SynBioFromLeukipposInstitute

by
Osella M, Lagomarsino MC.

"Gene networks exhibiting oscillatory dynamics are widespread in biology. The minimal regulatory designs giving rise to oscillations have been implemented synthetically and studied by mathematical modeling. However, most of the available analyses generally neglect the coupling of regulatory circuits with the cellular "chassis" in which the circuits are embedded. For example, the intracellular macromolecular composition of fast-growing bacteria changes with growth rate. As a consequence, important parameters of gene expression, such as ribosome concentration or cell volume, are growth-rate dependent, ultimately coupling the dynamics of genetic circuits with cell physiology. This work addresses the effects of growth rate on the dynamics of a paradigmatic example of genetic oscillator, the repressilator. Making use of empirical growth-rate dependencies of parameters in bacteria, we show that the repressilator dynamics can switch between oscillations and convergence to a fixed point depending on the cellular state of growth, and thus on the nutrients it is fed. The physical support of the circuit (type of plasmid or gene positions on the chromosome) also plays an important role in determining the oscillation stability and the growth-rate dependence of period and amplitude. This analysis has potential application in the field of synthetic biology, and suggests that the coupling between endogenous genetic oscillators and cell physiology can have substantial consequences for their functionality."
http://bit.ly/Zi1wQ6


In this connection some interesting piece of history:
"Shortly after François Jacob and Jacques Monod developed their first model of gene regulation, Goodwin proposed the first model of a genetic oscillator, showing that regulatory interactions among genes allowed periodic fluctuations to occur. Shortly after this model became published, he also formulated a general theory of complex gene regulatory networks using statistical mechanics. In its simplest form, Goodwin's oscillator involves a single gene that represses itself. Goodwin equations were originally formulated in terms of conservative (Hamiltonian) systems, thus not taking into account dissipative effects that are required in a realistic approach to regulatory phenomena in biology. Many versions have been developed since then. The simplest (but realistic) formulation considers three variables, X, Y and Z indicating the concentrations of RNA, protein and end product which generates the negative feedback loop.The equations are (see fig below) 
and closed oscillations can occur for n>8 and behave limit cycles: after a perturbation of the system's state, it returns to its previous attractor. A simple modification of this model, adding other terms introducing additional steps in the transcription machinery allows to find oscillations for smaller n values. Goodwin's model and its extensions have been widely used over the years as the basic skeleton for other models of oscillatory behavior, including circadian clocks, cell division or physiological control systems."
http://bit.ly/14YpWxz

by
Dante W. Romanini , Pamela Peralta-Yahya , Vanessa Mondol , and Virginia W. Cornish 

"Genetic recombination is central to the generation of molecular diversity and enhancement of evolutionary fitness in living systems. Methods such as DNA shuffling that recapitulate this diversity mechanism in vitro are powerful tools for engineering biomolecules with useful new functions by directed evolution. Synthetic biology now brings demand for analogous technologies that enable the controlled recombination of beneficial mutations in living cells. Thus, here we create a Heritable Recombination system centered around a library cassette plasmid that enables inducible mutagenesis via homologous recombination and subsequent combination of beneficial mutations through sexual reproduction in Saccharomyces cerevisiae. Using repair of nonsense codons in auxotrophic markers as a model, Heritable Recombination was optimized to give mutagenesis efficiencies of up to 6% and to allow successive repair of different markers through two cycles of sexual reproduction and recombination. Finally, Heritable Recombination was employed to change the substrate specificity of a biosynthetic enzyme, with beneficial mutations in three different active site loops crossed over three continuous rounds of mutation and selection to cover a total sequence diversity of 1013. Heritable Recombination, while at an early stage of development, breaks the transformation barrier to library size and can be immediately applied to combinatorial crossing of beneficial mutations for cell engineering, adding important features to the growing arsenal of next generation molecular biology tools for synthetic biology."

http://bit.ly/ZhYrzI

by
Erika Check Hayden

"Tools for data-sharing promise to improve chances of connecting mutations with symptoms of rare diseases.

For the first five months of Harrison Harkins’ life, doctors had little idea about what was causing his spinal malformation and inability to gain weight. But in November 2011, Matthew Bainbridge, a computational biologist at Baylor College of Medicine in Houston, Texas, found a clue. After analysing genetic data from Harrison and his parents, Bainbridge discovered that the child had an abnormal version of a gene called ASXL3. But Bainbridge had no easy access to records of other children with ASXL3 mutations, and could not be sure that this mutation was the culprit. So he did what many scientists do: he networked. A Dutch team put Bainbridge in touch with German researchers who were treating another boy with an ASXL3 mutation — and symptoms similar to Harrison’s. After finding two further cases in an internal Baylor database, Bainbridge felt that the connection was concrete. He describes the syndrome seen in all four children, and probably caused by ASXL3 mutations, in a paper published on 5 February (M. N. Bainbridge et al. Genome Med. 5, 11; 2013)....."

http://bit.ly/14WYXCx

*

by
Sagt CM.

"Systems metabolic engineering is based on systems biology, synthetic biology, and evolutionary engineering and is now also applied in industry. Industrial use of systems metabolic engineering focuses on strain and process optimization. Since ambitious yields, titers, productivities, and low costs are key in an industrial setting, the use of effective and robust methods in systems metabolic engineering is becoming very important. Major improvements in the field of proteomics and metabolomics have been crucial in the development of genome-wide approaches in strain and process development. This is accompanied by a rapid increase in DNA sequencing and synthesis capacity. These developments enable the use of systems metabolic engineering in an industrial setting. Industrial systems metabolic engineering can be defined as the combined use of genome-wide genomics, transcriptomics, proteomics, and metabolomics to modify strains or processes. This approach has become very common since the technology for generating large data sets of all levels of the cellular processes has developed quite fast into robust, reliable, and affordable methods. The main challenge and scope of this mini review is how to translate these large data sets in relevant biological leads which can be tested for strain or process improvements. Experimental setup, heterogeneity of the culture, and sample pretreatment are important issues which are easily underrated. In addition, the process of structuring, filtering, and visualization of data is important, but also, the availability of a genetic toolbox and equipment for medium/high-throughput fermentation is a key success factor. For an efficient bioprocess, all the different components in this process have to work together. Therefore, mutual tuning of these components is an important strategy."

http://1.usa.gov/Vdlbgo

fig 
Cellular metabolism can be optimized for industrial use.http://en.wikipedia.org/wiki/Metabolic_engineering ;

by
Emma Frowa & Jane Calverta 

"Synthetic biology represents a recent attempt to bring engineering principles and practices to working with biology. In practice, the nature of the relationship between engineering and biology in synthetic biology is a subject of ongoing debate. The disciplines of biology and engineering are typically seen to involve different ways of knowing and doing, and to embody different assumptions and objectives. Tensions between these approaches are playing out as the field of synthetic biology is being established. Here, we study negotiations between engineering and biology through the International Genetically Engineered Machine (iGEM) competition. This undergraduate competition has been important in launching and bootstrapping the field of synthetic biology, and serves as a test-bed for the engineering approach. We show how a number of issues that iGEM teams must grapple with – including standardization, design, intellectual property, and the imagination of the social – involve the negotiation of engineering, biology, and other disciplines (including computer science), in ways more complex than the engineering rhetoric of synthetic biology implies. We suggest that a new moral economy for synthetic biology is being created, in which epistemic and institutional values, conventions, and practices are being negotiated and (re)defined."

http://bit.ly/12BC8FS

by
Kouprina N, Samoshkin A, Erliandri I, Nakano M, Lee HS, Fu H, Iida Y, Aladjem M, Oshimura M, Masumoto H, Earnshaw WC, Larionov V.

"Human artificial chromosomes (HACs) represent a novel promising episomal system for functional genomics, gene therapy and synthetic biology. HACs are engineered from natural and synthetic alphoid DNA arrays upon transfection into human cells. The use of HACs for gene expression studies requires the knowledge of their structural organization. However, none of de novo HACs constructed so far has been physically mapped in detail. Recently we constructed a synthetic alphoid(tetO)-HAC that was successfully used for expression of full-length genes to correct genetic deficiencies in human cells. The HAC can be easily eliminated from cell populations by inactivation of its conditional kinetochore. This unique feature provides a control for phenotypic changes attributed to expression of HAC-encoded genes. This work describes organization of a megabase-size synthetic alphoid DNA array in the alphoid(tetO)-HAC that has been formed from a ~50 kb synthetic alphoid(tetO)-construct. Our analysis showed that this array represents a 1.1 Mb continuous sequence assembled from multiple copies of input DNA, a significant part of which was rearranged before assembling. The tandem and inverted alphoid DNA repeats in the HAC range in size from 25 to 150 kb. In addition, we demonstrated that the structure and functional domains of the HAC remains unchanged after several rounds of its transfer into different host cells. The knowledge of the alphoid(tetO)-HAC structure provides a tool to control HAC integrity during different manipulations. Our results also shed light on a mechanism for de novo HAC formation in human cells."

http://bit.ly/VX5wV9

*Synthetic biology of antibiotic production*

Talk by 
Eriko Takano 

"Speaker abstract

Streptomyces bacteria are well known for their ability to produce an immense diversity of secondary metabolites, including many antibiotics. The underlying biosynthetic machinery is a particularly interesting target for synthetic biology, due to its inherent modularity at multiple levels1,2. A treasure trove of antibiotic biosynthesis gene clusters has been identified by genome sequencing, typically 20–50 per genome3,4. We can use synthetic biology to re-engineer the bacterial genomes to awaken this multitude of cryptic antibiotic clusters. We have already demonstrated the potential of this strategy by awakening the cryptic/orphan CPK gene cluster5, which produces a novel antibacterial compound. Generalizing this approach using standardized molecular modules will become a central tool for discovering new bioactive compounds, ranging from anti-cancer drugs to antibiotics6."

http://bit.ly/14UxvFm

by
Jayodita Sanghvi, Jonathan R. Karr, Derek Macklin, Miriam Gutschow, Jared Jacobs, Benjamin Bolival, John Glass, Nacyra Assad-Garcia and Markus W. Covert

 

Abstarct (SBE’s 6th International Conference on Bioengineering and Nanotechnology)

"Researchers have spent years uncovering the molecular mechanisms of the numerous processes in a cell, from metabolism to cell division. Still, an integrated comprehension of this knowledge remains a challenge. We have attempted to collate the community's understanding and have built the first comprehensive computational model of a single living cell. This model of the simplest known self-replicating organism, Mycoplasma genitalium, describes all of the known gene functions and molecular interactions. It includes data from over 900 publications and over 1900 parameters. The model incorporates the cross talk between 28 cellular processes including metabolism, transcription, translation, replication, and cell division. Each process was first independently modeled using different mathematical representations, such as linear optimization, ordinary differential equations, probabilistic binding, and geometry, that were best fit for the individual cellular processes. Then, the 28 processes were integrated together at a one-second timestep. The whole-cell model has been validated and benchmarked against existing physiological data, and we have performed additional experiments to further validate the model. The resulting simulations predict cell behavior in response to genomic and environmental perturbations as well as phenotypes that lead to questions that have never before been addressed. The model is able to predict kinetic rates, new gene annotations, chromosomal occupancy, energy usage, new forms of cell cycle regulation, and much more.

 We hope that that this whole-cell model and expansions on this model will accelerate biological discovery and bioengineering by serving as tools to guide synthetic biology. Further, in combination with the recent de novo synthesis and transplantation of Mycoplasma genomes to produce a synthetic cell (Gibson et al., 2008; Gibson et al., 2010), whole-cell models raise the possibility of computer-aided rational design of novel microorganisms."

http://bit.ly/12EhiB6

Fig
http://en.wikipedia.org/wiki/Mycoplasma_genitalium

by
Roland Pease

"Synthetic biologists have developed DNA modules that perform logic operations in living cells. These ‘genetic circuits’ could be used to track key moments in a cell’s life or, at the flick of a chemical switch, change a cell’s fate, the researchers say. Their results are described this week in Nature Biotechnology1.

 Synthetic biology seeks to bring concepts from electronic engineering to cell biology, treating gene functions as components in a circuit. To that end, researchers at the Massachusetts Institute of Technology (MIT) in Cambridge have devised a set of simple genetic modules that respond to inputs much like the Boolean logic gates used in computers. “These developments will more readily enable one to create programmable cells with decision-making capabilities for a variety of applications,” says James Collins, a synthetic biologist at Boston University in Massachusetts who was not involved in the study...."

http://bit.ly/VSSUOR

*Carbon-Nanotube-Embedded Hydrogel Sheets for Engineering Cardiac Constructs and Bioactuators*

by
Su Ryon Shin , Sung Mi Jung , Momen Zalabany , Keekyoung Kim , Pinar Zorlutuna , Sang bok Kim , Mehdi Nikkhah , Masoud Khabiry , Mohamed Azize , Jing Kong , Kai-tak Wan , Tomas Palacios , Mehmet R Dokmeci , Hojae Bae , Xiaowu (Shirley) Tang , and Ali Khademhosseini

"We engineered functional cardiac patches by seeding neonatal rat cardiomyocytes onto carbon nanotube (CNT) incorporated photocrosslinkable gelatin methacrylate (GelMA) hydrogel. The resulting cardiac constructs showed excellent mechanical integrity and advanced electrophysiological functions. Specifically, myocardial tissues cultured on 50 μm thick CNT-GelMA showed 3 times higher spontaneous synchronous beating rates and 85% lower excitation threshold, compared to those cultured on pristine GelMA hydrogels. Our results indicate that the electrically conductive and nanofibrous networks formed by CNTs within a porous gelatin framework is the key characteristics of CNT-GelMA leading to improved cardiac cell adhesion, organization, and cell-cell coupling. Centimeter-scale patches were released from glass substrates to form 3D biohybrid actuators, which showed controllable linear cyclic contraction/extension, pumping, and swimming actuations. In addition, we demonstrate for the first time that cardiac tissues cultured on CNT-GelMA resist damage by a model cardiac inhibitor as well as a cytotoxic compound. Therefore, incorporation of CNTs into gelatin, and potentially other biomaterials, could be useful in creating multifunctional cardiac scaffolds for both therapeutic purposes and in vitro studies. These hybrid materials could also be used for neuron and other muscle cells to create tissue constructs with improved organization, electroactivity, and mechanical integrity."

http://bit.ly/Y5x7O4


see also by the same last author

*Carbon Nanotube Reinforced Hybrid Microgels as Scaffold Materials for Cell Encapsulation*
http://bit.ly/Y5xQ1U

is also source of the fig

by
Anne Trafton

"MIT engineers have created genetic circuits in bacterial cells that not only perform logic functions, but also remember the results, which are encoded in the cell’s DNA and passed on for dozens of generations.

 The circuits, described in the Feb. 10 online edition of Nature Biotechnology, could be used as long-term environmental sensors, efficient controls for biomanufacturing, or to program stem cells to differentiate into other cell types. “Almost all of the previous work in synthetic biology that we’re aware of has either focused on logic components or on memory modules that just encode memory. We think complex computation will involve combining both logic and memory, and that’s why we built this particular framework to do so,” says Timothy Lu, an MIT assistant professor of electrical engineering and computer science and biological engineering and senior author of the Nature Biotechnology paper. Lead author of the paper is MIT postdoc Piro Siuti. Undergraduate John Yazbek is also an author.  More than logic Synthetic biologists use interchangeable genetic parts to design circuits that perform a specific function, such as detecting a chemical in the environment. In that type of circuit, the target chemical would generate a specific response, such as production of green fluorescent protein (GFP).   Circuits can also be designed for any type of Boolean logic function, such as AND gates and OR gates. Using those kinds of gates, circuits can detect multiple inputs. In most of the previously engineered cellular logic circuits, the end product is generated only as long as the original stimuli are present: Once they disappear, the circuit shuts off until another stimulus comes along.  Lu and his colleagues set out to design a circuit that would be irreversibly altered by the original stimulus, creating a permanent memory of the event. To do this, they drew on memory circuits that Lu and colleagues designed in 2009. Those circuits depend on enzymes known as recombinases, which can cut out stretches of DNA, flip them, or insert them. Sequential activation of those enzymes allows the circuits to count events happening inside a cell. Lu designed the new circuits so that the memory function is built into the logic gate itself. With a typical cellular AND gate, the two necessary inputs activate proteins that together turn on expression of an output gene. However, in the new circuits, the inputs stably alter regions of DNA that control GFP production. These regions, known as promoters, recruit the cellular proteins responsible for transcribing the GFP gene into messenger RNA, which then directs protein assembly. For example, in one circuit described in the paper, two DNA sequences called terminators are interposed between the promoter and the output gene (GFP, in this case). Each of these terminators inhibits the transcription of the output gene and can be flipped by a different recombinase enzyme, making the terminator inactive.  Each of the circuit’s two inputs turns on production of one of the recombinase enzymes needed to flip a terminator. In the absence of either input, GFP production is blocked. If both are present, both terminators are flipped, resulting in their inactivation and subsequent production of GFP.  Once the DNA terminator sequences are flipped, they can’t return to their original state — the memory of the logic gate activation is permanently stored in the DNA sequence. The sequence also gets passed on for at least 90 generations. Scientists wanting to read the cell’s history can either measure its GFP output, which will stay on continuously, or if the cell has died, they can retrieve the memory by sequencing its DNA. Using this design strategy, the researchers can create all two-input logic gates and implement sequential logic systems. “It’s really easy to swap things in and out,” says Lu, who is also a member of MIT’s Synthetic Biology Center. “If you start off with a standard parts library, you can use a one-step reaction to assemble any kind of function that you want.” Long-term memory Such circuits could also be used to create a type of circuit known as a digital-to-analog converter. This kind of circuit takes digital inputs — for example, the presence or absence of single chemicals — and converts them to an analog output, which can be a range of values, such as continuous levels of gene expression.  For example, if the cell has two circuits, each of which expresses GFP at different levels when they are activated by their specific input, those inputs can produce four different analog output levels. Moreover, by measuring how much GFP is produced, the researchers can figure out which of the inputs were present. That type of circuit could offer better control over the production of cells that generate biofuels, drugs or other useful compounds. Instead of creating circuits that are always on, or using promoters that need continuous inputs to control their output levels, scientists could transiently program the circuit to produce at a certain level. The cells and their progeny would always remember that level, without needing any more information.  Used as environmental sensors, such circuits could also provide very precise long-term memory. “You could have different digital signals you wanted to sense, and just have one analog output that summarizes everything that was happening inside,” Lu says. This platform could also allow scientists to more accurately control the fate of stem cells as they develop into other cell types. Lu is now working on engineering cells to follow sequential development steps, depending on what kinds of inputs they receive from the environment.  Michael Jewett, an assistant professor of chemical and biological engineering at Northwestern University, says the new design represents a “huge advancement in DNA-encoded memory storage.” “I anticipate that the innovations reported here will help to inspire larger synthetic biology efforts that push the limits of engineered biological systems,” says Jewett, who was not involved in the research."http://bit.ly/Y3KzUJ

this is a comment to:
https://plus.google.com/u/0/106140549977596536572/posts/Ae1bKTr1Bqz