DNASeq of different Mycoplasma pneumoniae isolated clones presenting a genomic deletion produced by a multi-recombinase engineering protocol

We developed a multi recombinase engineering rationale, that combines oligonucleotide recombineering with the selective capacity of antibiotic resistance via transient insertion of selector plasmids. We tested this method in Mycoplasma pneumoniae, a bacterium with a very inefficient native recombination machinery. A wide variety of targeted genome modifications were carried out. We did whole genome sequencing of some clones to confirm that the engineering method is not mutagenic and ensure that genome modifications only occurred at the intended loci. Specifically we sequenced clones carrying 1 kb deletion at 4 different chromosomal locations (i.e., M129-GP35-PtetCre ?1kbmpn088::lox scar, M129-GP35-PtetCre ?1kbmpn256::lox scar, M129-GP35-PtetCre ?1kbmpn440::lox scar, M129-GP35-PtetCre ?1kbmpn583::lox scar), a clone carrying a 30 kb deletion (M129-GP35-PtetCre ?30kbNE region::pLoxPuro) and a clone carrying a 5.5 kb deletion that was complemented with the two essential genes found in this area (M129-GP35 ?5.5kbmpn633-mpn638::mpn636-637lox scar)