Extended Data Figure 4. FtsA cysteine mutant strains: generation and absence of phenotype changes.

a, Workflow for REXER⁷²-based strain construction. PCR products containing the 3x HA-tag or cysteine point mutations were inserted into a shuttle vector by Golden Gate assembly⁷⁷. Assembled shuttle vectors were transformed into the donor strain, conjugated, and excised in vivo using Cas9. Targeting constructs contain homology regions for λ Red mediated recombination into the target locus. Recombinants were selected for neoR and tetR markers and against the pheS* marker. Strains were cured of the helper plasmid pKW20 by growth in absence of selection. b, Growth of strains containing single or double cysteine point mutations and a 3x HA-tag in the endogenous ftsA gene, and a kanamycin resistance cassette inserted after the lpxC gene. Parent strains and the original MG1655 strain are also shown. SW: sandwich fusion. c, Growth curves of the same strains in liquid LB medium. Plotted are traces of technical octuplicates (coloured) with mean (black). d, DIC images of the same strains in exponential phase (OD₆₀₀ = 0.2-0.3) demonstrating the absence of elongated cells. Similar results were achieved in biological triplicate, of which one was imaged using DIC and two were imaged using phase contrast.