Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension

Horton, Robert M.; Hunt, Henry D.; Ho, Steffan N.; Pullen, Jeffrey K.; Pease, Larry R.

doi:10.1016/0378-1119(89)90359-4

Cited by 2,870 publications

(1,978 citation statements)

References 11 publications

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“…Schematics of the anthrax toxin-Hoc recombinants. A. Anthrax toxin antigens and their domains (red, green and blue) were fused to the N-or C-termini of Hoc (yellow) via a linker (pink) by the PCR-based SOE strategy [24,25]. Insertion of the fusions into the plasmid vector resulted in the attachment of hexa-histidine tag (grey) to the N-terminus of each recombinant.…”

Section: Discussionmentioning

confidence: 99%

“…Toxin-Hoc fusions were generated by employing the basic principles of PCR-based Splicing by Overlap Extension (SOE) strategy as described previously [24,25]. The N-terminal Hoc fusions include full-length clones, PA-Hoc, LF-Hoc, and EF-Hoc, and domain clones, LFnHoc and LFc-Hoc (LFn refers to the N-terminal domain of LF that interacts with PA63; LFc refers to the C-terminal domain of LF that has the MAPKK protease activity; see Fig.…”

Section: Construction Of Anthrax Toxin-hoc Fusion Recombinantsmentioning

confidence: 99%

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Multicomponent anthrax toxin display and delivery using bacteriophage T4

Shivachandra

Peachman

et al. 2007

Vaccine

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We describe a multicomponent antigen display and delivery system using bacteriophage T4. Two dispensable outer capsid proteins, Hoc (highly antigenic outer capsid protein, 155 copies) and Soc (small outer capsid protein, 810 copies), decorate phage T4 capsid. These proteins bind to the symmetrically localized capsid sites, which appear following prohead assembly and expansion. We hypothesized that multiple antigens fused to Hoc can be displayed on the same capsid and such particles can elicit broad immunological responses. Anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), and their functional domains, were fused to Hoc with an N-terminal hexa-histidine tag and the recombinant proteins were over-expressed in E. coli and purified. Using a defined in vitro assembly system, the anthrax-Hoc fusion proteins were efficiently displayed on T4 capsid, either individually or in combinations. All of the 155 Hoc binding sites can be occupied by one antigen, or they can be split among two or more antigens by varying their molar ratio in the binding reaction. Immunization of mice with T4 phage carrying PA, LF, and EF elicited strong antigen-specific antibodies against all antigens as well as lethal toxin neutralization titers. The triple antigen T4 phage elicited stronger PA-specific immune responses than the phage displaying PA alone. These features offer novel avenues to develop customized multicomponent vaccines against anthrax and other pathogenic diseases.

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Section: Discussionmentioning

confidence: 99%

Section: Construction Of Anthrax Toxin-hoc Fusion Recombinantsmentioning

confidence: 99%

Multicomponent anthrax toxin display and delivery using bacteriophage T4

Shivachandra

Peachman

et al. 2007

Vaccine

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“…Another reason for their popularity is that overlap regions can easily be added by PCR. The mechanism of action of these methods varies greatly: Circular Polymerase Extension Cloning (CPEC) is an evolution of Overlap Extension PCR (OE-PCR 44 ) and is essentially a high-fidelity PCR amplification, where template and primers are replaced by the DNA fragments to be assembled into a plasmid 45 . As these are designed to share homology at their ends, the parts anneal to each other during PCR and act as primers for one another's amplification, until eventually a nicked circular molecule is generated.…”

Section: Long Overlap-based Assemblymentioning

confidence: 99%

Bricks and blueprints: methods and standards for DNA assembly

Casini

Storch

Baldwin

et al. 2015

Nat Rev Mol Cell Biol

248

186

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PrefaceDNA assembly is a key part of constructing gene expression systems and even whole chromosomes. In the past decade a plethora of powerful new DNA assembly methods including Gibson assembly, Golden Gate and LCR have been developed. In this Innovation article we discuss these methods and standards such as MoClo, GoldenBraid, MODAL and PaperClip, which have been developed to facilitate a streamlined assembly workflow, aid material exchange, and the creation of modular, reusable DNA parts. IntroductionOur capacity to cut and paste DNA from different sources and to assemble it into gene constructs has been one of the key drivers of biological research and biotechnology over the past four decades. However, despite countless advances in molecular biology, the assembly of DNA parts into new constructs remains a craft that is both time consuming and unpredictable. The decreasing costs of gene synthesis promises to alleviate these limitations by providing custom-made double-stranded DNA fragments typically between 200 and 2000 bp in length 1 . Nonetheless, gene synthesis does not eliminate the need for DNA assembly, which remains necessary for the production of constructs beyond one kilobase in size, both in research labs and at gene synthesis companies. DNA assembly also enables carrying out projects with more complex experimental needs and is especially valuable for building diverse plasmid libraries and creating multicomponent systems, and has even been used to construct synthetic cells 2 .Addressing the limitations of DNA assembly methods has been one of the key goals of synthetic biology, a scientific discipline focused on the construction and testing of new or redesigned versions of genes, gene networks, pathways and cells 3,4 . In order to tackle projects of increasing scale and complexity, researchers have invested significant effort into developing new tools for DNA assembly and into matching them with improved, lower-cost gene synthesis (for reviewes on gene synthesis see REFS. 1,5 1,5 ), as well as a suite of important new tools for genome editing (Box 1). With these combined advances the field is now at a point where gene synthesis and DNA assembly can empower even undergraduate students to construct entire eukaryotic chromosomes 6 . This acceleration in the scale of DNA assembly enables construction projects too complex to be drawn out on the back of an envelope, which instead require an engineering approach. In the past decade important 1 assembly methods such as Gibson assembly and Golden Gate have been developed 7,8 , which define new protocols for joining together DNA parts. Alongside these methods, researchers have also developed various physical standards such as MODAL (Modular Overlap-Directed Assembly with Linkers) 9 and MoClo (Modular Cloning system) 12 that define rules for the format of DNA parts that can be used with them. These physical standards facilitate the re-use of parts between experiments, exchange of parts between research groups and importantly provide modularity in construction. ...

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“…The ptb-buk fragment was generated by EcoRI and NotI digestion of pCDF-PB 29 . The B. subtilis ptb and buk genes were cloned into an artificial operon using splicing by overlap extension PCR 54,55 to mimic the natural C. acetobutylicum ptb-buk operon. The E. coli genes ycdW, aceA and aceK were similarly cloned into an artificial operon to mimic the structure of the natural aceB-aceA-aceK operon in E. coli, with the ycdW gene replacing aceB (see Supplementary Methods).…”

Section: Methodsmentioning

confidence: 99%

A platform pathway for production of 3-hydroxyacids provides a biosynthetic route to 3-hydroxy-γ-butyrolactone

et al. 2013

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The replacement of petroleum feedstocks with biomass to produce platform chemicals requires the development of appropriate conversion technologies. 3-Hydroxy-g-butyrolactone has been identified as one such chemical; however, there are no naturally occurring biosynthetic pathways for this molecule or its hydrolyzed form, 3,4-dihydroxybutyric acid. Here we design a novel pathway to produce various chiral 3-hydroxyacids, including 3,4-dihydroxybutyric acid, consisting of enzymes that condense two acyl-CoAs, stereospecifically reduce the resulting b-ketone and hydrolyze the CoA thioester to release the free acid. Acetyl-CoA serves as one substrate for the condensation reaction, whereas the second is produced intracellularly by a pathway enzyme that converts exogenously supplied organic acids. Feeding of butyrate, isobutyrate and glycolate results in the production of 3-hydroxyhexanoate, 3-hydroxy-4-methylvalerate and 3,4-dihydroxybutyric acid þ 3-hydroxy-gbutyrolactone, respectively, molecules with potential uses in applications from materials to medicines. We also unexpectedly observe the condensation reaction resulting in the production of the 2,3-dihydroxybutyric acid isomer, a potential value-added monomer.

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Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension

Cited by 2,870 publications

References 11 publications

Multicomponent anthrax toxin display and delivery using bacteriophage T4

Multicomponent anthrax toxin display and delivery using bacteriophage T4

Bricks and blueprints: methods and standards for DNA assembly

A platform pathway for production of 3-hydroxyacids provides a biosynthetic route to 3-hydroxy-γ-butyrolactone

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