host e coli strain mg1655  (ATCC)

Journal: bioRxiv

Article Title: Programmable domestication of thermophilic bacteria through removal of non-canonical defense systems

doi: 10.64898/2026.03.21.713436

Figure Lengend Snippet: Plasmid artificial modification–assisted conjugative engineering (PACE). Methylation-matched plasmids produced in engineered E. coli donor strains are transferred to Geobacillus recipients via pRK24-mediated conjugation. While host-specific methylation enables restriction evasion, intracellular defense systems limit plasmid establishment in wild strains. b, Architecture of the native Type II-C CRISPR–Cas system (GeoCas9EF) adapted for genome editing. A 21-bp spacer sgRNA cassette targets genomic loci adjacent to a protospacer-adjacent motif (PAM). c, Sequence logo of the preferred PAM recognized by GeoCas9EF in G. stearothermophilus EF60045, indicating a consensus 5’-NNNNCAAA- 3’ motif. d, CRISPR-mediated genome editing strategy. A conjugative plasmid carrying GeoCas9EF, sgRNA, and homologous repair arms enables genome modification via Cas9-induced cleavage and homologous recombination. Single-crossover (SCO) intermediates are resolved into double-crossover (DCO) mutants or false positives. Promotor optimization for Cas9 expression in strain SJEF4-2 is shown. e, PCR validation of representative gene deletions generated by CRISPR editing in SJEF4-2 and EF60045, targeting pyrimidine biosynthesis loci (e.g., pyrR and pyrFE ). Expected fragment sizes are indicated. f, Transformation efficiencies following sequential removal of endogenous defense systems. In EF60045 and SJEF4-2, deletion of native plasmids and non-canonical defense modules, including Wadjet II, BREX, CBASS, Gabija, Abi, and SspBCDE, dramatically increased electroporation efficiency (CFU µg ¹ DNA) and conjugation efficiency (transconjugants per recipient), revealing these systems as dominant barriers to genetic domestication.

Article Snippet: The broad-host-range conjugative plasmid pRK24 (Addgene plasmid # 51950, courtesy of Farren Isaacs) was first introduced into E. coli MC variants and selected on tetracycline (10 μg/mL).

Techniques: Plasmid Preparation, Modification, Methylation, Produced, Conjugation Assay, CRISPR, Sequencing, Homologous Recombination, Expressing, Biomarker Discovery, Generated, Transformation Assay, Electroporation

Journal: Nature Communications

Article Title: Co-occurrence of diverse defense systems shapes complex microbe-virus relationships in deep-sea cold seeps

doi: 10.1038/s41467-025-68174-6

Figure Lengend Snippet: a Types and proportions of verified defense systems and candidate defense systems in cold seep microbial genomes, including their primary mechanisms of action and corresponding system counts. “DS” denotes defense systems and “num” represents the total count. “Type num” refers to the number of distinct defense system types within each mechanism category. b Plaque assays demonstrating phage infection of Escherichia coli B (ATCC ® 11303™) transformed with plasmids carrying various candidate defense systems or an empty vector (control) using a small volume drop method. The bacteria described above were mixed in the molten agar matrix and ten-fold serial dilutions of phages T7, T5, and T4 were spotted onto bacterial lawns. All the assays are repeated at least 3 times with similar results. Predicted protein domains of the tested systems are shown on the right. Detailed data on annotated defense systems are provided in Supplementary Data – .

Article Snippet: Recombinant vectors containing these candidate systems were transformed into the host strain E. coli B (ATCC ® 11303TM), which naturally lacks these systems.

Techniques: Infection, Transformation Assay, Plasmid Preparation, Control, Bacteria