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