Precise Control Of Synthetic Genome Rearrangement

Development of synthetic biology has expanded from the design of simple genetic circuits to the rapid construction of complex and multi-level artificial biological system.The precise control of genetic circuits is the building block of artificial biological systems.The rapid synthesis of large fragments of DNA is the basic tool for constructing artificial synthetic systems.The rational design and rapid evolution of genomes are important to improve the adaptability of artificial synthetic systems.Biocontainment systems are crucial for preventing the potential risk of artificial synthetic systems.This manuscript focuses on the following two aspects:First,Rapid construction of artificial biological systems and precise control of genome rearrangement for evolution;Second,design of biocontainment system for reducing the potential risks of artificial biological systems.Rapid assembly of DNA overlapping multifragments(RADOM)is developed for rapid construction of artificial biological system and precise control of genome rearrangement and evolution.The method can be used not only for synthesis of yeast genome,but also construction of the exogenous metabolic pathway.And then an"AND Gate"genetic circuits was designed for precise control of Synthetic chromosome rearrangement and modification by loxP-mediated evolution(SCRaMbLE),which generates combinatorial genomic diversity through rearrangements at designed recombinase sites.SCRaMbLE can be used to increase carotenoids production of yeast,deep sequencing of which revealed functional genes and new knowledge.SCRaMbLE of heterozygous diploid genome can decrease the lethality rate and enhance the diversity of genome rearrangement.Moreover,Multiplex SCRaMbLE Iterative Cycling(MuSIC)strategy is developed to iteratively accumulate multiple rearrangements through multiple cycles of SCRaMbLE.The strategy increased the production yield of carotenoids up to 38.8-fold through 5 iterative cycles of SCRaMbLE within 40 days.Deletions,duplications,inversions and translocations were observed in the diploid strains with the desired phenotypes.The orthogonal ribosome biofirewall systems is developed for safety control of artificial synthetic systems.The activation circuit is a genetic AND gate based on activation of the encrypted pathway by the orthogonal ribosome in response to specific environmental signals.The degradation circuit is a genetic NOT gate with an output of I-SceI homing endonuclease,which conditionally degrades the orthogonal ribosome genes.The activation circuit can be flexibly incorporated into genetic circuits and metabolic pathways for encryption.The plasmid-based encryption of the deoxychromoviridans pathway and the genome-based encryption of lacZ are tightly regulated and can decrease the expression to 7.3%and 7.8%,respectively.The degradation circuit can decrease the expression levels of the target plasmids and the orthogonal rRNA(O-rRNA)plasmids to 0.76%in nonsterile soil medium.Orthogonal ribosome biofirewall is a versatile platform that can be useful in biosafety research and in the biotechnology industry.In conclusion,this work tried to explore strategies of rapid evolution of artificial synthetic systems and principles of biocontainment systems.Based on the rapid DNA assemble ability and SCRaMbLE,artificial synthetic system can be constructed and evolved rapidly.Base on the precise control ability of "AND Gate"genetic circuits,artificial synthetic system can be used under safety control.The results of this work improved the ability of design and regulating artificial synthetic systems.