Enzymatic assembly of DNA molecules up to several hundred kilobases

Gibson, Daniel G.; Young, L. S.; Chuang, Ronald Y.; Venter, J. Craig; Hutchison, Clyde A.; Smith, Hamilton O.

doi:10.1038/nmeth.1318

Cited by 8,612 publications

(7,560 citation statements)

References 17 publications

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“…Briefly, a linear PCR product containing two random 25 nucleotide “barcode” regions flanked by loxP and lox2272 sites along with common linker sequences for priming was combined with a gateway compatible vector at a SacI restriction site through in vitro DNA assembly (Gibson et al , 2009). This barcoded destination vector pool was transformed into One Shot ccdB Survival T1R Competent Cells (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

A framework for exhaustively mapping functional missense variants

Weile

Sun

Coté

et al. 2017

Molecular Systems Biology

166

286

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Although we now routinely sequence human genomes, we can confidently identify only a fraction of the sequence variants that have a functional impact. Here, we developed a deep mutational scanning framework that produces exhaustive maps for human missense variants by combining random codon mutagenesis and multiplexed functional variation assays with computational imputation and refinement. We applied this framework to four proteins corresponding to six human genes: UBE2I (encoding SUMO E2 conjugase), SUMO1 (small ubiquitin‐like modifier), TPK1 (thiamin pyrophosphokinase), and CALM1/2/3 (three genes encoding the protein calmodulin). The resulting maps recapitulate known protein features and confidently identify pathogenic variation. Assays potentially amenable to deep mutational scanning are already available for 57% of human disease genes, suggesting that DMS could ultimately map functional variation for all human disease genes.

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Section: Methodsmentioning

confidence: 99%

A framework for exhaustively mapping functional missense variants

Weile

Sun

Coté

et al. 2017

Molecular Systems Biology

166

286

View full text Add to dashboard Cite

show abstract

“…Using SnapGene (GSL Biotech LLC) software DNA fragments to join were designed to have overhangs at either ends and were PCR‐amplified by gene‐specific primers comprising 5′ extensions. The Gibson assembly reaction was performed using a custom‐made enzyme mix (Gibson et al , 2009). The resulting pENTR constructs were sub‐cloned into the pPW vector ( Drosophila Genomics Resource Center, Bloomington, IN) using the Gateway cloning system (Thermo Fisher Scientific) following the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Downregulation of basal myosin‐ II is required for cell shape changes and tissue invagination

et al. 2018

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Tissue invagination drives embryo remodeling and assembly of internal organs during animal development. While the role of actomyosin‐mediated apical constriction in initiating inward folding is well established, computational models suggest relaxation of the basal surface as an additional requirement. However, the lack of genetic mutations interfering specifically with basal relaxation has made it difficult to test its requirement during invagination so far. Here we use optogenetics to quantitatively control myosin‐II levels at the basal surface of invaginating cells during Drosophila gastrulation. We show that while basal myosin‐II is lost progressively during ventral furrow formation, optogenetics allows the maintenance of pre‐invagination levels over time. Quantitative imaging demonstrates that optogenetic activation prior to tissue bending slows down cell elongation and blocks invagination. Activation after cell elongation and tissue bending has initiated inhibits cell shortening and folding of the furrow into a tube‐like structure. Collectively, these data demonstrate the requirement of myosin‐II polarization and basal relaxation throughout the entire invagination process.

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“…However there are many microorganisms that produce useful secondary metabolites, which are not amenable to such genetic manipulation. The rise of synthetic biology has provided access to larger synthetic DNA constructs, rapid DNA capture,7, 8, 9 editing,10, 11 assembly,12, 13 and other advances 14, 15. The prospect of using these new tools to assemble de novo biosynthetic pathways in well‐characterized heterologous host strains16, 17 for diversification and optimization of natural products, derived from less tractable microorganisms, is an attractive goal.…”

mentioning

confidence: 99%

De novo Biosynthesis of “Non‐Natural” Thaxtomin Phytotoxins

2018

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Thaxtomins are diketopiperazine phytotoxins produced by Streptomyces scabies and other actinobacterial plant pathogens that inhibit cellulose biosynthesis in plants. Due to their potent bioactivity and novel mode of action there has been considerable interest in developing thaxtomins as herbicides for crop protection. To address the need for more stable derivatives, we have developed a new approach for structural diversification of thaxtomins. Genes encoding the thaxtomin NRPS from S. scabies, along with genes encoding a promiscuous tryptophan synthase (TrpS) from Salmonella typhimurium, were assembled in a heterologous host Streptomyces albus. Upon feeding indole derivatives to the engineered S. albus strain, tryptophan intermediates with alternative substituents are biosynthesized and incorporated by the NRPS to deliver a series of thaxtomins with different functionalities in place of the nitro group. The approach described herein, demonstrates how genes from different pathways and different bacterial origins can be combined in a heterologous host to create a de novo biosynthetic pathway to “non‐natural” product target compounds.

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Enzymatic assembly of DNA molecules up to several hundred kilobases

Cited by 8,612 publications

References 17 publications

A framework for exhaustively mapping functional missense variants

A framework for exhaustively mapping functional missense variants

Downregulation of basal myosin‐ II is required for cell shape changes and tissue invagination

De novo Biosynthesis of “Non‐Natural” Thaxtomin Phytotoxins

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