Building S.cerevisiae Chromosome XIV
Synthetic DNA is manufactured to incorporate several design features to be consistent throughout the genome of the synthetic yeast. Some of these features include the conversion of all stop codons to TAA and the insertion of loxPsym sites flanking all non-essential genes. In our laboratory we start with chunks of DNA up to 10 kb in length with “sticky” overhangs and co-transform our strain with a series of 4 chunks.
The end most chunk contains marker LEU2 or URA3, alternating between successive megachunks allowing selection of integration with each round.
Following phenotypic screening, the correct integration of synthetic DNA chunks is screened by real time PCR analysis. PCR Tag primers are designed to differentiate between wt and syn DNA. The entire length of the 750 kb chromosome will be made “synthetic” by this iterative process.
Figures by Tom Williams and Natalie Curach