A detailed description of the Sc2.0 design is in Richardson et al 2017 Science.

Planned alterations to native chromosome sequence are potentially infinite in number and so a great deal of thought must be given to the specific alterations to be incorporated into the synthetic chromosome(s). In general, we are making modest changes predicted to have minimal or no impact on fitness of the organism under laboratory conditions.

These changes are introduced in a stepwise, “bottom up” approach. The advantage of a stepwise iterative approach is that the “investment” made in the project at any given stage is limited to about 30kb, so if a particular segment is incompatible with viability, or gives rise to a slow-growth phenotype, the segment can be re-synthesized after the nature of the growth defect is mapped and diagnosed (by singling transforming in the 10kb pieces, or by mapping which segments of a given 30 kb chunk are incorporated during transformation [PCRTags]. This very practical strategy is in contrast to a “home-run” strategy in which a more aggressive design is conceived, a lot of work invested, and then one hopes the outcome is a viable cell. A key design principle is that we will not attempt to make individual changes likely to result in a decrease in fitness. The cumulative effect of such changes is likely to result in an unviable yeast cell within a relatively low number of cycles. Nevertheless, we are building in a system to allow subsequent evolution of a minimal genome, as well as faster growing variants and variants with a potentially infinite variety of biological adaptations by building into the genome a “SCRaMbLE” controllable genome evolution system that can be controlled by the experimentalist at will.

Design

Alterations

We will introduce rather conservative changes to the genome sequence, initially deleting or relocating genomic features using strategies we feel, from first principles, are unlikely to reduce fitness significantly. At the same time, we will be introducing site-specific recombination sequences, allowing subsequent in vitro evolution of the yeast strains generated. This opens up a whole new dimension – not just one synthetic genome, but whole populations of synthetic genomes will be available for analysis and future study.
Design

Methodology

The strategy to build the Sc2.0 genome is based on a hierarchical assembly plan devised specifically for this project. Building blocks (~750bp) are assembled into minichunks (~3kb), which are assembled into into chunks (~10kb), which are subsequently assembled into megachunks (30-50kb) that can finally be introduced into yeast to replace the corresponding wild type sequence. Here we will describe the design and workflow associated with assembling chunks into megachunks and yeast transformation.
Design

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.
Design

Principles

Planned alterations to native chromosome sequence are potentially infinite in number and so a great deal of thought must be given to the specific alterations to be incorporated into the synthetic chromosome(s). In general, we are making modest changes predicted to have minimal or no impact on fitness of the organism under laboratory conditions. The synthetic DNA encoding the designer changes are introduced in a stepwise, “bottom up” approach. The advantage here is that the “investment” made in the project at any given stage is limited to 30-60kb, so if a particular segment is incompatible with viability, or gives rise to a slow-growth phenotype, the segment can be re-synthesized after the nature of the growth defect is mapped and diagnosed (by singly transforming in the 10kb pieces, or by mapping which segments of a given 30-60 kb “megachunk” are incorporated during transformation [PCRTags].