We present a synthetic gene circuit for decoupling cell growth from metabolite production through autonomous regulation of enzymatic pathways by integrated modules that sense nutrient and substrate. The two-layer circuit allows Escherichia coli to selectively utilize target substrates in a mixed pool; channel metabolic resources to growth by delaying enzymatic conversion until nutrient depletion; and activate, terminate, and re-activate conversion upon substrate availability. We developed two versions of controller, both of which have glucose nutrient sensors but differ in their substrate-sensing modules. One controller is specific for hydroxycinnamic acid and the other for oleic acid. Our hydroxycinnamic acid controller lowered metabolic stress 2-fold and increased the growth rate 2-fold and productivity 5-fold, whereas our oleic acid controller lowered metabolic stress 2-fold and increased the growth rate 1.3-fold and productivity 2.4-fold. These results demonstrate the potential for engineering strategies that decouple growth and production to make bio-based production more economical and sustainable.
The molecular components of transcriptional regulation are modular. Transcription factors have domains for specific functions such as DNA binding, dimerization, and protein-protein interactions associated with transcriptional activation and repression. Similarly, promoters are modular. They consist of combinations of cis-acting elements that are the binding sites for transcription factors. It is this promoter architecture that largely determines the expression pattern of a gene. The modular nature of promoters is supported by the observation that many cis-acting elements retain their activities when they are taken out of their native promoter context and used as building blocks in synthetic promoters. We therefore have a large collection of cis-acting elements to use in building synthetic promoters and many minimal promoters upon which to build them. This review discusses what we have learned concerning how to use these building blocks to make synthetic promoters. It has become clear that we can increase the strength of a promoter by adding increasing numbers of cis-acting elements. However, it appears that there may be a sweet spot with regard to inducibility as promoters with increasing numbers of copies of an element often show increased background expression. Spacing between elements appears important because if elements are placed too close together activity is lost, presumably due to reduced transcription factor binding due to steric hindrance. In many cases, promoters that contain combinations of cis-acting elements show better expression characteristics than promoters that contain a single type of element. This may be because multiple transcription factor binding sites in the promoter places it at the end of multiple signal transduction pathways. Finally, some cis-acting elements form functional units with other elements and are inactive on their own. In such cases, the complete unit is required for function in a synthetic promoter. Taken together, we have learned much about how to construct synthetic promoters and this knowledge will be crucial in both designing promoters to drive transgenes and also as components of defined regulatory networks in synthetic biology.
Biology is the world’s greatest manufacturing platform, according to MIT spinout Ginkgo Bioworks. The synthetic-biology startup is re-engineering yeast to act as tiny organic “factories” that produce chemicals for the flavor, fragrance, and food industries, with aims of making products more quickly, cheaply, and efficiently than traditional methods. “We see biology as a transformative technology,” says Ginkgo co-founder Reshma Shetty PhD ’08, who co-invented the technology at MIT. “It is the most powerful and sophisticated manufacturing platform on the planet, able to self-assemble incredible structures at a scale that is far out of reach of the most cutting-edge human technology.” Similar to how beer is brewed — where yeast eats sugars and creates alcohol and flavors through fermentation — Ginkgo’s “hacked” yeast eats fatty acids and produces desired chemicals that recreate certain scents and flavors during fermentation. Those chemicals can then be extracted and used in a number of different products.
Steven Burgess steps down as managing editor at the PLOS SynBio Community. His parting review is typically excellent. Great piece on Oxitec’s sterile mosquitos, and the human challenge of testing them in Key West. NIH reconsiders its moratorium on human-animal chimera research. A PBS article on using CRISPR in grapes turns into a remarkably comprehensive overview of the opportunities and challenges in editing agricultural plants. Is NgAgo’s gene editing reproducible? Jury’s still out, but preliminary evidence isn’t good. DARPA launches an Engineered Living Materials initiative, with the goal of designing biomaterials that grow into the shape of whatever structure/component. Open for proposals through September! Interesting warning about over-hyping technology: a piece on embryonic stem cells and the challenges of treating disease with them. If you’re in New York and want to build stuff with bio, you can now take a crash course in making custom biomaterials, put on by New York’s Genspace DIYbio lab. If you find yourself in Cambridge, check out Cafe Synthetique, the local synbio salon. Sounds like a GeneMods sister organization! Cool profile in STAT of David Baker and co.’s plans to solve problems in the world (mostly diseases, in this piece) with computationally designed proteins. Highlights from the 2016 BioDesign Challenge (from last month, but good enough to post anyway). Industry news:
Engineered Cas9 with lower off-target effects goes commercial: high-fidelity Cas9 variant from Joung lab licensed to Editas. Total cost of CRISPR patent fight exceeds $20 million and counting. DOD gives Ginkgo $2 million to develop probiotic vaccine for traveler’s diarrhea. Books and Longreads
I just discovered BioCoder, a quarterly newsletter about synthetic biology from technology media company O’Reilly Media, and I’m really enjoying it. Carl Zimmer gets his genome sequenced and analyzed by some of the best researchers in the field. A Game of Genomes chronicles his adventure. A must-read. Ed Yong’s fantastic book about the microbes that precede, surround, live on, and comprise us, I Contain Multitudes, came out. I cannot recommend enough that you get your hands (or ears, it’s on Audible!) on this book. Now, onto the research papers!
Easily and quickly sensing the presence of small molecules is one of the rate-limiting challenges in metabolic engineering. Now, Savage Lab reports rapid construction of metabolite biosensors using domain insertion profiling. Baker Lab does it again, reporting rationally designed, two-component, 120 subunit, icosahedral protein complexes.. Electrochemical across membranes fundamentally underly pretty much all biological energy production. Fotiadis Lab has now engineered a photo-switchable, light-driven proton pump. Genetic circuits:
A Lu Lab collaboration programs yeast and builds a mini bioreactor to produce single doses of multiple biological therapeutics on command. CRISPR/Gene Editing:
A Joung Lab collaboration relaxes Staphylococcus aureus Cas9’s PAM specificity, expanding the sites this smaller programmable nuclease can target. It’s been a bumper crop month for making molecular recordings in the genomes of cells. Science published three papers (summarized here): Church Lab reported molecular recordings produced using the actual CRISPR parts of CRISPR (as opposed to Cas9); Lu Lab used recombinases to build state machines in living cells; and a collaboration between Shendure and Schier Labs used Cas9 to record the differentiation and trace the lineage of all the cells in a zebrafish. Then, Lu Lab developed a method to continuously record the presence of Cas9 in human cells using a self-targeting guide RNA. Metabolic Engineering
Jewett Lab takes a step toward easier natural product mining by expressing non-ribosomal peptide synthetases and producing small-molecule peptides in a cell-free lysate system. Building biology to understand it
A massively recoded, 57-codon E. coli? Not quite yet, but Church Lab computationally designs the genome, synthesizes the parts, and makes progress on assembling and testing them. Venter Institute edits and interrogates bacterial ribosome genes, on a synthetic bacterial genome in yeast. GenomeWeb has a summary if you don’t have time to read the whole paper. Review article in ACS SynBio argues that mammalian artificial chromosomes (MACs) are the way of the future.
Powered by a chemical reaction controlled by microfluidics, the 3D-printed 'octobot' has no electronics
By Leah Burrows, SEAS Communications
(CAMBRIDGE, Massachusetts) — A team of Harvard University researchers with expertise in 3D printing, mechanical engineering, and microfluidics has demonstrated the first autonomous, untethered, entirely soft robot. This small, 3D-printed robot — nicknamed the octobot — could pave the way for a new generation of completely soft, autonomous machines.
Eventbrite - ThinkSTEAM presents Synthetic Biology Workshop with Columbia University's iGEM Team - Saturday, October 1, 2016 at Lasker Biomedical Research Building, New York, NY. Find event and ticket information.
Algae (a term used to group many photosynthetic organisms into a rather heterologous mash-up) do not have a kind place in the public imagination. Take for example the following passage from Stephen King’s Pet Semetary:
“Dead fields under a November sky, scattered rose petals brown and turning up at the edges, empty pools scummed with algae, rot, decomposition, dust… “
Leaving aside their use in a horror novel as a way to set an image of decay, algae attract significant scientific attention, and are even considered cool (well, at least by some). But when I tell people I work on algal Synbio, one question that eventually comes up (or is silently implied) is why do I bother, when there are more prominent and better-developed systems? In this blog I will try to partly address this point of view, as well as provide a small perspective on the subject.
The practical benefits of algae for humans date back to the days of early photosynthesis, when they produced the oxygenic atmosphere we currently breathe. Moreover, algal forms were the ancestors of chloroplasts and land plants. Today, algae are found in almost every environment, in having various forms and ecological roles. But what is their place in synthetic biology?
In terms of algal synthetic biology and biotechnology, there are two main research domains: eukaryotic microalgae and cyanobacteria.
The model for studying eukaryotic microalgae is Chlamydomonas reinhardtii—although the use of more species is being explored. C. reinhardtii can be transformed both via nuclear and chloroplast transformation, each having different potentials (e.g. random insertion vs. homologous recombination, eukaryotic vs. prokaryotic translation and protein maturation, etc.)
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.”
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