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: iScience

Article Title: Origin of replication discovery for environmentally isolated Pantoea strain enables expression of heterologous proteins, pathways and products

doi: 10.1016/j.isci.2026.115031

Figure Lengend Snippet: Magic pool BarSeq results for all origin sequences tested The y axis denotes the names of the origins of replication, with predicted origins in green, and origins found in literature in black. Colors in each time point, or source, square represent the fraction of barcode counts associated with that origin versus the total fraction of barcodes in that sample (see legend). The number present in each box represents the total number of unique barcodes detected for that particular origin of replication at that time point, or for that source. (A) Plate-based sources for the magic pool conjugations with the 3 hosts. “Plate” is indicative of cells harvested directly from an agar plate, while “Outgrowth” is indicative of an overnight of growth in liquid media after scraping colonies off of an agar plate. (B) Liquid-based time points for magic pool conjugations with the 3 hosts. Time points 1, 2, 3 and 4 represent 24, 48, 72, and 96 h respectively. Note that the recipient strain E. coli BW25113 is part of the recipient set along with Pantoea MT58 and Brevundimonas sp , and is different from the conjugation strain E. coli WM3064, which was used as the donor. Note also that the 3845 hit for Brevundimonas sp. was analyzed due to a single bar code enrichment and represents an artifact rather than a real candidate.

Article Snippet: The plasmid DNA that was isolated from the E. coli pir + cells, and subsequently used to transform into the E. coli WM3064 conjugation donor, was sequenced by Plasmidsaurus using long-read sequencing (once with PacBio sequencing technology and once with in-house using Nanopore sequencing) with custom analysis and annotation.

Techniques: Conjugation Assay