SpeedyGenes: an improved gene synthesis method for the efficient production of error-corrected, synthetic protein libraries for directed evolution

Currin, Andrew; Swainston, Neil; Day, Philip J.; Kell, Douglas B.

doi:10.1093/protein/gzu029

Cited by 44 publications

(49 citation statements)

References 54 publications

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“…Thus we have described methods for the controlled generation and assembly of DNA/protein sequences [288,294,[389][390][391] designed to navigate these very large search spaces 'intelligently' [324]. While specificity is largely (but not at all completely) based on residues at or near the active site, we note in particular that raising k cat requires contributions from residues that may be very distant from the active site (e.g.…”

Section: Synthetic Biology For Efflux Transporter Engineeringmentioning

confidence: 99%

Control of metabolite efflux in microbial cell factories: current advances and future prospects

Kell¹

2018

Preprint

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The yield and ease of purification of biotechnological products are typically greatly enhanced if they can be secreted into the external medium. Although not universally appreciated, however, small molecule biotechnological products do not ‘float across’ the phospholipid bilayer portion of biological membranes, and thus they need transporters to assist their passage into the extramembrane and extracellular spaces. Some of these transporters may be reversible, equilibrative transporters that might more normally be used for uptake, while others may have an efflux directionality imposed on them via suitable energy coupling mechanisms. Despite the energetic costs of small molecule synthesis, natural evolution does in fact provide a number of mechanisms by which the secretion of such products can actually enhance fitness. Where available these provide useful starting points, and assaying for such activities is crucial. In particular, in a systems biology approach, we first need to identify such/suitable activities before we can seek to increase them. They may then be improved through promoter engineering, via manipulation of control elements, or by directed evolution of the transporter proteins themselves. Modern methods of synthetic biology provide enormous opportunities for all kinds of efflux transporter engineering; they are just beginning to be realised.

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Section: Synthetic Biology For Efflux Transporter Engineeringmentioning

confidence: 99%

Control of metabolite efflux in microbial cell factories: current advances and future prospects

Kell¹

2018

Preprint

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“…Some of the selected enzymes, such as T7 endonuclease I, display the capacity to cleave mismatch regions in hetero-duplex nucleic acids and have been applied in mutation screening [11,15] and to a lesser degree in repair systems applied in artificial gene synthesis [2,7]. The endonucleases were of prokaryotic or eukaryotic origins (Table 1).…”

Section: Cleavage Activity Of Recombinant Endonucleasesmentioning

confidence: 99%

“…Gene synthesis is also changing established paradigms within the recombinant DNA technology field, in particular for heterologous gene expression, vaccine development, gene therapy and molecular engineering. In recent years, improvements in artificial DNA production methodologies have originated more robust, simple and cost-effective gene assembly technologies [2,3]. In addition, exploitation of the latest high-throughput molecular technologies supported the large-scale and low-cost production of DNA sequences [4].…”

Section: Introductionmentioning

confidence: 99%

T7 Endonuclease I Mediates Error Correction in Artificial Gene Synthesis

et al. 2016

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Efficacy of de novo gene synthesis largely depends on the quality of overlapping oligonucleotides used as template for PCR assembly. The error rate associated with current gene synthesis protocols limits the efficient and accurate production of synthetic genes, both in the small and large scales. Here, we analysed the ability of different endonuclease enzymes, which specifically recognize and cleave DNA mismatches resulting from incorrect impairments between DNA strands, to remove mutations accumulated in synthetic genes. The gfp gene, which encodes the green fluorescent protein, was artificially synthesized using an integrated protocol including an enzymatic mismatch cleavage step (EMC) following gene assembly. Functional and sequence analysis of resulting artificial genes revealed that number of deletions, insertions and substitutions was strongly reduced when T7 endonuclease I was used for mutation removal. This method diminished mutation frequency by eightfold relative to gene synthesis not incorporating an error correction step. Overall, EMC using T7 endonuclease I improved the population of error-free synthetic genes, resulting in an error frequency of 0.43 errors per 1 kb. Taken together, data presented here reveal that incorporation of a mutation-removal step including T7 endonuclease I can effectively improve the fidelity of artificial gene synthesis.

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“…The PcaV libraries were constructed using the SpeedyGenes gene synthesis method [49,50]. PcaV WT and library genes were designed with the GeneGenie software [51], allowing the generation of WT or variant libraries by overlap-extension PCR (OE-PCR) using 10 overlapping oligonucleotides.…”

Section: Pcav Libraries Design and Constructionmentioning

confidence: 99%

Directed evolution of the PcaV allosteric transcription factor to generate a biosensor for aromatic aldehydes

Machado

Currin

Dixon

2019

Preprint

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Background:Genetically encoded biosensors are useful tools for the detection of metabolites and industrially valuable molecules, and present many potential applications in biotechnology and biomedicine. However, the most common approach to develop biosensors relies on employing a limited set of naturally occurring allosteric transcript factors (aTFs). Therefore, altering the substrate specificity of aTFs towards the detection of new effectors is an important goal. Results:Here, the PcaV repressor, a member of the MarR aTF family, was used to develop a biosensor for the detection of hydroxyl-substituted benzoic acids, including protocatechuic acid (PCA). The PCA biosensor was further subjected to directed evolution to alter its substrate specificity towards vanillin and other closely related aromatic aldehydes, to generate the Van2 biosensor. Substrate recognition of Van2 was explored in vitro using a range of biochemical and biophysical analyses, and extensive in vivo genetic-phenotypic analysis was performed to determine the role of each amino acid change upon biosensor performance. Conclusions:This is the first study to report directed evolution of a member of the MarR aTF family, and demonstrates the plasticity of the PCA biosensor by altering its substrate specificity to generate a biosensor for aromatic aldehydes.

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SpeedyGenes: an improved gene synthesis method for the efficient production of error-corrected, synthetic protein libraries for directed evolution

Cited by 44 publications

References 54 publications

Control of metabolite efflux in microbial cell factories: current advances and future prospects

Control of metabolite efflux in microbial cell factories: current advances and future prospects

T7 Endonuclease I Mediates Error Correction in Artificial Gene Synthesis

Directed evolution of the PcaV allosteric transcription factor to generate a biosensor for aromatic aldehydes

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