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.