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.

In recent years, next-generation sequencing (NGS) technology has greatly reduced the cost of sequencing whole genomes, whereas the cost of sequence verification of plasmids via Sanger sequencing has remained high. Consequently, industrial-scale strain engineers either limit the number of designs or take short cuts in quality control. Here, we show that over 4000 plasmids can be completely sequenced in one Illumina MiSeq run for less than $3 each (15× coverage), which is a 20-fold reduction over using Sanger sequencing (2× coverage). We reduced the volume of the Nextera tagmentation reaction by 100-fold and developed an automated workflow to prepare thousands of samples for sequencing. We also developed software to track the samples and associated sequence data and to rapidly identify correctly assembled constructs having the fewest defects. As DNA synthesis and assembly become a centralized commodity, this NGS quality control (QC) process will be essential to groups operating high-throughput pipelines for DNA construction.

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.

Almond leaf scorch (ALS) disease is caused by the bacterium Xylella fastidiosa and transmitted by xylem-feeding insects. Reports of increased incidence of ALS-diseased trees in California prompted surveys in three almond [Prunus dulcis (Mill.) D. A. Webb]-growing regions, from June 2003 to September 2005, to determine insect vector species composition and abundance. For comparison, sampling in and near vineyards in the San Joaquin Valley, California, also was completed. Sampling in or near almond orchards collected >42,000 Cicadomorpha of which 4.8% were xylem feeders, including 1912 grass sharpshooter, Draeculacephala minerva Ball; five Xyphon fulgida Nottingham; and a single spittlebug, Philaenus spumarius L. The most abundant vector was D. minerva. Season-long sampling indicated that D. minerva was a year-round resident in and/or near almonds in the Sacramento Valley, but not in the San Joaquin Valley. Similarly, D. minerca was rare in vineyards in the San Joaquin Valley, but was abundant in irrigated pastures near vineyards. D. minerva was most frequently collected along orchard margins, and peak densities were observed in summer, the period of time when bacterial titers are reported to increase in infected trees. Screening of D. minerva for presence of X.fastidiosa found that 1.1% of insects collected near almond orchards and 4.5% of insects collected from pastures tested positive. The X. fastidiosa subspecies and genotype detected in insects collected from orchards matched those collected from ALS-diseased almond trees in the same orchard. Of the few X. fulgida and P. spumarius collected, none tested positive for X. fastidiosa. Results are discussed with respect to X. fastidiosa vector control and detection methods.

No abstract