Expanding the Product Profile of a Microbial Alkane Biosynthetic Pathway
by Matthew Harger, Lei Zheng, Austin Moon, Casey Ager, Ju Hye An, Chris Choe, Yi-Ling Lai, Benjamin Mo, David Zong, Matthew D. Smith, Robert G. Egbert, Jeremy H. Mills, David Baker, Ingrid Swanson Pultz, and Justin B. Siegel
"Microbially produced alkanes are a new class of biofuels that closely match the chemical composition of petroleum-based fuels. Alkanes can be generated from the fatty acid biosynthetic pathway by the reduction of acyl-ACPs followed by decarbonylation of the resulting aldehydes. A current limitation of this pathway is the restricted product profile, which consists of n-alkanes of 13, 15, and 17 carbons in length. To expand the product profile, we incorporated a new part, FabH2 from Bacillus subtilis, an enzyme known to have a broader specificity profile for fatty acid initiation than the native FabH of Escherichia coli. When provided with the appropriate substrate, the addition of FabH2 resulted in an altered alkane product profile in which significant levels of n-alkanes of 14 and 16 carbons in length are produced. The production of even chain length alkanes represents initial steps toward the expansion of this recently discovered microbial alkane production pathway to synthesize complex fuels. This work was conceived and performed as part of the 2011 University of Washington international Genetically Engineered Machines (iGEM) project." http://bit.ly/11oAPcS
"Gene expression in chloroplasts is highly regulated during translation by sequence and secondary-structure elements in the 5′ untranslated region (UTR) of mRNAs. These chloroplast mRNA 5′ UTRs interact with nuclear-encoded factors to regulate mRNA processing, stability, and translation initiation. Although several UTR elements in chloroplast mRNAs have been identified by site-directed mutagenesis, the complete set of elements required for expression of plastid mRNAs remains undefined. Here we present a synthetic biology approach using an arrayed oligonucleotide library to examine in vivo hundreds of designed variants of endogenous UTRs from Chlamydomonas reinhardtii and quantitatively identify essential regions through next-generation sequencing of thousands of mutants. We validate this strategy by characterizing the relatively well-studied 5′ UTR of the psbD mRNA encoding the D2 protein in photosystem II and find that our analysis generally agrees with previous work identifying regions of importance but significantly expands and clarifies the boundaries of these regulatory regions. We then use this strategy to characterize the previously unstudied psaA 5′ UTR and obtain a detailed map of regions essential for both positive and negative regulation. This analysis can be performed in a high-throughput manner relative to previous site-directed mutagenesis methods, enabling compilation of a large unbiased data set of regulatory elements of chloroplast gene expression. Finally, we create a novel synthetic UTR based on aggregate sequence analysis from the libraries and demonstrate that it significantly increases accumulation of an exogenous protein, attesting to the utility of this strategy for enhancing protein production in algal chloroplasts."
"This paper deals with the development of a new simulator that will be very helpful to establish new accurate and predictive design-oriented models for the BioBricks used in synthetic biology. The simulator uses the principle of the game-of-life: molecules can move on a grid and, at every iteration, binding and dissociation rules are applied when two molecules are on same node. The principle is elementary but it can highlight interesting biological phenomenon. Those can be modeled by mathematical equations to achieve design-oriented models. In this case, the simulator also helps to make to link between mathematical parameters and the microscopic parameters. A first version of the software has been implemented in MATLAB. It permits to retrieve very interesting results, such as the Hill's equation and the properties of Hill's coefficient."
by Mahmud Hussain , Nimish Gera , Andrew B. Hill , and Balaji M. Rao
"The use of binding proteins from non-immunoglobulin scaffolds has become increasingly common in biotechnology and medicine. Typically, binders are isolated from a combinatorial library generated by mutating a single scaffold protein. In contrast, here we generated a “superlibrary” or “library-of-libraries” of 4 × 108 protein variants by mutagenesis of seven different hyperthermophilic proteins; six of the seven proteins have not been used as scaffolds prior to this study. Binding proteins for five different model targets were successfully isolated from this library. Binders obtained were derived from five out of the seven scaffolds. Strikingly, binders from this modestly sized superlibrary have affinities comparable or higher than those obtained from a library with 1000-fold higher sequence diversity but derived from a single stable scaffold. Thus scaffold diversification, i.e., randomization of multiple different scaffolds, is a powerful alternate strategy for combinatorial library construction."
*Test-tube data:* *Archives could last for thousands of years when stored in DNA instead of magnetic tapes and hard drives*
"LIKE all the best ideas, this one was born in a pub. Nick Goldman and Ewan Birney of the European Bioinformatics Institute (EBI) near Cambridge, were pondering what they could do with the torrent of genomic data their research group generates, all of which has to be archived.
The volume of data is growing faster than the capacity of the hard drives used to hold it. “That means the cost of storage is rising, but our budgets are not,” says Dr Goldman. Over a few beers, the pair began wondering if artificially constructed DNA might be one way to store the data torrent generated by the natural stuff. After a few more drinks and much scribbling on beer mats, what started out as a bit of amusing speculation had turned into the bones of a workable scheme. After some fleshing out and a successful test run, the full details were published this week in Nature....."
A CRISPR-Cas system is harnessed to introduce template-driven mutations in S. pneumoniae and E. coli at high efficiency without requiring selectable markers.
Gerd Moe-Behrens's insight:
Congratulation +David Bikard
*RNA-guided editing of bacterial genomes using CRISPR-Cas systems*
by Wenyan Jiang,David Bikard,David Cox,Feng Zhang& Luciano A Marraffini
"Here we use the clustered, regularly interspaced, short palindromic repeats (CRISPR)–associated Cas9 endonuclease complexed with dual-RNAs to introduce precise mutations in the genomes of Streptococcus pneumoniae and Escherichia coli. The approach relies on dual-RNA:Cas9-directed cleavage at the targeted genomic site to kill unmutated cells and circumvents the need for selectable markers or counter-selection systems. We reprogram dual-RNA:Cas9 specificity by changing the sequence of short CRISPR RNA (crRNA) to make single- and multinucleotide changes carried on editing templates. Simultaneous use of two crRNAs enables multiplex mutagenesis. In S. pneumoniae, nearly 100% of cells that were recovered using our approach contained the desired mutation, and in E. coli, 65% that were recovered contained the mutation, when the approach was used in combination with recombineering. We exhaustively analyze dual-RNA:Cas9 target requirements to define the range of targetable sequences and show strategies for editing sites that do not meet these requirements, suggesting the versatility of this technique for bacterial genome engineering."
"The hottest new field in biotech is synthetic biology: Scientists can now re-program life at the cellular level, just like a computer program. Syn-bio experts (also known as bio-hackers) are re-programming the DNA in viruses and creating novel life forms that can replicate and grow just like natural single cell organisms. Joining Robert Tercek in the discussion are Andrew Hessel, Distinguished Research Scientist with the Bio/Nano Programmable Matter Group at Autodesk, and Dr. William Hurlbut, Physician and Consulting Professor at Stanford University. Inventing the Future is a live news program featuring coming trends that will shape society. In today's world, success means knowing "What's Next After What's Next?" Lead by Robert Tercek, Inventing the Future offers insight into the future of the world after tomorrow..."
Müller K, Engesser R, Metzger S, Schulz S, Kämpf MM, Busacker M, Steinberg T, Tomakidi P, Ehrbar M, Nagy F, Timmer J, Zubriggen MD, Weber W "Growth and differentiation of multicellular systems is orchestrated by spatially restricted gene expression programs in specialized subpopulations. The targeted manipulation of such processes by synthetic tools with high-spatiotemporal resolution could, therefore, enable a deepened understanding of developmental processes and open new opportunities in tissue engineering. Here, we describe the first red/far-red light-triggered gene switch for mammalian cells for achieving gene expression control in time and space. We show that the system can reversibly be toggled between stable on- and off-states using short light pulses at 660 or 740 nm. Red light-induced gene expression was shown to correlate with the applied photon number and was compatible with different mammalian cell lines, including human primary cells. The light-induced expression kinetics were quantitatively analyzed by a mathematical model. We apply the system for the spatially controlled engineering of angiogenesis in chicken embryos. The system's performance combined with cell- and tissue-compatible regulating red light will enable unprecedented spatiotemporally controlled molecular interventions in mammalian cells, tissues and organisms."
Zürcher E, Tavor-Deslex D, Lituiev D, Enkeli K, Tarr PT, Müller B "Cytokinins are classic plant hormones that orchestrate plant growth, development, and physiology. They affect gene expression in target cells by activating a multistep phosphorelay network. Type-B response regulators, acting as transcriptional activators, mediate the final step in the signaling cascade. Previously, we have introduced a synthetic reporter, TCS (Two Component signaling Sensor)::GFP, which reflects the transcriptional activity of type-B response regulators. TCS::GFP was instrumental in uncovering novel roles of cytokinin, and deepening our undestanding of existing functions. However, TCS-mediated expression of reporters is weak in some developmental contexts, where cytokinin signaling has a documented role, such as in the shoot apical meristem, or in the vasculature of Arabidopsis thaliana. In addition, we observed that GFP expression becomes rapidly silenced in TCS::GFP transgenic plants. Here, we present an improved version of the reporter, TCS new (TCSn), which, compared to TCS, is more sensitive to phosphorelay signaling in Arabidopsis and maize cellular assays, while retaining its specificity. Transgenic Arabidopsis TCSn::GFP plants exhibit strong and dynamic GFP expression patterns consistent with known cytokinin functions. In addition, GFP expression has been stable over generations, allowing crosses with different genetic backgrounds. Thus, TCSn represents a significant improvement to report the transcriptional output profile of phosphorelay signaling networks in Arabidopsis, maize, and likely other plants that display common response regulator DNA-binding specificities."
By: Caroline Channing January 23, 2013 3:37 PM. The Manchester Institute of Biotechnology is hosting a joint Academia/Industry discussion meeting on the topic of "Future Perspectives in Synthetic Biology" on Friday 8th February 2013.
Synthetic Biology is a practical application of Systems Biology
*Video*: *Systems as the driver for synthetic biology*
"We now possess the ability to read and write DNA. These tools are not only revolutionizing biotechnology but also the basic life sciences as well. The challenge is that we are still learning the grammar. In other words, we often do not know which genetic perturbations to make in order to alter the behavior of an organism. As a result, synthetic biology still involves much trial and error. Moreover, we are still far from the point where we can engineer new organisms from scratch – rather, we need to alter the physiology of existing ones. Even then, we still need to understand how these organisms function in an integrated manner. In this talk, I will discuss the application systems biology to synthetic biology as a general strategy for overcoming many of these challenges. I will first review some of our previous work applying comparative genomics to inform biological design. I will then discuss our work applying systems biology approaches to improve organisms for fuel and chemical production. I will conclude by discussing our recent work developing tools for engineering non-model organisms with unique properties.
Dr. Christopher Rao is an associate professor of chemical and biomolecular engineering at the University of Illinois, Urbana-Champaign, a Robert W. Schaefer Professional Scholar, and a member of the Energy Biosciences Institute. He received his B.S. degree in Chemical Engineering from Carnegie Mellon University in 1994 and his Ph.D. degree in Chemical Engineering from the University of Wisconsin in 2000 under the guidance of Dr. James Rawlings. Prior to joining UIUC in 2005, he was a postdoctoral fellow at the Howard Hughes Medical Institute and University of California, Berkeley working under the guidance of Dr. Adam Arkin. Dr. Rao has authored and co-authored 58 research articles. In addition, he has given invited lectures in more than 55 international meetings and institutions. Dr. Rao has received numerous research and teaching awards and honors, including American Institute of Chemical Engineers (AIChE) Computing and Systems Technology Outstanding Young Research Award (2012), Petit Scholar from UIUC College of Liberal Arts and Sciences (2011), International Federation of Automatic Control (IFAC) High Impact Paper Award (2010), UIUC School of Chemical Sciences Excellence in Teaching Award (2008), American Institute of Chemical Engineers (AIChE) Computing and Systems Technology W. David Smith Award (2007), and National Science Foundation CAREER Award (2007). Dr. Rao is an editor for PLoS Computational Biology and PLoS One. The goal of his research is to understand how cells employ feedback control in decision-making processes. In addition, he develops experimental and computational tools for synthetic biology."
Jeffrey A. Dietrich, David L. Shis, Azadeh Alikhani, and Jay D. Keasling Continued advances in metabolic engineering are increasing the number of small molecules being targeted for microbial production. Pathway yields and productivities, however, are often suboptimal, and strain improvement remains a persistent challenge given that the majority of small molecules are difficult to screen for and their biosynthesis does not improve host fitness. In this work, we have developed a generalized approach to screen or select for improved small-molecule biosynthesis using transcription factor-based biosensors. Using a tetracycline resistance gene 3′ of a small-molecule inducible promoter, host antibiotic resistance, and hence growth rate, was coupled to either small-molecule concentration in the growth medium or a small-molecule production phenotype. Biosensors were constructed for two important chemical classes, dicarboxylic acids and alcohols, using transcription factor-promoter pairs derived from Pseudomonas putida, Thauera butanivorans, or E. coli. Transcription factors were selected for specific activation by either succinate, adipate, or 1-butanol, and we demonstrate product-dependent growth in E. coli using all three compounds. The 1-butanol biosensor was applied in a proof-of-principle liquid culture screen to optimize 1-butanol biosynthesis in engineered E. coli, identifying a pathway variant yielding a 35% increase in 1-butanol specific productivity through optimization of enzyme expression levels. Lastly, to demonstrate the capacity to select for enzymatic activity, the 1-butanol biosensor was applied as synthetic selection, coupling in vivo 1-butanol biosynthesis to E. coli fitness, and an 120-fold enrichment for a 1-butanol production phenotype was observed following a single round of positive selection."
by Anita Loeschcke, Annette Markert, Susanne Wilhelm, Astrid Wirtz, Frank Rosenau, Karl-Erich Jaeger, and Thomas Drepper
"Secondary metabolites represent a virtually inexhaustible source of natural molecules exhibiting a high potential as pharmaceuticals or chemical building blocks. To gain broad access to these compounds, sophisticated expression systems are needed that facilitate the transfer and expression of large chromosomal regions, whose genes encode complex metabolic pathways. Here, we report on the development of the novel system for the transfer and expression of biosynthetic pathways (TREX), which comprises all functional elements necessary for the delivery and concerted expression of clustered pathway genes in different bacteria. TREX employs (i) conjugation for DNA transfer, (ii) randomized transposition for its chromosomal insertion, and (iii) T7 RNA polymerase for unimpeded bidirectional gene expression. The applicability of the TREX system was demonstrated by establishing the biosynthetic pathways of two pigmented secondary metabolites, zeaxanthin and prodigiosin, in bacteria with different metabolic capacities. Thus, TREX represents a valuable tool for accessing natural products by allowing comparative expression studies with clustered genes."
by Shen-Long Tsai, Nancy A. DaSilva , and Wilfred Chen
"A new adaptive strategy was developed for the ex vivo assembly of a functional tetravalent designer cellulosome on the yeast cell surface. The design is based on the use of (1) a surface-bound anchoring scaffoldin composed of two divergent cohesin domains, (2) two dockerin-tagged adaptor scaffoldins to amplify the number of enzyme loading sites based on the specific dockerin–cohesin interaction with the anchoring scaffoldin, and (3) two dockerin-tagged enzymatic subunits (the endoglucanse Gt and the β-glucosidase Bglf) for cellulose hydrolysis. Cells displaying the tetravalent cellulosome on the surface exhibited a 4.2-fold enhancement in the hydrolysis of phosphoric acid swollen cellulose (PASC) compared with free enzymes. More importantly, cells displaying the tetravalent celluosome also exhibited an 2-fold increase in ethanol production compared with cells displaying a divalent cellulosome using a similar enzyme loading. These results clearly indicate the more crucial role of enzyme proximity than just simply increasing the enzyme loading on the overall cellulosomal synergy. To the best of our knowledge, this is the first report that exploits the natural adaptive assembly strategy in creating artificial cellulosome structures. The unique feature of the anchoring and the adaptor scaffoldin strategy to amplify the number of enzymatic subunits can be easily extended to more complex cellulosomal structures to achieve an even higher level of enzyme synergy."
"Scientists report that they have developed a method that cuts down the time it takes to make new ‘parts’ for microscopic biological factories from two days to only six hours.
The scientists, from Imperial College London, say their research brings them another step closer to a new kind of industrial revolution, where parts for these biological factories could be mass-produced. These factories have a wealth of applications including better drug delivery treatments for patients, enhancements in the way that minerals are mined from deep underground and advances in the production of biofuels.
Professor Paul Freemont, Co- Director of the Centre for Synthetic Biology and Innovation at Imperial College London and principle co-investigator of the study, which is published today in the journal Nucleic Acids Research, says:
“Before the industrial revolution most items were made by hand, which meant that they were slower to manufacture, more expensive to produce and limited in number. We are at a similar juncture in synthetic biology, having to test and build each part from scratch, which is a long and slow process. We demonstrate in our study a new method that could help to rapidly scale up the production and testing of biological parts.”
Parts made up of DNA are re-engineered by scientists and put into cells to make biological factories. However, a major bottleneck in synthetic biology is the lack of parts from which to build new types of factories. To build parts using the current time-consuming method, scientists have to re-engineer DNA in a cell and observe how it works. If it functions according to their specifications, then the scientists store the part specifications in a catalogue....."
Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology
by James Chappell, Kirsten Jensen and Paul S. Freemont
"A bottleneck in our capacity to rationally and predictably engineer biological systems is the limited number of well-characterized genetic elements from which to build. Current characterization methods are tied to measurements in living systems, the transformation and culturing of which are inherently time-consuming. In order to address this we have validated a completely in vitro approach for the characterization of DNA regulatory elements using E. coli extract cell-free systems. Importantly, we demonstrate that characterization in cell-free systems correlates and is reflective of performance in vivo for the most frequently used DNA regulatory elements. Moreover, we devise a rapid and completely in vitro method to generate DNA templates for cell-free systems, bypassing the need for DNA template generation and amplification from living cells. This in vitro approach is significantly quicker than current characterization methods and is amenable to high-throughput techniques, providing a valuable tool for rapidly prototyping libraries of DNA regulatory elements for synthetic biology."
"A challenge in biology is to understand how complex molecular networks in the cell execute sophisticated regulatory functions. Here we explore the idea that there are common and general principles that link network structures to biological functions, principles that constrain the design solutions that evolution can converge upon for accomplishing a given cellular task. We describe approaches for classifying networks based on abstract architectures and functions, rather than on the specific molecular components of the networks. For any common regulatory task, can we define the space of all possible molecular solutions? Such inverse approaches might ultimately allow the assembly of a design table of core molecular algorithms that could serve as a guide for building synthetic networks and modulating disease networks."
"Scientists in the field of tissue engineering are now applying the principles of cell biology, material science and biomedical engineering to create biological substitutes that will restore and maintain normal function in diseased and injured tissues/organs [1-3]. Tissue-engineered scaffolds should (i) facilitate the localization and delivery of tissue-specific cells to precise sites in the body, (ii) maintain a three-dimensional architecture that permits the formation of new tissues, and (iii) guide the development of new tissues with appropriate function. ..."
Steve Jurvetson, a leading Silicon Valley venture capitalists: "we’ll see remarkable progress in…synthetic biology"
*What does the future of innovation look like*? *Thought leaders at the intersection give us a glimpse*
"Writing and data science both involve telling stories and there are many stories to be told in Silicon Valley. In these articles, I attempt to convey a human portrait of the tech world by offering a personal account of some of the events that I attend. Recently, I went to The Intersection, a conference on innovation and social change.
MOUNTAIN VIEW, Calif. – “I want you to go up to that lady in red and ask her if she knows what a maglev train is.” Josiah, an outgoing and precocious 18-year old, ...
Steve Jurvetson, one of Silicon Valley’s leading venture capitalists, then had a session on the science of innovation. Citing typical Kurzweilian arguments about semi-conductors, he argues that the pace of innovation was expanding exponentially. “In the coming years, we’ll see remarkable progress in artificial intelligence, synthetic biology, space exploration, and 3D-printing,” he predicts."
"Genomics, the topic of this year's lecture series at the University of Arizona's College of Science, is not an inherently controversial topic.
It is the realm of scientists who sequence and assemble the entire set of DNA contained in each cell of an organism. Genomics provides a road map for researchers in a variety of endeavors - biology, evolution, immunology, pharmacology, medicine, agriculture and more. It has the potential to unlock the mysteries of our ancestry, feed the world, improve health, cure disease and lengthen productive lives. In the public realm it also raises fears about the misuse of medical information, the danger of synthetic biology and concerns about our food supply, expressed in terms like "super weeds" and "Frankenfoods." That is partly the reason for the lecture series, said Dr. Fernando Martinez, the pediatrician and asthma researcher who heads the UA's Bio5 Research Institute and will give the initial lecture Wednesday. "The time has come for society to understand the genome," said Martinez. He predicts that within the next 10 years, a personalized genome map will be made available to the parents of each child born in the United States....."
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