Phenotype Monitoring

We use haploid strains allowing simple growth (fitness) tests to be performed after each cycle of incorporating a synthetic segment. A straightforward and relatively sensitive way for us to monitor fitness on an ongoing basis is to examine colony size when the strain is plated out on the appropriate selective and non-selective media and compared to the parental strain(s). However, while this method will readily identify instances in which alterations lead to relatively major fitness defects, minor fitness defects may not be detected and certainly the spectrum of growth conditions examined will be small. We will also perform competitive growth assays to evaluate whether minute changes in fitness have occurred.

We also use more elaborate phenotyping tools, in particular transcript profiling to determine whether the incorporation of synthetic chromosome induces changes in gene expression globally .  This type of analysis is typically performed after ~10 cycles of incorporating synthetic megachunks, or ~300-500kb of synthetic DNA.