e coli mg1655 rare  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: SspA is a transcriptional regulator of CRISPR adaptation in E. coli

doi: 10.1093/nar/gkae1244

Figure Lengend Snippet: CRISPRi screen identifies adaptation host factors. ( A ) Overview of the CRISPR adaptation process, highlighting key known host factors. ( B ) Schematic of the CRISPRi adaptation host factor screen. ( C ) Binned coverage plot of sgRNAs across the Escherichia coli genome. sgRNA occupancy was calculated as the difference between the normalised (post/pre-screen) binned sgRNA counts per base of the experimental (+dCas9) and paired control (–dCas9) conditions. Regions of the genome with high (‘enriched’) sgRNA coverage are interpreted to be genomic loci that positively regulate CRISPR adaptation; regions of the genome with low (or negative, i.e. ‘depleted’) sgRNA coverage are interpreted to be genomic loci that negatively regulate CRISPR adaptation. The highest-ranking regions with attributable genes are labelled; other labelled loci are the Ori and Ter regions, the murA gene and the CRISPR-II array. n = 9 biological replicates. ( D ) Volcano plot showing log 2 fold change for all genes versus their adjusted –log 10 P -values ( n = 9 biological replicates). The horizontal dashed line represents an adjusted P -value of 0.05; the vertical lines represent log 2 fold changes of –0.75 and 0.75. Genes targeted by sgRNAs differentially enriched that were selected for individual validation are labelled on the plot. ( E ) Top: deep-sequencing based measurement of the rates of new spacer acquisition in Keio knockouts harbouring pSCL565, after growth for 48h in liquid culture without induction of Cas1-Cas2 expression. Acquisition rates are shown relative to the wild-type parental strain. Open circles represent biological replicates ( n ≥ 3), bars are the mean (one-way ANOVA effect of strain P < 0.0001; Sidak's corrected multiple comparisons for wild-type versus knockouts, Δ pcnB P = 0.00217, Δ sspA P = 0.000102, polA ΔKlenow P < 0.0001; others ns). Bottom: representative agarose gel for the data shown. Expansions of the CRISPR array can be seen as higher sized bands above the parental array length. Additional statistical details in .

Article Snippet: Escherichia coli K-12 MG1655 and LC-E75 ( ) (derivative of MG1655, Addgene #115925) were used for the CRISPRi screen.

Techniques: CRISPR, Control, Biomarker Discovery, Sequencing, Expressing, Agarose Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: SspA is a transcriptional regulator of CRISPR adaptation in E. coli

doi: 10.1093/nar/gkae1244

Figure Lengend Snippet: Features of spacers acquired in knockout strains. ( A ) Prespacer substrates for CRISPR adaptation arise from a variety of sources. ( B ) Breakdown of percent normalised spacer count (total number of new spacers/number of CRISPR arrays sequenced × 100) according to spacer origin ( E. coli or plasmid) and strain of interest after naïve CRISPR adaptation assays. b-e: Δ polA : polA ΔKlenow fragment mutant. ( C ) Breakdown of percent of spacer attributable to each spacer origin ( E. coli or plasmid) and strain of interest after naïve CRISPR adaptation assays. ( D ) Breakdown of percent normalised spacer count (total number of new spacers / number of CRISPR arrays sequenced × 100) according to spacer origin ( E. coli or plasmid) and strain of interest after prespacer electroporation CRISPR adaptation assays. ( E ) Breakdown of percent of spacer attributable to each spacer origin ( E. coli or plasmid) and strain of interest after prespacer electroporation CRISPR adaptation assays. ( F ) Motifs in the 15-bp up- and downstream of the newly acquired spacer in its source location. ( G ) Binned coverage plot of newly acquired spacer across the E. coli genome (outer, purple) and pSCL565 plasmid (inner, tan) for the wild-type strain (top-left) and derivatives. See for the full set. h. qPCR-based measurement of the relative copy number of pSCL565 Ori and cas1 sequences in the wild-type and polA ΔKlenow mutant. Delta CT values in . Open circles represent biological replicates ( n ≥ 3), bars are the mean (one-way ANOVA effect of strain and target P < 0.0001; Sidak's corrected multiple comparisons for wild-type versus Δ sspA , CDF ori copy number P < 0.0001, cas1 copy number P < 0.0001). Additional statistical details in .

Article Snippet: Escherichia coli K-12 MG1655 and LC-E75 ( ) (derivative of MG1655, Addgene #115925) were used for the CRISPRi screen.

Techniques: Knock-Out, CRISPR, Plasmid Preparation, Mutagenesis, Electroporation

Journal: Nucleic Acids Research

Article Title: SspA is a transcriptional regulator of CRISPR adaptation in E. coli

doi: 10.1093/nar/gkae1244

Figure Lengend Snippet: SspA regulates CRISPR adaptation independently of H-NS. ( A ) Deep-sequencing based measurement of the rates of new spacer acquisition in strains pre-immunised with either a T or NT defence plasmid, harvested 3 h post λ vir infection in liquid culture and growth at 30°C. Open circles represent biological replicates ( n ≥ 3), bars are the mean (one-way ANOVA effect of strain P < <0.0001; Sidak's corrected multiple comparisons for wild-type + T versus knockouts + T, Δ sspA P = 082553, Δ hns P < 0.0001, Δ sspA Δ hns P = 0.999999; Δ sspA + T versus knockouts + T, Δ hns P < 0.0001, Δ sspA Δ hns P = 0.154762; Δ hns + T versus Δ hns + NT P < 0.0001; Δ hns + T versus Δ sspA Δ hns + T P < 0.0001). ( B ) Breakdown of normalised spacer count (total number of new spacers / number of CRISPR arrays sequenced) according to spacer origin ( E. coli , lambda or plasmid) and strain of interest. ( C ) Binned coverage plot of Δ hns + T newly acquired spacers across the lambda genome (outer, purple). The location of the T immunisation spacer is shown on the lambda genome; ‘missing in λ vir ’ indicates a genomic region missing in our strain of λ vir . ( D ) Percent of spacers acquired that are on the same strand as the T immunisation spacer, according to the spacer source ( E. coli or lambda). ( E ) Schematic of the sspAB and hns operonic rescue plasmids. All plasmids are low (∼5) copy, and encode either 1. The sspAB operon, 2. The hns operon, or 3. both, under their native regulation. ( F ) Schematic of the CRISPR adaptation assays in wild-type, sspA and/or hns mutant strains. Strains were electroporated with pSCL565 and rescue plasmids 1., 2. or 3. (see E ), and assessed for their ability to acquire new spacers into the endogenous CRISPR I array. ( G ) PCR-based detection of new spacer acquisition into the CRISPR I array of wild-type, of WT, Δ sspA :: FRT , Δ hns :: FRT and Δ sspA :: FRT Δ hns :: FRT strains harbouring pSCL565 and rescue plasmids 1., 2. or 3. (see E ), after growth for 48 h in liquid culture. Open circles represent biological replicates ( n ≥ 3), bars are the mean. Horizontal dashed line represents the mean rate of spacer acquisition in the wild-type strain (one-way ANOVA effect of strain P < <0.0001; Sidak's corrected multiple comparisons for wild-type versus knockouts, Δ sspA P < 0.0001, Δ hns P < 0.0001, Δ sspA Δ hns P < 0.0001; Δ sspA versus knockouts, Δ hns P = 0.714182, Δ sspA Δ hns P = 0.002269, Δ sspA + sspAB rescue P < 0.0001; Δ hns versus knockouts, Δ sspA Δ hns P < 0.0001, Δ hns + hns rescue P < 0.0001; Δ sspA Δ hns versus Δ sspA Δ hns + sspA & hns rescues P < 0.0001). ( H ) PCR-based detection of new spacer acquisition into the CRISPR I array of WT, Δ sspA :: FRT , Δ hns :: FRT , Δ sspA :: FRT Δcas3-Cascade::Cm R or Δ hns :: FRT Δcas3-Cascade::Cm R strains harbouring pSCL565 after growth for 48h in liquid culture. Open circles represent biological replicates ( n ≥ 3), bars are the mean (one-way ANOVA effect of strain P < 0.0001; Sidak's corrected multiple comparisons for wild-type versus knockouts, Δ sspA P < 0.0001, Δ hns P < 0.0001, Δ sspA Δ cas3-cascade P < 0.0001, Δ hns Δ cas3-cascade P = 0.125466; Δ sspA versus Δ hns P = 0.004161; Δ sspA versus Δ sspA Δ cas3 - cascade

Article Snippet: Escherichia coli K-12 MG1655 and LC-E75 ( ) (derivative of MG1655, Addgene #115925) were used for the CRISPRi screen.

Techniques: CRISPR, Sequencing, Plasmid Preparation, Infection, Mutagenesis

Journal: Nucleic Acids Research

Article Title: SspA is a transcriptional regulator of CRISPR adaptation in E. coli

doi: 10.1093/nar/gkae1244

Figure Lengend Snippet: sspA is a transcriptional regulator of CRISPR adaptation. A. sspAB operon, proteins and function. Bottom left: crystal structure of an SspA dimer in complex with E. coli RNAP-promoter open complex, showing the conserved SspA PHP 84-86 residues interacting with RNAP and σ 70 (PDB 7DY6 ). Top right: crystal structure of SspB escorting an SsrA-tagged substrate being delivered to the ClpXP protease complex (PDB 8ET3 ). B. Schematic of the sspAB operon of WT, Δ sspA :: kan R and Δ sspB :: kan R strains. kan R : kanamycin resistance cassette. C. Deep-sequencing based measurement of the rates of new spacer acquisition in strains harbouring pSCL565 and, in the case of the Δ sspA :: kan R , either an empty plasmid or a low (∼5) copy plasmid encoding the sspAB operon, after growth for 48 h in liquid culture. Adaptation rates are shown relative to the wild-type parental strain. The Δ ihfA strain was used as a negative control, as it is required for in vivo spacer acquisition in the E. coli type I-E CRISPR system ( , ). Open circles represent biological replicates ( n ≥ 3), bars are the mean. Horizontal dashed line represents the mean rate of spacer acquisition in the wild-type strain (one-way ANOVA effect of strain P < 0.0001; Sidak's corrected multiple comparisons for wild-type versus knockouts, Δ sspA P < 0.0001, Δ sspB P = 0.109807; Δ sspA versus Δ sspB P < 0.0001). D. Schematic of the sspAB operon variant rescue plasmids. All plasmids are low (∼5) copy, and encode variants of the sspAB operon under its native regulation. Frameshift mutants of SspA (AN 5-6 > AQ 5-6 GCC|AAC > GCT| CAA |C) and SspB (PR 9-10 > PS 9-10 CCA|CGT > CCA| TCG |T) encode sequences with single base insertions to cause protein translation to terminate early. The SspA PHP 84-86 > AAA 84-86 mutant is RNAP-binding deficient and thus does not enable the shift in promoter use (σ 70 → σ S ) . A single sspA rescue plasmid yielded no transformants into the Δ sspA :: kan R strain over multiple attempts. E. Top: deep-sequencing based measurement of the rates of new spacer acquisition in strains harbouring pSCL565 and, in the case of the Δ sspA :: kan R , either an empty plasmid or a low (∼5) copy plasmid encoding variants of the sspAB operon as described in d., after growth for 48 h in liquid culture. Adaptation rates are shown relative to the wild-type parental strain. Open circles represent biological replicates ( n ≥ 3), bars are the mean. Horizontal dashed line represents the mean rate of spacer acquisition in the wild-type strain (one-way ANOVA effect of strain P < 0.0001; Sidak's corrected multiple comparisons for wild-type versus knockouts, Δ sspA P < 0.0001, Δ sspA + empty plasmid P < 0.0001, Δ sspA + sspAB rescue P = 1, Δ sspA + sspA * (PHP84-86 > AAA84-86) & sspB rescue P < 0.0001; Δ sspA versus rescues, Δ sspA + empty vector P = 0.997758, Δ sspA + sspA * (PHP84-86 > AAA84-86) & sspB P = 0.334315, Δ sspA + sspAB P < 0.0001, Δ sspA + sspB P = 0.892991, Δ sspA + sspA * & sspB * (frameshifted) P = 1). Bottom: representative agarose gel for the data shown. Expansions of the CRISPR array can be seen as higher sized bands above the parental array length. F. RT-qPCR of the fold-change in RNA copy number of cas1 and cas2 in the Δ sspA versus WT strains, measured relative to mreB and GAPDH . g. Volcano plot showing the log 2 fold change in expression of genes in the Δ sspA versus WT strains versus their adjusted –log 10 P -values ( n = 3 biological replicates). The horizontal dashed line represents an adjusted P -value of 0.05; the vertical lines represent log 2 fold changes of –0.75 and 0.75. We identified nearly a thousand genes that were differentially expressed. Additional statistical details in .

Article Snippet: Escherichia coli K-12 MG1655 and LC-E75 ( ) (derivative of MG1655, Addgene #115925) were used for the CRISPRi screen.

Techniques: CRISPR, Sequencing, Plasmid Preparation, Negative Control, In Vivo, Variant Assay, Mutagenesis, Binding Assay, Agarose Gel Electrophoresis, Quantitative RT-PCR, Expressing

Journal: Nucleic Acids Research

Article Title: SspA is a transcriptional regulator of CRISPR adaptation in E. coli

doi: 10.1093/nar/gkae1244

Figure Lengend Snippet: H-NS regulates CRISPR interference downstream of SspA. ( A ) Model for SspA-mediated regulation of CRISPR-Cas defence. Phage infection triggers upregulation of SspA , which in turn induces a global transcriptional shift towards σ S -regulated promoters. This results in H-NS downregulation ( , ), induction of CRISPR-Cas mediated defence through de-repression Cas gene expression ( , ), leading to increased rates of CRISPR adaptation and interference. ( B ) Schematic of the sspAB and hns operons of WT, Δ sspA :: FRT , Δ hns :: FRT and Δ sspA :: FRT Δ hns :: FRT strains. FRT : flippase recognition target, a scar left after the removal of resistance cassettes. ( C ) Schematic of the CRISPR interference-mediated defence assays in pre-immunised E. coli strains. Top: schematic of the CRISPR-I immunisation (defence) plasmids. All plasmids are low (∼5) copy and encode an E. coli CRISPR-I array with a first spacer encoding either a Target (complementary to the λ genome ( , )), or a Non-Target (NT) spacer. Bottom: The experimental strains were electroporated with either the T or NT plasmid, and infected to varying titres of λ vir . Note that the strains encode a complete endogenous E. coli Type I-E CRISPR-Cas system. ( D ) Representative plaque assays of λ vir on experimental strains (described above) pre-immunised with either T or NT defence plasmids. Strains were infected with λ vir and grown on plates at 30°C for 16 h. Full plaque assay plates for n = 3 biological replicates in . ( E ) Efficiency of plating of λ vir on experimental strains; raw plaque counts and subsequent analysis in . Open circles represent biological replicates ( n ≥ 3) of individual plaque assays, bars are the mean (one-way ANOVA effect of strain P = 0.033454; Sidak's corrected multiple comparisons for wild-type versus knockouts, Δ sspA P = 0.181757, Δ hns P = 0.043319, ΔsspA Δhns P = 0.043316; for Δ hns versus Δ sspA Δhns P = 1). E. Anti-phage defence and growth in overnight liquid culture of experimental strains, post λ vir infection (MOI: 0.1). Hue around solid line (mean) represents the standard deviation across three biological replicates.

Article Snippet: Escherichia coli K-12 MG1655 and LC-E75 ( ) (derivative of MG1655, Addgene #115925) were used for the CRISPRi screen.

Techniques: CRISPR, Infection, Gene Expression, Plasmid Preparation, Plaque Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Exploring the Potential of Glutathione Reductase Overexpression to Improve Tellurium Nanoparticle Production in Escherichia coli

doi: 10.3390/ijms26041549

Figure Lengend Snippet: Figure 2. Spectral and molecular weight evaluation for purified GR enzymes: ⎯E. coli GR, ⎯BNF22 GR, ⎯BNF08 GR, and ⎯MF01 GR. (A) SDS-PAGE gel. (B) CD spectra. (C) Results of the size exclusion chromatography. The molecular weight of GR enzymes was determined to be ≈110 kDa. (D) Absorption spectra under reduced conditions. (E) Fluorescence emission spectra.

Article Snippet: Assays of GR overexpression were performed in E. coli MG1655 (DE3); the strain was purchased from Addgene under the bacterial strain reference number #37854 [50].

Techniques: Molecular Weight, Purification, SDS Page, Circular Dichroism, Size-exclusion Chromatography, Fluorescence

Journal: Chembiochem

Article Title: Biosensor‐Guided Engineering of a Baeyer‐Villiger Monooxygenase for Aliphatic Ester Production

doi: 10.1002/cbic.202400712

Figure Lengend Snippet: LuxAB‐based HT assays. ( A ) Aliphatic esters ( 1–11 b ) are cleaved by BS2 and the corresponding alcohols are oxidized by AlkJ to aldehydes. The latter are sensed by LuxAB expressed in the same cell. ( B ) Primary alcohols ( 1–11 c ) are added directly, oxidized, and the corresponding aldehydes detected as before; co‐factors are omitted for clarity. Bioluminescence signals monitored over time before (0 min) and after the addition (3–15 min) of 1 mM substrate to RCs of E. coli RARE (OD 600 ≈10.0), co‐expressing BS2 from pET28a, AlkJ and LuxAB from pLA1. The XCO value was determined in the presence of 1 % ( ν/ν ) dimethylformamide (DMF) as described below. ^[24] Heat‐maps show the mean fold‐increase in bioluminescence above background of biological replicates (n=3); n.a.=not available ( 7 b , 10 b , 1 c , and 4 c ).

Article Snippet: E. coli RARE was a gift by the Prather group but is also available from Addgene (#61440).

Techniques: Expressing

Journal: Chembiochem

Article Title: Biosensor‐Guided Engineering of a Baeyer‐Villiger Monooxygenase for Aliphatic Ester Production

doi: 10.1002/cbic.202400712

Figure Lengend Snippet: Activity of BVMOs and variants in vivo . ( A ) Formation of ester 6 b from the benchmark ketone 6 a monitored by LuxAB‐based assays (green) and confirmed by GC/FID analysis (magenta). The variants C57A, S188A, and the corresponding double‐mutant exhibited similar activities than BVMO Halo wild‐type (WT). E. coli TOP10 transformed with the empty pBAD vector was used as negative control (NC) and did not convert 6 a ; the cyclohexanone monooxygenase from Acinetobacter sp. (CHMO Acineto ) was used as additional control for the oxidation of aliphatic ketones. ^[59] The fold‐increase in bioluminescence and the concentration of 6 b are given as mean values ± SD of biological replicates (n≥2). Biosensing conditions are described below. Biotransformations were performed at 25 °C in E. coli RCs as indicated (OD 600 ≈10.0); 5 mM substrate load, 5 % ( ν/ν ) DMF as co‐solvent. For time‐resolved production of 6 b and recovery of material, see Figure S4. ( B ) Conversion of ketone 5 a (light pink) into the desired ester 5 b (magenta). Biotransformation conditions as in ( A ); overall conversion of 5 a was lower than for 6 a . The composition of reaction mixtures [mM] and recovery of material [%] are represented as mean values ± SD of biological replicates (n≥2). Reduced recoveries attributed to low solubility and/or volatility of compounds. ^[24]

Article Snippet: E. coli RARE was a gift by the Prather group but is also available from Addgene (#61440).

Techniques: Activity Assay, In Vivo, Mutagenesis, Transformation Assay, Plasmid Preparation, Negative Control, Control, Concentration Assay, Solvent, Solubility