Biophysical Society TV talks to Pamela Silver, PhD, the Chair & Speaker of the Synthetic Biology Symposium about why it was important to feature this topic a...
The combinatorial nature of many important mathematical problems, including nondeterministic-polynomial-time (NP)-complete problems, places a severe limitation on the problem size that can be solved with conventional, sequentially operating electronic computers. There have been significant efforts in conceiving parallel-computation approaches in the past, for example: DNA computation, quantum computation, and microfluidics-based computation. However, these approaches have not proven, so far, to be scalable and practical from a fabrication and operational perspective. Here, we report the foundations of an alternative parallel-computation system in which a given combinatorial problem is encoded into a graphical, modular network that is embedded in a nanofabricated planar device. Exploring the network in a parallel fashion using a large number of independent, molecular-motor-propelled agents then solves the mathematical problem. This approach uses orders of magnitude less energy than conventional computers, thus addressing issues related to power consumption and heat dissipation. We provide a proof-of-concept demonstration of such a device by solving, in a parallel fashion, the small instance {2, 5, 9} of the subset sum problem, which is a benchmark NP-complete problem. Finally, we discuss the technical advances necessary to make our system scalable with presently available technology.
Genetic circuits, composed of complex networks of interacting molecular machines, enable living systems to sense their dynamic environments, perform computation on the inputs and formulate appropriate outputs. By rewiring and expanding these circuits with novel parts and modules, synthetic biologists have adapted living systems into vibrant substrates for engineering. Diverse paradigms have emerged for designing, modeling, constructing, and characterizing such artificial genetic systems. In this paper, we first provide an overview of recent advances in the development of genetic parts and highlight key engineering approaches. We then review the assembly of these parts into synthetic circuits from the perspectives of digital & analog logic, systems biology, and metabolic engineering, three areas of particular theoretical and practical interest. Finally, we discuss notable challenges that the field of synthetic biology still faces in achieving reliable and predictable forward-engineering of artificial biological circuits.
Mathematical modeling of biochemical pathways is an important resource in Synthetic Biology, as the predictive power of simulating synthetic pathways represents an important step in the design of synthetic metabolons. In this paper, we are concerned with the mathematical modeling, simulation, and optimization of metabolic processes in biochemical microreactors able to carry out enzymatic reactions and to exchange metabolites with their surrounding medium. The results of the reported modeling approach are incorporated in the design of the first microreactor prototypes that are under construction. These microreactors consist of compartments separated by membranes carrying specific transporters for the input of substrates and export of products. Inside the compartments of the reactor multienzyme complexes assembled on nano-beads by peptide adapters are used to carry out metabolic reactions. The spatially resolved mathematical model describing the ongoing processes consists of a system of diffusion equations together with boundary and initial conditions. The boundary conditions model the exchange of metabolites with the neighboring compartments and the reactions at the surface of the nano-beads carrying the multienzyme complexes. Efficient and accurate approaches for numerical simulation of the mathematical model and for optimal design of the microreactor are developed. As a proof-of-concept scenario, a synthetic pathway for the conversion of sucrose to glucose-6-phosphate (G6P) was chosen. In this context, the mathematical model is employed to compute the spatio-temporal distributions of the metabolite concentrations, as well as application relevant quantities like the outflow rate of G6P. These computations are performed for different scenarios, where the number of beads as well as their loading capacity are varied. The computed metabolite distributions show spatial patterns, which differ for different experimental arrangements. Furthermore, the total output of G6P increases for scenarios where microcompartimentation of enzymes occurs. These results show that spatially resolved models are needed in the description of the conversion processes. Finally, the enzyme stoichiometry on the nano-beads is determined, which maximizes the production of glucose-6-phosphate.
Genetic invasions threaten nearly all cells and genomes. Bacteria and archaea “remember” encounters with invaders through an adaptive immunity strategy involving clustered, regularly interspaced, short palindromic repeats (CRISPRs). These repeats can store snippets of an invader's genome as “spacers” (1), which then constitute a heritable memory that can instruct an immune response against future encounters with foreign DNAs carrying those same sequences. The ability to form new memories of invasion depends on the ability of a CRISPR to incorporate new spacers. The mechanistic basis for this acquisition from DNA is increasingly understood (2) and has often been assumed to be the sole mode of CRISPR adaptation. On page 932 of this issue, Silas et al. (3) describe an intriguing twist on bacterial adaptive immunity, identifying a subset of CRISPR systems with the remarkable ability to incorporate new spacers directly from RNA.
When you hear “gene editing,” do you think cancer cures? Designer babies? What about new fuel sources or drought-tolerant crops? Gene editing with a technique known as CRISPR/Cas9, hailed as a scientific breakthrough, could mean all those things.
There’s a possibility, of course, that CRISPR/Cas9 may not live up to expectations. That’s happened before. Twenty-five years ago, recombinant DNA was supposed to transform society in a way that, well, didn’t happen. But any past biotech disappointments are not stopping a slew of Bay Area CRISPR startups from taking the leap into the new world of DNA tinkering and synthetic biology. They are, in fact, everywhere, from biotech labs in Emeryville to kitchen counters in Burlingame.
“It’s something that people read about, it’s something people see that’s cutting edge,” says Josiah Zayner, a former synthetic biologist at NASA. “Yet it’s so accessible, it’s something that you can do in your home, on your kitchen table.”
Zayner has considered himself a “biohacker” for a long time. Now it’s his full-time job.
Nitrogen fixation has been established in protokaryotic model Escherichia coli by transferring a minimal nif gene cluster composed of 9 genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV) from Paenibacillus sp. WLY78. However, the nitrogenase activity in the recombinant E. coli 78-7 is only 10 % of that observed in wild-type Paenibacillus. Thus, it is necessary to increase nitrogenase activity through synthetic biology. RESULTS: In order to increase nitrogenase activity in heterologous host, a total of 28 selected genes from Paenibacillus sp. WLY78 and Klebsiella oxytoca were placed under the control of Paenibacillus nif promoter in two different vectors and then they are separately or combinationally transferred to the recombinant E. coli 78-7. Our results demonstrate that Paenibacillus suf operon (Fe-S cluster assembly) and the potential electron transport genes pfoAB, fldA and fer can increase nitrogenase activity. Also, K. oxytoca nifSU (Fe-S cluster assembly) and nifFJ (electron transport specific for nitrogenase) can increase nitrogenase activity. Especially, the combined assembly of the potential Paenibacillus electron transporter genes (pfoABfldA) with K. oxytoca nifSU recovers 50.1 % of wild-type (Paenibacillus) activity. However, K. oxytoca nifWZM and nifQ can not increase activity. CONCLUSION: The combined assembly of the potential Paenibacillus electron transporter genes (pfoABfldA) with K. oxytoca nifSU recovers 50.1 % of wild-type (Paenibacillus) activity in the recombinant E. coli 78-7. Our results will provide valuable insights for the enhancement of nitrogenase activity in heterogeneous host and will provide guidance for engineering cereal plants with minimal nif genes.
An exciting new scientific frontier—synthetic biology—took center stage as a celebrated scientist from the Massachusetts Institute of Technology (MIT) recently spoke at the headquarters of the Office of Naval Research ...
Morphology and function of the nervous system is maintained via well-coordinated processes both in central and peripheral nervous tissues, which govern the homeostasis of organs/tissues. Impairments of the nervous system induce neuronal disorders such as peripheral neuropathy or cardiac arrhythmia. Although further investigation is warranted to reveal the molecular mechanisms of progression in such diseases, appropriate model systems mimicking the patient-specific communication between neurons and organs are not established yet. In this study, we reconstructed the neuronal network in vitro either between neurons of the human induced pluripotent stem (iPS) cell derived peripheral nervous system (PNS) and central nervous system (CNS), or between PNS neurons and cardiac cells in a morphologically and functionally compartmentalized manner. Networks were constructed in photolithographically microfabricated devices with two culture compartments connected by 20 microtunnels. We confirmed that PNS and CNS neurons connected via synapses and formed a network. Additionally, calcium-imaging experiments showed that the bundles originating from the PNS neurons were functionally active and responded reproducibly to external stimuli. Next, we confirmed that CNS neurons showed an increase in calcium activity during electrical stimulation of networked bundles from PNS neurons in order to demonstrate the formation of functional cell-cell interactions. We also confirmed the formation of synapses between PNS neurons and mature cardiac cells. These results indicate that compartmentalized culture devices are promising tools for reconstructing network-wide connections between PNS neurons and various organs, and might help to understand patient-specific molecular and functional mechanisms under normal and pathological conditions.
The CRISPR-associated (Cas) protein Cas9 is a molecular scissor for cutting DNA. The first step in the cutting reaction is the RNA-guided unwinding of the DNA double helix. Jiang et al. determined the structures of Cas9 bound to DNA unwound by the targeting RNA (see the Perspective by Chen and Bailey). Cas9 bends the DNA to allow guide RNA infiltration into the double helix. The two separated DNA strands, one bound to RNA, are subsequently positioned in the dual active sites of the protein for cutting.
Science, this issue p. 867; see also p. 811 Abstract
Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)–associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding to form an RNA-DNA hybrid and a displaced DNA strand inside the protein. The role of this R-loop structure in positioning each DNA strand for cleavage by the two Cas9 nuclease domains is unknown. We determine molecular structures of the catalytically active Streptococcus pyogenes Cas9 R-loop that show the displaced DNA strand located near the RuvC nuclease domain active site. These protein-DNA interactions, in turn, position the HNH nuclease domain adjacent to the target DNA strand cleavage site in a conformation essential for concerted DNA cutting. Cas9 bends the DNA helix by 30°, providing the structural distortion needed for R-loop formation.
Can scientists seriously program genetic switches into microbes, to make them into “smart” drugs that adjust to real-time conditions in the body? A little spinout from MIT just got $40 million to see how far it can go with the concept.
Cambridge, Mass.-based Synlogic is announcing today it has raised $40 million in a Series B venture financing. The deal was led by OrbiMed Healthcare Fund Management, and included Deerfield Management, Atlas Venture and New Enterprise Associates. The company has now raised about $70 million total, since it pulled in a Series A deal worth about $30 million in July 2013.
The financing comes just days after Synlogic struck a partnership with AbbVie. The big drugmaker wants to see if it can use the little company’s synthetic biology techniques to engineer live microbial drugs against inflammatory bowel diseases, specifically Crohn’s disease and ulcerative colitis.
The scientific idea, as I described last week here at Forbes, sounds more than a little wild and futuristic. Synlogic essentially wants to take a live bacterium, like a probiotic, and program in some specific genetic circuitry that no other drug on the market has today. With the genetic circuits carefully calibrated, the bacterium can sense the ever-changing conditions it encounters in the body, and adjust the drug dose based on what’s needed in real-time. Such a drug could hit the market by 2025, said CEO Jose-Carlos Gutierrez-Ramos. (For more on the science, subscribers can read “Why a Pfizer R&D Exec Joined Synlogic to Make Therapeutic Synthetic Life” in Timmerman Report.)
“Synlogic is at the intersection of two exciting areas of science–applying synthetic biology techniques to engineer microbes to act as therapeutics,” said Ed Mathers, a partner with NEA, in an email. “Best part here is that the microbes are harmless unless they encounter the ‘quote/unquote’ toxin at which point a switch turns them on to act! Simplistic, but elegant.”
The company plans to use the money to advance its lead drug candidates against phenylketonuria (PKU) and urea cycle disorders. Both of them are on schedule to enter their first human clinical trials in the first quarter of 2017, Gutierrez-Ramos said.
In another step for synthetic biology, genes designed in the lab and not seen in nature have been used by researchers to rescue bacterial cells from death
Source: PLOS iGEM Collection – Open For Submissions PLOS is pleased to announce that submissions from the iGEM 2015 Jamboree can now be made to the PLOS iGEM Collection.
Terminators, which signal the end of transcription processes, are typically placed behind the last coding sequence of an operon to prevent interference between transcript units in most biologically synthetic systems. Here, we seek to extend the usability of terminators in genetic system design by using terminators as regulatory genetic parts. Terminators with different impacts on their upstream and downstream genes are characterized in detail via dynamic modeling to predict the behavior of the overall genetic system. Some nonlinear effects of terminators observed in our terminator measurements potentially facilitate regulation of gene expression. Through dynamic modeling in silico, we find that such genetic systems may behave like genetic filters. In agreement with the simulations, we successfully implement genetic high-pass and bandpass filters in vivo, demonstrating the potential of using terminators as regulatory parts. The genetic bandpass filter in this work is implemented through the interdependence between genetic parts, in which the termination efficiency of a terminator varies with the strength of the upstream promoter. This design strategy for a bandpass filter requires fewer base pairs than the conventional strategy of concatenating high-pass and low-pass filters. Our results show that this novel utilization of terminators as regulatory parts may provide a new perspective for efficient design of genetic circuits. We believe that further exploration of the complicated dynamics of terminators is important in the development of synthetic biology.
Ruthie Weiss’s basketball team seemed to be minutes away from its fourth straight loss. But even as she stood on the sidelines for a brief rest, the nine-year-old had not given up. She convinced the coach to put her back in the game. Then, she charged out onto the court, caught a pass from a teammate and drove straight to the basket. Swish! Ruthie scored a quick two points, putting her team in the lead. As the game clock wound down, she scored again, clinching the victory. The team had earned its first win of the season, and celebrated as if it had just taken the national championship. A couple of parents from the opposing team even stopped by to congratulate Ruthie, who had scored all of her team’s 13 points: “Wow, she’s unbelievable!” they told her mum and dad.
Modern synthetic gene regulatory networks emerge from iterative design-build-test cycles that encompass the decisions and actions necessary to design, build, and test target genetic systems. Historically, such cycles have been performed manually, with limited formal problem-definition and progress-tracking. In recent years, researchers have devoted substantial effort to define and automate many sub-problems of these cycles and create systems for data management and documentation that result in useful tools for solving portions of certain workflows. However, biologists generally must still manually transfer information between tools, a process that frequently results in information loss. Furthermore, since each tool applies to a different workflow, tools often will not fit together in a closed-loop and, typically, additional outstanding sub-problems still require manual solutions. This thesis describes an attempt to create a tool that harnesses many smaller tools to automate a fully closed-loop decision-making process to design, build, and test synthetic biology networks and use the outcomes to inform redesigns. This tool, called Phoenix, inputs a performance-constrained signal-temporal-logic (STL) equation and an abstract genetic-element structural description to specify a design and then returns iterative sets of building and testing instructions. The user executes the instructions and returns the data to Phoenix, which then processes it and uses it to parameterize models for simulation of the behavior of compositional designs. A model-checking algorithm then evaluates these simulations, and returns to the user a new set of instructions for building and testing the next set of constructs. In cases where experimental results disagree with simulations, Phoenix uses grammars to determine where likely points of design failure might have occurred and instructs the building and testing of an intermediate composition to test where failures occurred. A design tree represents the design hierarchy displayed in the user interface where progress can be tracked and electronic datasheets generated to review results. Users can validate the computations performed by Phoenix by using them to create sets of classic and novel temporal synthetic genetic regulatory functions in E. coli.
From Sunday July 3rd to Saturday July 9th of this year, the University of Edinburgh, Imperial College London and the BBSRC are hosting a 5-day workshop (the Synthetic Genomes Conference + a 2-day international conference (the annual Sc2.0) in Edinburgh, UK. The course is suitable for people in research, industry, students, people in DIYBio and more.
The course will consist of a hands-on workshop taught by international experts covering recent breakthroughs in synthetic biology and synthetic genomes, including synthetic chromosome design and assembly, CRISPR-mediated genome engineering, SCRaMbLE-mediated synthetic genome rearrangement, and more. Furthermore, the 5th annual Sc2.0 conference includes major speakers in synthetic biology and yeast research, including Jef Boeke from NYU, Jim Collins from MIT and HMS, Pam Silver from HMS, Jasper Rine from UC Berkeley, and more.
The cost of the course is £420 and covers accommodation, meals, and attendance for both the workshop and the conference. There are also a limited amount of travel awards and bursaries available. For more information on registration details and about the conference, please visit the Synthetic Genome Summer course website. Also send further enquiries to b.blout@imperial.ac.uk.
Epigenetics has been investigated in cancer initiation, and development, especially, since the appearance of epigenomics. Epigenetics may be defined as the mechanisms that lead to heritable changes in gene function and without affecting the sequence of genome. These mechanisms explain how individuals with the same genotype produce phenotypic differences in response to environmental stimuli. Recently, with the accumulation of high-throughput next-generation sequencing (NGS) data, a key goal of systems biology is to construct networks for different cellular levels to explore whole cellular mechanisms. At present, there is no satisfactory method to construct an integrated genetic and epigenetic cellular network (IGECN), which combines NGS omics data with gene regulatory networks (GRNs), microRNAs (miRNAs) regulatory networks, protein-protein interaction networks (PPINs), and epigenetic regulatory networks of methylation using high-throughput NGS data. RESULTS: We investigated different kinds of NGS omics data to develop a systems biology method to construct an integrated cellular network based on three coupling models that describe genetic regulatory networks, protein-protein interaction networks, microRNA (miRNA) regulatory networks, and methylation regulation. The proposed method was applied to construct IGECNs of gastric cancer and the human immune response to human immunodeficiency virus (HIV) infection, to elucidate human defense response mechanisms. We successfully constructed an IGECN and validated it by using evidence from literature search. The integration of NGS omics data related to transcription regulation, protein-protein interactions, and miRNA and methylation regulation has more predictive power than independent datasets. We found that dysregulation of MIR7 contributes to the initiation and progression of inflammation-induced gastric cancer; dysregulation of MIR9 contributes to HIV-1 infection to hijack CD4+ T cells through dysfunction of the immune and hormone pathways; dysregulation of MIR139-5p, MIRLET7i, and MIR10a contributes to the HIV-1 integration/replication stage; dysregulation of MIR101, MIR141, and MIR152 contributes to the HIV-1 virus assembly and budding mechanisms; dysregulation of MIR302a contributes to not only microvesicle-mediated transfer of miRNAs but also dysfunction of NF-κB signaling pathway in hepatocarcinogenesis. CONCLUSION: The coupling dynamic systems of the whole IGECN can allow us to investigate genetic and epigenetic cellular mechanisms via omics data and big database mining, and are useful for further experiments in the field of systems and synthetic biology.
Engineered synthetic biological devices have been designed to perform a variety of functions from sensing molecules and bioremediation to energy production and biomedicine. Notwithstanding, a major limitation of in vivo circuit implementation is the constraint associated to the use of standard methodologies for circuit design. Thus, future success of these devices depends on obtaining circuits with scalable complexity and reusable parts. Here we show how to build complex computational devices using multicellular consortia and space as key computational elements. This spatial modular design grants scalability since its general architecture is independent of the circuit’s complexity, minimizes wiring requirements and allows component reusability with minimal genetic engineering. The potential use of this approach is demonstrated by implementation of complex logical functions with up to six inputs, thus demonstrating the scalability and flexibility of this method. The potential implications of our results are outlined.
While James R. Clapper, Director of National Intelligence, calls genome editing a “national security threat”, bioethicists warn of CRISPR-created superbabies, and prominent researchers argue whether patents trump papers, I prefer to quietly look at applications of the technology that aren’t dramatic enough to enter the endless news cycle, but elegantly reveal the power of the technology.
I found one such report in the current issue of Genome Research, about ectrodactyly (“absence of digits”), more colorfully known as split-hand/foot malformation. It affects about 1 in 18,000 newborns, and is also seen in toads, frogs, salamanders, mice, rabbits, cows, chickens, marmosets, dogs, cats, and manatees.
Over the past 3 years, the technique known as CRISPR—clustered regularly interspaced short palindromic repeats—has revolutionized the field of genome editing (1). A single enzyme called CRISPR-associated protein 9 (Cas9) can be programmed with CRISPR-derived RNAs (crRNAs) to introduce double-stranded DNA (dsDNA) breaks at specific sites in the genome. On page 867 of this issue, Jiang et al. (2) describe structures of Streptococcus pyogenes Cas9 captured in a state poised for DNA cleavage. This new snapshot, together with those from previous structures, explains how binding to a dsDNA target allosterically activates DNA cleavage by the Cas9 endonuclease.
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