Publications by authors named "William J Blake"

11 Publications

  • Page 1 of 1

Synthetic chromosome arms function in yeast and generate phenotypic diversity by design.

Nature 2011 Sep 14;477(7365):471-6. Epub 2011 Sep 14.

High Throughput Biology Center, Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, Maryland 21205, USA.

Recent advances in DNA synthesis technology have enabled the construction of novel genetic pathways and genomic elements, furthering our understanding of system-level phenomena. The ability to synthesize large segments of DNA allows the engineering of pathways and genomes according to arbitrary sets of design principles. Here we describe a synthetic yeast genome project, Sc2.0, and the first partially synthetic eukaryotic chromosomes, Saccharomyces cerevisiae chromosome synIXR, and semi-synVIL. We defined three design principles for a synthetic genome as follows: first, it should result in a (near) wild-type phenotype and fitness; second, it should lack destabilizing elements such as tRNA genes or transposons; and third, it should have genetic flexibility to facilitate future studies. The synthetic genome features several systemic modifications complying with the design principles, including an inducible evolution system, SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution). We show the utility of SCRaMbLE as a novel method of combinatorial mutagenesis, capable of generating complex genotypes and a broad variety of phenotypes. When complete, the fully synthetic genome will allow massive restructuring of the yeast genome, and may open the door to a new type of combinatorial genetics based entirely on variations in gene content and copy number.
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http://dx.doi.org/10.1038/nature10403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3774833PMC
September 2011

Pairwise selection assembly for sequence-independent construction of long-length DNA.

Nucleic Acids Res 2010 May 1;38(8):2594-602. Epub 2010 Mar 1.

Codon Devices, Inc., One Kendall Square, Building 300, Cambridge, MA 02139, USA.

The engineering of biological components has been facilitated by de novo synthesis of gene-length DNA. Biological engineering at the level of pathways and genomes, however, requires a scalable and cost-effective assembly of DNA molecules that are longer than approximately 10 kb, and this remains a challenge. Here we present the development of pairwise selection assembly (PSA), a process that involves hierarchical construction of long-length DNA through the use of a standard set of components and operations. In PSA, activation tags at the termini of assembly sub-fragments are reused throughout the assembly process to activate vector-encoded selectable markers. Marker activation enables stringent selection for a correctly assembled product in vivo, often obviating the need for clonal isolation. Importantly, construction via PSA is sequence-independent, and does not require primary sequence modification (e.g. the addition or removal of restriction sites). The utility of PSA is demonstrated in the construction of a completely synthetic 91-kb chromosome arm from Saccharomyces cerevisiae.
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http://dx.doi.org/10.1093/nar/gkq123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860126PMC
May 2010

Creation of a type IIS restriction endonuclease with a long recognition sequence.

Nucleic Acids Res 2009 May 20;37(9):3061-73. Epub 2009 Mar 20.

Codon Devices, Inc, Cambridge, MA 02139, USA.

Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6 nt from the target site to generate homogeneous, 5', four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases.
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http://dx.doi.org/10.1093/nar/gkp182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2685105PMC
May 2009

Phenotypic consequences of promoter-mediated transcriptional noise.

Mol Cell 2006 Dec;24(6):853-65

Department of Biomedical Engineering, Massachusetts, USA.

A more complete understanding of the causes and effects of cell-cell variability in gene expression is needed to elucidate whether the resulting phenotypes are disadvantageous or confer some adaptive advantage. Here we show that increased variability in gene expression, affected by the sequence of the TATA box, can be beneficial after an acute change in environmental conditions. We rationally introduce mutations within the TATA region of an engineered Saccharomyces cerevisiae GAL1 promoter and measure promoter responses that can be characterized as being either highly variable and rapid or steady and slow. We computationally illustrate how a stable transcription scaffold can result in "bursts" of gene expression, enabling rapid individual cell responses in the transient and increased cell-cell variability at steady state. We experimentally verify computational predictions that the rapid response and increased cell-cell variability enabled by TATA-containing promoters confer a clear benefit in the face of an acute environmental stress.
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http://dx.doi.org/10.1016/j.molcel.2006.11.003DOI Listing
December 2006

Origins of extrinsic variability in eukaryotic gene expression.

Nature 2006 Feb 21;439(7078):861-4. Epub 2005 Dec 21.

Department of Bioengineering, University of California San Diego, La Jolla, California 92093, USA.

Variable gene expression within a clonal population of cells has been implicated in a number of important processes including mutation and evolution, determination of cell fates and the development of genetic disease. Recent studies have demonstrated that a significant component of expression variability arises from extrinsic factors thought to influence multiple genes simultaneously, yet the biological origins of this extrinsic variability have received little attention. Here we combine computational modelling with fluorescence data generated from multiple promoter-gene inserts in Saccharomyces cerevisiae to identify two major sources of extrinsic variability. One unavoidable source arising from the coupling of gene expression with population dynamics leads to a ubiquitous lower limit for expression variability. A second source, which is modelled as originating from a common upstream transcription factor, exemplifies how regulatory networks can convert noise in upstream regulator expression into extrinsic noise at the output of a target gene. Our results highlight the importance of the interplay of gene regulatory networks with population heterogeneity for understanding the origins of cellular diversity.
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http://dx.doi.org/10.1038/nature04281DOI Listing
February 2006

And the noise played on: stochastic gene expression and HIV-1 infection.

Cell 2005 Jul;122(2):147-9

Center for BioDynamics and Department of Biomedical Engineering, Boston University, MA 02215, USA.

Stochastic gene expression has been implicated in a variety of cellular processes, including cell differentiation and disease. In this issue of Cell, take an integrated computational-experimental approach to study the Tat transactivation feedback loop of HIV-1. They show that fluctuations in a key regulator, Tat, in an isogenic population of infected cells result in two distinct expression states corresponding to latent and productive HIV-1 infection. These findings demonstrate the importance of stochastic gene expression in molecular "decision-making."
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http://dx.doi.org/10.1016/j.cell.2005.07.006DOI Listing
July 2005

Stochasticity in gene expression: from theories to phenotypes.

Nat Rev Genet 2005 Jun;6(6):451-64

Department of Cellular and Molecular Medicine and Ottawa Institute of Systems Biology, University of Ottawa, 451 Smyth Road, Ottawa, Ontario K1H8M5, Canada.

Genetically identical cells exposed to the same environmental conditions can show significant variation in molecular content and marked differences in phenotypic characteristics. This variability is linked to stochasticity in gene expression, which is generally viewed as having detrimental effects on cellular function with potential implications for disease. However, stochasticity in gene expression can also be advantageous. It can provide the flexibility needed by cells to adapt to fluctuating environments or respond to sudden stresses, and a mechanism by which population heterogeneity can be established during cellular differentiation and development.
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http://dx.doi.org/10.1038/nrg1615DOI Listing
June 2005

Molecular biology. Signal processing in single cells.

Science 2005 Mar;307(5717):1886-8

Lipper Center for Computational Genetics and Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.

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http://dx.doi.org/10.1126/science.1110797DOI Listing
March 2005

Synthetic biology evolves.

Trends Biotechnol 2004 Jul;22(7):321-4

Department of Biomedical Engineering, Boston University, 44 Cummington Street, Boston, MA 02215, USA.

Synthetic biology is advancing rapidly as biologists, physicists and engineers are combining their efforts to understand and program cell function. By characterizing isolated genetic components or modules, experimentalists have paved the way for more quantitative analyses of genetic networks. A recent paper presents a method of computational, or in silico, evolution in which a set of components can evolve into networks that display desired behaviors. An integrated approach that includes a strategy of in silico design by evolution, together with efforts exploiting directed evolution in vivo, is likely to be the next step in the evolution of synthetic biology.
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http://dx.doi.org/10.1016/j.tibtech.2004.04.008DOI Listing
July 2004

The engineering of gene regulatory networks.

Annu Rev Biomed Eng 2003 ;5:179-206

Center for BioDynamics, Department of Biomedical Engineering, and Bioinformatics Program, Boston University, Boston, Massachusetts 02215, USA.

The rapid accumulation of genetic information and advancement of experimental techniques have opened a new frontier in biomedical engineering. With the availability of well-characterized components from natural gene networks, the stage has been set for the engineering of artificial gene regulatory networks with sophisticated computational and functional capabilities. In these efforts, the ability to construct, analyze, and interpret qualitative and quantitative models is becoming increasingly important. In this review, we consider the current state of gene network engineering from a combined experimental and modeling perspective. We discuss how networks with increased complexity are being constructed from simple modular components and how quantitative deterministic and stochastic modeling of these modules may provide the foundation for accurate in silico representations of gene regulatory network function in vivo.
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http://dx.doi.org/10.1146/annurev.bioeng.5.040202.121553DOI Listing
January 2004

Noise in eukaryotic gene expression.

Nature 2003 Apr;422(6932):633-7

Center for BioDynamics, Center for Advanced Biotechnology, Bioinformatics Program, and Department of Biomedical Engineering, Boston University, 44 Cummington Street, Boston, Massachusetts 02215, USA.

Transcription in eukaryotic cells has been described as quantal, with pulses of messenger RNA produced in a probabilistic manner. This description reflects the inherently stochastic nature of gene expression, known to be a major factor in the heterogeneous response of individual cells within a clonal population to an inducing stimulus. Here we show in Saccharomyces cerevisiae that stochasticity (noise) arising from transcription contributes significantly to the level of heterogeneity within a eukaryotic clonal population, in contrast to observations in prokaryotes, and that such noise can be modulated at the translational level. We use a stochastic model of transcription initiation specific to eukaryotes to show that pulsatile mRNA production, through reinitiation, is crucial for the dependence of noise on transcriptional efficiency, highlighting a key difference between eukaryotic and prokaryotic sources of noise. Furthermore, we explore the propagation of noise in a gene cascade network and demonstrate experimentally that increased noise in the transcription of a regulatory protein leads to increased cell-cell variability in the target gene output, resulting in prolonged bistable expression states. This result has implications for the role of noise in phenotypic variation and cellular differentiation.
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http://dx.doi.org/10.1038/nature01546DOI Listing
April 2003