Journal: ACS Omega

Article Title: A Study of CRISPR Ribonucleoprotein Displacement in Cell-Free Systems

doi: 10.1021/acsomega.4c09275

Figure Lengend Snippet: System outline for control of CRISPR interference via displacement of bound CRISPR ribonucleoproteins (RNPs). (a) Scheme of proposed reactions in the cell-free TXTL system. Relative plasmid concentrations in our experiments: [p_deGFP] = 1× , [p_dCas9] = 3× , [p_rsg9] = variable, [p_Rx] = variable. Here, and elsewhere, we use the notation “p_X” for the plasmid that expresses product X. (b) Detailed scheme for proposed displacement-mediated removal of a CRISPR RNP from target DNA (reaction vi from part (a)) via RNA strand displacement across the RNA:DNA hybrid region where the spacer binds to its target.

Article Snippet: E. coli cell-free TXTL extract was purchased from Daicel Arbor Biosciences (Ann Arbor, MI).

Techniques: Control, CRISPR, Plasmid Preparation

Journal: ACS Omega

Article Title: A Study of CRISPR Ribonucleoprotein Displacement in Cell-Free Systems

doi: 10.1021/acsomega.4c09275

Figure Lengend Snippet: Further investigation of remover RNA activity against coexpressed and preincubated removable sgRNAs. (a) New RNA species used in the experiments presented in this figure. Here and henceforth, we used removable sgRNAs (rsgRNAs) with a 40 nt linear binding region; the corresponding complementary remover RNA is thus called R40. (b) Experiment outline for preincubation of CRISPR RNPs with target DNA prior to the addition of plasmid-expressed remover RNA that has been separately incubated for the first 2 h of the experiment. (c) End point data from a one-pot experiment, presented as percent fluorescence recovery, normalized between the off-target (sgNT) and on-target (rsg9 only) controls. Modest fluorescence recovery is again observed. Negative fluorescence recovery percentages indicate lower end point fluorescence than the rsg9 positive control, which could be attributed to load on the TXTL system. Bars show means of three replicates; error bars are one standard deviation. Note that the y -axes of this and similar subsequent plots have been chosen to provide maximal clarity on the fluorescence recovery values across their relevant ranges. (d) End point data from a preincubation experiment, presented as percent fluorescence recovery. Significantly lower fluorescence recovery is observed, which suggests difficulty displacing the preincubated, transcribed rsgRNAs from their targets. Bars show means of three replicates; error bars are one standard deviation. Unprocessed end point data corresponding to this figure is presented in Figure S5 .

Article Snippet: E. coli cell-free TXTL extract was purchased from Daicel Arbor Biosciences (Ann Arbor, MI).

Techniques: Activity Assay, Binding Assay, CRISPR, Plasmid Preparation, Incubation, Fluorescence, Positive Control, Standard Deviation

Journal: ACS Omega

Article Title: A Study of CRISPR Ribonucleoprotein Displacement in Cell-Free Systems

doi: 10.1021/acsomega.4c09275

Figure Lengend Snippet: Initial study of removal of CRISPR RNPs using synthetic sgRNAs, in a one-pot experiment. (a) Scheme of proposed reactions in the cell-free TXTL system when synthetic sgRNAs are used. The only difference is that a finite quantity of the synthetic sgRNA is provided at the start, rather than it being synthesized continually from a plasmid during the experiment. We hypothesized that using a fixed supply of sgRNA would make it easier for our antisense remover RNAs to operate, as there would be less sgRNA to remove overall. (b) Fluorescence timecourse data showing performance of removers against synthetic sgRNAs. The R40 and R0 removers are as outlined in Figure a. The observed recovery of the deGFP fluorescence signal in the presence of R40 (remover with linear binding region) could indicate displacement-mediated removal of CRISPR RNPs, as no such signal is seem with R0 (control remover without linear binding region). Line shows mean of three replicates; shaded area is one standard deviation above and below the mean. (c) End point fluorescence data from the experiment shown in part (b). (d) End point fluorescence data from part (c), expressed as percentage fluorescence recovery by normalizing between the rsg9 negative control and sgNT positive control.

Article Snippet: E. coli cell-free TXTL extract was purchased from Daicel Arbor Biosciences (Ann Arbor, MI).

Techniques: CRISPR, Synthesized, Plasmid Preparation, Fluorescence, Binding Assay, Control, Standard Deviation, Negative Control, Positive Control

Journal: bioRxiv

Article Title: A truncation library for the soluble production of squalene synthase from Candida albicans in Escherichia coli

doi: 10.1101/2025.02.06.636915

Figure Lengend Snippet: SDS-PAGE analysis of the expression of SQS variants in E. coli . ‘ω’ denotes C-terminal truncations and ‘Δ’ denotes N-terminal truncations. ‘-C’, negative control (empty pET-28b(+)); ‘M’, PageRuler Prestained Protein Ladder. Expected sizes for SQS variants range from 42.0-45.7 kDa.

Article Snippet: The ligation mixture was transformed into E. coli TOP10 cells (Thermo Scientific) using a heat-shock method and plated onto low-salt Luria Bertani (LB, 10 g.l -1 tryptone, 5 g.l -1 yeast extract, 5 g.l -1 NaCl) agar plates containing 30 mg.l -1 kanamycin.

Techniques: SDS Page, Expressing, Negative Control

Journal: LWT

Article Title: Escherichia coli virulence inhibition by cell-free supernatants from mangrove forest bacteria producing quorum sensing inhibitor

doi: 10.1016/j.lwt.2023.115182

Figure Lengend Snippet: Fig. 5. Inhibitory effect of 1/2MIC of S. warneri MG1_8-CFS extract on E. coli biofilm formation and cell attachment in 48 h cultivation.

Article Snippet: The minimum inhibitory concentration (MIC) of the cell-free supernatant (CFS) extract against E. coli ATCC 25922 was 25 mg/mL.

Techniques: Cell Attachment Assay

Journal: LWT

Article Title: Escherichia coli virulence inhibition by cell-free supernatants from mangrove forest bacteria producing quorum sensing inhibitor

doi: 10.1016/j.lwt.2023.115182

Figure Lengend Snippet: Fig. 4. The effect of S. warneri MG1_8-CFS extract on E. coli biofilm formation. (A) Crystal violet quantification of biofilm formation of E. coli strains. (B) Biofilm inhibition percentage caused by sub-MIC of S. warneri MG1_8-CFS extract. Different letters are significantly different (Tukey test, P ≤0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The minimum inhibitory concentration (MIC) of the cell-free supernatant (CFS) extract against E. coli ATCC 25922 was 25 mg/mL.

Techniques: Inhibition

Journal: LWT

Article Title: Escherichia coli virulence inhibition by cell-free supernatants from mangrove forest bacteria producing quorum sensing inhibitor

doi: 10.1016/j.lwt.2023.115182

Figure Lengend Snippet: Fig. 6. AI-2 signaling production and interference by S. warneri MG1_8-CFS extract at 1/2MIC concentration of biofilm forming E. coli and V. campbellii ATCC BAA- 1119 (Positive control).

Article Snippet: The minimum inhibitory concentration (MIC) of the cell-free supernatant (CFS) extract against E. coli ATCC 25922 was 25 mg/mL.

Techniques: Concentration Assay, Positive Control

Journal: LWT

Article Title: Escherichia coli virulence inhibition by cell-free supernatants from mangrove forest bacteria producing quorum sensing inhibitor

doi: 10.1016/j.lwt.2023.115182

Figure Lengend Snippet: Fig. 7. Swimming motility of biofilm forming E. coli in the presence of S. warneri MG1_8-CFS extract at 1/2MIC concentration and control.

Article Snippet: The minimum inhibitory concentration (MIC) of the cell-free supernatant (CFS) extract against E. coli ATCC 25922 was 25 mg/mL.

Techniques: Concentration Assay, Control