Linkage and association studies have mapped thousands of genomic regions that contribute to phenotypic variation, but narrowing these regions to the underlying causal genes and variants has proven much more challenging. Resolution of genetic mapping is limited by the recombination rate. We developed a method that uses CRISPR (clustered, regularly interspaced, short palindromic repeats) to build mapping panels with targeted recombination events. We tested the method by generating a panel with recombination events spaced along a yeast chromosome arm, mapping trait variation, and then targeting a high density of recombination events to the region of interest. Using this approach, we fine-mapped manganese sensitivity to a single polymorphism in the transporter Pmr1. Targeting recombination events to regions of interest allows us to rapidly and systematically identify causal variants underlying trait differences.
Systems metabolic engineering, which recently emerged as metabolic engineering integrated with systems biology, synthetic biology, and evolutionary engineering, allows engineering of microorganisms on a systemic level for the production of valuable chemicals far beyond its native capabilities. Here, we review the strategies for systems metabolic engineering and particularly its applications in Escherichia coli. First, we cover the various tools developed for genetic manipulation in E. coli to increase the production titers of desired chemicals. Next, we detail the strategies for systems metabolic engineering in E. coli, covering the engineering of the native metabolism, the expansion of metabolism with synthetic pathways, and the process engineering aspects undertaken to achieve higher production titers of desired chemicals. Finally, we examine a couple of notable products as case studies produced in E. coli strains developed by systems metabolic engineering. The large portfolio of chemical products successfully produced by engineered E. coli listed here demonstrates the sheer capacity of what can be envisioned and achieved with respect to microbial production of chemicals. Systems metabolic engineering is no longer in its infancy; it is now widely employed and is also positioned to further embrace next-generation interdisciplinary principles and innovation for its upgrade. Systems metabolic engineering will play increasingly important roles in developing industrial strains including E. coli that are capable of efficiently producing natural and nonnatural chemicals and materials from renewable nonfood biomass.
Synthetic biology is characterized by the development of novel and powerful DNA fabrication methods and by the application of engineering principles to biology. The current study describes Terminator Operon Reporter (TOR), a new gene assembly technology based on the conditional activation of a reporter gene in response to sequence errors occurring at the assembly stage of the synthetic element. These errors are monitored by a transcription terminator that is placed between the synthetic gene and reporter gene. Switching of this terminator between active and inactive states dictates the transcription status of the downstream reporter gene to provide a rapid and facile readout of the accuracy of synthetic assembly. Designed specifically and uniquely for the synthesis of protein coding genes in bacteria, TOR allows the rapid and cost-effective fabrication of synthetic constructs by employing oligonucleotides at the most basic purification level (desalted) and without the need for costly and time-consuming post-synthesis correction methods. Thus, TOR streamlines gene assembly approaches, which are central to the future development of synthetic biology.
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.”
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...
The bioengineering of individual microbial organisms or microbial communities has great potential in agriculture, bioremediation and industry. Understanding community level drivers can improve community level functions to enhance desired outcomes in complex environments, whereas individual microbes can be reduced to a programmable biological unit for specific output goals. While understanding the bioengineering potential of both approaches leads to a wide range of potential uses, public acceptance of such technology may be the greatest hindrance to its application. Public perceptions and expectations of "naturalness", as well as notions of disgust and dread, may delay the development of such technologies to their full benefit. We discuss these bioengineering approaches and draw on the psychological literature to suggest strategies that scientists can use to allay public concerns over the implementation of this technology
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.
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...
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.
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.
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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