Assembly of DNA parts into DNA constructs is a foundational technology in the emerging field of synthetic biology. An efficient DNA assembly method is particularly important for high-throughput, automated DNA assembly in biofabrication facilities and therefore we investigated one-step, scarless DNA assembly via ligase cycling reaction (LCR). LCR assembly uses single-stranded bridging oligos complementary to the ends of neighboring DNA parts, a thermostable ligase to join DNA backbones, and multiple denaturation-annealing-ligation temperature cycles to assemble complex DNA constructs. The efficiency of LCR assembly was improved ca. 4-fold using designed optimization experiments and response surface methodology. Under these optimized conditions, LCR enabled one-step assembly of up to 20 DNA parts and up to 20 kb DNA constructs with very few single-nucleotide polymorphisms (<1 per 25 kb) and insertions/deletions (<1 per 50 kb). Experimental comparison of various sequence-independent DNA assembly methods showed that circular polymerase extension cloning (CPEC) and Gibson isothermal assembly did not enable assembly of more than four DNA parts with more than 50% of clones being correct. Yeast homologous recombination and LCR both enabled reliable assembly of up to 12 DNA parts with 60-100% of individual clones being correct, but LCR assembly provides a much faster and easier workflow than yeast homologous recombination. LCR combines reliable assembly of many DNA parts via a cheap, rapid, and convenient workflow and thereby outperforms existing DNA assembly methods. LCR assembly is expected to become the method of choice for both manual and automated high-throughput assembly of DNA parts into DNA constructs.
DNA assembly from building blocks remains a cornerstone in synthetic biology, whether it be for gene synthesis (?1 kb), pathway engineering (?10 kb) or synthetic genomes (>100 kb). Despite numerous advances in the techniques used for DNA assembly, verification of the assembly is still a necessity, which becomes cost-prohibitive and a logistical challenge with increasing scale. Here we describe for the first time a comprehensive, high-throughput solution for structural DNA assembly verification by restriction digest using exhaustive in silico enzyme screening, rolling circle amplification of plasmid DNA, capillary electrophoresis and automated digest pattern recognition. This low-cost and robust methodology has been successfully used to screen over 31 000 clones of DNA constructs at <$1 per sample.
Frequently during evolution, new phenotypes evolved due to novelty in gene regulation, such as that caused by genome rewiring. This has been demonstrated by comparing common regulatory sequences among species and by identifying single regulatory mutations that are associated with new phenotypes. However, while a single mutation changes a single element, gene regulation is accomplished by a regulatory network involving multiple interactive elements. Therefore, to better understand regulatory evolution, we have studied how mutations contributed to the adaptation of cells to a regulatory challenge. We created a synthetic genome rewiring in yeast cells, challenged their gene regulation, and studied their adaptation. HIS3, an essential enzyme for histidine biosynthesis, was placed exclusively under a GAL promoter, which is induced by galactose and strongly repressed in glucose. Such rewired cells were faced with significant regulatory challenges in a repressive glucose medium. We identified several independent mutations in elements of the GAL system associated with the rapid adaptation of cells, such as the repressor GAL80 and the binding sites of the activator GAL4. Consistent with the extraordinarily high rate of cell adaptation, new regulation emerged during adaptation via multiple trajectories, including those involving mutations in elements of the GAL system. The new regulation of HIS3 tuned its expression according to histidine requirements with or without these significant mutations, indicating that additional factors participated in this regulation and that the regulatory network could reorganize in multiple ways to accommodate different mutations. This study, therefore, stresses network plasticity as an important property for regulatory adaptation and evolution.
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