DNA Synthesis Automation
To 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.
This project will be part of the synthetic yeast genome consortium (Sc2.0, www.syntheticyeast.org), which needs to synthesize 12Mbp DNA de novo. 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.
Computer Assisted Design
CAD tools are instrumental in any engineering fields; however synthetic biology as an engineering field is lagging behind in this regard. We are collaborating with the world’s best CAD company, Autodesk, Inc., to develop a cutting edge design tool named Autogene. We developed Autogene 1.0 as a proof of principle, and it won the best software award in the International Genetically Engineered Machine Competition (iGEM) 2012 (http://2012.igem.org/Team:Johns_Hopkins-Software). We are now looking for motivated students with strong computational background to continue this project and take it to the next level.
The philosophy of Autogene is: Scan, Modify and Print. On the Scan level, we will partner with BGI, the world largest sequencing center, to develop a comprehensive biological sequence feature database, and will continue leveraging Autodesk's cloud computing power to develop state of the art feature finding algorithms. On the Modify/Design level, we will focus on design of novel pathways to be integrated into our NeoChromosome (a synthetic yeast chromosome). And on the Print/Synthesis level, we are hoping to connect Autogene directly to our DNA synthesis automation platform so that a designer DNA sequence can be quickly fabricated and characterised.
This project is in the context of the international synthetic yeast consortium (Sc2.0, www.syntheticyeast.org), which aims to re-design and synthesize a designer yeast genome de novo (for details see Dymond, Nature 2010). The goal of this project is to design and build the world’s first Neochromosome in yeast, hosting all of the tRNAs which have been removed from all 16 chromosomes.
tRNA genes have been show to be hotspots for DNA instability for several reasons. Firstly, tRNA genes are often the target of 5’ upstream retrotransposon incorporation. Also, tRNA genes are transcribed heavily, which can lead to single strand breaks during DNA synthesis, when RNA polymerase III and DNA polymerase collide and stall. Furthermore, homologous regions upstream of tRNA genes, due to retrotransposon insertions, can aid homologous recombination of chromosomal regions. Thus in Sc2.0, all tRNA genes will be moved to their own chromosome (“Party Chromosome”), so that the effect of their absence throughout the rest of the genome can be observed. 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 to observe whether there is a preference for tRNA gene relative location and copy number.