We define a new inversion-based machine called a permuton of n genetic elements, which allows the n elements to be rearranged in any of the n·(n – 1)·(n – 2)···2 = n! distinct orderings. We present two design algorithms for architecting such a machine. We define a notion of a feasible design and use the framework to discuss the feasibility of the permuton architectures. We have implemented our design algorithms in a freely usable web-accessible software for exploration of these machines. Permutation machines could be used as memory elements or state machines and explicitly illustrate a rational approach to designing biological systems.
About the Cover: The cover artwork shows an artist's rendition of programmable biology: a brittle star of the species Gorgonocephalus arcticus occupies the central processor slot on a motherboard. Artwork by Ana Vásquez based on an illustration by naturalist Ernst Haeckel.
The ability to record molecular events in vivo would enable monitoring of signaling dynamics within cellular niches and critical factors that orchestrate cellular behavior. We present a self-contained analog memory device for longitudinal recording of molecular stimuli into DNA mutations in human cells. This device consists of a self-targeting guide RNA (stgRNA) that repeatedly directs Streptococcus pyogenes Cas9 nuclease activity toward the DNA that encodes the stgRNA, thereby enabling localized, continuous DNA mutagenesis as a function of stgRNA expression. We demonstrate programmable and multiplexed memory storage in human cells triggered by exogenous inducers or inflammation, both in vitro and in vivo. This tool, Mammalian Synthetic Cellular Recorder Integrating Biological Events (mSCRIBE), provides a unique strategy for investigating cell biology in vivo and enables continuous evolution of targeted DNA sequences.
Many of the most important potential applications of Synthetic Biology will require the ability to design and implement high performance feedback control systems that can accurately regulate the dynamics of multiple molecular species within the cell. Here, we argue that the use of design strategies based on combining ultrasensitive response dynamics with negative feedback represents a natural approach to this problem that fully exploits the strongly nonlinear nature of cellular information processing. We propose that such feedback mechanisms can explain the adaptive responses observed in one of the most widely studied biomolecular feedback systems—the yeast osmoregulatory response network. Based on our analysis of such system, we identify strong links with a well-known branch of mathematical systems theory from the field of Control Engineering, known as Sliding Mode Control. These insights allow us to develop design guidelines that can inform the construction of feedback controllers for synthetic biological systems.
Synthetic biologists report the most far-reaching rewiring yet of a bacterial genome. The feat, described today in Science, involved repurposing 3.8% of the base pairs of the bacterium Escherichia coli1.
The scientists replaced 7 of its 64 genetic codons — sequences that code for amino acids — with others that produce the same components. They were able to reduce the number of codons by synthesizing the DNA in 55 fragments, each of which was 50,000 base pairs long. They have yet to reassemble those pieces into a functioning E. coli.
Despite that, the team, led by researchers at Harvard Medical School in Boston, Massachusetts, say that it is a major step in the push to engineer organisms with new properties, such as resistance to infection by viruses. The synthetic biologists, including George Church at Harvard, reported their results on 18 August in Science1. They say the work also serves as a prototype for the Human Genome Project—Write, in which scientists aim to synthesize a human genome.
The biosynthetic microbe could wire future nanoelectronics after Navy-funded researchers supercharged its conductivity.
A microbe so common it’s found everywhere from the muddy bottom of the Potomac River to soil hundreds of meters into the earth could one day be wiring the military’s nanotechnology and sensing toxic chemicals from unmanned vehicles.
The bacteria, called Geobacter, thrive where organic life normally couldn’t in an “unprecedented” way, said Derek Lovley, a microbiology professor at the University of Massachusetts Amherst working with the U.S. Navy.
Here’s how it works. Rather than expelling electrons through oxygen-based respiration, Geobacter grow hair-like protein filaments that transfer electrons out of the cell onto surrounding iron minerals. Over the past year, Lovley and a team of researchers genetically modified those protein filaments to supercharge their conductivity, as part of an ongoing collaboration with the Office of Naval Research. The researchers tweaked two of the protein’s amino acids, halving their size and increasing their conductivity 2,000-fold, ONR officials announced Tuesday:
“Research like Dr. Lovley’s could lead to the development of new electronic materials to meet the increasing demand for smaller, more powerful computing devices,” said Linda Chrisey, a program officer in ONR’s Warfighter Performance Department, which sponsors the research. “Being able to produce extremely thin wires with sustainable materials has enormous potential application as components of electronic devices such as sensors, transistors and capacitors.”
The filaments conduct electricity the same way copper does, making them promising alternatives for wiring in the military’s future nanoelectronics. Although this first test comes nowhere near copper’s conductivity, the modified Geobacter pili already are as effective as man-made alternatives like carbon nanotubes, Lovley said. Better yet, they have none of the issues associated with manufacturing large quantities of the carbon nanotubes, which has proved difficult to scale up due to resource constraints and laborious purification processes.
“We usually grow [Geobacter] on acetate, or acetic acid, which is basically what’s in vinegar—those kind of cheap and renewable resources,” he said. “And it’s very stable for a protein … so for example, they’re stable in boiling water. Stable at a very basic or very high pH. For a protein for they’re remarkably robust and stable.”
The bacteria could pair with other synthetic biological innovations the military is pursuing, including transmitting electricity to other bioengineered microbes producing butanol as an alternative fuel for the military’s remote outposts.
Beyond wiring and transistor applications, Lovley said he envisions a future with “the wire itself being a sensor.” If there’s a chemical the military wants to detect without exposing troops to—say, one found in explosives or toxic pollutants—researchers could further modify Geobacter so the nanowire itself binds with that chemical whenever the two come into contact, Lovley said. The altered bacteria could then be added to a silicon chip on an unmanned vehicle.
Though Lovley and his team discovered Geobacter’s conductive nanowires a decade ago, it was only last year that they started brainstorming in earnest with colleagues in polymer science about modifying the bacteria’s properties.
“The idea that there was a microbe that would make a wire to conduct electricity out of the cell was pretty revolutionary, and there was a lot of controversy,” he said. “So we really spent like nine of those 10 years studying the biological role.”
ONR’s Rear Adm. Mat Winter said the Navy has been sponsoring the Geobacter research for many years among the three-to-four thousand grants it awards to academic partners annually.
“It’s important to keep that volume focused” on basic research, said Winter,, at the Center for Strategic and International Studies last month. That creates “solution space so that ideas that can be knitted together for capabilities to emerge.”
In Geobacter’s case, this has paid off. Before genetically engineering the microbe’s filaments, Lovley’s team had already discovered its ability to serve as microbial fuel cells. The Navy is exploring applications of that now, including sustainably powering sensors embedded on the ocean floor.
“We continue to focus on advanced materials in our laboratories and understanding how we can do microbial energy, where we’re taking the positive electrons that are made on the microbes on the seabed, and we’re capturing those, and we’re hooking up some red and black connectors, and we’re gathering the electricity,” Winter said at CSIS. “So we’re not there yet, but … machines at the nano-level are going to be an incredible game-changer.”
One of the goals of synthetic biology is to develop programmable artificial gene networks that can transduce multiple endogenous molecular cues to precisely control cell behavior. Realizing this vision requires interfacing natural molecular inputs with synthetic components that generate functional molecular outputs. Interfacing synthetic circuits with endogenous mammalian transcription factors has been particularly difficult. Here, we describe a systematic approach that enables integration and transduction of multiple mammalian transcription factor inputs by a synthetic network. The approach is facilitated by a proportional amplifier sensor based on synergistic positive autoregulation. The circuits efficiently transduce endogenous transcription factor levels into RNAi, transcriptional transactivation, and site-specific recombination. They also enable AND logic between pairs of arbitrary transcription factors. The results establish a framework for developing synthetic gene networks that interface with cellular processes through transcriptional regulators.
From individual cells deciding how to differentiate during development, to social insects intricately coordinating their actions when scavenging for food; the ability to perform complex computations and process information enables life. The Biocompute Lab explores biology from this perspective, focusing on the molecular-scale mechanisms that individual cells and groups of cells use to perform such tasks. We apply tools and methods from the field of synthetic biology to create new living systems from the ground-up. By studying these artificial systems using novel techniques we are developing that exploit next-generation sequencing, microfluidics and computational modelling, we aim to better understand the rules governing how biological parts are best pieced together to perform useful computations. Understanding the computational architecture of cells opens new ways of reprogramming cells to tackle problems spanning the sustainable production of materials to novel therapeutics. It also provides key insights into how biology controls the complex processes and structures that sustain life. The Biocompute Lab falls within BrisSynBio, a BBSRC/EPSRC Synthetic Biology Research Centre at the University of Bristol.
An MIT spinout, Synlogic, is aiming to create a new class of medicines, by re-programming bacteria found in the gut as “living therapeutics.”
Based on research by its co-founders, MIT professors Tim Lu and Jim Collins, Synlogic creates so-called synthetic biotics, which sense and correct metabolic abnormalities that underlie some major diseases and rare genetic disorders.
Human intestines are filled with trillions of bacteria, collectively called the microbiota, that carry out vital health functions. Synlogic’s synthetic biotics — capsules, liquid suspensions, or other dosage forms that can be taken regularly — augment the microbiota with new metabolic capabilities or complement lost functionality in organs such as the liver.
“Over the past decade or so, the intricate connections between microbes and our bodies have become clearer and clearer, and it’s well known now that the bacteria that live in our gut have a major influence on human health,” says Lu, an associate professor of electrical engineering and computer science and of biological engineering, and head of MIT’s Synthetic Biology Group, who serves as scientific advisor for Synlogic. “We leverage that interface as a way of treating human disease.”
Last month, Synlogic raised an additional $40 million in venture capital and secured its first industry partnership with pharmaceutical giant AbbVie. For the partnership, Synlogic will collaborate with AbbVie to develop synthetic biotics for the potential treatment of inflammatory bowel disease, which may include probiotic microbes programmed to detect intestinal inflammation, and produce anti-inflammatory molecules or break down pro-inflammatory effectors.
Two of Synlogic’s main candidate drugs, expected to enter clinical trials during the next 12 months, treat rare genetic metabolic disorders. One drug candidate is for treating urea cycle disorder (UCD), which is caused by an enzyme deficiency that leads to a buildup of toxic ammonia in the blood. The other is for treating phenylketonuria (PKU), which involves a dangerous excess of the amino acid phenylalanine due to a mutation in another metabolic enzyme. In both cases, Synlogic’s drugs process and flush out the toxic metabolites from the body.
Think of Synlogic’s drugs like biological thermostats, says Collins, the Termeer Professor of Medical Engineering and Science in MIT’s Department of Biological Engineering and Institute for Medical Engineering and Science, who also chairs Synlogic’s scientific advisory board. Instead of identifying and regulating the temperature of a room, he says, “The synthetic biotics detect and regulate the amount of an enzyme or metabolic byproduct in a patient’s body.”
Programming E. coli
For more than a decade at Boston University and MIT, Collins and Lu (who is Collins’ former student) have been developing “genetic circuits” for bacteria, which include on/off switches made with synthetic DNA or RNA sequences that instruct the bacteria to count, store memory, and even perform logic.
Collins and Lu have used these genetic circuits to program bacteria to seek and cure infection. In 2011, this approach earned Collins funding from the Bill and Melinda Gates Foundation to engineer bacteria to detect cholera and produce targeted antimicrobial peptides to treat it.
A few years ago, Lu and Collins, along with several venture capitalists, launched Synlogic to focus on commercializing “a new class of therapeutics based on living cells,” Collins says. In 2014, Synlogic came out of stealth mode, securing $30 million in funding from venture firms and the Gates Foundation.
Since then, Synlogic has worked primarily on programming E. coli Nissle, a strain of bacteria derived from the gut that is also used widely and safely as a probiotic. The programmed E. coli Nissle, Lu says, provides greater precision, safety, and efficacy for disease treatment, compared with traditional methods.
For inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis, for instance, current treatments include small-molecule drugs or antibodies with anti-inflammatory properties. But the challenge is getting the right dosage, Lu says. “If you apply too little, it’s not going to work. If you apply too much, you have a chance you may immunosuppress the patient and cause side effects,” he says.
Synlogic can “program microbes to detect inflammation and make anti-inflammation molecules at the site of inflammation, as well as produce molecules that positively impact immune system function,” Collins says.
Then there’s the more rare but debilitating urea cycle disorder, which affects 2,000 to 6,000 people in the United States and impairs their ability to processes ammonia. If ammonia builds up too much and reaches the brain, it can lead to brain damage, coma, and death. The best available treatment option for people with UCD today is a liver transplant.
Synlogic aims to treat UCD with a daily biotic that functions in a surprising way: “It can decrease the ammonia in the bloodstream, without even contacting the blood,” Lu says.
Ammonia levels in the bloodstream, he explains, are dependent on ammonia production in the large intestine. Synlogic’s biotic converts intestinal ammonia into an amino acid, which is flushed out of the body through the stool, thereby dramatically reducing the flow of ammonia to the blood and reducing ammonia levels in the bloodstream.
Synlogic’s biotic for PKU, which affects 13,000 people in the United States, functions in a similar way, to regulate the processing and extraction of phenylalanine. PKU patients must adhere to a lifelong, extremely low-protein diet that can result in serious developmental disorders, because they can’t eat normal foods that contain phenylalanine — including many meat, dairy, and seafood products. “If we can degrade phenylalanine with convenient administration of this probiotic, that will change the course of this disease,” Lu says.
Collins says Synlogic has potential to treat many other rare genetic metabolic disorders. But the recent AbbVie deal, he says, also “opens up possibilities of using these microbes to produce biologics or other small molecules to treat a range of conditions.” These include cardiovascular disease and autoimmune, oncology, and central nervous system disorders, which have been linked to metabolic dysregulation.
Reaching clinical efficacy
Part of the reason that probiotic treatments are not used for serious diseases is their lack of clinically validated efficacy. Synlogic, on the other hand, aims to overcome these efficacy issues with potent and precision-programmed synthetic biotics, Lu says.
For example, in engineering the safe and easily programmable E. coli Nissle, Synlogic engineers have designed the microbe to consume a massive amount of toxic metabolites. The E. coli Nissle strain that forms the basis of Synlogic’s UCD program, for instance, can consume orders of magnitude more ammonia than natural E. coli can, Lu says. “For this treatment to work for patients, you want the max performance you can squeeze out of any one of these biotics,” he says.
Based on their preclinical data, Synlogic’s treatments have the potential to reach “clinical levels” of efficacy not seen often in synthetic biology, says Collins: “Synlogic is programming these probiotic microbes to consume ammonia or phenylalanine for example, and they are reaching levels that are expected to be clinically meaningful, which is quite remarkable.”
RNA is involved in a wide-range of important molecular processes in the cell, serving diverse functions: regulatory, enzymatic, and structural. Together with its ease and predictability of design, these properties can lead RNA to become a useful handle for biological engineers with which to control the cellular machinery. By modifying the many RNA links in cellular processes, it is possible to reprogram cells toward specific design goals. We propose that RNA can be viewed as a molecular programming language that, together with protein-based execution platforms, can be used to rewrite wide ranging aspects of cellular function. In this review, we catalogue developments in the use of RNA parts, methods, and associated computational models that have contributed to the programmability of biology. We discuss how RNA part repertoires have been combined to build complex genetic circuits, and review recent applications of RNA-based parts and circuitry. We explore the future potential of RNA engineering and posit that RNA programmability is an important resource for firmly establishing an era of rationally designed synthetic biology.
Changing environments pose a challenge to living organisms. Cells need to gather and process incoming information, adapting to changes in predictable ways. This requires in particular the presence of memory, which allows different internal states to be stored. Biological memory can be stored by switches that retain information on past and present events. Synthetic biologists have implemented a number of memory devices for biological applications, mostly in single cells. It has been shown that the use of multicellular consortia provides interesting advantages to implement biological circuits. Here we show how to build a synthetic biological memory switch using an eukaryotic consortium. We engineered yeast cells that can communicate and retain memory of changes in the extracellular environment. These cells were able to produce and secrete a pheromone and sense a different pheromone following NOT logic. When the two strains were cocultured, they behaved as a double-negative-feedback motif with memory. In addition, we showed that memory can be effectively changed by the use of external inputs. Further optimization of these modules and addition of other cells could lead to new multicellular circuits that exhibit memory over a broad range of biological inputs.
Life on Earth is incredibly diverse. Yet, underneath that diversity, there are a number of constants and highly conserved processes: all life is based on DNA and RNA; the genetic code is universal; biology is limited to a small subset of potential chemistries. A vast amount of knowledge has been accrued through describing and characterizing enzymes, biological processes and organisms. Nevertheless, much remains to be understood about the natural world. One of the goals in Synthetic Biology is to recapitulate biological complexity from simple systems made from biological molecules–gaining a deeper understanding of life in the process. Directed evolution is a powerful tool in Synthetic Biology, able to bypass gaps in knowledge and capable of engineering even the most highly conserved biological processes. It encompasses a range of methodologies to create variation in a population and to select individual variants with the desired function–be it a ligand, enzyme, pathway or even whole organisms. Here, we present some of the basic frameworks that underpin all evolution platforms and review some of the recent contributions from directed evolution to synthetic biology, in particular methods that have been used to engineer the Central Dogma and the genetic code.
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