Journal: bioRxiv

Article Title: Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene

doi: 10.1101/710293

Figure Lengend Snippet: Hierarchy of P. putida MMR system A. The pyrF reporter gene used to assess MMR hierarchy in P. putida EM42 is outlined. Locus tag and chromosomal coordinates are shown. The four PYR_X oligos introduce the same Stop codon (red dot), thus rendering a pyrF -strain which is uracil auxotroph and 5FOA resistant, but bear a different degenerated position each (yellow dot, the genomic nucleotide that pairs with oligonucleotide sequence is depicted inside), generating three mismatches per oligonucleotide. Pictures are not drawn to scale B. P. putida EM42/pSEVA2514- rec2 (WT strain-wild-type MMR system) was subjected to recombineering with an equimolar mixture of oligos PYR_C, PYR_A, PYR_G and PYR_T. After selection of minimal media plus Ura/5FOA, 500 pyrF -colonies were streaked in the same media and the streaks re-suspended in water, then pelleted and the whole genomic content extracted. pyrF gene was PCR amplified from the gDNA and sequenced by Illumina deep sequencing. Sequences were analysed to verify the presence of single mutations on the four degenerated positions targeted by PYR oligonucleotides. The relative frequencies of incorporated mutations were plotted and labelled with the original mismatch and the base change originated. The values are the mean of two independent experiments, bars representing standard deviations

Article Snippet: Genomic DNA samples of P. putida EM42 query strains were sequenced in Macrogen Inc. (Korea).

Techniques: Introduce, Sequencing, Selection, Amplification