Figure 3

Replacement of Mcc dnaA gene in the mycoplasma genome. (A) Schematic representation of replacement of the Mmc dnaA gene with an orthologous gene from Mcc. Here, the dnaA gene in the Mmc genome (indicated by an orange arrow) was replaced by the Mcc orthologue (purple arrow) using TREC-IN. Diagnostic primers to confirm correct insertion of the cassettes and seamless replacement of the endogenous dnaA gene are indicated by dashed arrows (see Additional file 1: Figure S3 for primer information). (B) PCR screening to confirm replacement of the Mmc dnaA gene with an orthologous gene from Mcc. (a) DNA from yeast colonies after selection on SD-His-Ura were amplified using primers DG1F/DG1R (left junction; expected size, 222 bp) and DG2F/DG2R (right junction; expected size, 438 bp). (b) DNA from yeast colonies after selection on geneticin were amplified using primers DG2F/DG4R (left junction; expected size, 285 bp), DG6F/DG5R (middle junction; expected size, 615 bp) and DG3F/DG2R (right junction; expected size, 446 bp). (c) DNA from yeast colonies after selection on 5-FOA were amplified using primers DG1F/DG5R (left junction; expected size, 388 bp) and DG3F/DG2R (right junction; expected size, 446 bp). A representative of 5 colonies is shown for each transformation and for each junction.