Journal: bioRxiv

Article Title: Design and construction towards a pan-microbial toolkit

doi: 10.1101/2024.02.23.581749

Figure Lengend Snippet: (A). The POSSUM module provides a collection of DNA sequences which can be combined through Golden Gate assembly to create libraries of plasmid variants that can be screened for function in target microbes. (B) Illustration of the Loop Assembly method employed by the POSSUM module. Alternating rounds of BsaI and SapI Golden Gate assemblies combine up to four parts at a time into destination vectors. Assembly levels alternate between Odd and Even destination vectors. A detailed description of all POSSUM module Level 0 parts is provided in Supplementary Table 4 . oriT : conjugation origin of transfer; RBS: ribosome binding site; CDS: coding sequence; NGS: next-generation sequencing.

Article Snippet: All Golden Gate assembly destination vectors possessing the ColE1 ORI (pGLDV_1-17, pGLDV_21) were generated by Clonal Gene Synthesis (Twist Biosciences).

Techniques: Plasmid Preparation, Conjugation Assay, Binding Assay, Sequencing, Next-Generation Sequencing

Journal: bioRxiv

Article Title: Design and construction towards a pan-microbial toolkit

doi: 10.1101/2024.02.23.581749

Figure Lengend Snippet: (A) Schematic showing the design of ORI-marker plasmids. (B) Schematic illustrating the final Golden Gate assembly step used for constructing ORI-marker plasmid libraries. An equimolar pool of barcoded ORI Level 1 plasmids is combined with a pre-assembled destination vector carrying a selection marker (in this case gentamicin, GEN). A SapI Golden Gate assembly reaction replaces the YFP dropout part with the pooled barcoded ORIs. Assembly products are electroporated directly into an E. coli conjugation donor strain to create an input library. (C) Amplicon-sequencing data showing the abundance of the 23 individual ORIs within six ORI-marker libraries (pGL2_217-222). Each ORI library carries a different antibiotic selection marker (Y-axis). Amplicon-sequencing was performed on plasmid DNA extracted from the conjugation donor (‘input library’). The scatter plot illustrates the percentage of reads mapping to each ORI barcode within the libraries. The vertical dotted line represents the expected abundance of each ORI if library distribution was perfectly even.

Article Snippet: All Golden Gate assembly destination vectors possessing the ColE1 ORI (pGLDV_1-17, pGLDV_21) were generated by Clonal Gene Synthesis (Twist Biosciences).

Techniques: Marker, Plasmid Preparation, Selection, Conjugation Assay, Amplification, Sequencing