Skip to main content
. 2023 Jun 27;12(7):1989–2003. doi: 10.1021/acssynbio.3c00065

Figure 5.

Figure 5

Multiplex Tn integrations in B. thailandensis and temperature-sensitive pCAST. (A) Multiplexing schematic, both pDonor are simultaneously transformed into the cell. (B) Relative frequency table of multiplex targeting with auxotrophy/fluorescence disruption and genotyping results for B. thailandensis in co-transformed targets trpA-1 and eyfp-2. (C) Tn-Seq results for eyfp-2 (yellow) in chromosome I and trpA-1 (red) in chromosome II. Read frequency presented as a percentage across both chromosomes (bin size = 1 Kbp). Zoomed-in view of transposon insertion of both gene targets start at the PAM site (0 bp) (bin size = 1 bp). (D) Map of sgRNA targets in B. thailandensis genes motA and class A β-lactamase (β-lac) encoding BTH_RS07435. (E) Relative frequency table for B. thailandensis pRO16Sh5 temperature-sensitive pCAST with phenotype (n = mutants collected) and genotype (n = 24) results. Phenotype results reflect disruption of tryptophan production (trpA-1), motility (motA-1), or carbenicillin 100 μg/mL resistance (class A β-lactamase). (F) Relative frequency table for P. putida pRO16Sh2 temperature-sensitive pCAST with auxotrophy (n = mutants collected) and genotype (n = 24) results. (G) Motility assay for pRO16Sh5 B. thailandensismotA-1 in semisolid LB 0.3% agar, successful gene disruption results in loss of motility halo. (H) Schematic for iterative transformations. The pDonors are transformed consecutively into the cell, selecting for the transposon and pCAST in between transformations. (I) Recovery plate of serA-1 transformations for P. putida with a violacein transposon integration. (J) Relative frequency table for iterative transformation for all three bacteria with genotype results. The initial target, with the corresponding antibiotic marker in parenthesis, is given followed by the second mutation.