TO KEEP OLD trains running, operators had to keep a firm grip on a dead man’s switch. If the operator became incapacitated or, well, dead, his hand would loosen, the brakes would engage, and the train wouldn’t turn into a runaway—no active intervention required. That was 20th century engineering.
In the 21st century, where scientists are as likely to engineer microbes as locomotives, “Deadman” is a kill switch created by MIT biologists to prevent engineered microbes from running out of control in the wild. Deadman and another microbial kill switch called Passcode are the newest of the increasingly sophisticated ways biologists hope to control microbes they’re building to cure diseases or clean up oil and toxic spills. Without those controls, the bugs will never leave the lab. “The biggest enemy we have is uncertainty,” says Karmella Haynes, a synthetic biologist at Arizona State University. “We don’t have a practical way to prove tomorrow that GMOs are absolutely dangerous or absolutely safe. The appropriate response to uncertainty is, let’s arm ourselves with an engineering solution.”
The simplest kill switches simply deleted a gene that made some molecule critical to the life of the organism. Without scientists feeding that molecule to the microbe, it died. The risk was that microbes in the wild might find an unexpected source for it.
But earlier this year, scientists at Harvard and Yale described kill switches that force the engineered microbe to rely on lab-made molecules that do not exist in nature. “This year could be characterized as the year of the kill switch,” says James Collins, an MIT synthetic biologist who led the most recent study, published in Nature Chemical Biology.
Collins and his colleagues designed Deadman so that any microbe with it would need a small molecule—a drug, if you will—to repress a genetic circuit for suicide. No small molecule, and the bug goes kaput. Passcode, though, is even cooler. The microbe relies on some combination of three small molecules. It might need, for example, molecules A and B, but the complete absence of C. And you can change the combination. Hence: Passcode.
“That will allow you to really scale up the different combinations for small molecules—or cocktails, if you will,” says Farren Isaacs, a molecular biologist at Yale who was not involved in the research. The fact that Passcode is easily changed will make companies happy for another reason: Keeping engineered microbes secret is hard. Anyone who gets ahold of your proprietary microbe could sequence its genome and copy it. With Passcode, the real secret sauce is the cocktail of small molecules that keep your microbe alive.
In addition to his job at MIT, Collins is a cofounder and scientific advisor to Synlogic, a Cambridge-based company that wants to engineer microbes that can attack pathogens such as cholera. But you don’t want the engineered microbes to stick around forever in the body, and you especially don’t want them to stick around if the patient has an unexpected bad reaction. Collins’ kill switch technology seems to fix all that.
So far, Collins’ paper shows only that Passcode works in E. coli, but based on past experiments, it’s likely to work in other common bacteria as well. A potential roadblock, though, is that bacteria are wily: After four days, subsequent generations became adapted to escape the Deadman and Passcode kill switches. Collins says that a commercial kill switch would probably combine several different strategies and toxins for extra layers of security.
As paradoxical as it might sound, synthetic biology companies have good reason to want to kill their products under the right circumstances: protect the public from possible dangers and protect it from competitors. That’s two for the price of one technology, even better than the dead man’s switch of trains.
Biocontainment systems that couple environmental sensing with circuit-based control of cell viability could be used to prevent escape of genetically modified microbes into the environment. Here we present two engineered safeguard systems known as the 'Deadman' and 'Passcode' kill switches. The Deadman kill switch uses unbalanced reciprocal transcriptional repression to couple a specific input signal with cell survival. The Passcode kill switch uses a similar two-layered transcription design and incorporates hybrid LacI-GalR family transcription factors to provide diverse and complex environmental inputs to control circuit function. These synthetic gene circuits efficiently kill Escherichia coli and can be readily reprogrammed to change their environmental inputs, regulatory architecture and killing mechanism.
Synthetic biology is a multi-disciplinary field that applies engineering techniques to biological systems. While the foundations of synthetic biology were laid in the 1990s with the burgeoning of genomics and automated sequencing, synthetic biology as a discipline, emerged in the early 2000s with the development of simple gene regulatory circuits. Since then, the field has grown beyond making simple circuits to developing diagnostic tools.
Shimyn Slomovic, Keith Pardee, and James J. Collins of MIT and Harvard discuss the current trends in synthetic biology to make diagnostic tools, and what additional hurdles must be overcome before these methods can be applied in the field or in the clinical setting. Their article is part of a special hundredth anniversary series in the Proceedings of the National Academy of Sciences commemorating exceptional research published in the journal over the last century.
The rational design of diagnostic tools in synthetic biology uses natural regulation and detection systems to design sensors. Ideally, these sensors are selective, sensitive, and provide some kind of output, such as fluorescence or a color change, when it has detected the target cell, environmental cue, or pathogen. Slomovic, Pardee, and Collins discuss three broad categories of synthetic biology-based diagnostic tools: whole-cell biosensing, in vitro diagnostics, and in vivo diagnostics.
Whole-cell biosensing
Microorganisms can be employed as a kind of biosensor. One well-known example is photosynthetic cyanobacteria incorporated into hardware. The cyanobacteria are redox active. Electron transfer through the hardware will produce a current, but herbicides quench its redox activity. The lack of current serves as an indicator for the presence of herbicides in places such as natural water reservoirs.
More recent research involves tailoring microorganisms using genetic engineering. Engineering methods allow scientists to design bacteria that are selective for a particular substance. For example, bacteria have been engineered to detect arsenic in water. These genetically engineered bacterial cells have been integrated into circuits and other mechanical components to produce a hybrid device. However, hybrid devices have lost popularity, giving way to plate readers which have a higher throughput.
RNA-based biosensing can detect metabolites or RNA sequences that are specific to a particular disease. RNA biosensing works by releasing stalled RNA translation using a synthetic piece of RNA that contains a complementary portion of the target sequence. One of the difficulties with this technique, however, is that the RNA sensing portions are confined within the bacterial cell and are not accessible to the external environment.
In vitro diagnostics
Bacteriophages are nature's bacteria sensors. They are viruses that home in on particular bacteria and infect them. Engineered bacteriophages are relatively inexpensive to make and can be designed to provide output indicators, such as bioluminescence, when it has detected the presence of a particular bacterial strain. Bacteriophages have been used both as sensors and as alternatives to antibiotics. For example, phages allow scientists to detect the presence of bacteria associated with the bubonic plague in a human blood sample in a matter of hours.
Paper-based diagnostics are a cheap, mobile method that shows promise for eventual field use. Using commercially available bacterial or mammalian transcription/translation systems, scientists are able to freeze-dry these systems on to paper or other porous materials. These systems target certain RNA sequences in a similar way to the RNA-based biosensing, mentioned above, but the circuitry is on paper rather than within the cell. The system becomes active when rehydrated, allowing the freeze-dried material to be easily transported. Recent tests with an Ebola virus disease detection system used color-changing enzymes for easy output.
In vivo diagnostics
Real-time biosensing is attractive for monitoring changes in the organism's internal environment. This can be done with engineered bacteria that are administered to the organism. One example is the use of engineered E. coli to monitor the gut microbiome. In mouse studies, E. coli. were engineered to detect and record exposure of the gut microbiome to a particular drug.
Mammalian cells can also function as in vivo biosensors, and they have the added benefit of possibly serving as both the sensor and the therapy. These cells utilize synthetic gene networks that can either change or monitor the organism's cellular environment. Additionally, cellular biosensing allows the synthetic circuit to be isolated from the environment but also able to interact with it.
One way that mammalian cells have been used for diagnostics is to monitor the expression profiles of certain cells. Cells express, or "turn on", some genes but not others. The particular set of genes that are turned on provides an expression profile. Some cancer cells, cells that have been infected by a virus, or cells whose immune system is malfunctioning (autoimmune) have different expression profiles from normal cells. These cells can be targeted and identified using engineered mammalian cells. This has the added benefit of potentially destroying the diseased cell either by turning on the cell's self-destruct genes or by drug targeting.
Unfortunately, the biggest difficulty with in vivo diagnostics is delivering the engineered cell or synthetic genetic construct to the right place. Slomovic, Pardee, and Collins point out that this is where the field of nanobiotechnology comes into play. Recent research shows that some nanoparticles will home in on certain cells types, and others have been looking at lipid-based vesicles as another possibility for targeted delivery.
Slomovic, Pardee, and Collins point out that "success in synthetic biology will continue to be found at the interfaces between disciplines." Synthetic biology, while still a relatively new field, draws from years of work in genetics, microbiology, engineering, and nanotechnology, and by combining these fields, researchers have been able to make formidable progress in biosensing and in vitro and in vivo diagnostics.
More information: Shimyn Slomovic et al. Synthetic biology devices for in vitro and in vivo diagnostics, Proceedings of the National Academy of Sciences (2015). DOI: 10.1073/pnas.1508521112
Abstract There is a growing need to enhance our capabilities in medical and environmental diagnostics. Synthetic biologists have begun to focus their biomolecular engineering approaches toward this goal, offering promising results that could lead to the development of new classes of inexpensive, rapidly deployable diagnostics. Many conventional diagnostics rely on antibody-based platforms that, although exquisitely sensitive, are slow and costly to generate and cannot readily confront rapidly emerging pathogens or be applied to orphan diseases. Synthetic biology, with its rational and short design-to-production cycles, has the potential to overcome many of these limitations. Synthetic biology devices, such as engineered gene circuits, bring new capabilities to molecular diagnostics, expanding the molecular detection palette, creating dynamic sensors, and untethering reactions from laboratory equipment. The field is also beginning to move toward in vivo diagnostics, which could provide near real-time surveillance of multiple pathological conditions. Here, we describe current efforts in synthetic biology, focusing on the translation of promising technologies into pragmatic diagnostic tools and platforms.
The efficiency of many cellular processes relies on the defined interaction among different proteins within the same metabolic or signaling pathway. Consequently, a spatial colocalization of functionally interacting proteins has frequently emerged during evolution. This concept has been adapted within the synthetic biology community for the purpose of creating artificial scaffolds. A recent advancement of this concept is the use of peptide motifs and their cognate adaptor domains. SH2, SH3, GBD, and PDZ domains have been used most often in research studies to date. The approach has been successfully applied to the synthesis of a variety of target molecules including catechin, D-glucaric acid, H2, hydrochinone, resveratrol, butyrate, gamma-aminobutyric acid, and mevalonate. Increased production levels of up to 77-fold have been observed compared to non-scaffolded systems. A recent extension of this concept is the creation of a covalent linkage between peptide motifs and adaptor domains, which leads to a more stable association of the scaffolded systems and thus bears the potential to further enhance metabolic productivity.
Synthetic biology — which involves engineering biological systems for new uses — has become an increasingly prominent, and promising, field of study in colleges and universities worldwide. Research has yielded, for example, viruses that attack harmful bacteria, yeasts that produce biofuels, and engineered microorganisms capable of detecting toxins in the environment, among many other innovations. Yet high school students rarely learn about synthetic biology at all, says Natalie Kuldell, an instructor of biological engineering at MIT. The issue, she says, is lack of accessible, hands-on curricula for such a rapidly developing field. “With subjects like physics, for instance, you can demonstrate engineering by building Rube Goldberg machines or model bridges,” Kuldell says. “But it’s hard to think how to bring engineering to biology for high school students.” In partnership with high school teachers, Kuldell launched the BioBuilder Educational Foundation in 2011 to provide schools with lab kits and lesson plans — adapted from her own MIT curriculum and MIT research labs — to boost interest and innovation in the field. Today, more than 400 teachers in 43 states are using the formal BioBuilder curriculum. Some have also adapted the coursework for middle school students. Many other teachers worldwide incorporate some of the material, which is freely available online, into their lesson plans. Recently, BioBuilder and Boston’s Museum of Science received a National Science Foundation (NSF) grant to develop synthetic biology kits to send to more than 200 other museums around the country next summer. “It’s certainly taken off,” Kuldell says, adding: “Teachers love learning, and love teaching, and BioBuilder lets them do both.” A new side of biology To use BioBuilder, students start off reading comic books and watching animations that present a specific research problem and experiment. Then it’s off to the lab, where groups of students are provided kits to conduct their own experiments. BioBuilder offers five labs, which include engineering bacteria to emit specific colors or odors, or building circuits. Kits include all necessary tools and materials, such as droppers, centrifuge tubes, circuit parts, and bacteria such as E. coli to study. Teachers can decide which BioBuilder lessons to teach, and how. Some teachers run full semester-long classes, and some pick and choose a few lessons each year; others use it as an afterschool program or final project. David Mangus, head of a new biotechnology program at Brockton High School in Brockton, Massachusetts, has been using several BioBuilder lessons as well as the “Biology by Design” unit — where students must write mock grant proposals — as capstone projects. BioBuilder, he says, has shown students a new side of biology. “It’s a different way of thinking about life,” he says. “It’s what we can do with it, instead of just experiencing it.” A favorite lab among students, Kuldell says, is called “Eau That Smell,” which involves examining bacteria engineered to smell like ripe bananas at a certain stage of their growth. Students must then modify the bacteria to emit that specific odor at different growth stages. “They do a lot of quantitative and qualitative measurements, start thinking about what data they believe, how to improve the system, and how to better engineer a system,” she says. Many students also enjoy “What a Colorful World,” a lab where students study E. coli programmed to change colors. In it, students must consider how different cellular chassis (structures) might be engineered to control the emitted color, “just as a car’s chassis must be tailored to the engine it will house,” Kuldell says. When the labs are complete, students may share any data through an online portal run by BioBuilder. Kuldell also hosts an afterschool BioBuilderClub for students and teachers around the world, where students can engineer their own biotechnology designs. Students also have the opportunity to present their work to each other and to synthetic biologists throughout the year. This encourages students to really get involved and “own their data,” Kuldell says. Impact beyond the classroom Over the years, BioBuilder has served as a launchpad for notable projects and influenced the career choices of some students. Last year, a team of high school students from the Taipei American School in Taiwan used their BioBuilder classwork to engineer an extracellular protein that binds to and inhibits Granzyme B, an enzyme that causes tissue damage. For their project, the students won the grand prize for the high school track at this year’s International Genetically Engineered Machine (iGEM) Competition in Boston, the world’s top synthetic biology competition for college and high school students. At Brockton High School, sophomore Aysha Cheretakis, one of Mangus’ students, has become a founding member of the school’s BioBuilderClub, which is currently engineering an organism to detect Rohypnol (a drug commonly referred to as “roofies”) in drinks, which they hope will be used in crime labs. Cheretakis became interested in synthetic biology after the “real-life laboratory experience” of BioBuilder’s “What a Colorful World” lab. “It’s not just stuff we read out of a book. In the lab we can apply our knowledge to the real world,” she says. For Michael Sheets, a graduate of Tyngsborough High School in Tyngsborough, Massachusetts, BioBuilder was the gateway to studying microbiology at Olin College. The program, he adds, also gave him a “fantastic boost” in his college research: “The knowledge and skills I gained have stayed with me and been immensely useful in my education and internships since, and I’m sure will continue to aid me throughout my career.” Building BioBuilder Kuldell launched BioBuilder as part of a NSF grant, called SynBERC, which provided 10 years of funding for synthetic biology research. The grant funded Kuldell’s partnership with several area high school teachers who came to MIT for summer research. Together they developed an online educational platform for synthetic biology, based on research at MIT and elsewhere. Teachers who were interested in bringing those lessons to their labs and classrooms came to a professional development workshop at MIT and returned to their schools across the nation to pilot the program. Soon the BioBuilder curriculum and professional development program grew in popularity and Kuldell turned to the MIT Venture Mentoring Service (VMS) for support in launching the program as a nonprofit. Among other things, they helped Kuldell form BioBuilder’s successful “training the trainers” model, where high school teachers not only learn the curriculum at workshops but also learn to teach the curriculum to other teachers at their own schools. “VMS were so important in helping me think about how to make something sustainable, how to speak about it, how to bring in partners, and develop partnerships,” she says. “They were invaluable.” Next up for Kuldell is establishing a teaching lab for BioBuilder at LabCentral, a shared incubator for biotech startups in Cambridge. BioBuilder recently moved headquarters there, after years of operating out of MIT’s Department of Biological Engineering. The proposed BioBuilder@LabCentral Innovation Lab would help connect students and teachers with entrepreneurs in the area. She also hopes to launch a program there where community college students can fulfill part of a synthetic biology certificate program to help them land jobs at biotech firms. “The same way we make connections with teachers in middle and high schools, we hope to make connections with students to get them from coursework into careers,” she says.
Jeff Carroll inherited the DNA mutation that causes Huntington’s disease. It means that in a decade or two, he’ll lose control over his body and slowly go mad, just like his mother did.
That’s the reason Carroll, 38, says he’d be in favor of gene editing embryos. He says the idea of correcting DNA errors in the next generation has no “ick factor” for him at all. “I have no compunctions about it,” says Carroll, who is a neuroscientist at Western Washington University, in Bellingham. “I am saying, please, please do mess with our DNA.”
This week in Washington, D.C., hundreds of scientists and ethicists are meeting at the National Academies of Science to debate whether society should sanction or prohibit “germ-line engineering,” or altering the DNA of sperm, eggs, or embryos to correct genetic defects before children are born.
It’s a controversial idea, but now easier than ever to do because of a powerful technology called CRISPR. Many experts at the meeting seem to be leaning toward endorsing an indefinite moratorium on any effort to create gene-modified babies, calling the technology too new, too unsafe, and having too few medical uses, a position that has been endorsed by the Obama administration.
But when MIT Technology Review reached out to several families who’ve dealt with devastating genetic illnesses, all said they approved of using the technology as quickly as possible. That could create a potential clash between desperate families and cautious scientists and politicians.
To some, gene editing means the possible end of inherited genetic disease. In the future, seeing a case of Tay-Sachs or cystic fibrosis might be just as rare as finding a case of polio is today. Others warn of a slippery slope toward “consumer eugenics” and out-of-control changes to the gene pool. “Although gene editing is in its infancy, it is likely that the pressure to use it will increase,” says David Baltimore, a Nobel Prize-winning professor at the California Institute of Technology who is leading the deliberations in Washington (see “Everything You Need to Know About CRISPR Gene Editing’s Monster Year”).
The debate so far is highly technical and abstract. But that will change once patients get involved. Sharon Terry, CEO of a patient group called the Genetic Alliance, says her in-box has been filled with e-mails from families, with many expressing views like, “Hell yes!” although others still haven’t heard about CRISPR, a technology developed only three years ago.
Promoters are indispensable elements of a standardized parts collection for synthetic biology. Regulated promoters of a wide variety of well-defined induction ratios and expression strengths are highly interesting for many applications. Exemplarily, we discuss the application of published genome scale transcriptomics data for the primary selection of methanol inducible promoters of the yeast Pichia pastoris (Komagataella sp.). Such a promoter collection can serve as an excellent toolbox for cell and metabolic engineering, and for gene expression to produce heterologous proteins.
WHEN CHINESE SCIENTISTS announced in April they had edited human embryos using a new genetic tool called Crispr, the headlines blared “designer babies,” and the world woke up to Crispr’s power. But for those looking closely at the data, the paper was a relief.
Crispr did not work very well to edit the human embryos—nonviable ones, to reduce the ethical problems with the research. In fact, it worked terribly. Out of 54 embryos the team altered with Crispr and tested, only four ended up with the right genetic tweaks. And even these four embryos had off-target mutations, places where Crispr edited the DNA even though it shouldn’t have. Off-target mutations in the wrong gene can be dangerous, and the paper was proof it was too unsafe to “fix” viable human embryos with Crispr. At least for now.
Today, on the first day of an international summit on human gene editing in Washington, DC, MIT researchers announced that they have tweaked a Crispr protein to reduce those off-target effects. It’s the latest in a series of improvements to the Crispr system that, together, are inching the error rate down toward practically zero.
As long as Crispr creates too many off-target mutations, it’s easy to assert that the technology is not predictable, and that editing human embryos is therefore not ethical. Of the five steps Crispr co-inventor Jennifer Doudna recently proposed in Nature for oversight of human gene editing, the first is adopting standards for measuring efficiency and off-target effects. But if gene editing with Crispr is safe, do the ethics change? That’s a big question for the DC meeting—one that Crispr scientists have often tiptoed around.
“Safety is something we all agree is important. There’s a language for that that’s acceptable to all sides,” says Josephine Johnston, director of research at the Hastings Center. “But the other kinds of concerns that people have about this work are much more difficult to have conversations about.” Those concerns are not scientific but social, including questions about consent of unborn babies and the roles of humans in making permanent genetic changes. As the Crispr technology improves, the safety concerns are peeling away to reveal the ethical concerns at the core.
On face of it, the MIT paper is yet another molecular nuts and bolts study with a barely scrutable title (“Rationally engineered Cas9 nucleases with improved specificity”). Crispr is essentially a pair of DNA scissors, and Cas9 is the protein in the Crispr system that unzips DNA and runs along looking for its target by matching the DNA sequence against a snippet of its guide RNA. When Cas9 finds its target, snip snip. The problem is that Cas9 will sometimes think it’s found a target even when up to five of the guide RNA’s approximately 20 letters do not match the DNA—hence the off-target mutations.
Feng Zhang, another scientist with a claim to CRISPR’s invention as a gene-editing technique, and his colleagues at MIT changed one part of Cas9 slightly so its guide RNA binds fewer mismatched DNA sequences. This improved Cas9’s specificity by about 25-fold at the sites they tested. Zhang’s lab, along with that of other labs of Harvard, have come up with several other ways to make Cas9 better behaved, such as pairing up Cas9s so they only work in twos and both guide RNAs match.
With these improvements, Harvard geneticist George Church estimates that Crispr’s error rate, best-case scenario, could be just 1 in 300 trillion letters of DNA. (The rate can vary quite widely in different types of cells and with different guide RNA designs.) Even at the higher end, that’s comparable to the spontaneous mutation rate in humans, says Church.
He asserts that off-target mutations are already a solved problem. With typical Churchian flair, he says, “I’m beginning to feel like this is hitting a fly with sledgehammer. I think the thing is already dead.” Even if not all scientists agree about off-target mutations yet, solving the problem of Crispr’s non-specificity is looking more like a question of when rather than if.
A conversation about the ethics of human modification has been kicking around since the 1980s, when scientists started using cruder forms of gene editing. “For decades, we’ve been able to say it’s not there yet, so we’re not going to do [human gene editing]. It was an easy way to stop the conversation,” says Debra Mathews, a bioethicist at Johns Hopkins University. “We’re now at a point where it is precise enough that we do actually just have to have the conversation.” Over the next three days in DC, scientists and policymakers will have to do just that.
Protein synthesis is essential for bacterial growth and survival. Its study in Escherichia coli helped uncover features conserved among bacteria as well as universally. The pattern of discovery and the identification of some of the longest-known components of the protein synthesis machinery, including the ribosome itself, tRNAs, and translation factors proceeded through many stages of successively more refined biochemical purifications, finally culminating in the isolation to homogeneity, identification, and mapping of the smallest unit required for performing the given function. These early studies produced a wealth of information. However, many unknowns remained. Systems biology approaches provide an opportunity to investigate protein synthesis from a global perspective, overcoming the limitations of earlier ad hoc methods to gain unprecedented insights. This chapter reviews innovative systems biology approaches, with an emphasis on those designed specifically for investigating the protein synthesis machinery in E. coli.
Introducing random mutations into DNA with in vitro techniques is easy, but getting this mutagenized DNA back into cells often is not. Performing mutagenesis directly in cells allows researchers to avoid this bottleneck, but current in vivo methods show only modest mutation rates. To improve these rates, Badran and Liu created and tested several plasmids that include genes affecting mutation frequency. The final plasmid tested, MP6, encodes dominant negative variants of a DNA-proofreading enzyme, a protein that impairs mismatch repair, a cytidine deaminase that increases bas…
These are some awesome books to get you started in synthetic biology and the business/economics thereof. There’s no Dummies How To book (yet) but these all factored into how I think about this still-emerging field. I also recommend setting up Google Alerts for synbio and watching the incoming stories in real time. Short on reading time? Begin with Rob Carlson’s Biology is Technology or Juan Enriquez’s poetic As the Future Catches You.
Juan Enriquez, As the Future Catches You Kevin Kelly, What Technology Wants Chris Anderson, The Long Tail* Clay Shirky, Here Comes Everybody* Regenesis, George Church J. Craig Venter, A Life Decoded Chris Anderson, Free* Marcus Wohlsen, Biopunk Rob Carlson, Biology is Technology J. Craig Venter, Life at the Speed of Light
The engineering ideal of synthetic biology presupposes that organisms are composed of standard, interchangeable parts with a predictive behaviour. In one word, organisms are literally recognized as machines. Yet living objects are the result of evolutionary processes without any purposiveness, not of a design by external agents. Biological components show massive overlapping and functional degeneracy, standard-free complexity, intrinsic variation and context dependent performances. However, although organisms are not full-fledged machines, synthetic biologists may still be eager for machine-like behaviours from artificially modified biosystems.
Are you interested in Synthetic Biology? The Massachusetts Institute of Technology is offering a free online course developed by Adam Arkin and Ron Weiss named “Principles of Synthetic Biology.” Th...
Molecular and synthetic biology have made better tools for the understanding of our bodies, the treatment of human diseases, the production of our food sources ...
By Grant Vousden-Dishington Founder @johncumbers kicking off #SBBSF15pic.twitter.com/xh0FXOVouL — SynBioBeta (@SynBioBeta) November 4, 2015 Two months ago, PLOS Synbio covered and sponsored the iGEM 2015 Giant Jamboree, where John Cumbers was...
Synthetic Biology Open Language (SBOL) Visual is a graphical standard for genetic engineering. It consists of symbols representing DNA subsequences, including regulatory elements and DNA assembly features. These symbols can be used to draw illustrations for communication and instruction, and as image assets for computer-aided design. SBOL Visual is a community standard, freely available for personal, academic, and commercial use (Creative Commons CC0 license). We provide prototypical symbol images that have been used in scientific publications and software tools. We encourage users to use and modify them freely, and to join the SBOL Visual community: http://www.sbolstandard.org/visual.
Tim Lu's synthetic-biology research at Massachusetts Institute of Technology in Cambridge combines biological engineering with electronics and computer science to create bacteria that make structural proteins containing tiny semi-conductors called quantum dots. He explains how genome-editing techniques are furthering his research and their role in treating disease.
Many advances in synthetic biology require the removal of a large number of genomic elements from a genome. Most existing deletion methods leave behind markers, and as there are a limited number of markers, such methods can only be applied a fixed number of times. Deletion methods that recycle markers generally are either imprecise (remove untargeted sequences), or leave scar sequences which can cause genome instability and rearrangements. No existing marker recycling method is automation-friendly. We have developed a novel openly available deletion tool that consists of: 1) a method for deleting genomic elements that can be repeatedly used without limit, is precise, scar-free, and suitable for automation; and 2) software to design the method’s primers. Our tool is sequence agnostic and could be used to delete large numbers of coding sequences, promoter regions, transcription factor binding sites, terminators, etc in a single genome. We have validated our tool on the deletion of non-essential open reading frames (ORFs) from S. cerevisiae. The tool is applicable to arbitrary genomes, and we provide primer sequences for the deletion of: 90% of the ORFs from the S. cerevisiae genome, 88% of the ORFs from S. pombe genome, and 85% of the ORFs from the L. lactis genome.
The RNA-guided endonuclease Cas9 is a versatile genome editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that “enhanced specificity” SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome editing applications requiring a high level of specificity.
Synthetic biology: Elegans control by auxin Nature.com A fundamental strategy used to study the function of proteins in a complex biological system is to make them 'appear' and 'disappear' at will, and then analyse the consequences.
Researchers at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT have engineered changes to the revolutionary CRISPR-Cas9 genome editing system that significantly cut down on “off-target” editing errors. The refined technique addresses one of the major technical issues in the use of genome editing. The CRISPR-Cas9 system works by making a precisely targeted modification in a cell's DNA. The protein Cas9 alters the DNA at a location that is specified by a short RNA whose sequence matches that of the target site. While Cas9 is known to be highly efficient at cutting its target site, a major drawback of the system has been that, once inside a cell, it can bind to and cut additional sites that are not targeted. This has the potential to produce undesired edits that can alter gene expression or knock a gene out entirely, which might lead to the development of cancer or other problems. In a paper published today in Science, Feng Zhang and his colleagues report that changing three of the approximately 1,400 amino acids that make up the Cas9 enzyme from S. pyogenes dramatically reduced “off-target editing” to undetectable levels in the specific cases examined. Zhang is the W.M. Keck Career Development Professor in Biomedical Engineering in MIT’s departments of Brain and Cognitive Sciences and Biological Engineering, and a member of both the Broad Institute and McGovern Institute. Zhang and his colleagues used knowledge about the structure of the Cas9 protein to decrease off-target cutting. DNA, which is negatively charged, binds to a groove in the Cas9 protein that is positively charged. Knowing the structure, the scientists were able to predict that replacing some of the positively charged amino acids with neutral ones would decrease the binding of “off target” sequences much more than “on target” sequences. After experimenting with various possible changes, Zhang’s team found that mutations in three amino acids dramatically reduced “off-target” cuts. For the guide RNAs tested, “off-target” cutting was so low as to be undetectable. The newly-engineered enzyme, which the team calls “enhanced” S. pyogenes Cas9, or eSpCas9, will be useful for genome editing applications that require a high level of specificity. The Zhang Lab is immediately making the eSpCas9 enzyme available for researchers worldwide. The team believes the same charge-changing approach will work with other recently described RNA-guided DNA targeting enzymes, including Cpf1, C2C1, and C2C3, which Zhang and his collaborators reported on earlier this year. The prospect of rapid and efficient genome editing raises many ethical and societal concerns, says Zhang, who is speaking this morning at the International Summit on Gene Editing in Washington. “Many of the safety concerns are related to off-target effects,” he says. “We hope the development of eSpCas9 will help address some of those concerns, but we certainly don’t see this as a magic bullet. The field is advancing at a rapid pace, and there is still a lot to learn before we can consider applying this technology for clinical use.”
Synthetic biology is associated with great expectations as well as grave concerns. Currently, due to the small number of concrete applications, an assessment of innovations in synthetic biology has to start with an analysis of early indicators of both the opportunities and the risk-relevant features of expected applications.
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