-assisted continuous and non-continuous evolution

doi: 10.1038/s41596-020-00410-3

Figure Lengend Snippet: (a) In a fixed-volume vessel, the “lagoon”, selection phage 3 encoding an evolving protein of interest (POI) trigger gIII expression from an accessory plasmid 6 in host E. coli cells, resulting in the production of the essential phage protein, pIII. The lagoon is continuously diluted with a culture of fresh host cells. All replicating DNA within the lagoon is mutagenized via an engineered mutagenesis plasmid 7 to provide genetic diversity. Only the SP containing genes encoding functional POI variants are capable of replicating faster than the rate of dilution, allowing them to persist in the lagoon. (b) Gene variants on the SP that encode active POIs trigger the expression of gIII from the AP, typically through the activation or recruitment of an RNA polymerase (RNAP). The MP expresses mutagenic genes under the control of the arabinose promoter; induction of mutagenesis occurs upon addition of arabinose to the growth media. (c) Established PACE selection strategies for various POI (blue) activities, including RNA polymerase activity (upper left), protein:DNA binding (upper middle), protein solubility (upper right), proteolysis of specific amino acid sequences (middle left), protein:protein binding( middle right), base editing (lower left), and incorporation of non-canonical amino acids by orthogonal aminoacyl-tRNA synthetases (lower right), and base editing (lower right). (d) Negative selections can be performed in PACE by linking the expression of a dominant-negative version of gIII, gIII-neg, to undesired activity, such that phage propagation requires SP encoding POI variants with the desired activity and lacking the undesired activity.

Article Snippet: E. coli S2060 (Addgene 105064), derived from DH10β, genotype F’ proA+B+ Δ(lacIZY) zzf∷Tn10 lacIQ1 PN25-tetR luxCDE Ppsp(AR2) lacZ luxR Plux groESL / endA1 recA1 galE15 galK16 nupG rpsL ΔlacIZYA araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) proBA∷pir116 araE201 ΔrpoZ Δflu ΔcsgABCDEFG ΔpgaC λ– E. coli S2208, defined as strain S2060 transformed with plasmid pJC175e

Techniques: Selection, Expressing, Plasmid Preparation, Mutagenesis, Functional Assay, Activation Assay, Control, Activity Assay, Binding Assay, Solubility, Protein Binding, Dominant Negative Mutation

Journal: Nature protocols

Article Title: Phage-assisted continuous and non-continuous evolution

doi: 10.1038/s41596-020-00410-3

Figure Lengend Snippet: Troubleshooting table

Article Snippet: E. coli S2060 (Addgene 105064), derived from DH10β, genotype F’ proA+B+ Δ(lacIZY) zzf∷Tn10 lacIQ1 PN25-tetR luxCDE Ppsp(AR2) lacZ luxR Plux groESL / endA1 recA1 galE15 galK16 nupG rpsL ΔlacIZYA araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) proBA∷pir116 araE201 ΔrpoZ Δflu ΔcsgABCDEFG ΔpgaC λ– E. coli S2208, defined as strain S2060 transformed with plasmid pJC175e

Techniques: Plasmid Preparation, Sequencing, Selection, Transferring, Cloning, Activity Assay, Concentration Assay, Infection, Mutagenesis, Inhibition, Expressing, Isolation

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