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Engineering an allosteric transcription factor to respond to new ligands

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Noah D Taylor, Alexander S Garruss, Rocco Moretti, Sum Chan, Mark A Arbing, Duilio Cascio, Jameson K Rogers, Farren J Isaacs, Sriram Kosuri, David Baker, Stanley Fields, George M Church & Srivatsan Raman

"Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. We engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol and sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG). The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits."

http://bit.ly/1SfHZuq
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Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system

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Young Je Lee, Allison Hoynes-O'Connor, Matthew C. Leong and Tae Seok Moon

"A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA–asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions."

http://bit.ly/1Ks4iKJ
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Kernel Architecture of the Genetic Circuitry of the Arabidopsis Circadian System

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Mathias Foo, David E. Somers, Pan-Jun Kim

"A wide range of organisms features molecular machines, circadian clocks, which generate endogenous oscillations with ~24 h periodicity and thereby synchronize biological processes to diurnal environmental fluctuations. Recently, it has become clear that plants harbor more complex gene regulatory circuits within the core circadian clocks than other organisms, inspiring a fundamental question: are all these regulatory interactions between clock genes equally crucial for the establishment and maintenance of circadian rhythms? Our mechanistic simulation for Arabidopsis thaliana demonstrates that at least half of the total regulatory interactions must be present to express the circadian molecular profiles observed in wild-type plants. A set of those essential interactions is called herein a kernel of the circadian system. The kernel structure unbiasedly reveals four interlocked negative feedback loops contributing to circadian rhythms, and three feedback loops among them drive the autonomous oscillation itself. Strikingly, the kernel structure, as well as the whole clock circuitry, is overwhelmingly composed of inhibitory, rather than activating, interactions between genes. We found that this tendency underlies plant circadian molecular profiles which often exhibit sharply-shaped, cuspidate waveforms. Through the generation of these cuspidate profiles, inhibitory interactions may facilitate the global coordination of temporally-distant clock events that are markedly peaked at very specific times of day. Our systematic approach resulting in experimentally-testable predictions provides insights into a design principle of biological clockwork, with implications for synthetic biology."

http://bit.ly/1Q9C5VS
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DNA nanobots will target cancer cells in the first human trial using a terminally ill patient

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DANIEL KORN

"The very mention of “nanobots” can bring up a certain future paranoia in people—undetectable robots under my skin? Thanks, but no thanks. Professor Ido Bachelet of Israel’s Bar-Ilan University confirms that while tiny robots being injected into a human body to fight disease might sound like science fiction, it is in fact very real.

Cancer treatment as we know it is problematic because it targets a large area. Chemo and radiation therapies are like setting off a bomb—they destroy cancerous cells, but in the process also damage the healthy ones surrounding it. This is why these therapies are sometimes as harmful as the cancer itself. Thus, the dilemma with curing cancer is not in finding treatments that can wipe out the cancerous cells, but ones that can do so without creating a bevy of additional medical issues. As Bachelet himself notes in a TEDMED talk: “searching for a safer cancer drug is basically like searching for a gun that kills only bad people.”

This is where nanobots come in—rather than take out every cell in the area they’re distributed to, they’re able to recognize and interact with specific molecules. This means that new drugs don’t even need to be developed; instead, drugs that have already been proven to be effective for cancer treatment but too toxic for regular use can be used in conjunction with nanobots to control said toxicity."

http://bit.ly/1PxdJcL
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Synthetic biology: applying biological circuits beyond novel therapies

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Anton Dobrin,  Pratik Saxena and    Martin Fussenegger

"Synthetic biology, an engineering, circuit-driven approach to biology, has developed whole new classes of therapeutics. Unfortunately, these advances have thus far been undercapitalized upon by basic researchers. As discussed herein, using synthetic circuits, one can undertake exhaustive investigations of the endogenous circuitry found in nature, develop novel detectors and better temporally and spatially controlled inducers. One could detect changes in DNA, RNA, protein or even transient signaling events, in cell-based systems, in live mice, and in humans. Synthetic biology has also developed inducible systems that can be induced chemically, optically or using radio waves. This induction has been re-wired to lead to changes in gene expression, RNA stability and splicing, protein stability and splicing, and signaling via endogenous pathways. Beyond simple detectors and inducible systems, one can combine these modalities and develop novel signal integration circuits that can react to a very precise pre-programmed set of conditions or even to multiple sets of precise conditions. In this review, we highlight some tools that were developed in which these circuits were combined such that the detection of a particular event automatically triggered a specific output. Furthermore, using novel circuit-design strategies, circuits have been developed that can integrate multiple inputs together in Boolean logic gates composed of up to 6 inputs. We highlight the tools available and what has been developed thus far, and highlight how some clinical tools can be very useful in basic science. Most of the systems that are presented can be integrated together; and the possibilities far exceed the number of currently developed strategies."

http://rsc.li/1QUl5bS
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A review of SynBio 2015

The Gene Editing Tsunami

 by Steven Burgess 

* PLOS Synthetic Biology Community*

"When we decided to crowdsource a review of the year from the synthetic biology community we weren’t sure what the response would be – but it has been fantastic! So good in fact, that we have decided to split the result into several parts. In this installment we have picked out 10 scientific highlights, and this will be followed by pieces on business, ethics and the future. We hope you enjoy reading, so much happened this year there are some things you might have missed!

The Gene Editing Tsunami

“I put my head together with some of Oxford’s top synthetic biology PhD students and we all agreed that this was the year of CRISPR – gene drives, embryonic editing, immuno-safe pig organs and more.”
Max Jamilly, PhD student in synthetic biology, Oxford

“2015 was definitely the year of CRISPR. Hard to not note its importance now and for the future.”
Dr. Charles Ebikeme, scientist and writer

“The maturation of the CRISPR-Cas system based technologies excites me, because they have huge potential to fundamentally enhance our targeted genome editing toolbox.”
Dr. Gerd Moe-Behrens, Leukippos institute

Can you hear that deafening roar approaching? CRISPR,CRISPR,CRISPR! In 2015 there was no escaping the exponential rise of the genome editing technique known as CRISPR-Cas9, which allows precise editing of DNA, is quick, cheap and easy to use, and works in almost every species it has been tried in. You could barely open a webpage without seeing some click bait linking to the latest bright use of the technology, ranginf from CRISPR mediated epigenome editing to optogenetics. The propaganda appeared to work, and by far the greatest number of replies to our survey nominated CRISPR developments – you are obviously still excited… and so are we!!! There is a reason for this; the best quote I heard explaining it came during the GARNet/OpenPlant CRISPR workshop in September, when Prof. Holger Putcha aptly described the technology as a tsunami, up there with PCR in terms of its impact upon molecular biology. When major news outlets began to pick up the story, and you see tabloids in the UK talking about “crispr”, you know this technology is going to be important for everyone – not just scientists. We will return to specific examples of how gene editing has been making waves, but first we should calm down a little and reflect that other exciting science has being going on too.'

http://bit.ly/1NprNSV
The gene editing tsunami and the ten synbio highlights of 2015 crowdsourced from the synthetic biology community,
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Biosensors on demand

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Anonymous

"Biosensors are powerful tools in synthetic biology for engineering metabolic pathways or controlling synthetic and native genetic circuits in bacteria. Scientists have had difficulty developing a method to engineer “designer” biosensor proteins that can precisely sense and report the presence of specific molecules, which has so far limited the number and variety of biosensor designs able to precisely regulate cell metabolism, cell biology, and synthetic gene circuits.
But new research published in Nature Methods ("Engineering an allosteric transcription factor to respond to new ligands") by a team at Harvard’s Wyss Institute for Biologically Inspired Engineering and Harvard Medical School (HMS) has leveraged combination of computational protein design, in vitro synthesis and in vivo testing to establish a first-of-its-kind strategy for identifying custom-tailored biosensors.
"Our original motivation for developing customizable biosensors was to get a life or death feedback loop for metabolic engineering," said George Church, Ph.D., Wyss Institute Core Faculty member, Professor of Genetics at HMS, Professor of Health Sciences and Technology at Harvard and the Massachusetts Institute of Technology (MIT), and the senior author on the study. "This would essentially give us 'Darwinian evolution on steroids', where colonies of bacteria genetically programmed to output a desirable commodity molecule would rapidly become more efficient from generation to generation as only the best metabolic producers will be 'self-identified' for survival."
"This advance represents a powerful new way for us to access the chemical diversity of the biosphere by mining for new pathways to make useful molecules," said Srivatsan Raman, Ph.D., formerly a Postdoctoral Fellow at the Wyss Institute and HMS and currently Assistant Professor of Biochemistry at University of Wisconsin-Madison, who is the corresponding author on the study.
To develop the method, researchers chose as their test case a natural regulatory protein from E. coli called LacI. LacI is an allosteric transcription factor (aTF), which becomes active in response to sensing "inducer" metabolites or molecules in the bacterium’s environment, thereby triggering expression of a downstream gene. Using LacI, the team set out to develop a framework for re-engineering new biosensor variants that would respond to four inducer molecules (lactitol, sucralose, gentiobiose, and fucose) that cannot be metabolized by natural E. coli. Sucralose, for example, is a completely synthetic sugar molecule sold commercially as Splenda®.
To synthesize and identify the custom-made LacI variants for sensing these four new inducers, the team designed a novel workflow incorporating a combinatorial synthesis strategy that relies on computational protein design and the Wyss Institute’s custom DNA synthesis resources to build a variant library of potential new biosensor designs comprising hundreds of thousands of mutated LacI proteins.
Then, to identify the variants with the most specific responses to the four target molecules of interest, the team engineered groups of E. coli bacteria to express green fluorescent protein (GFP) when the desired molecule was detected, thereby making the bacteria fluoresce. Performing high-throughput in vivo screening of the sensor library in the engineered E. coli, the team identified the most effective variants by their high fluorescence, then filtered them out and genetically sequenced them to reveal the DNA profiles and design maps for transforming aTFs into custom-tailored sensors with high specificity.
The results are striking in that an optimized engineered aTF sensor can be identified for sensing any arbitrary molecule using this approach, opening new doors in synthetic biology by putting allosteric proteins in the control of genetic engineers.
"The LacI protein we chose to re-design into a custom biosensor is only one of thousands of different allosteric transcription factors that exist in nature," said Noah Taylor, a graduate researcher at the Wyss Institute who recently finished his Ph.D. in Biological and Biomedical Sciences at HMS, and the first author on the study. "The ability to engineer LacI using nothing more than sequence and structure information suggests we could find tens, hundreds, or even thousands of other biosensors that respond to different molecules."
Biosensors built using this approach provide feedback on how much of a certain metabolite is present inside a cell. Metabolically engineered bacteria can be outfitted with these custom aTFs, enabling them to monitor their own bioproduction of a desired chemical, pharmaceutical or biofuel. This allows sophisticated designs in which the lack of sufficient product could result in the death of an individual cell, eliminating it from the culture. In this way, powerful evolutionary methods can be harnessed for metabolic engineering.
Sensitive detection of metabolites within cells also presents a new paradigm for the way scientists can interrogate single cells. Until now, it has been very challenging to study the metabolic state of a single individual cell. But designer biosensors could be utilized as custom responders to metabolites of interest, giving insight into the metabolic states of live cells in close to real time.
"We are now utilizing the method to find biosensors for a variety of high-value targets, particularly those that can aid in protecting the environment," said Alexander Garruss, co-author on the study, who is a graduate researcher at the Wyss Institute and a Ph.D. candidate in Bioinformatics and Integrative Genomics at HMS.
Beyond measuring metabolites within cells, the combinatorial synthesis approach paves a path forward toward designing countless new and highly specific biological sensors for novel applications such as environmental monitoring, medical diagnostics, bioremediation, and precision gene therapies.
"The team’s ability to engineer custom biosensors for virtually any molecule is another triumph showing the power of synthetic biology, and its ability to generate valuable new tools to advance medicine and protect our environment," said Wyss Institute Founding Director Donald Ingber, M.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and the Vascular Biology Program at Boston Children’s Hospital and Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences."
http://bit.ly/1PCiEFw


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Orientational nanoparticle assemblies and biosensors

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Wei Ma, Liguang Xu, Libing Wang, , Hua Kuang, Chuanlai Xu, 

"Assemblies of nanoparticles (NPs) have regional correlated properties with new features compared to individual NPs or random aggregates. The orientational NP assembly contributes greatly to the collective interaction of individual NPs with geometrical dependence. Therefore, orientational NPs assembly techniques have emerged as promising tools for controlling inorganic NPs spatial structures with enhanced interesting properties. The research fields of orientational NP assembly have developed rapidly with characteristics related to the different methods used, including chemical, physical and biological techniques. The current and potential applications, important challenges remain to be investigated. An overview of recent developments in orientational NPs assemblies, the multiple strategies, biosensors and challenges will be discussed in this review."

http://bit.ly/1KouYw3
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Towards enabling engineered microbial-electronic systems: RK2-based conjugal transfer system for Shewanella synthetic biology

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M. Hajimorad  and  J.A. Gralnick

"Synthetic biology has been traditionally associated with electronics through the application of circuit design concepts towards the genetic engineering of microbes. Due to recent advances in the understanding of extracellular electron transfer in the bacterium Shewanella oneidensis (Shewanella), synthetic biology advances now have the potential of being used towards electronics applications. Towards this end, there is a need for tools that enable the systematic optimisation of genetic circuits in Shewanella. With the introduction of an RK2 origin of transfer cassette, we show that a modular plasmid system constructed prior for synthetic biology efforts in the bacterium Escherichia coli (E. coli) can be ported to Shewanella. In the process, it is also shown that different replication origins can be maintained in Shewanella and that multiple-plasmid strains can be realised in the bacterium. The results suggest that parts accumulated from E. coli synthetic biology efforts over the past decade and a half may be able to be ported to Shewanella, enabling the future engineering of systems where microbes interface with electronics (e.g. biosensors)."

http://bit.ly/1PtFF0F
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Fully integrated wearable sensor arrays for multiplexed in situ perspiration analysis

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Wei Gao, Sam Emaminejad, Hnin Yin Yin Nyein, Samyuktha Challa, Kevin Chen, Austin Peck, Hossain M. Fahad, Hiroki Ota, Hiroshi Shiraki, Daisuke Kiriya, Der-Hsien Lien, George A. Brooks, Ronald W. Davis & Ali Javey

"Wearable sensor technologies are essential to the realization of personalized medicine through continuously monitoring an individual’s state of health1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. Sampling human sweat, which is rich in physiological information13, could enable non-invasive monitoring. Previously reported sweat-based and other non-invasive biosensors either can only monitor a single analyte at a time or lack on-site signal processing circuitry and sensor calibration mechanisms for accurate analysis of the physiological state14, 15, 16, 17, 18. Given the complexity of sweat secretion, simultaneous and multiplexed screening of target biomarkers is critical and requires full system integration to ensure the accuracy of measurements. Here we present a mechanically flexible and fully integrated (that is, no external analysis is needed) sensor array for multiplexed in situ perspiration analysis, which simultaneously and selectively measures sweat metabolites (such as glucose and lactate) and electrolytes (such as sodium and potassium ions), as well as the skin temperature (to calibrate the response of the sensors). Our work bridges the technological gap between signal transduction, conditioning (amplification and filtering), processing and wireless transmission in wearable biosensors by merging plastic-based sensors that interface with the skin with silicon integrated circuits consolidated on a flexible circuit board for complex signal processing. This application could not have been realized using either of these technologies alone owing to their respective inherent limitations. The wearable system is used to measure the detailed sweat profile of human subjects engaged in prolonged indoor and outdoor physical activities, and to make a real-time assessment of the physiological state of the subjects. This platform enables a wide range of personalized diagnostic and physiological monitoring applications."

http://bit.ly/1JRZXAP
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Inner Workings: DNA for data storage and computing

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Megan Scudellari

"On the surface, genetic and electrical engineering appear to have little in common. One field revolves around carbon and the other is built upon silicon; one makes RNA from DNA and the other converts AC to DC.

But some creative biologists have begun to apply the concepts of electrical engineering to living cells. “We view ourselves as biological programmers,” says Timothy Lu, a member of the Synthetic Biology Group at the Massachusetts Institute of Technology (MIT). Lu and others are engineering circuits into bacterial cells, literally programming them for functions, such as data storage and computation. DNA’s straightforward, self-replicating helices are easy to amplify, modify, and are generally quite stable, says Lu. And since each position of DNA can encode four different pieces of information—A, T, G, or C—instead of just two, as with classic binary silicon systems, DNA could someday, in principle, store more data in less space. “The same properties that make DNA a great genetic code for living organisms also makes it an interesting substrate to engineer,” "

http://bit.ly/1UgwyA7
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Implementation of a genetic logic circuit: bio-register

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Chun-Liang Lin , Ting-Yu Kuo, Yang-Yi Chen

"We introduce an idea of synthesizing a class of genetic registers based on the existing sequential biological circuits, which are composed of fundamental biological gates. In the renowned literature, biological gates and genetic oscillator have been unveiled and experimentally realized in recent years. These biological circuits have formed a basis for realizing a primitive biocomputer. In the traditional computer architecture, there is an intermediate load-store section, i.e. a register, which serves as a part of the digital processor. With which, the processor can load data from a larger memory into it and proceed to conduct necessary arithmetic or logic operations. Then, manipulated data are stored back to the memory by instruction via the register. We propose here a class of bio-registers for the biocomputer. Four types of register structures are presented. In silicon experiments illustrate results of the proposed design."

http://bit.ly/1RMxq1E
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