‘Shotgun DNA synthesis’ for the high-throughput construction of large DNA molecules

Kim, Hwangbeom; Han, Hyojun; Ahn, Jae Pyung; Lee, Joongoo; Cho, Namjin; Jang, Hoon; Kim, Hyoki; Kwon, Sunghoon; Bang, Duhee

doi:10.1093/nar/gks546

Cited by 40 publications

(34 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Following the early demonstrations of gene assembly using microarray-derived oligo pools, 3–5 exciting developments have been made to improve the quality and efficiency of microarray-based oligo synthesis and gene assembly. 6–10 We have previously developed a microarray inkjet synthesizer to synthesize pools of thousands of codon-usage variants for protein expression optimization at low cost and high throughput. 6 …”

Section: Introductionmentioning

confidence: 99%

High-throughput screening of microchip-synthesized genes in programmable double-emulsion droplets

Chan

Tian

et al. 2017

Nanoscale

View full text Add to dashboard Cite

The rapid advances of synthetic biology and biotechnology increasingly demand high-throughput screening technology, such as screening of the functionalities of synthetic genes for optimization of protein expression. Compartmentalization of single cells in water-in-oil (W/O) emulsion droplets allows screening of a vast number of individualized assays, and recent advances in automated microfluidic devices further help realize the potential of droplet technology for high-throughput screening. However these single-emulsion droplets are incompatible with aqueous phase analysis and the inner droplet environment cannot easily communicate with the external phase. We present a high-throughput, miniaturized screening platform for microchip-synthesized genes using microfluidics-generated water-in-oil-in-water (W/O/W) double emulsion (DE) droplets that overcome these limitations. Synthetic gene variants of fluorescent proteins are synthesized with a custom-built microarray inkjet synthesizer, which are then screened for expression in Escherichia coli (E. coli) cells. Bacteria bearing individual fluorescent gene variants are encapsulated as single cells into DE droplets where fluorescent signals are enhanced by 100 times within 24 h of proliferation. Enrichment of functionally-correct genes by employing error correction method is demonstrated by screening DE droplets containing fluorescent clones of bacteria with the red fluorescent protein (rfp) gene. Permeation of isopropyl β-D-1-thiogalactopyranoside (IPTG) through the thin oil layer from the external solution initiates target gene expression. The induced expression of the synthetic fluorescent proteins from at least ~100 bacteria per droplet generates detectable fluorescent signals to enable fluorescence-activated cell sorting (FACS) of the intact droplets. This technology obviates time- and labor-intensive cell culture typically required in conventional bulk experiment.

show abstract

Section: Introductionmentioning

confidence: 99%

High-throughput screening of microchip-synthesized genes in programmable double-emulsion droplets

Chan

Tian

et al. 2017

Nanoscale

View full text Add to dashboard Cite

show abstract

“…In one such application, Matzas et al (2010) used Roche 454 sequencing combined with a beadpicking robot to selectively remove oligonucleotide sequences (attached to beads) from the sequencing array that showed perfect sequences that were then used for gene synthesis. Kim et al (2012) used random sequence tags to mark individual synthetic sequences in a 454 sequencing reaction and used these tags to selectively amplify correct sequences from the sequenced pool. In a similar approach, Schwartz et al (2012) used Illumina sequencing barcodes to "dial-out" correct sequences via selective amplification of the sequence-verified bar-coded sequences.…”

Section: Error Correction and Sequence Validationmentioning

confidence: 99%

Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology

Hughes

Ellington

2017

Cold Spring Harb Perspect Biol

323

235

View full text Add to dashboard Cite

The chemical synthesis of DNA oligonucleotides and their assembly into synthons, genes, circuits, and even entire genomes by gene synthesis methods has become an enabling technology for modern molecular biology and enables the design, build, test, learn, and repeat cycle underpinning innovations in synthetic biology. In this perspective, we briefly review the techniques and technologies that enable the synthesis of DNA oligonucleotides and their assembly into larger DNA constructs with a focus on recent advancements that have sought to reduce synthesis cost and increase sequence fidelity. The development of lower-cost methods to produce high-quality synthetic DNA will allow for the exploration of larger biological hypotheses by lowering the cost of use and help to close the DNA read -write cost gap.DNA neither cares nor knows. DNA just is. And we dance to its music.-Richard Dawkins River Out of Eden: A Darwinian Life

show abstract

“…We then took the median value (to minimise effects of outliers) of the log2 fold change over all of the dilutions to determine the fitness for that barcode . edian(f , , , Assembly Retrieval by Dialout Amplification The presence of a unique barcode on each assembly allows us to retrieve them from the library using PCR amplification 13,15 . We attempted to amplify 48 unique orthologs and 12 gain-of function mutants.…”

Section: Complementation Assaymentioning

confidence: 99%

“…In these efforts, the ability to isolate and concentrate DNA from the background pool complexity was paramount for robust assemblies 11 . Previous efforts to multiplex such assemblies have not isolated reactions from one another, and thus suffered from short assembly lengths, highly-biased libraries, the inability to scale, and constraints on sequence homology [12][13][14][15] . DropSynth works by pulling down only those oligos required for a particular gene's assembly onto barcoded microbeads from a complex oligo pool.…”

mentioning

confidence: 99%

Multiplexed Gene Synthesis in Emulsions for Exploring Protein Functional Landscapes

Plesa

Sidore

Lubock

et al. 2017

Preprint

View full text Add to dashboard Cite

Next-generation sequencing has engendered an expanding suite of functional assays that can test sequence-function relationships at unprecedented scales in pooled formats (multiplex). Such assays are currently constrained by the short length of oligonucleotide (oligo) pools, which limit potential applications. Here we report a simple, low-cost, and scalable method called DropSynth that assembles gene libraries from oligo pools for use in multiplexed functional assays. DropSynth utilizes a library of barcoded beads to isolate and concentrate oligos needed for a gene's synthesis in a pooled format. These bead-bound oligos are then emulsified, processed, and assembled into genes within the emulsion droplets. We synthesized~1000 phylogenetically diverse orthologs of phosphopantetheine adenylyltransferase (PPAT) and tested their fitness in a multiplexed functional assay. While the majority of orthologs complement, those that do not are broadly distributed across the phylogenetic tree. Synthetic errors in our assemblies allow us to explore local landscapes around the designed orthologs revealing constrained mutations for complementing orthologs as well as gain-of-function mutations for low-fitness orthologs. This broad mutational scanning approach is complementary to deep mutational scanning and helps us understand proteins by probing evolutionarily divergent sequences that share function.peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/163550 doi: bioRxiv preprint first posted online Jul. 14, 2017;The rapid progress in DNA sequencing has provided a glimpse of the vast diversity of sequence and function that nature has explored. Unfortunately our ability to explore the functional aspects of diverse sequences remains minuscule. ) that link a function to a next generation sequencing (NGS)-based output to allow testing of thousands to millions of sequences for function in a pooled manner. Mutagenesis and deep mutational scanning combined with multiplexed functional assays are now able to systematically probe all single and double amino acid substitutions for a protein's function 3,4 . Despite the power of these approaches, the sequence space explored in such experiments is minute when compared to the evolutionary distance between even highly-homologous protein sequences. Our ability to sample a broad diversity of sequences is bottlenecked by two limitations in existing approaches for producing the libraries to be tested. First, low-cost microarray-based oligo libraries allow for large libraries of designed <200 nucleotide (nt) sequences 5 , which is far below the typical length of a protein and limits many other applications. Secondly, gene synthesis is capable of creating long-length sequences, but its costs currently make it prohibitively expensive for sampling large libraries of designed sequences [6][7][8][9] .Here we develop a new gene synthesis method we term DropSynth, a multiplexed...

show abstract

‘Shotgun DNA synthesis’ for the high-throughput construction of large DNA molecules

Cited by 40 publications

References 21 publications

High-throughput screening of microchip-synthesized genes in programmable double-emulsion droplets

High-throughput screening of microchip-synthesized genes in programmable double-emulsion droplets

Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology

Multiplexed Gene Synthesis in Emulsions for Exploring Protein Functional Landscapes

Contact Info

Product

Resources

About