Journal: Current biology : CB

Article Title: A conserved PDZ Binding Motif in aPKC interacts with Par-3 and mediates cortical polarity

doi: 10.1016/j.cub.2019.12.055

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Doe Lab N/A D. melanogaster: elav-Gal4, UAS-mCD8:GFP, hs:flp; FRT-G13, tubPGal80 Bloomington Drosophila Stock Center RRID:BDSC_5145 Oligonucleotides Recombinant DNA pCMV mammalian expression plasmid ThermoFisher 10586014 pMal C4X bacterial expression plasmid Addgene 75288 pGex 4Ti bacterial expression plasmid Amersham 27458001 pUASTattB fly cloning and transformation plasmid Addgene EF362409.1 Software and Algorithms ImageJ GraphPad Other PD10 Desalting columns 95017–001 VivaSpin 20 sample concentrators MWCO 30kD VWR 95056–130 VivaSpin 20 sample concentrators MWCO 10kD VWR 95056–128 Shaker Flasks – 125mL VWR 89095–258 Open in a separate window KEY RESOURCES TABLE Par-3 and Par-6/aPKC interact through a PDZ2 – PDZ Binding Motif interaction The Par-3 PDZ2 interaction with the aPKC PBM is conserved across metazoan The aPKC PBM is required for cortical polarity in Drosophila neuroblasts

Techniques: Diagnostic Assay, Recombinant, Construct, Variant Assay, Expressing, Blocking Assay, Plasmid Preparation, Clone Assay, Transformation Assay, Software

Journal: Cell reports

Article Title: Systematic dissection of σ 70 sequence diversity and function in bacteria

doi: 10.1016/j.celrep.2021.109590

Figure Lengend Snippet: (A) Evolutionary conservation of primary σ 70 (by Jensen-Shannon divergence) based on alignment of 4,702 σ 70 sequences. Residue positions are based on E. coli σ 70 with different domains shown (σ 70 1 , σ 70 2 , σ 70 3 , and σ 70 4 ). (B) Distribution of amino acid substitution counts of σ 70 orthologs for domains 2–4 compared to the E. coli σ 70 sequence. Colors in each bar correspond to ortholog host phylogeny at the phylum level, with the exception of Proteobacteria, which are separated at the class level. (C) Cumulative distribution of orthologs clustered to 90% sequence identity for each domain. (D) Pairwise comparisons of substitution count ratios between σ 70 domains. Dashed lines denote 1:1 ratio.

Article Snippet: E. coli ECNR2 , Addgene / , 26931.

Techniques: Residue, Sequencing

Journal: Cell reports

Article Title: Systematic dissection of σ 70 sequence diversity and function in bacteria

doi: 10.1016/j.celrep.2021.109590

Figure Lengend Snippet: (A) Fitness landscape of E. coli σ 70 at residues 379–613 profiled by MAGE-seq. Columns of the heatmap correspond to positions along the σ 70 protein and rows correspond to all 20 amino acid residues plus stop codons (*). Open circles denote the wild-type E. coli σ 70 residue at each position. Gray squares denote data not available. Regions of structured alpha helices, relative solvent accessibility, and average fitness at each residue position are displayed above the heatmap. (B) Histogram of the distribution of fitness effects (DFEs) for each σ 70 domain. Dotted lines denote fitness of 0.95, deemed as the separation between neutral and detrimental fitness. (C) Scatterplot of σ 70 evolutionary conservation and mean fitness for each residue position. Neutral residues (fitness ≥ 0.95) are displayed in gray, while detrimental residues (fitness < 0.95) are colored by their respective domains. Colored dash lines indicate linear regressions of detrimental residue positions in each domain. (D) Protein structure of σ 70 (ribbon model) bound to an open DNA complex (stick model) using PDB: 6CA0. Red color scale represents mean fitness at each residue position on the σ 70 structure; dark gray regions are residues not profiled with MAGE-seq.

Article Snippet: E. coli ECNR2 , Addgene / , 26931.

Techniques: Residue, Solvent

Journal: Cell reports

Article Title: Systematic dissection of σ 70 sequence diversity and function in bacteria

doi: 10.1016/j.celrep.2021.109590

Figure Lengend Snippet: (A) DFEs of residue variants observed in natural ortholog sequences (top) compared to DFEs of all possible single-residue mutations in σ 70 (bottom). Fitness threshold of 0.95 is designated by the dotted line. (B) Plot of residue fitness in σ 70 orthologs versus binned 16S phylogenetic distance to E. coli showing higher fraction of deleterious fitness variants at greater evolutionarily distance from E. coli . (C) Blue boxplots show expected aggregate fitness (EAF) distributions of natural orthologs (pink) with increasing binned number of residue differences to E. coli σ 70 . Fitness at 0.95 is denoted by the dotted line. Gray boxplots show null EAF distributions of synthetically generated σ 70 sequences with random mutations at each residue difference bin. (D) EAF for each σ 70 domain against the total binned number of residue differences across domains 2–4.

Article Snippet: E. coli ECNR2 , Addgene / , 26931.

Techniques: Residue, Generated

Journal: Cell reports

Article Title: Systematic dissection of σ 70 sequence diversity and function in bacteria

doi: 10.1016/j.celrep.2021.109590

Figure Lengend Snippet: E. coli strains with σ 70 orthologs

Article Snippet: E. coli ECNR2 , Addgene / , 26931.

Techniques: Residue

Journal: Cell reports

Article Title: Systematic dissection of σ 70 sequence diversity and function in bacteria

doi: 10.1016/j.celrep.2021.109590

Figure Lengend Snippet: (A) Residue-level fitness map in the 15 σ 70 orthologs measured in E. coli . (B) Plot of EAF of each σ 70 ortholog and their measured fitness in E. coli . Measured fitness values represent the average from two independent fitness competition experiments. (C) Plot of fitness differential (measured fitness minus EAF) and proportion of residue differences of orthologs from E. coli RpoD. (D) Number of differentially expressed genes in Mx σ 70 and Ou σ 70 transcriptomes compared to Ec σ 70 . (E) The number and grouping of essential genes that are differentially upregulated or downregulated in Mx σ 70 and Ou σ 70 .

Article Snippet: E. coli ECNR2 , Addgene / , 26931.

Techniques: Residue

Journal: Cell reports

Article Title: Systematic dissection of σ 70 sequence diversity and function in bacteria

doi: 10.1016/j.celrep.2021.109590

Figure Lengend Snippet:

Article Snippet: E. coli ECNR2 , Addgene / , 26931.

Techniques: Virus, Recombinant, Reverse Transcription, SYBR Green Assay, Sequencing, Software

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