Smith HO: Enzymatic assembly of DNA molecules up to several hundred kilobases

The J. Craig Venter Institute, Synthetic Biology Group, Rockville, Maryland, USA.
Nature Methods (Impact Factor: 32.07). 05/2009; 6(5):343-5. DOI: 10.1038/nmeth.1318
Source: PubMed


We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.

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Available from: Daniel G Gibson, May 12, 2014
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    • "The same PCR conditions were used for a further 30 rounds and the product was purified using a QiaQuick PCR cleanup kit (Qiagen, Hilden, Germany ) and eluted in EB. Gibson assembly [47] was used to insert the assembled gene into gelpurified pETFLAG vector digested with NdeI/XhoI (NEB, Waltham, MA). A 1.5μL aliquot of cut vector at 20ng/μL was added to 1μL of assembled gene and 7.5μL of Gibson enzyme mix (all enzymes from NEB, Waltham, MA). "
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    ABSTRACT: Broadly neutralizing antibodies targeting a highly conserved region in the hemagglutinin (HA) stem protect against influenza infection. Here, we investigate the protective efficacy of a protein (HB36.6) computationally designed to bind with high affinity to the same region in the HA stem. We show that intranasal delivery of HB36.6 affords protection in mice lethally challenged with diverse strains of influenza independent of Fc-mediated effector functions or a host antiviral immune response. This designed protein prevents infection when given as a single dose of 6.0 mg/kg up to 48 hours before viral challenge and significantly reduces disease when administered as a daily therapeutic after challenge. A single dose of 10.0 mg/kg HB36.6 administered 1-day post-challenge resulted in substantially better protection than 10 doses of oseltamivir administered twice daily for 5 days. Thus, binding of HB36.6 to the influenza HA stem region alone, independent of a host response, is sufficient to reduce viral infection and replication in vivo. These studies demonstrate the potential of computationally designed binding proteins as a new class of antivirals for influenza.
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    • "The synthetic fragment P lepA –sp containing the constitutive promoter P lepA , the strong RBS (Shine–Dalgarno sequence) from gsiB, and the signal peptide of yweA (Zhang et al., 2013) was used to express H6LHyal. The lox71–zeo–lox66 cassettes, P lepA –sp, H6LHyal, and two homology arms were ligated into pBluescript SK( þ ) to generate the plasmid pSKIZH with one-step assembly (Gibson et al., 2009). The plasmid was then used to generate strain E168TH from the B. subtilis strain E168T. "
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    ABSTRACT: Low-molecular-weight hyaluronan (LMW-HA) has attracted much attention because of its many potential applications. Here, we efficiently produced specific LMW-HAs from sucrose in Bacillus subtilis. By coexpressing the identified committed genes (tuaD, gtaB, glmU, glmM, and glmS) and downregulating the glycolytic pathway, HA production was significantly increased from 1.01 g L−1 to 3.16 g L−1, with a molecular weight range of 1.40×106–1.83×106 Da. When leech hyaluronidase was actively expressed after N-terminal engineering (1.62×106 U mL−1), the production of HA was substantially increased from 5.96 g L−1 to 19.38 g L−1. The level of hyaluronidase was rationally regulated with a ribosome-binding site engineering strategy, allowing the production of LMW-HAs with a molecular weight range of 2.20×103–1.42×106 Da. Our results confirm that this strategy for the controllable expression of hyaluronidase, together with the optimization of the HA synthetic pathway, effectively produces specific LMW-HAs, and could also be used to produce other LMW polysaccharides.
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    • "The second plasmid constitutively expresses Cas9 and tracrRNA on a p15A vector using an AmpR antibiotic marker. Plasmid construction was performed by PCR-amplifying the Cas9 and tracrRNA expression cassettes from the pdCas9 plasmid [8] and assembling with a PCR-amplified p15A vector fragment using Gibson's method [83]. The third plasmid expresses the precrRNA using an IPTG-inducible P tac promoter on a ColE1 vector using a CmR antibiotic marker, and was constructed by PCR-amplifying the precrRNA cassette from pdCas9 and assembling it with a PCR-amplified ColE1 fragment using Gibson's method. "
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