Sc2.0Our group is heavily involved with the synthetic yeast genome consortium (Sc2.0, www.syntheticyeast.org), which aims to re-design and synthesize a 12 Mb designer yeast genome de novo (for details see Dymond, Nature 2010). Our group will design and synthesize at least one chromosome in house, so the success of this project will be of great significance and highly valuable to the field of synthetic biology.
Evolution of Synthetic Yeast genomes (IESY)The Induced Evolution of Synthetic Yeast genomes (IESY) project builds on pioneering developments from the Saccharomyces cerevisiae 2.0 (Sc2.0) consortium. IESY represents an innovative concept in synthetic biology to explore combinatory diversity in pre-programmed synthetic genomes to evolve new and useful functions. Using the system for Synthetic Chromosome Rearrangement and Modification by LoxP-mediated evolution (SCRaMbLE) we are able to generate high-fitness yeast cells with synthetic chromosomes under specific conditions. The SCRaMbLE system can generate genome diversity with the capability of genome minimization and the ability to produce new biological functions through rapid evolution of genome content, copy number and gene order.
tRNA NeochromosomeThis project is part of the international synthetic yeast consortium. The goal of this project is to design and build a tRNA neochromosome in yeast, hosting all of the tRNAs which will be removed from the synthetic yeast genome.
tRNA genes have been shown to be hotspots for DNA instability for several reasons. First, tRNA genes are often the target of 5’ upstream retrotransposon incorporation. Second, tRNA genes are transcribed heavily, which can lead to DNA damage caused by collisions between the replication and transcriptional machineries. Furthermore, retrotransposon insertions produce highly-homologous repeats leading to homologous recombination between chromosomal regions.
Thus, as part of Sc2.0, all tRNA genes will be moved to their own dedicated chromosome (a “Party Chromosome”). All introns will also be removed from tDNA, as another goal of the project is to observe the effect of a yeast genome with fewer introns. rox Recombination sites will be placed between the tRNA genes, which will enable us ‘SCRaMbLE’ the neochromosome and observe whether there is a preference for tRNA copy number under different conditions.
Neochromosomes to Understand Chromosome Segregation(In collaboration with the Adele Marston lab’) This project aims to better understand the structure and function of the budding yeast pericentromere and its role in chromosome segregation. Failure to accurately separate sister chromatids lead many medical conditions such as birth defects and cancer. Budding yeast, as a simple eukaryote, is a good model organism to study cell division. With the help of synthetic chromosomes, this project will study the effect of transcription on cohesin localisation and movement across the pericentromere and how this influence sister chromatid cohesion.
DNA Synthesis Automation and Technology Development
DNA Synthesis AutomationTo close the gap between design and fabrication in synthetic biology, we are proposing to automate the DNA synthesis process, utilizing liquid handling robots and other laboratory automation equipment. The first goal is to implement basic molecular biology operations on a liquid handler, such as cherry picking, PCR, gel electrophoresis, cloning, bacterial and yeast transformation, plasmid DNA miniprep, and colony screening. In each step of molecular biology experiments, there will be failures, and how to reschedule these failures in an optimized fashion is a bigger challenge. Therefore, the second goal is to develop a workflow management system which schedules experiments based on given optimization objectives, such as cost, total time and minimal hands-on time.
EMMATraditional cloning methods often require laborious manual design and assembly of plasmids using tailored sequential cloning steps. This process can be protracted, complicated, expensive and error-prone. In our lab, we have developed an extensible mammalian modular assembly kit (EMMA), that facilitate the efficient design and production of bespoke vectors relieving a current bottleneck for researchers. The method supports assembly of combinatorial libraries, and hierarchical assembly for production of larger multigenetic cargos. We are also investigating new efficient ways of delivering megabase-size DNA constructs into mammalian cells. EMMA is compatible with automated production and provides new opportunities for mammalian synthetic biology.
DNA Recombinase toolkit for synthetic biologySite-specific DNA recombinases are a family of DNA enzymes that can recognize specific DNA sequences and drive DNA recombination between specific sites to achieve deletion, inversion, integration or transposition of target DNA fragments. The Sc2.0 synthetic yeast genome is designed to contain numerous LoxP sites located throughout, enabling random genome rearrangement for accelerated yeast evolution, a process called SCRaMbLE-(Synthetic Chromosome Recombination and Modification by LoxP-mediated Evolution).
Our group is also constructing a tRNA Neochromosome that contains all tRNA genes relocated from the synthetic yeast genome. For the purpose of SCRaMbLE, a recombination system based on Dre/rox achieves orthogonal evolution of the neochromosome.