2.2 Plasmid construction and genomic modification
Deletion and expression plasmids were cloned as described in detail in the supplementary information. Plasmids derived from pEMG and pSEVA412S were transformed into E. coli DH5α λpir cells, pBG-based plasmids into E. coli PIR2. Integration at the attTn7 -site was achieved by four-parental patch mating as described by Wynands et al. [5]. Genomic deletions were realized using the I-SceI-based method by Martinez-Garcia and de Lorenzo [10] using a streamlined protocol adapted from Wynands et al. [5]. Genomic modifications were verified by colony PCR.