How are we doing genomic modifications? Which methods are we using from the beginning to the end of the complete process? This flowchart gives you a detailed overview. Together with the given protocols you can repeat it down to every little pipetting- step we made!
Planning of the whole cassette and its fragments in geneious, designing primers with overhangs (which ensure the later assembly of the composite cassette) for it and synthesize them via IDT.
Produce the fragments via PCR, so the overhangs generate the homologous parts of each of the fragments.
Transformation and Homologous Recombination
(A) Transformation of the fragments into the yeast cells. (B) The homologous parts of the fragments ensure that the fragments are assembled in the right order after transformation inside the cell. (C) Integration of the composite cassette into the part of one chromosome, encoded by the recombination sites at the 5'- and 3'- ends of the whole cassette.
After growth on the auxotrophy- plates verification through verification- PCR. Because the lengths of the cassettes were often quite long for one PCR, often multiple primer pairs were used. When the PCR has been successful and the gel showed the awaited length, sequencing of the introduced gene is given in order.
Cre- loxP Treatment
After complete verification, the marker gene between the loxp- sites is removed through the cre protein, for further transformation and for avoidance of antibiotic resistant cells.