Multiplex automated genome engineering (MAGE) for the optimization of metabolic pathways, construction of new genetic codes, and evolution of synthetic organisms

New tools advance our understanding of the world by enabling us to answer age-old questions. In turn, a better understanding of nature's complexity will inevitably produce newer and deeper questions that require even more advanced tools to answer them. This thesis aims to contribute to this cyclical nature of intellectual inquiry. We specifically focus on the technological barriers associated with the manipulation of an organism's genome, which has limited our ability to test the ever-growing pool of bioinformatics-derived hypotheses in the post-genomics era. Chapter 1 reviews key topics on the relationship between the genome and the genetic code, with emphasis on evolution and mutability, based on past literature. Chapter 2 describes a new platform, Multiplex Automate Genome Engineering (MAGE), to rapidly engineer and evolve genomes of living organisms using an array of semi-rational, combinatorial, and high-throughput approaches. We demonstrated the utility of MAGE by optimizing the 1-deoxy-D-xylulose-5-phosphate (DXP) biosynthesis pathway to overproduce the industrially important isoprenoid lycopene in Escherichia coli. Chapter 3 describes the use of MAGE to modify the Escherichia coli chromosome at over 300 specific locations. The multiplexed engineering of many chromosomal positions by using a coupled selection revealed cooperativity during lambda-Red allelic replacement events of a replicating genome. A quantitative roadmap for constructing synthetic genomes by engineering and synthesis is presented. Chapter 4 describes a combined strategy of using MAGE and a conjugation-based genome assembly approach to construct a recoded E. coli genome. The first instantiation of the recoded E. coli (rE.coli) involved the substitution of the amber codon with the ochre codon. The broader goals involve the reassignment of multiple codons to create genomes that are multi-phage resistant and can be used for the stable incorporation of non-standard amino acids. We outline progress towards achieving these objectives and highlight possible challenges and solutions. Chapter 5 describes the utilization of MAGE to building synthetic proteomes in vitro by systematically introducing poly-histidine purification tags into all essential components of the protein translation system in vivo. Co-purification of these factors from one strain allowed the reconstruction of multi-enzymatic catalysis in a single-pot reaction system. Chapter 6 details the construction of automated instrumentations for MAGE. New devices that decrease the cost of manual labor for constructing and manipulating genomes will hopefully revolutionize the way biology is studied and used in both academic and industrial settings. We finally conclude with some closing remarks of the future prospects for genome engineering to expand our understanding of fundamental cellular and evolutionary processes through construction and engineering of biological systems with useful properties.