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Programmable on-chip DNA compartments as artificial cells

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by
Eyal Karzbrun, Alexandra M. Tayar, Vincent Noireaux, Roy H. Bar-Ziv

"The assembly of artificial cells capable of executing synthetic DNA programs has been an important goal for basic research and biotechnology. We assembled two-dimensional DNA compartments fabricated in silicon as artificial cells capable of metabolism, programmable protein synthesis, and communication. Metabolism is maintained by continuous diffusion of nutrients and products through a thin capillary, connecting protein synthesis in the DNA compartment with the environment. We programmed protein expression cycles, autoregulated protein levels, and a signaling expression gradient, equivalent to a morphogen, in an array of interconnected compartments at the scale of an embryo. Gene expression in the DNA compartment reveals a rich, dynamic system that is controlled by geometry, offering a means for studying biological networks outside a living cell."

 http://bit.ly/1qaF0k3

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ComplexInsight's curator insight, August 15, 2014 2:36 AM

At Radcliffe Institute of Advanced Study at Harvard, Roy Bar-Ziv is expanding his research to explore the paradigm of programmable on-chip DNA compartments as artificial cells, in which the essential reactions of living cells encoded in DNA take place inside miniaturized compartments fabricated in silicon. Understanding the emergent properties of these compartments may lead to assembly of artificial cells capable of computation, autonomous sensing, and replication, with applications in future technologies.

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Terminator Operon Reporter: combining a transcription termination switch with reporter technology for improved gene synthesis and synthetic biology applications

Terminator Operon Reporter: combining a transcription termination switch with reporter technology for improved gene synthesis and synthetic biology applications | SynBioFromLeukipposInstitute | Scoop.it
Synthetic biology is characterized by the development of novel and powerful DNA fabrication methods and by the application of engineering principles to biology.
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Enhancing antibody folding and secretion by tailoring the Saccharomyces cerevisiae endoplasmic reticulum

The yeast Saccharomyces cerevisiae provides intriguing possibilities for synthetic biology and bioprocess applications, but its use is still constrained by cellular characteristics that limit the product yields. Considering the production of advanced biopharmaceuticals, a major hindrance lies in the yeast endoplasmic reticulum (ER), as it is not equipped for efficient and large scale folding of complex proteins, such as human antibodies.
RESULTS:
Following the example of professional secretory cells, we show that inducing an ER expansion in yeast by deleting the lipid-regulator gene OPI1 can improve the secretion capacity of full-length antibodies up to fourfold. Based on wild-type and ER-enlarged yeast strains, we conducted a screening of a folding factor overexpression library to identify proteins and their expression levels that enhance the secretion of antibodies. Out of six genes tested, addition of the peptidyl-prolyl isomerase CPR5 provided the most beneficial effect on specific product yield while PDI1, ERO1, KAR2, LHS1 and SIL1 had a mild or even negative effect to antibody secretion efficiency. Combining genes for ER enhancement did not induce any significant additional effect compared to addition of just one element. By combining the Δopi1 strain, with the enlarged ER, with CPR5 overexpression, we were able to boost the specific antibody product yield by a factor of 10 relative to the non-engineered strain.
CONCLUSIONS:
Engineering protein folding in vivo is a major task for biopharmaceuticals production in yeast and needs to be optimized at several levels. By rational strain design and high-throughput screening applications we were able to increase the specific secreted antibody yields of S. cerevisiae up to 10-fold, providing a promising strain for further process optimization and platform development for antibody production.
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Tal Danino’s Microuniverses are made with bacteria #ArtTuesday

Tal Danino’s Microuniverses are made with bacteria #ArtTuesday | SynBioFromLeukipposInstitute | Scoop.it
From The Creators Project: Tal Danino grows various strains of bacteria into detailed, microscopic patterns that pull you in close.
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Being an Entrepreneur is No Longer a Choice

Being an Entrepreneur is No Longer a Choice | SynBioFromLeukipposInstitute | Scoop.it
Being an Entrepreneur is No Longer a Choice https://t.co/IGkir2heQW #emprendedores
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Programmable materials find strength in molecular repetition | Penn State University

Programmable materials find strength in molecular repetition | Penn State University | SynBioFromLeukipposInstitute | Scoop.it
Synthetic proteins based on those found in a variety of squid species' ring teeth may lead the way to self-healing polymers carefully constructed for specific toughness and stretchability that might have applications in textiles, cosmetics and...
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A Synthetic Biologist's Beautiful Palette of Bacteria Art | The Creators Project

A Synthetic Biologist's Beautiful Palette of Bacteria Art
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Is the World Ready for Synthetic Life? Scientists Plan to Create Whole Genomes

Is the World Ready for Synthetic Life? Scientists Plan to Create Whole Genomes | SynBioFromLeukipposInstitute | Scoop.it
Last weekend, an invite-only group of about 150 experts convened privately at Harvard. Behind closed doors, they discussed the prospect of designing and building an entire human genome from scratch, using only a computer, a DNA synthesizer and raw materials.

The artificial genome would then be inserted into a living human cell to replace its natural DNA. The hope is that the cell “reboots,” changing its biological processes to operate based on instructions provided by the artificial DNA.

In other words, we may soon be looking at the first “artificial human cell.”

But the goal is not just Human 2.0. The project, “HGP-Write: Testing Large Synthetic Genomes in Cells,” also hopes to develop powerful new tools that push synthetic biology into exponential growth on an industrial scale. If successful, we won’t only have the biological tools to design humans as a species — we would have the ability to redesign the living world.

Manufacturing Life

At its core, synthetic biology is a marriage between engineering principles and biotechnology. If DNA sequencing is about reading DNA, genetic engineering is about editing DNA, synthetic biology is about programming new DNA — regardless of its original source — to build new forms of life.

Synthetic biologists view DNA and genes as standard biological bricks that can be used interchangeably to create and modify living cells.

The field has a plug-and-play mentality, says Dr. Jay Kiesling, a pioneer of synthetic engineering at the University of California at Berkeley. “When your hard drive dies, you can go to the nearest computer store, buy a new one, swap it out,” he says, “Why shouldn’t we use biological parts in the same way?”

To accelerate the field, Kiesling and colleagues are putting together a database of standardized DNA pieces — dubbed “BioBricks” — that can be used as puzzle pieces to assemble genetic material completely new to nature.

To Kiesling and others in the field, synthetic biology is like developing a new programming language. Cells are hardware, while DNA is the software that makes them run. With enough knowledge about how genes work, synthetic biologists believe that they will be able to write genetic programs from scratch, allowing them to build new organisms, alter nature and even guide the course of human evolution.

Similar to genetic engineering, synthetic biology gives scientists the power to tinker with natural DNA. The difference is mostly scale: genetic editing is a cut-and-paste process that adds foreign genes or changes the letters in existing genes. Often, only a few sites are changed.

Synthetic biology, on the other hand, creates genes from scratch. This allows scientists far more opportunities to make extensive changes to known genes, or even design their own. The possibilities are nearly endless.

Biodrugs, Biofuels, BioCrops

The explosion of synthetic biology in the past decade has already churned out results that thrilled both scientists and corporations.

Back in 2003, Kiesling published one of the earliest proof-of-concept studies demonstrating the power of the approach. He focused on a chemical called artemisinin, a powerful anti-malaria drug extracted from sweet wormwood that’s often the last line of defense against the disease.

Yet despite numerous attempts at cultivating the plant, yields remain extremely low.

Kiesling realized that synthetic biology offered a way to bypass the harvesting process altogether. By introducing the right genes into bacteria cells, he reasoned, the cells could turn into artemisinin-manufacturing machines, thus providing an abundant new source for the drug.

Getting there was tough. The team had to build an entirely new metabolic pathway into the cell, allowing it to process chemicals otherwise unknown to the cell.

Through trial-and-error, the team pasted together part of dozens of genes from several organisms into a custom DNA package. When they inserted the package into E. Coli, a bacteria commonly used in the lab to produce chemicals, it created a new pathway in the bacteria that allowed it to secrete artemisinin.

With more tinkering to increase efficacy, Kiesling and his team were able to bring up production by a factor of a million and reduce the drug’s price more than 10-fold.

Artemisinin was only the first step in a much larger program. The drug is a hydrocarbon, which belongs to a family of molecules often used to make biofuels. So why not use the same process to manufacture biofuels? By swapping out genes used to make artemisinin with those coding biofuel hydrocarbons, the team has already engineered multiple microbes capable of converting sugar to fuel.

Agriculture is another field poised to benefit from synthetic biology. Theoretically, we could take genes used to fix nitrogen from bacteria, put it into cells from our crops to completely alter their natural growth process. With the right combination of genes, we may be able to grow nutrition-packed crops — directed by an artificial genome — that require less water, land, energy and fertilizers.

Synthetic biology may even be used to produce completely new foods, such as flavorings created through fermentation with engineered yeast, or vegan cheeses and other animal-free milk products.

“We need to reduce carbon emissions and toxic inputs, use less land and water, combat pests, and increase soil fertility,” says Dr. Pamela Ronald, a professor at UC Davis. Synthetic biology may give us the tools to get there.

Re-creating Life

Practical applications aside, one of the ultimate goals of synthetic biology is to create a synthetic organism made exclusively from custom-designed DNA.

The main roadblock right now is technological. DNA synthesis is currently expensive, slow and prone to errors. Most existing techniques can only make DNA strands that are roughly 200 letters long, whereas genes are usually over ten times as long. The human genome contains roughly 20,000 genes that make proteins.

That said, costs for DNA synthesis have been rapidly dropping over the past decade.

According to Dr. Drew Endy, a geneticist at Stanford University, the cost of sequencing an individual letter has plummeted from $4 in 2003 to a mere 3 cents now. The estimated cost of printing all 3 billion letters of the human genome at the moment is a staggering $90 million, although that is expected to drop to $100,000 within 20 years if the trend continues.

An increasingly reasonable price tag has already opened doors to whole-genome synthesis.

Back in the 90s, Craig Venter, best known for his leading role in sequencing the human genome, began investigating the minimal set of genes required to make life. Together with colleagues at the Institute for Genomic Research, Venter removed genes from a bacterium Mycoplasma genitalium to identify those critical to life.

In 2008, Venter pieced together these “essential genes” and built the entire new “minimal” genome from a soup of chemicals using DNA synthesis.

Several years later, Venter transplanted the artificial genome into a second bacterium. The genes took over and “rebooted” the cell, allowing it to grow and self-replicate — the first living organism with a completely synthetic genome.

From Bacteria to Human

The new venture, if funded, would replicate Venter’s experiments using our own genome. Given that the human genome is nearly 5,000 times larger than Venter’s bacterium, it’s hard to say just how much more difficult the synthesis might be.

Even if that goal fails, however, the field is still bound to take a quantum leap forward. According to Dr. George Church, a leading geneticist at Harvard Medical School, the project could generate technological advances that improve our general ability to synthesize long strings of DNA — regardless of origin.

In fact, Church stressed that the project’s main goal is advancing technology.

But many are skeptical. According to Endy, who was invited to the meeting but decided to bow out, the project was originally named “HGP2: The Human Genome Synthesis Project,” and its primary goal was “to synthesize a complete human genome in a cell line within a period of 10 years.”

It’s perhaps not a surprise that news of the meeting caused a stir.

Regardless of its actual goals, the project raises the prospect of building custom-designed humans, or even semi-humans who have computers as parents.

The associated risks are easy to imagine and undoubtedly terrifying: how safe is it to directly manipulate or build life? How likely are accidents that unleash new organisms on an unprepared world? Who owns and has access to the technology? Would it breed new discrimination or further separate the 1% from the 99%?

“You can’t possibly begin to do something like this if you don’t have a value system in place that allows you to map concepts of ethics, beauty, and aesthetics onto our own existence,” says Endy.

“Given that human genome synthesis is a technology that can completely redefine the core of what now joins all of humanity together as a species, we argue that discussions of making such capacities real…should not take place without open and advance consideration of whether it is morally right to proceed,” he said.
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Breakthrough Holds Promise for Medicine: Synthetic Nanomotors Controlled Inside Live Human Cells

Breakthrough Holds Promise for Medicine: Synthetic Nanomotors Controlled Inside Live Human Cells | SynBioFromLeukipposInstitute | Scoop.it
Penn State University chemists and engineers have placed tiny synthetic motors inside live human cells, propelled them with ultrasonic waves, and steered them magnetically.
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Synthetic Human Genome Project

Synthetic Human Genome Project | SynBioFromLeukipposInstitute | Scoop.it
In a closed door meeting at Harvard on Tuesday nearly 150 scientists got together to organize the human synthetic genome project.
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Editorial: Engineering Synthetic Metabolons: From Metabolic Modeling to Rational Design of Biosynthetic Devices

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Engineering yeast shikimate pathway towards production of aromatics: rational design of a chassis cell using systems and synthetic biology - UQ eSpace

Aromatic building blocks are amongst the most important bulk feedstocks in the chemical industry. As these compounds are commonly derived from petrochemistry, obtaining them is becoming more and more a matter of costs and sustainability. Biochemistry gives rise to a wealth of compounds that can potentially replace current petroleum-based chemicals or be used for novel materials. The aromatic compounds para-aminobenzoic acid (pABA) and para-hydroxybenzoic acid (pHBA) and the aromatics derived compound cis,cis-muconic acid (ccMA) can be precursors for, but are not limited to, terephthalic or/and adipic acid. These are essential feedstocks for the production of PET and nylon. The three compounds can be derived from the shikimate pathway, an anabolic pathway leading to the biosynthesis of aromatic amino acids, present in certain prokaryotes and eukaryotes, including fungi. By combination of metabolic modelling with genetic engineering, a microbial production system based on the yeast Saccharomyces cerevisiae can be designed, which effectively channels flux into the target compounds.

In order to develop a competitive bio-based process, yields, titers and rates need to be maximized. While productivity or rates in a process can be altered using genetic engineering, carbon yield, and pathway feasibility are stoichiometrically and thermodynamically predetermined. Both limitations need to be considered when designing a microbial production system.

For formation of adipic acid and precursors many bio-based routes exists. More rational than just picking one for in vivo studies rather all available biochemical pathways were examined in silico using metabolic modelling. To compare theoretical yields and reaction thermodynamics an interface that allowed network-embedded thermodynamic analysis of elementary flux modes was developed. This allowed distinguishing between thermodynamically feasible and infeasible flux distributions. Feasible maximum theoretical product carbon yields were substantially different in E. coli and S. cerevisiae metabolic models and ranged from 32% to 92%. Further, many pathways appeared to be restricted by a thermodynamic equilibrium lying on the substrate side, some even infeasible.

The only routes that deliver significant product yields and were thermodynamically favoured were shikimate pathway based. Being currently of strong scientific interest, recent implementations of these pathways in E. coli and S. cerevisiae were evaluated and strain construction strategies optimized, using the concept of constrained minimal cut-sets. Especially in S. cerevisiae a single non-obvious knock-out target allowed coupling of growth to product formation; in particular, the deletion of the pyruvate kinase reaction resulted in a minimum yield constraint of 28%.

Though unique to shikimate pathway, this strategy is transferrable to other products which are derived from chorismate and also involve the formation of pyruvate as a by-product. This applies to pHBA. With further optimizations, the strategy was applied in vivo for the production of this compound in S. cerevisiae. As the pyruvate kinase knock-out entails a growth defect on glucose, a synthetic circuit was used, which allowed conditional knock-down and activation of the determined genetic modifications and by this dynamic control of the phenotype. Thus, production could be separated from growth and it could be proven that the in silico determined genetic intervention strategy holds valid in vivo, resulting in a 1.1 mM final product titer.

Further, production of pABA from shikimate pathway in S. cerevisiae was investigated and optimized: Several alleles from different yeast strains of the genes (ABZ1 and ABZ2) for pABA formation from chorismate were screened, using a strain genetically engineered to channel flux to chorismate. ABZ1 of AWRI1631 and ABZ2 of QA23 delivered the highest pABA production. To further increase production, the impact of carbon-source on product yield was investigated in silico using metabolic modelling. It was found that combined glycerol-ethanol was a superior carbon-source than glucose, glycerol or ethanol alone, especially when employed in molar ratios between 1:2 and 2:1. This could be confirmed in vivo with carbon yields reaching up to 2.64%. A fed-batch process on glycerol-ethanol delivered total aromatics titers as high as 1.73 mM.

It could be shown that feasibility and viability of adipic acid production greatly depends on the pathway and the organism chosen for engineering. Weaknesses of existing strain construction strategies for ccMA production could be identified and a radical optimization strategy was determined.

Inferences from metabolic modelling were proven experimentally for pHBA and pABA production. The obtained concentrations and yields are the highest in S. cerevisiae to date and among the highest in a microbial production system, underlining th
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Full speed ahead to the City on the Hill

Full speed ahead to the City on the Hill | SynBioFromLeukipposInstitute | Scoop.it
Landscape architects and ecological engineering scientists can design networks of self-sustaining and functioning natural ecosystems within the urban matrix. Urban parks, green roofs, and vegetation corridors will be more common and better connected to ensure the continuity of the natural ecosystem in the urban environment. The paradigm needs to shift from planting trees and building parks within the city to building the city around the natural environment.
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DIY Biology catches on with the UK’s first BioHackacthon

DIY Biology catches on with the UK’s first BioHackacthon | SynBioFromLeukipposInstitute | Scoop.it
The hackathon phenomena is knocking on biology’s door. The first UK Biohackathon will be held at the University of Cambridge in June, to learn and create with SynBio.

Hackathons are massively popular events in computer programming, joining people during a short period to create a hands-on project. The concept has now spread to biology – more specifically to synthetic biology.

This emerging area has a lot of potential, from biofuels to perfumes to healthcare. It already attracted a large bio-hacking community, the famous iGEM competitions and a specialized incubator.
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What does it mean for researchers, journalists and the public when secrecy surrounds science?

What does it mean for researchers, journalists and the public when secrecy surrounds science? | SynBioFromLeukipposInstitute | Scoop.it
Did you hear about the secret meeting earlier this month at Harvard Medical School? The one where scientists schemed to create a parentless human being from scratch? Maybe you read one of the skeptical news articles, or the stories illustrated with images from the dystopian sci-fi classic “Blade Runner” or of a robot Frankenstein. One blogger compared the meeting to a gathering of “Bond villains.”

The press coverage was suspicious and critical. Why would a bunch of scientists need to exclude the media and the public from a meeting about something as ethically fraught as synthesizing a human genome?

Three weeks later, the exact details of what happened are still being contested. I’m a researcher in synthetic biology, and I learned of the project from reading the newspaper. I reached out to the meeting’s organizers, who – for reasons I’ll explain – declined to comment for this article. But in conversations with meeting invitees, as well as some critics, I’ve found that much of the press coverage was misleading, and says more about the relationship between journalists and scientists than the meeting itself.

What really happened behind closed doors when over 130 scientists, industry leaders and ethicists convened to talk about synthesizing a human genome? How did these sessions end up so widely misunderstood by the media and the public?

Open doors versus science publishing protocols

The May 10 meeting was titled “HGP-Write: Testing Large Synthetic Genomes in Cells.” HGP refers to the Human Genome Project, the world’s largest collaborative biological effort that resulted in the sequencing of the full human genome in 2003.

Those invited say the organizers hoped to inspire scientists and the public with a new grand challenge project: to advance from reading genomes to writing them, by manufacturing them from individual DNA building blocks. In an invitation dated March 30, the hosts proposed a bold collaborative effort to “synthesize a complete human genome within a cell line.” Panels tackled whether such an effort is worthwhile, as well as the ethical, technological and economic challenges.

The conversation was not intended to be restricted. The meeting organizers – Harvard geneticist George Church; New York University systems geneticist Jef Boeke; Andrew Hessel, of the Bio/Nano research group at Autodesk, Inc.; and Nancy J. Kelley, a lawyer specializing in biotechnology consulting – had plans to engage the broader scientific community, as well as industry, policy makers and the public. They made a video recording of the entire meeting, originally intended to be live-streamed over the Internet. They planned to apply for federal funding, which would invite regulatory oversight. And they submitted a white paper to a major peer-reviewed journal explaining the scientific, technological and ethical aspects of the project.

But the publication of the paper was delayed – the authors haven’t disclosed why, although editors commonly ask for revisions as part of the peer review process. (As of this writing, it has not yet come out.) The organizers are prohibited from discussing the paper in public until it is published – a common journal policy known as an embargo. In deference to the embargo, they declined to comment in detail for this article.

News of the delay came just days before the meeting, and, with dozens of attendees en route, the hosts made a fateful decision. They chose to proceed, but to close the doors to most journalists and ask attendees to delay public discussion until the embargo lifts. (At least one journalist was there – Simone Ross, co-founder of Techonomy Media, confirmed her attendance to me.) “I’m not sure that was the best idea,” Dr. Church told STAT News of the decision to proceed out of the public eye.

The secrecy bred suspicion. “Would it be OK to sequence and then synthesize Einstein’s genome?” asked Stanford bioengineer Drew Endy and Northwestern bioethicist Laurie Zoloth in a joint essay. In theory, an artificial human genome could be used to generate a living human without biological parents. “This idea is an enormous step for the human species, and it shouldn’t be discussed only behind closed doors,” STAT News quoted Dr. Zoloth.

Beyond qualms about the science itself, some observers were concerned that the organizers' decisions - which included seeking industry partners and private funding - were quiet moves towards “privatiz[ing] the current conversation about heritable genetic modification.”
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Dynamic DNA polymers can be reversed using biocompatible techniques - Scienmag

Dynamic DNA polymers can be reversed using biocompatible techniques - Scienmag | SynBioFromLeukipposInstitute | Scoop.it
DNA-based straight and branched polymers or nanomaterials that can be created and dissolved using biocompatible methods are now possible thanks to the work of Penn State biomedical engineers.
Synthe..
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Medicinal Microbes: Making Drugs with Bugs - Science Cafe

Bacteria are usually considered to be a threat to humans. Yet, bacteria are also capable of stunning feats of organic chemistry unmatched by the efforts of ...
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Author interview with Jonathan Young - Computational Biologist at UT Austin - Overleaf Blog

Author interview with Jonathan Young - Computational Biologist at UT Austin - Overleaf Blog | SynBioFromLeukipposInstitute | Scoop.it
Jonathan is a graduate student at UT Austin, and we recently asked him about his work and his experiences using Overleaf
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Biology Is Key To Unlocking The Future Of Design

Biology Is Key To Unlocking The Future Of Design | SynBioFromLeukipposInstitute | Scoop.it
Biology Is Key To Unlocking The Future Of Design
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A guide to CRISPR gene activation : Wyss Institute at Harvard

A guide to CRISPR gene activation : Wyss Institute at Harvard | SynBioFromLeukipposInstitute | Scoop.it
Wyss Institute at Harvard
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Scientists Want to Bring Back Woolly Mammoths — But Why?

Scientists Want to Bring Back Woolly Mammoths — But Why? | SynBioFromLeukipposInstitute | Scoop.it
Two teams are working on "de-extinction" projects that could one day lead to another of the giant mammals walking the Earth.
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JBEI, UCSD scientists develop systems biology-based workflow to improve biofuels productivity

JBEI, UCSD scientists develop systems biology-based workflow to improve biofuels productivity | SynBioFromLeukipposInstitute | Scoop.it
Researchers at the US Department of Energy (DOE)’s Joint BioEnergy Institute (JBEI), in collaboration with researchers at the University of California, San Diego, have developed a workflow that integrates various “omics” data and genome-scale...
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Programming Surface Chemistry with Engineered Cells - ACS Synthetic Biology

We have developed synthetic gene networks that enable engineered cells to selectively program surface chemistry. E. coli were engineered to upregulate biotin synthase, and therefore biotin synthesis, upon biochemical induction. Additionally, two different functionalized surfaces were developed that utilized binding between biotin and streptavidin to regulate enzyme assembly on programmable surfaces. When combined, the interactions between engineered cells and surfaces demonstrated that synthetic biology can be used to engineer cells that selectively control and modify molecular assembly by exploiting surface chemistry. Our system is highly modular and has the potential to influence fields ranging from tissue engineering to drug development and delivery.
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Influence of Pichia pastoris cellular material on polymerase chain reaction performance as a synthetic biology standard for genome monitoring

Advances in synthetic genomics are now well underway in yeasts due to the low cost of synthetic DNA. These new capabilities also bring greater need for quantitating the presence, loss and rearrangement of loci within synthetic yeast genomes. Methods for achieving this will ideally; i) be robust to industrial settings, ii) adhere to a global standard and iii) be sufficiently rapid to enable at-line monitoring during cell growth. The methylotrophic yeast Pichia pastoris (P. pastoris) is increasingly used for industrial production of biotherapeutic proteins so we sought to answer the following questions for this particular yeast species. Is time-consuming DNA purification necessary to obtain accurate end-point polymerase chain reaction (e-pPCR) and quantitative PCR (qPCR) data? Can the novel linear regression of efficiency qPCR method (LRE qPCR), which has properties desirable in a synthetic biology standard, match the accuracy of conventional qPCR? Does cell cultivation scale influence PCR performance? To answer these questions we performed e-pPCR and qPCR in the presence and absence of cellular material disrupted by a mild 30s sonication procedure. The e-pPCR limit of detection (LOD) for a genomic target locus was 50 pg (4.91x103 copies) of purified genomic DNA (gDNA) but the presence of cellular material reduced this sensitivity sixfold to 300 pg gDNA (2.95x104 copies). LRE qPCR matched the accuracy of a conventional standard curve qPCR method. The presence of material from bioreactor cultivation of up to OD600 = 80 did not significantly compromise the accuracy of LRE qPCR. We conclude that a simple and rapid cell disruption step is sufficient to render P. pastoris samples of up to OD600 = 80 amenable to analysis using LRE qPCR which we propose as a synthetic biology standard.
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Full speed ahead to the City on the Hill

Full speed ahead to the City on the Hill | SynBioFromLeukipposInstitute | Scoop.it
With the help of synthetic biology, the city of the future can potentially be engineered less like a conglomerate of machines and more like a natural system. Cells can be engineered to sense and respond to environmental signals. Communication networks through sensors can be embedded in the city infrastructure. Protocell technology, a new material that possesses some of the properties of living systems, can be manipulated to grow architecture. We might see self-repairing architecture.
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Stanford-NIST collaboration aims to give the bio-economy a big boost by measuring tiny things

Stanford-NIST collaboration aims to give the bio-economy a big boost by measuring tiny things | SynBioFromLeukipposInstitute | Scoop.it
The Joint Initiative for Metrology in Biology, located at Stanford, will bring together academic, government and industrial scientists to improve the measurement techniques, or metrology, of molecular products and processes to facilitate advances...
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