Gene synthesis by integrated polymerase chain assembly and PCR amplification using a high-speed thermocycler

TerMaat, Joel R.; Pienaar, Elsje; Whitney, Scott E.; Mamedov, T. G.; Subramanian, Anuradha

doi:10.1016/j.mimet.2009.09.015

Cited by 16 publications

(15 citation statements)

References 14 publications

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“…Optimization and improvements of existing DNA assembly methods were reported previously for techniques such as SLIC 29 , PCA 30 and LCR 31 . SLIC was a development of LIC that allowed bypassing of strict DNA sequence requirements of the vector ends by omitting the addition of specific dNTPs to the exonuclease reaction step.…”

Section: Discussionmentioning

confidence: 99%

RapGene: a fast and accurate strategy for synthetic gene assembly in Escherichia coli

Zampini

Stevens

Pachebat

et al. 2015

Sci Rep

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The ability to assemble DNA sequences de novo through efficient and powerful DNA fabrication methods is one of the foundational technologies of synthetic biology. Gene synthesis, in particular, has been considered the main driver for the emergence of this new scientific discipline. Here we describe RapGene, a rapid gene assembly technique which was successfully tested for the synthesis and cloning of both prokaryotic and eukaryotic genes through a ligation independent approach. The method developed in this study is a complete bacterial gene synthesis platform for the quick, accurate and cost effective fabrication and cloning of gene-length sequences that employ the widely used host Escherichia coli.

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Section: Discussionmentioning

confidence: 99%

RapGene: a fast and accurate strategy for synthetic gene assembly in Escherichia coli

Zampini

Stevens

Pachebat

et al. 2015

Sci Rep

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“…Oligonucleotides with regions of degeneracy corresponding to the proposed variable regions were ordered from IDT and assembled into double-stranded de novo genes by polymerase cycling assembly (PCA) and PCR [24]. The number of PCA and PCR cycles were optimized to yield the greatest yield and diversity of correct sequences [27]. NEBuilder HiFi DNA Assembly was performed to introduce the de novo genes into a suitable vector for expression in E. coli.…”

Section: Design and Construction Of A Cavity Library Into The Structumentioning

confidence: 99%

A Strategy for Combinatorial Cavity Design in De Novo Proteins

Karas

Hecht

2020

Life

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Protein sequence space is vast; nature uses only an infinitesimal fraction of possible sequences to sustain life. Are there solutions to biological problems other than those provided by nature? Can we create artificial proteins that sustain life? To investigate these questions, we have created combinatorial collections, or libraries, of novel sequences with no homology to those found in living organisms. Previously designed libraries contained numerous functional proteins. However, they often formed dynamic, rather than well-ordered structures, which complicated structural and mechanistic characterization. To address this challenge, we describe the development of new libraries based on the de novo protein S-824, a 4-helix bundle with a very stable 3-dimensional structure. Distinct from previous libraries, we targeted variability to a specific region of the protein, seeking to create potential functional sites. By characterizing variant proteins from this library, we demonstrate that the S-824 scaffold tolerates diverse amino acid substitutions in a putative cavity, including buried polar residues suitable for catalysis. We designed and created a DNA library encoding 1.7 × 106 unique protein sequences. This new library of stable de novo α-helical proteins is well suited for screens and selections for a range of functional activities in vitro and in vivo.

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“…In a following approach gfp was synthesized by either RapGene or polymerase cycling assembly 26 (PCA) ( Fig. 3b ).…”

Section: Resultsmentioning

confidence: 99%

Terminator Operon Reporter: combining a transcription termination switch with reporter technology for improved gene synthesis and synthetic biology applications

Zampini

Mur

Stevens

et al. 2016

Sci Rep

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Synthetic biology is characterized by the development of novel and powerful DNA fabrication methods and by the application of engineering principles to biology. The current study describes Terminator Operon Reporter (TOR), a new gene assembly technology based on the conditional activation of a reporter gene in response to sequence errors occurring at the assembly stage of the synthetic element. These errors are monitored by a transcription terminator that is placed between the synthetic gene and reporter gene. Switching of this terminator between active and inactive states dictates the transcription status of the downstream reporter gene to provide a rapid and facile readout of the accuracy of synthetic assembly. Designed specifically and uniquely for the synthesis of protein coding genes in bacteria, TOR allows the rapid and cost-effective fabrication of synthetic constructs by employing oligonucleotides at the most basic purification level (desalted) and without the need for costly and time-consuming post-synthesis correction methods. Thus, TOR streamlines gene assembly approaches, which are central to the future development of synthetic biology.

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Gene synthesis by integrated polymerase chain assembly and PCR amplification using a high-speed thermocycler

Cited by 16 publications

References 14 publications

RapGene: a fast and accurate strategy for synthetic gene assembly in Escherichia coli

RapGene: a fast and accurate strategy for synthetic gene assembly in Escherichia coli

A Strategy for Combinatorial Cavity Design in De Novo Proteins

Terminator Operon Reporter: combining a transcription termination switch with reporter technology for improved gene synthesis and synthetic biology applications

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