SynBioFromLeukipposInstitute

by

Tina Toni, Bruce Tidor

"Biological systems are inherently variable, with their dynamics influenced by intrinsic and extrinsic sources. These systems are often only partially characterized, with large uncertainties about specific sources of extrinsic variability and biochemical properties. Moreover, it is not yet well understood how different sources of variability combine and affect biological systems in concert. To successfully design biomedical therapies or synthetic circuits with robust performance, it is crucial to account for uncertainty and effects of variability. Here we introduce an efficient modeling and simulation framework to study systems that are simultaneously subject to multiple sources of variability, and apply it to make design decisions on small genetic networks that play a role of basic design elements of synthetic circuits. Specifically, the framework was used to explore the effect of transcriptional and post-transcriptional autoregulation on fluctuations in protein expression in simple genetic networks. We found that autoregulation could either suppress or increase the output variability, depending on specific noise sources and network parameters. We showed that transcriptional autoregulation was more successful than post-transcriptional in suppressing variability across a wide range of intrinsic and extrinsic magnitudes and sources. We derived the following design principles to guide the design of circuits that best suppress variability: (i) high protein cooperativity and low miRNA cooperativity, (ii) imperfect complementarity between miRNA and mRNA was preferred to perfect complementarity, and (iii) correlated expression of mRNA and miRNA – for example, on the same transcript – was best for suppression of protein variability. Results further showed that correlations in kinetic parameters between cells affected the ability to suppress variability, and that variability in transient states did not necessarily follow the same principles as variability in the steady state. Our model and findings provide a general framework to guide design principles in synthetic biology."

 http://bit.ly/10lPH9m

by Shamees Aden

"London designer and researcher Shamees Aden has developed a prototype for running shoes that would be 3D-printed from synthetic biological material and could repair themselves overnight.
Shamees Aden's Protocells trainer would be 3D-printed to the exact size of the user's foot from a material that would fit like a second skin. It would react to pressure and movement created when running, puffing up to provide extra cushioning where required. Aden developed the project in collaboration with Dr Martin Hanczyc, a professor at the University of Southern Denmark who specialises in protocell technology. Protocells are very basic molecules that are not themselves alive, but can be combined to create living organisms.
By mixing different types of these non-living molecules, scientists are attempting to produce artificial living systems that can be programmed with different behaviours, such as responsiveness to pressure, light and heat. "The cells have the capability to inflate and deflate and to respond to pressure," Aden told Dezeen at the Wearable Futures conference in London. "As you're running on different grounds and textures it's able to inflate or deflate depending on the pressure you put onto it and could help support you as a runner."
After a run, the protocells in the material would lose their energy and the shoes would be placed in a jar filled with protocell liquid, which would keep the living organisms healthy. The liquid could also be dyed any colour, causing the shoes to take on that colour as the cells rejuvenate. "You would take the trainers home and you would have to care for it as if it was a plant, making sure it has the natural resources needed to rejuvenate the cells," said the designer.
...._http://bit.ly/18mcNEg

by
Jared E. Toettcher, Orion D. Weinersend email, Wendell A. Lim

"Highlights

Optogenetic inputs can directly control the activity of the Ras/Erk moduleIndividual cells exhibit precise and reversible Ras/Erk dose-response behaviorRas efficiently transmits signals to Erk with dynamics ranging from minutes to hoursAn optogenetic/proteomic screen identifies dynamically gated Ras response modulesSummary The complex, interconnected architecture of cell-signaling networks makes it challenging to disentangle how cells process extracellular information to make decisions. We have developed an optogenetic approach to selectively activate isolated intracellular signaling nodes with light and use this method to follow the flow of information from the signaling protein Ras. By measuring dose and frequency responses in single cells, we characterize the precision, timing, and efficiency with which signals are transmitted from Ras to Erk. Moreover, we elucidate how a single pathway can specify distinct physiological outcomes: by combining distinct temporal patterns of stimulation with proteomic profiling, we identify signaling programs that differentially respond to Ras dynamics, including a paracrine circuit that activates STAT3 only after persistent (>1 hr) Ras activation. Optogenetic stimulation provides a powerful tool for analyzing the intrinsic transmission properties of pathway modules and identifying how they dynamically encode distinct outcomes...."

 http://bit.ly/19xvY9b

by
Sun ZZ, Yeung E, Hayes CA, Noireaux V, Murray RM.

"Accelerating the pace of synthetic biology experiments requires new approaches for rapid prototyping of circuits from individual DNA regulatory elements. However, current testing standards require days to weeks due to cloning and in vivo transformation. In this work, we first characterized methods to protect linear DNA strands from exonuclease degradation in an Escherichia coli based transcription-translation cell-free system (TX-TL), as well as mechanisms of degradation. This enabled the use of linear DNA PCR products in TX-TL. We then compared expression levels and binding dynamics of different promoters on linear DNA and plasmid DNA. We also demonstrated assembly technology to rapidly build circuits entirely in vitro from separate parts. Using this strategy, we prototyped a four component genetic switch in under 8 h entirely in vitro. Rapid in vitro assembly has future applications for prototyping multiple component circuits if combined with predictive computational models."

 http://bit.ly/1bOWl07

by
Richard A. Stein

"“Science is more than a body of knowledge, it’s a way of thinking,” remarked Carl Sagan, and probably his words were never more powerfully relevant than for portraying one of the newest biomedical felds, systems biology.

A recent symposium inaugurating the depart- ment of systems biology at Columbia Univer- sity Medical Center comes at a very auspi- cious time, one in which biomedical sciences, chemistry, physics, engineering, bioinformat- ics, and computer sciences are converging to shape a vibrant new discipline.
As Lee Goldman, M.D., M.P.H., execu- tive vice president for health and biomedical sciences at the Columbia University College of Physicians and Surgeons pointed out dur- ing the opening remarks, this new feld “rep- resents so much about our future.”
Within a relatively short time, we have re- alized the possibility of sequencing and map- ping the genome of virtually any organism. Concomitantly, as increasingly sophisticated technologies allow whole-genome sequences to be completed within hours to days, navigat- ing the vast datasets has become the foremost challenge, opening a gap in our ability to un- derstand and interpret their significance....."

 http://bit.ly/18dDwmj

by
Paula Tuulia Jouhten

"Cell factories are commonly microbial organisms utilized for bioconversion of renewable resources to bulk or high value chemicals. Introduction of novel production pathways in chassis strains is the core of the development of cell factories by synthetic biology. Synthetic biology aims to create novel biological functions and systems not found in nature by combining biology with engineering. The workflow of the development of novel cell factories with synthetic biology is ideally linear which will be attainable with the quantitative engineering approach, high-quality predictive models, and libraries of well-characterized parts. Different types of metabolic models, mathematical representations of metabolism and its components, enzymes and metabolites, are useful in particular phases of the synthetic biology workflow. In this minireview, the role of metabolic modelling in synthetic biology will be discussed with a review of current status of compatible methods and models for the in silico design and quantitative evaluation of a cell factory."

http://bit.ly/1gaBRk7

by
Yunzi Luo,Hua Huang,Jing Liang,Meng Wang,Lu Lu,Zengyi Shao,Ryan E. Cobb& Huimin Zhao

"Polycyclic tetramate macrolactams (PTMs) are a widely distributed class of natural products with important biological activities. However, many of these PTMs have not been characterized. Here we apply a plug-and-play synthetic biology strategy to activate a cryptic PTM biosynthetic gene cluster SGR810-815 from Streptomyces griseus and discover three new PTMs. This gene cluster is highly conserved in phylogenetically diverse bacterial strains and contains an unusual hybrid polyketide synthase-nonribosomal peptide synthetase, which resembles iterative polyketide synthases known in fungi. To further characterize this gene cluster, we use the same synthetic biology approach to create a series of gene deletion constructs and elucidate the biosynthetic steps for the formation of the polycyclic system. The strategy we employ bypasses the traditional laborious processes to elicit gene cluster expression and should be generally applicable to many other silent or cryptic gene clusters for discovery and characterization of new natural products.

 http://bit.ly/IS5mcQ

comment:

*New method of DNA editing allows synthetic biologists to unlock secrets of a bacterial genome*

by
Anonymous

"A group of University of Illinois researchers, led by Centennial Chair Professor of the Department of Chemical and Biomolecular Engineering Huimin Zhao, has demonstrated the use of an innovative DNA engineering technique to discover potentially valuable functions hidden within bacterial genomes. Their work was reported in a Nature Communications article on December 5, 2013.

The genome of every bacterial species contains genes that can synthesize a diverse arsenal of compounds. These include natural antibiotics, antifungals, and other biochemicals that help the bacteria fight off unfriendly fellow microbes; such compounds are of potentially great medical importance. The genes encoding the enzymes a bacterium needs to create these compounds are often arranged in clusters. Each gene corresponds to one of a set of proteins that work together in a biochemical pathway to create one or a few products.If a colony of bacteria is producing a biologically active compound, sometimes referred to as a natural product, scientists can isolate it, study its structure and function, and discover its potential uses. Many natural products have already been discovered by screening the compounds produced by different bacterial and other microbial species.The compounds discovered so far, however, represent a small fraction of those that bacteria are capable of producing.Bacteria are masters at survival; their genomes represent a set of contingency plans for a wide array of environmental situations. Like a painter laying out a palette with only the colors needed that day, a bacterium will only express the genes and synthesize the compounds that will help it thrive in its current setting. Constant expression of the gene clusters that aren't useful in a given situation would be energetically wasteful.This conservation of energy is good for bacteria, but bad for researchers hoping to discover new natural products. This was the challenge that Zhao and colleagues hoped to address when they began their project. "Sequence analysis of bacterial genomes indicates that there are many cryptic or silent pathways that have not been discovered," Zhao said. " . . .they need the right signal to turn on expression of the whole gene cluster."Several strategies have been employed to trick cells into activating their little-used, "cryptic" gene clusters, such as culturing bacteria in a variety of harsh conditions or inserting sets of genes from one species of bacteria into the genome of another species. These techniques involve labor-intensive trial and error, with no guarantee of success.Zhao's group, rather than attempting to manipulate the environment, focused on reprogramming the control of gene expression within the cell. They used a genetic engineering method previously developed by Zhao's laboratory, called DNA assembler, to insert small sections of DNA between each gene in a cryptic gene cluster. The sections of DNA added were promoters, specialized regions that help control when and how much nearby genes are expressed. By adding the right promoters, Zhao and colleagues forced the cell to increase expression of every gene in the cluster.What makes Zhao's strategy possible is the ability of the DNA assembler method to join many different fragments of DNA in a single step. Previous methods for DNA editing limited researchers to making a series of sequential changes; the number of experimental steps required to add a promoter to each gene in even a small cluster would have been prohibitive. In contrast, Zhao said, "we can actually build the whole cluster, so that gives us ultimate flexibility, because we can add different promoters," ensuring that every gene within the cluster is consistently activated.For the study published in Nature Communications, Zhao and his coauthors modified a cryptic cluster of six genes from Streptomyces griseus, a species of soil bacterium. They added a promoter before each gene in the cluster to increase expression, and inserted the cluster into a related bacterial species, Streptomyces lividans, that is easier to grow in a laboratory setting.The resulting bacterial strain expressed all the genes in the previously silent cluster, and produced several previously unknown compounds. These compounds belonged to a class of natural products called polycyclic tetramate macrolactams or PTMs, many of which have useful biomedical applications. By examining the compounds produced by strains missing one of the six genes in the cluster, the researchers were able to discover the function of each gene's encoded protein, leading to a better understanding of how bacteria synthesize PTMs.Zhao sees the work as an important step toward a larger goal: to create a generalized, automated high-throughput method to reconstruct any biochemical pathway in a target experimental organism. Zhao is the leader of the recently formed Biosystems Design Research Theme at the Institute for Genomic Biology, University of Illinois, and development of this type of method is a major goal of the Theme."We want the technology platform established, then we can actually work on mammalian systems, on plant systems, on microorganisms," said Zhao. Yet his ultimate motivation is the discovery of potentially useful biochemicals: "It's very likely some of the compounds will turn into new drugs, and that's very exciting."A group of University of Illinois researchers, led by Centennial Chair Professor of the Department of Chemical and Biomolecular Engineering Huimin Zhao, has demonstrated the use of an innovative DNA engineering technique to discover potentially valuable functions hidden within bacterial genomes. Their work was reported in a Nature Communications article on December 5, 2013.The genome of every bacterial species contains genes that can synthesize a diverse arsenal of compounds. These include natural antibiotics, antifungals, and other biochemicals that help the bacteria fight off unfriendly fellow microbes; such compounds are of potentially great medical importance. The genes encoding the enzymes a bacterium needs to create these compounds are often arranged in clusters. Each gene corresponds to one of a set of proteins that work together in a biochemical pathway to create one or a few products.If a colony of bacteria is producing a biologically active compound, sometimes referred to as a natural product, scientists can isolate it, study its structure and function, and discover its potential uses. Many natural products have already been discovered by screening the compounds produced by different bacterial and other microbial species.The compounds discovered so far, however, represent a small fraction of those that bacteria are capable of producing.Bacteria are masters at survival; their genomes represent a set of contingency plans for a wide array of environmental situations. Like a painter laying out a palette with only the colors needed that day, a bacterium will only express the genes and synthesize the compounds that will help it thrive in its current setting. Constant expression of the gene clusters that aren't useful in a given situation would be energetically wasteful.This conservation of energy is good for bacteria, but bad for researchers hoping to discover new natural products. This was the challenge that Zhao and colleagues hoped to address when they began their project. "Sequence analysis of bacterial genomes indicates that there are many cryptic or silent pathways that have not been discovered," Zhao said. " . . .they need the right signal to turn on expression of the whole gene cluster."Several strategies have been employed to trick cells into activating their little-used, "cryptic" gene clusters, such as culturing bacteria in a variety of harsh conditions or inserting sets of genes from one species of bacteria into the genome of another species. These techniques involve labor-intensive trial and error, with no guarantee of success.Zhao's group, rather than attempting to manipulate the environment, focused on reprogramming the control of gene expression within the cell. They used a genetic engineering method previously developed by Zhao's laboratory, called DNA assembler, to insert small sections of DNA between each gene in a cryptic gene cluster. The sections of DNA added were promoters, specialized regions that help control when and how much nearby genes are expressed. By adding the right promoters, Zhao and colleagues forced the cell to increase expression of every gene in the cluster.What makes Zhao's strategy possible is the ability of the DNA assembler method to join many different fragments of DNA in a single step. Previous methods for DNA editing limited researchers to making a series of sequential changes; the number of experimental steps required to add a promoter to each gene in even a small cluster would have been prohibitive. In contrast, Zhao said, "we can actually build the whole cluster, so that gives us ultimate flexibility, because we can add different promoters," ensuring that every gene within the cluster is consistently activated.For the study published in Nature Communications, Zhao and his coauthors modified a cryptic cluster of six genes from Streptomyces griseus, a species of soil bacterium. They added a promoter before each gene in the cluster to increase expression, and inserted the cluster into a related bacterial species, Streptomyces lividans, that is easier to grow in a laboratory setting.The resulting bacterial strain expressed all the genes in the previously silent cluster, and produced several previously unknown compounds. These compounds belonged to a class of natural products called polycyclic tetramate macrolactams or PTMs, many of which have useful biomedical applications. By examining the compounds produced by strains missing one of the six genes in the cluster, the researchers were able to discover the function of each gene's encoded protein, leading to a better understanding of how bacteria synthesize PTMs.Zhao sees the work as an important step toward a larger goal: to create a generalized, automated high-throughput method to reconstruct any biochemical pathway in a target experimental organism. Zhao is the leader of the recently formed Biosystems Design Research Theme at the Institute for Genomic Biology, University of Illinois, and development of this type of method is a major goal of the Theme."We want the technology platform established, then we can actually work on mammalian systems, on plant systems, on microorganisms," said Zhao. Yet his ultimate motivation is the discovery of potentially useful biochemicals: "It's very likely some of the compounds will turn into new drugs, and that's very exciting.""

http://bit.ly/1aDluVn

by
Brynne C Stanton,Alec A K Nielsen,Alvin Tamsir,Kevin Clancy,Todd Peterson& Christopher A Voigt

"Genetic circuits perform computational operations based on interactions between freely diffusing molecules within a cell. When transcription factors are combined to build a circuit, unintended interactions can disrupt its function. Here, we apply 'part mining' to build a library of 73 TetR-family repressors gleaned from prokaryotic genomes. The operators of a subset were determined using an in vitro method, and this information was used to build synthetic promoters. The promoters and repressors were screened for cross-reactions. Of these, 16 were identified that both strongly repress their cognate promoter (5- to 207-fold) and exhibit minimal interactions with other promoters. Each repressor-promoter pair was converted to a NOT gate and characterized. Used as a set of 16 NOT/NOR gates, there are >1054 circuits that could be built by changing the pattern of input and output promoters. This represents a large set of compatible gates that can be used to construct user-defined circuits."

http://bit.ly/1aPORDL

"The cost and accuracy of genome sequencing have improved dramatically. George Church asks why so few people are opting to inspect their genome.

Readers of Nature, we can assume, are bright and insatiably curious. So why have so few obtained and interpreted their own genome sequence? We should avoid being judgementalof people who practise genomic modesty or who choose not to act on genome information, but we should also ask if we are providing adequate and equal access to education about the benefits and risks of genome information.For 7 years I led one of the teams registered to compete for the US$10-million Archon Genomics X Prize, and I was naturally disap- pointed by the abrupt cancellation of the competition in August. How- ever, the confusion surrounding the X Prize does provide an occasion to reflect on the problems and misunderstandings in genomics. The first is that genomics is seen as expensive. In fact, sequencing costs have plummeted — from $2.7 billion for the first human genome in 2003 down to $1,000 today. That’s not much more thanthe cost of a decent laptop, and much less than a car. However, people are reluctant to pay to have their genome sequenced — many feel that health care should be provided for free by insurance or the government and, indeed, this is our not-that- distant goal, as there are many in our community who would not benefit from genome information if it were not free. However, for those today who can afford a genome sequence, we would argue that, overall, the cost of sequencing is expected to be recovered over a lifetime through the avoid- ance of unnecessary diagnostics and therapeutics and time spent in waiting rooms and hospitals.Perhaps too many think that genomics isinaccurate. When it announced the cancella-tion, the X Prize Foundation claimed that “nocompany is sequencing whole genomes to theaccuracy the contest required”. Aside from thepre-judgemental weirdness, is this statement true? Haplotype phasing quality — a measure of accuracy — has improved from 350 kilobases in 2007 to 2,463 kilobases in 2013, and point errors have improved from 1 in 100,000 to 1 in 10 million — both well beyond the X Prize goals. Genetic analyses of tough tandem repeats are now common diagnostically...."
 http://bit.ly/H6SY7v ;

by
Koeppl H, Hafner M, Lu J.

"With recent improvements of protocols for the assembly of transcriptional parts, synthetic biological devices can now more reliably be assembled according to a given design. The standardization of parts open up the way for in silico design tools that improve the construct and optimize devices with respect to given formal design specifications. The simplest such optimization is the selection of kinetic parameters and protein abundances such that the specified design constraints are robustly satisfied. In this work we address the problem of determining parameter values that fulfill specifications expressed in terms of a functional on the trajectories of a dynamical model. We solve this inverse problem by linearizing the forward operator that maps parameter sets to specifications, and then inverting it locally. This approach has two advantages over brute-force random sampling. First, the linearization approach allows us to map back intervals instead of points and second, every obtained value in the parameter region is satisfying the specifications by construction. The method is general and can hence be incorporated in a pipeline for the rational forward design of arbitrary devices in synthetic biology."
 http://bit.ly/18slJ5K