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Scientific PublicationsDNA2.0 scientists are noted in italicsPlant J 2008 Identification of likely orthologs of tobacco salicylic acid-binding protein2 and their role in systemic acquired resistance in Arabidopsis thaliana. Vlot, Liu, Cameron, Park, Yang, Kumar, Zhou, Padukkavidana, Gustafsson, Pichersky, Klessig.
Arabidopsis orthologs to Salicylic acid-binding protein 2 (SABP2) from tobacco were identified and characterized. The orthologs (AtMES 1, 2, 7, and 9) were shown to be functionally homologous to SABP2 and redundant for synthesis of methyl salicylate, a signal for system acquired resistance. Nature 2008 451:704. Paleotemperature trend for Precambrian life inferred from resurrected proteins. Gaucher, Govindarajan, Ganesh.
Genes corresponding to since long extinct proteins were resurrected by DNA2.0 gene synthesis. The temperature optima of the resurrected genes closely follows the temperature calculated based on geological methods stretching back 3.5 billion years ago.
Bioinformatics 2007 23:2760. A syntactic model to design and verify synthetic genetic constructs derived from standard biological parts. Cai, Hartnett, Gustafsson, Peccoud.
Dr. Peccouds group at Virginia Tech takes the concept of DNA2.0's Gene Designer to the next logical step and create a context-free grammar algorithm to formalize the design principles of synthetic genetic constructs. The software, sequences and models are available at www.genocad.org. Check it out. BMC Biotechnol 2007 7:16. Engineering proteinase K using machine learning and synthetic genes. Liao, Warmuth, Govindarajan, Ness, Wang, Gustafsson, Minshull.
A synthetic biology approach to protein engineering that combines high throughput gene synthesis with machine learning-based design algorithms is described. The functionality of proteinase K is increased >20 fold while testing only 95 variants. The described method avoids the current protein engineering limitation of requiring high throughput screening for improved activity. Proc Natl Acad Sci Northpole 2006 12:25. Heterologous expression and functional characterization of the Santa Hoho2 gene. Claes, Reindeer, Nicolas, Tomte, Sridhar, Elf.
The Hoho2 gene responsible for facial hair formation of Santa Claus is isolated and shown to be an ortholog of human KRT6B. The Hoho2 gene was codon optimized and and the corresponding protein expressed in E. coli, reindeer and human. RNAi knockout constructs could be trans-complemented with an RNAi resistant Hoho2 variant.
BMC Bioinformatics 2006 7:285. Gene Designer: a synthetic biology tool for constructing artificial DNA segments. Villalobos, Ness, Gustafsson, Minshull, Govindarajan.
Gene Designer is a free stand-alone software made available by DNA2.0 for fast and easy design of synthetic DNA segments. Users can add, edit and combine genetic elements through an intuitive drag-and-drop graphic interface and a hierarchical DNA/Protein object map. Open reading frames can be codon optimized for protein expression in any host organism. Gene Designer also includes many additional features. Plant J 2006 45:863-8. Validation of RNAi silencing specificity using synthetic genes: salicylic acid-binding protein 2 is required for innate immunity in plants. Kumar, Gustafsson, Klessig.
An RNAi resistant homolog to SABP2 was designed by codon optimizing to be as distant as possible from the wt gene. The synthetic gene was expressed in tobacco and shown to trans-complement the RNAi knockout wt gene by Dr. Dan Klessig and coworkers at Cornell University. Genetic Engineering News 2005 25:32. Protein Expression Using Synthetic Genes. Gustafsson.
This publication describes the background to codon usage bias, synthetic genes, codon optimization and how this translational understanding now is applied to heterologous protein expression. Various gene design issues are addressed and evaluated. Curr Opin Chem Biol 2005 9:202-9. Predicting enzyme function from protein sequence. Minshull, Ness, Gustafsson, Govindarajan.
Information flow from protein function to DNA sequence can be captured and deconvoluted to gain a model for sequence-funtion correlation. The applicability of what is being learned from natural enzymes is discussed in the context of methods for catalyst design. ACS Symposium Series 900 Polymer Biocatalysis and Biomaterials 2005 (ISSN 0097-6156) 37-50. Empirical Biocatalyst Engineering: Escaping the Tyranny of High-Throughput Screening. Ness, Cox, Govindarajan, Gustafsson, Gross, Minshull.
Sequence-activity relationships is used in the design of proteins with specified properties. The availability gene synthesis make this a cost-effective technique for biocatalyst engineering. Because only small numbers of variants (<100) need to be tested, proteins engineered using sequence-activity relationships can be tested directly under final application conditions. This avoids the expenses and errors of developing and implementing high throughput surrogate screens. Trends Biotechnol 2004 22:346-53. Codon Bias and Heterologous Protein Expression. Gustafsson, Govindarajan, Minshull
The expression of functional proteins in heterologous hosts is a cornerstone of modern biotechnology. Improvements in the speed and cost of gene synthesis have facilitated the complete redesign of entire gene sequences to maximize the likelihood of high protein expression. Redesign strategies are discussed here, including modification of translation initiation regions, alteration of mRNA structural elements and use of different codon biases. Methods 2004 32:416-27. Engineered protein function by selective amino acid diversification. Minshull, Govindarajan, Ness, Gustafsson.
Differences between protein engineering methods are the ways in which amino acids are chosen for variation, the protocols followed for creating the variants, and how information regarding variant properties is used in creating subsequent variants. Tthese differences are discussed and examples are provided for how the experimental parameters of specific projects determine which method is most suitable. Curr Opin Biotechnol 2003 14:366-70. Putting engineering back into protein engineering: bioinformatic approaches to catalyst design. Gustafsson, Govindarajan, Minshull.
Mathematical and data-mining tools developed in other fields of engineering are now applied to analyze sequence-activity relationships of peptides and proteins and to assist in the design of proteins and peptides with specified properties. Decreasing costs to quickly synthesize statistically representative sets of proteins allow modern heuristic statistics to be applied to protein engineering. This provides an alternative approach to unreliable high-throughput surrogate screens. Nucleic Acids Res 1993 21:733-9 Quantative sequence-activity models (QSAM) - Tools for sequence design. Jonsson, Norberg, Carlsson, Gustafsson, Wold.
Models have been developed that allow the biological activity of a DNA segment to be altered in a desired direction. Partial least squares projections to latent structures (PLS) was used to establish a quantitative model between 25 E.coli promoters and their corresponding quantitative measure of in vivo strength. This quantitative sequence-activity model (QSAM) was used to generate two 68 bp fragments predicted to be more potent promoters than any of those on which the model originally was based. The optimized structures were experimentally verified to be strong promoters in vivo. |
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