e coli  (ATCC)

Journal: bioRxiv

Article Title: Toxin-based screening of C-terminal tags in Escherichia coli reveals the exceptional potency of ssrA-like degrons

doi: 10.1101/2024.01.29.576913

Figure Lengend Snippet: A) ATP-dependent proteases can initiate proteolysis by recognizing substrate degrons. B) Sequence-based recognition of substrates is poorly understood in bacteria. E. coli ’s five ATP-dependent proteases are depicted. C) Overview of the DEtox screening approach, which couples degron-directed toxin proteolysis to degron enrichment in liquid culture.

Article Snippet: Selection pressure was achieved through expression of the small (∼15 kDa) protein toxin VapC, from the VapBC toxin-antitoxin system of E. coli strain W (ATCC 9637) [ ].

Techniques: Sequencing, Bacteria

Journal: bioRxiv

Article Title: Toxin-based screening of C-terminal tags in Escherichia coli reveals the exceptional potency of ssrA-like degrons

doi: 10.1101/2024.01.29.576913

Figure Lengend Snippet: A ) Expression of untagged VapC with 1% arabinose arrests cell growth in wild-type E. coli and in a Δ clpA Δ clpX strain. B ) Appendage of the full-length ssrA tag to the C-terminus of VapC restores cell growth in wild-type but not Δ clpA Δ clpX cells. C ) A minimal ssrA tag (YALAA) is sufficient to rescue toxicity in wild-type but not Δ clpA Δ clpX cells. D ) No rescue is observed when the terminal Ala is substituted with Asp (YALAD).

Article Snippet: Selection pressure was achieved through expression of the small (∼15 kDa) protein toxin VapC, from the VapBC toxin-antitoxin system of E. coli strain W (ATCC 9637) [ ].

Techniques: Expressing

Journal: bioRxiv

Article Title: Toxin-based screening of C-terminal tags in Escherichia coli reveals the exceptional potency of ssrA-like degrons

doi: 10.1101/2024.01.29.576913

Figure Lengend Snippet: A ) Ec °ClpXP (0.25 μM) degrades GFP YALAA with a K M of 8.3 ± 0.9 μM, GFP YALAS with a K M of 65 ± 18 μM, and GFP YALAD with a K M of 820 ± 670 μM. Data were fit to a Michaelis-Menten equation. B ) Expression of VapC YALAA , VapC YALAS , VapC YALAD in E. coli results in growth rates that correlate with each tag’s respective K M . C ) A VapC library randomizing the last position of the minimal ssrA tag (VapC YALAX ) was transformed into wild-type E. coli and grown under inducing conditions in liquid culture. Sanger sequencing revealed random codon composition at 0 h but strong enrichment of Ala-encoding codons (GCT, GCG) after 6 h of growth.

Article Snippet: Selection pressure was achieved through expression of the small (∼15 kDa) protein toxin VapC, from the VapBC toxin-antitoxin system of E. coli strain W (ATCC 9637) [ ].

Techniques: Expressing, Transformation Assay, Sequencing

Journal: bioRxiv

Article Title: Toxin-based screening of C-terminal tags in Escherichia coli reveals the exceptional potency of ssrA-like degrons

doi: 10.1101/2024.01.29.576913

Figure Lengend Snippet: A ) A VapC library bearing five NNK randomized C-terminal codons (VapC 5X ) was transformed into wild-type E. coli and plated on 0% (-) and 1% (+) arabinose. Approximately 1% of library transformants support growth under inducing conditions, producing colonies of varying size. B ) Cell density of the transformed library was monitored over time in the absence and presence of inducer. Samples of induced culture were harvested at the indicated time points (red arrows) to assess library composition.

Article Snippet: Selection pressure was achieved through expression of the small (∼15 kDa) protein toxin VapC, from the VapBC toxin-antitoxin system of E. coli strain W (ATCC 9637) [ ].

Techniques: Transformation Assay

Journal: Molecules

Article Title: Cannabidiolic acid in Hemp Seed Oil Table Spoon and Beyond

doi: 10.3390/molecules27082566

Figure Lengend Snippet: Antimicrobial activities of CBDA against ATCC strains. a MIC, minimum inhibitory concentration values are expressed as concentration of CBDA. b MBC, minimum bactericidal concentration values are expressed as concentration of CBDA.

Article Snippet: E. coli ATCC 13762 , 0.5 , 8.

Techniques: Concentration Assay

Journal: Molecules

Article Title: Cannabidiolic acid in Hemp Seed Oil Table Spoon and Beyond

doi: 10.3390/molecules27082566

Figure Lengend Snippet: Time-kill kinetics of CBDA against E. coli ATCC 13762, S. aureus ATCC 6538, P. aeruginosa ATCC 27853 and E. faecalis ATCC 29212. Time killing curve of CBDA at MIC (◼), 2X MIC (▲), and 4X MIC (▼) values against ( A ) E. coli ATCC 13762, ( B ) S. aureus ATCC 6538, ( C ) P. aeruginosa ATCC 27853, and ( D ) E. faecalis ATCC 29212. NC (●): negative control. Data were expressed as the mean ± SD of three individual experiments. Calculated statistical significance by Student t test at 1, 2, 3, 5 h vs. 0 h was indicated * p < 0.05.

Article Snippet: E. coli ATCC 13762 , 0.5 , 8.

Techniques: Negative Control

Journal: Molecules

Article Title: Cannabidiolic acid in Hemp Seed Oil Table Spoon and Beyond

doi: 10.3390/molecules27082566

Figure Lengend Snippet: ( A ) Activity of CBDA against biofilms of E. coli ATCC 13762, S. aureus ATCC 6538, P. aeruginosa ATCC 27853 and E. faecalis ATCC 29212. CBDA was tested for inhibition of biofilm formation at concentrations corresponding to MIC, 2X MIC and 4X MIC against E. coli ATCC 13762, S. aureus ATCC 6538, P. aeruginosa ATCC 27853 and E. faecalis ATCC 29212. NC: negative control. Gentamycin (0.2 µg/mL) was used as positive control. Data were expressed as the mean ± SD of three individual experiments. Multiple comparisons were performed by ANOVA. Different letters (a, b, c) indicate significant differences at p < 0.05 analyzed by ANOVA. ( B ) Biofilms of S. aureus ATCC 6538 and P. aeruginosa ATCC 27853 were grown in 24-well plates for 24 h in the absence (NC) or presence of CBDA and stained with the LIVE/DEAD bacterial biofilm kit (SYTO-9 staining).

Article Snippet: E. coli ATCC 13762 , 0.5 , 8.

Techniques: Activity Assay, Inhibition, Negative Control, Positive Control, Staining

Journal: Molecules

Article Title: Cannabidiolic acid in Hemp Seed Oil Table Spoon and Beyond

doi: 10.3390/molecules27082566

Figure Lengend Snippet: Membrane permeabilization induced by CBDA in S. aureus ATCC 6538 and E. coli ATCC 13762. Dose–response curves of membrane permeabilization of ( A ) S. aureus ATCC 6538 and ( B ) E. coli ATCC 13762, measured by SYTOX Green fluorescence. Basal values at 0 time are coincident with basal fluorescence intensity axis, where measures are indicated in a non-proportional scale. Bacterial cells were suspended in distilled water and treated with CBDA for 0, 20, 40 and 60 min, whereupon fluorescence was measured. Values are means with standard error bars of triplicate measurements and correspond to one representative experiment of the three. Multiple comparisons were performed by ANOVA. Different letters (a, b) indicate significant differences at p < 0.05 analyzed by ANOVA. NC: negative control.

Article Snippet: E. coli ATCC 13762 , 0.5 , 8.

Techniques: Membrane, Fluorescence, Negative Control