Randomized BioBrick Assembly: A Novel DNA Assembly Method for Randomizing and Optimizing Genetic Circuits and Metabolic Pathways

The optimization of genetic circuits and metabolic pathways often inVolves, constructing various iterations of the same construct or using directed evolution to achieve the desired function. Alternatively, a method that randomizes individual parts in the same assembly reaction could be used for optimization by allowing for the ability to screen large numbers of individual clones expressing randomized circuits or pathways.. for optimal function: Here we describe a new assembly method, to randomize,igenetic circuits and metabolic pathways from modular DNA fragments derived from PCR-amplified BioBricks. As a proof-of-principle for this method, we successfully assembled CMY (Cyan, Magenta-Yellow) three-gene circuits using Gibson Assembly that express CFP, RFP, and YFP with independently randomized, promoters, ribosome binding sites, transcriptional terminators, and all parts randomized simultaneously. Sequencing results from 24 CMY circuits with various parts randomized show that 20/24 circuits are distinct and expression varies over a 200-fold range above background levels. We then adapted this method to randomize the same parts with enzyme coding sequences from the lycopene biosynthesis pathway instead of fluorescent proteins, designed to independently express each enzyme in the pathway from a different promoter. Lycopene production is improved using this randomization method by about 30% relative to the highest polycistronic-expressing pathway. These results demonstrate the potential of generating nearly 20,000 unique circuit or pathway combinations when three parts are permutated at each position in a three-gene circuit or pathway, and the methodology can likely be adapted to other circuits and pathways to maximize products of interest.