Fig. 4: Workflow for base editing-mediated (de)construction of complex bacterial phenotypes.

A To facilitate cloning procedures, a fluorescent protein marker was added to the pMBEC backbone, facilitating rapid counterselection of template vectors. The base editing-plasmid contains a constitutively-expressed msfGFP module flanked by BsaI recognition sites. This marker is replaced by either one spacer sequence or multiple gRNAs through Golden Gate assembly. B Generation of gRNAs through PCR for multiplexed genome editing. The Cas9 handle is amplified along with a Cas6 recognition sequence from a template vector, while the specific spacer sequence and Golden Gate-flanking motifs are introduced in the oligonucleotide sequences. The resulting gRNAs harbor unique BsaI sites that can be used to compose multiplex arrays by Golden Gate cloning. C pMBECs vectors enable deconstruction and construction of complex phenotypes by multiple genome editing. SacB-mediated curing of these plasmids facilitates multiple editing cycles upon confirmation and analysis of the resulting bacterial phenotypes.