If anyone understands the challenges associated with powering a eukaryotic cell with a designer genome built from scratch, it’s Leslie Mitchell, a postdoc in Jef Boeke’s lab at NYU. Mitchell has been leading experimental design and technological development for the Synthetic Yeast Genome Project (Sc2.0) since 2012. In this role, she collaborates with Sc2.0 International Consortium team members spanning 4 continents to provide remote mentorship and solve challenges associated with synthetic chromosome design features and assembly.
The goal of Sc2.0 is to design, build, assemble, and test the function of an entirely synthetic designer yeast genome. Earlier this month, the Sc2.0 International Consortium announced the completed design of the Sc2.0 genome, construction of five new synthetic chromosomes, and described the 3D organization of synthetic chromosomes in the nucleus, all of which earned them the March 10 cover story for Science. To date, 30% of the Sc2.0 genome has been constructed in cells, and the team hopes to have the entire genome – all 16 chromosomes – completed before the end of 2017.
According to Mitchell, the genome engineering effort of Sc2.0 can offer some powerful lessons for GP-write, both from an organizational and technical perspective.
Organizational Insights A well defined plan should be in place at the launch of the project, which all participants must agree upon, including funding, space, personnel, QA/standards, material transfers, publication policy, intellectual property, software, ownership of the project, training and education, and compliance with local laws. Additionally, because GP-write will involve global participation similar to Sc2.0, it will be important to maintain a collaborative and inclusive culture across international borders.
One organizational feature that has driven success of Sc2.0 is the distributive nature of the project. While Sc2.0 chromosome design is centralized, involving a close collaboration between yeast geneticists of Jef Boeke’s lab and computational biologists from Joel Bader’s lab at Johns Hopkins University, synthesis and assembly are parallelized between teams around the world. Not only does this partition the workload associated with chromosome assembly, it also distributes the financial burden as each team is responsible for obtaining their own funding.
Technical Insights Following a common design standard has been a key strategy for Sc2.0. All design features that are written into Sc2.0 chromosomes adhere to a set of overarching design principles, the goals of which are to direct growth to wild-type levels while simultaneously increasing genome flexibility and stability.
Another lesson learned from Sc2.0 is to take on a piece-by-piece assembly strategy. Such a strategy is practical in that it’s much easier to manipulate smaller segments of DNA in vitro, for example 30-60 kb segmented into 10 kb ‘chunks’. More importantly, this strategy allows the team to evaluate synthetic DNA function in cells along the way and quickly back track to any design features that negatively impact cell fitness. For the Sc2.0 project it is now clear that despite densely spaced clusters of edits (mean distance of ~400bp), the overall design is robust as few ‘bugs’, or designer changes that affect cell fitness, have been uncovered.
Sc2.0 has also highlighted limitations of gene synthesis and how these limitations may impact our ability to design and build synthetic genomes. While cost is a much-discussed issue associated with gene synthesis, a less publicized problem is that not all sequences can be easily synthesized de novo. It all comes down to sequence composition. For instance ‘low complexity DNA’ such as homopolymer runs and regions with extreme GC or AT richness can be particularly difficult to synthesize. Of course with enough time, money and effort most DNA sequences can eventually be built; de novo designed mammalian genome-scale synthesis however will require a drop in cost by orders of magnitude together with technology improvements to enable DNA synthesis without restrictions on sequence composition.
DNA delivery is another area ripe for technology development for GP-write. Yeast cells are easily transformed and incorporate DNA into their genome readily using their natural capacity for homologous recombination. Together these features have enabled an efficient Sc2.0 DNA delivery strategy encompassing segments of 30-60kb of designer DNA. The Sc2.0 strategy can applied on some level for GP-write, but there will be delivery challenges that will need to be addressed as we move into mammalian systems, in particular with respect to the delivery of increasingly large segments of designer DNA for targeted delivery.
An additional challenge that GP-write will face with respect to building artificial mammalian chromosomes is centromere engineering. Unlike budding yeast point centromeres, which are ~125 bp and easy to synthesize, mammalian centromeres are composed of megabases of highly repetitive sequences. Mitchell and her colleagues are currently trying to overcome this engineering challenge with the goal of building mammalian artificial chromosomes that will be stable over many generations of cells. Centromere engineering strategies starting from the ‘top-down’, by minimizing native chromosomes, as well as from the ‘bottom-up’, via de novo establishment of centromere function, will both be important to pursue.
With encoded features that enable genetic flexibility and increased genomic stability, Sc2.0 will be a designer genome with new capabilities. As a result, we will soon be able to ask new biological questions – about evolution, minimal genome sequences that support viability under different conditions, and the requirement of specific genomic features such as repeats and introns. Completion of Sc2.0 will represent a major stepping-stone for GP-write, which will build on the knowledge and technological advances of this project.
We invite you to join Mitchell and her colleagues at the next GP-write meeting on May 9-10th, 2017, which will be held at the New York Genome Center. Mitchell will be speaking on the topic of Genome Engineering Foundries at 11:30 am on May 10th. View the agenda.
Simbiotics is a spatially explicit multi-scale modeling platform for the design, simulation and analysis of bacterial populations. Systems ranging from planktonic cells and colonies, to biofilm formation and development may be modeled. Representation of biological systems in Simbiotics is flexible, user-defined processes may be in a variety of forms depending on desired model abstraction. Simbiotics provides a library of modules such as cell geometries, physical force dynamics, genetic circuits, metabolic pathways, chemical diffusion and cell interactions. Model defined processes are integrated and scheduled for parallel multi-thead and multi-CPU execution. A virtual lab provides the modeler with analysis modules and some simulated lab equipment, enabling automation of sample interaction and data collection. An extendable and modular framework allows for the platform to be updated as novel models of bacteria are developed, coupled with an intuitive user interface to allow for model definitions with minimal programming experience. Simbiotics can integrate existing standards such as SBML, and process microscopy images to initialise the 3D spatial configuration of bacteria consortia. Two case studies, used to illustrate the platform flexibility, focus on the physical properties of the biosystems modeled. These pilot case studies demonstrate Simbiotics versatility in modeling and analysis of natural systems and as a CAD tool for synthetic biology.
If we hope to one day leave Earth and explore the universe, our bodies are going to have to get a lot better at surviving the harsh conditions of space. Using synthetic biology, Lisa Nip hopes to harness special powers from microbes on Earth -- such as the ability to withstand radiation -- to make humans more fit for exploring space. "We're approaching a time during which we'll have the capacity to decide our own genetic destiny," Nip says. "Augmenting the human body with new abilities is no longer a question of how, but of when."
Rapid and reliable diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) is crucial for guiding effective patient treatment and preventing the spread of MRSA infections. Nonetheless, further simplification of MRSA detection procedures to shorten detection time and reduce labor relative to that of conventional methods remains a challenge. Here, we have demonstrated a Clustered regularly interspaced palindromic repeats (CRISPR)-mediated DNA-FISH method for the simple, rapid and highly sensitive detection of MRSA; this method uses CRISPR associated protein 9/single-guide RNA (dCas9/sgRNA) complex as a targeting material and SYBR Green I (SG I) as a fluorescent probe. A dCas9/sgRNA-SG I based detection approach has advantages over monoclonal antibody in conventional immunoassay systems due to its ability to interact with the target gene in a sequence-specific manner. The detection limit of MRSA was as low as 10 cfu/ml and was found to be sufficient to effectively detect MRSA. Unlike conventional gene diagnosis methods in which PCR must be accompanied or genes are isolated and analyzed, the target gene can be detected within 30 min with high sensitivity without performing a gene separation step by using cell lysates. We showed that the fluorescence signal of the MRSA cell lysate was more than 10-fold higher than that of methicillin-susceptible S. aureus (MSSA). Importantly, the present approach can be applied to any target other than MRSA by simply changing the single-guide RNA (sgRNA) sequence. Because dCas9/sgRNA-SG I based detection approach has proved to be easy, fast, sensitive, and cost-efficient, it can be applied directly at the point of care to detect various pathogens as well as MRSA in this study.
From 2008 to 2014 the United States invested around $820 million in synthetic biology research. About 0.07% as much money went to synthetic biology projects through crowdfunding sites like Kickstarter, Indiegogo, or Experiment.com. Of those crowdfunded projects, the Kickstarter campaign called Glowing Plants was by far the most well-funded. Glowing Plants, like its name suggests, aimed to genetically engineer plants that could light up using the genes from fireflies. After a tenuous four years of alternative funding and backup plans, the goal of making a brightly glowing plant is officially shelved. The question is whether Glowing Plants will continue to be the high mark for crowding funding of synthetic biology or whether there are future successes out there.
The ‘Glowing Plants’ Kickstarter is no more
In 2013, Antony Evans got well over seven times his $65,000 crowdfunding goal to engineer plants that light up. The pitch laid out an ambitious plan for how this fun glowing plant fit into the promise of synthetic biology. It was nothing like a technical grant proposal that a synthetic biology researcher usually submits for funding. It was selling the idea of a synthetic biology product that you could put on your desk. A fun novelty item that’s more play than work. Unfortunately, technical hurdles exist even for playful ideas.
If you go back through Glowing Plants updates you can track the arc of their ambitions.
April 23, 2013: Glow Plants project launches on Kickstarter
April 30, 2013: “How bright can we make our plants grow?”
August 13, 2014: “accepted by Y combinator!” They got to present at the tech accelerator’s Demo Day and pitch investors.
April 10, 2015: Should we switch plant species
May 20, 2016: “Help us build momentum on WeFunder!” This platformed helped to raise another $250,000 for the project with donors buying small shares
August 3, 1016: “5 out of 6 genes successfully integrated into a plant”
April 18, 2017: “Stopping work on the Glowing Plant” Contamination in the fragrant moss strains caused the team to downsize to still afford to get the planned shipments out. With the financial hit, their plan to put revenue toward the glowing plant research was ultimately halted. They conclude that “that despite that failure the project can still leave a positive legacy in inspiring people to learn more about synthetic biology and its benefits – and hopefully one day someone does finally make a Glowing Plant.”
There has been plenty of media attention for both the initial hype of Glowing Plants and the eventual decline into unrealized ideas, but that doesn’t mean there won’t be other high profile uses of crowdfunding for synthetic biology.
There’s still interest crowdfunding synthetic biology
Synthetic biology still makes it way onto a lot of the popular crowdfunding sites that have science: Kickstarter, Indiegogo, WeFunder and experiment.com. Yet none of these campaigns have reacher the financial backing that Glowing Plants got. Typical synthetic biology crowdfunding is raising a few thousand dollars and is primarily supporting educational activities.
The most common kind of crowdfunding success involves the International Genetically Engineered Machine (iGEM) competition that gives student the chance to work on and present their own synthetic biology projects. Some successfully funded research projects include $3001 for DNA memory, $2500 for detecting tuberculosis, $2189 for paper based pathogen detection, $1572 for detection of expired oxytocin medication, and $2120 for a high-school team studying crosstalk between neighboring genes. Typical federal grants for synthetic biology research on the orders of hundreds of thousands to millions of dollars over a few years.
But there’s still mismatche between synthetic biology and crowdfunding
Synthetic biology research is still expensive. For a synthetic biology product to be funded and hit the market takes an enormous amount of money. While iGEM teams can count student learning itself as a victory, any crowdfunding that promises a deliverable product will be in trouble. For instance, experiment.com has a collection of iGEM projects being funded.
Glowing Plant’s creator, Antony Evans, wrote a piece on equity crowdfunding and explained why the “inherent tension between promising rewards and the challenges and uncertainties of biological research” makes Kickstarter a bad model for scientific research. He sees equity crowdfunding as a more powerful tool in the biotech space. This newly legal mechanism called title III equity crowdfunding allows early stage startups to raise money from any ordinary person in return for shares in the company.
But no crowdfunding mechanism brings along the advisory input one would expect from a traditional investor. Good investors usually bring some expertise, guidance, or connections. Distributed funding is unlikely to bring that human capital. Even if some of your investors have those capabilities, none will have enough influence in the company to make an impact.
What do we need for successful crowdfunded synbio projects?
Synthetic biology will continue to get cheaper as DNA synthesis costs drop and the DIYBio community matures. That said, there are still many research projects that seem poorly suited for crowdfunding.
Some tangible product for the consumer – Crowdfunders like rewards. They don’t always have to be particular useful but Kickstart has shown that users are willing to put in money to feel a part of something and get a real thing as a token of that participation. Although equity crowdfunding puts the reward as share in the potential financial success, there will still need to be some product to ever have a return. That’s not a pressure on most research projects. Other side benefits other than the ultimate success of the design – Worthy achievements like student education, data useful to other projects, and new tools can come out of even unsuccessful projects and encourage more backers. Build on the work of others – Startup companies are often building out of intellectual property already developed in research labs. The challenge is to commercialize the innovation. Right now too many ideas are still being pitched for crowdfunding without any initial research backing the idea up. For synthetic biology, open science projects may provide enough genetic parts to let people use them off the shelf. Cheap ideas – If you want to take an idea to market without venture capital, it would have to be extremely low cost given the money currently out there in crowdfunding. The more likely approach is to use crowdfunding to get the attention and initial work done to attract traditional investors. Better metrics and transparency – Traditional investors have more power over and insight into the development of the startup. If equity crowdfunding is going to allow for people to make smart investments, the crowd needs more tools to properly assess the proposal. A success – A high profile success would go a long way to boost crowdfunding. Equity crowdfunding in particular needs a win to show that it’s actually possible to make money. Until then, it’s tough to see it attracting more than the early adopters. Ultimately, I think synthetic biology will get some victories in the crowdfunding arena. It’s unlikely to be a cure for cancer or global warming, but hopefully there’s a fun idea out there that can be made possible with small dollar backers and the power of biology.
Extra reading
See the appendix to the U.S. Trends in Synthetic Biology Research from the Wilson Center to calculate synthetic biology funding from 2008-2014.
Atlantic: “Whatever Happened to the Glowing Plant Kickstarter?”
MIT Tech Review: “Why Kickstarter’s Glowing Plant Left Backers in the Dark”
Synbiobeta: Review of RevBio’s Crowd Funding Campaign
Using new gene-editing technology, researchers have rewired mouse stem cells to fight inflammation caused by arthritis and other chronic conditions. Such stem cells, known as SMART cells (Stem cells Modified for Autonomous Regenerative Therapy), develop into ... Read more
Realizing the value of synthetic biology in biotechnology and medicine requires the design of molecules with specialized functions. Due to its close structure to function relationship, and the availability of good structure prediction methods and energy models, RNA is perfectly suited to be synthetically engineered with predefined properties. However, currently available RNA design tools cannot be easily adapted to accommodate new design specifications. Furthermore, complicated sampling and optimization methods are often developed to suit a specific RNA design goal, adding to their inflexibility. RESULTS: We developed a C ++ library implementing a graph coloring approach to stochastically sample sequences compatible with structural and sequence constraints from the typically very large solution space. The approach allows to specify and explore the solution space in a well defined way. Our library also guarantees uniform sampling, which makes optimization runs performant by not only avoiding reevaluation of already found solutions, but also by raising the probability of finding better solutions for long optimization runs. We show that our software can be combined with any other software package to allow diverse RNA design applications. Scripting interfaces allow the easy adaption of existing code to accommodate new scenarios, making the whole design process very flexible. We implemented example design approaches written in Python to demonstrate these advantages. AVAILABILITY: RNAblueprint, Python implementations and benchmark data sets are available at github: https://github.com/ribonets /.
The discovery of the CRISPR/Cas9 system in bacteria has ignited the world of gene editing. It has been applied to the modification of a growing range of organisms, with none found yet that can resist its powers. In particular, CRISPR technology has revolutionized the process for creating genetically modified mice—the workhorses for in vivo cancer research—allowing for shorter timelines and in many cases, more efficient procedures.
Once activated, 10 newly described CRISPR enzymes behave like Pac-Man to run amok and destroy RNA. The findings could be useful for detecting infectious viruses.
The new enzymes are variants of a CRISPR protein, Cas13a. In September, scientists reported in the journal Nature that Cas13a could be used to detect specific sequences of RNA, such as those from a virus. They showed that once it binds to its target RNA, it begins to indiscriminately cut up all RNA linked to a reporter molecule, making it fluoresce to allow signal detection.
Researchers subsequently paired CRISPR-Cas13a with RNA amplification, and showed that the system—which they dubbed Sherlock—could see viral RNA at extremely low concentrations to detect the presence of dengue and Zika, for example.
Such a system could be used to detect any type of RNA, including RNA distinctive of cancer cells, researchers say.
The Gordon Research Seminar on Synthetic Biology is a unique forum for graduate students, post-docs, and other scientists with comparable levels of experience and education to present and exchange new data and cutting edge ideas.
3D bioprinting is a pretty large category, and it isn't just about manufacturing organs. Scientists and researchers all over the world are using 3D printing technology to build tissue lattices, matrices, and scaffolds, for the purpose of regenerating living tissue or engineering it.
Simplified methods to assemble DNA fragments by independent cloning sequence have helped in the progress of synthetic biology, allowing some biotechnological processes to become economically viable by genetic improvement of microorganisms. We compared three methods of assembling six DNA fragments: PCR fusion-based, isothermal NEBuilder and circular polymerase extension cloning (CPEC). Double and triple fusion occurs directly with the PCR products using PCR fusion-based and NEBuilder methods. For multiple fragments the results showed higher efficiency by the CPEC method which allowed assembly of six fragments previously purified by agarose gel extraction, after a sequence of 20 annealing/extension cycles without any primer.
Here, I have explained LBS and Cello languages. Also, I have demonstrated how to use Cello Online Platform to create your own genetic circuit easily. [S
Tissues are the organizational units of function in metazoan organisms. Tissues comprise an assortment of cellular building blocks, soluble factors, and extracellular matrix (ECM) composed into specific three-dimensional (3-D) structures. The capacity to reconstitute tissues in vitro with the structural complexity observed in vivo is key to understanding processes such as morphogenesis, homeostasis, and disease. In this article, we describe DNA-programmed assembly of cells (DPAC), a method to fabricate viable, functional arrays of organoid-like tissues within 3-D ECM gels. In DPAC, dissociated cells are chemically functionalized with degradable oligonucleotide “Velcro,” allowing rapid, specific, and reversible cell adhesion to a two-dimensional (2-D) template patterned with complementary DNA. An iterative assembly process builds up organoids, layer-by-layer, from this initial 2-D template and into the third dimension. Cleavage of the DNA releases the completed array of tissues that are captured and fully embedded in ECM gels for culture and observation. DPAC controls the size, shape, composition, and spatial heterogeneity of organoids and permits positioning of constituent cells with single-cell resolution even within cultures several centimeters long.
Clostridium saccharoperbutylacetonicum N1-4 is well known as a hyper-butanol-producing strain. However, the lack of genetic engineering tools hinders further elucidation of its solvent production mechanism and development of more robust strains. In this study, we set out to develop an efficient genome engineering system for this microorganism based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (CRISPR-Cas9) system. First, the functionality of the CRISPR-Cas9 system previously customized for Clostridium beijerinckii was evaluated in C. saccharoperbutylacetonicum by targeting pta and buk, two essential genes for acetate and butyrate production, respectively. pta and buk single and double deletion mutants were successfully obtained based on this system. However, the genome engineering efficiency was rather low (the mutation rate is <20%). Therefore, the efficiency was further optimized by evaluating various promoters for guide RNA (gRNA) expression. With promoter PJ23119, we achieved a mutation rate of 75% for pta deletion without serial subculturing as suggested previously for C. beijerinckii. Thus, this developed CRISPR-Cas9 system is highly desirable for efficient genome editing in C. saccharoperbutylacetonicum. Batch fermentation results revealed that both the acid and solvent production profiles were altered due to the disruption of acid production pathways; however, neither acetate nor butyrate production was eliminated with the deletion of the corresponding gene. The butanol production, yield, and selectivity were improved in mutants, depending on the fermentation medium. In the pta buk double deletion mutant, the butanol production in P2 medium reached 19.0 g/liter, which is one of the highest levels ever reported from batch fermentations.
IMPORTANCE An efficient CRISPR-Cas9 genome engineering system was developed for C. saccharoperbutylacetonicum N1-4. This paves the way for elucidating the solvent production mechanism in this hyper-butanol-producing microorganism and developing strains with desirable butanol-producing features. This tool can be easily adapted for use in closely related microorganisms. As also reported by others, here we demonstrated with solid data that the highly efficient expression of gRNA is the key factor determining the efficiency of CRISPR-Cas9 for genome editing. The protocol developed in this study can provide essential references for other researchers who work in the areas of metabolic engineering and synthetic biology. The developed mutants can be used as excellent starting strains for development of more robust ones for desirable solvent production.
Transcriptional control can be used to program cells to label proteins with non-canonical amino acids by regulating the expression of orthogonal aminoacyl tRNA synthetases (aaRSs). However, we cannot yet program cells to control labeling in response to aaRS and ligand binding. To identify aaRSs whose activities can be regulated by interactions with ligands, we used a combinatorial approach to discover fragmented variants of Escherichia coli methionyl tRNA synthetase (MetRS) that require fusion to associating proteins for maximal activity. We found that these split proteins could be leveraged to create ligand-dependent MetRS using two approaches. When a pair of MetRS fragments was fused to FKBP12 and the FKBP-rapamycin binding domain of mTOR, and mutations were introduced that direct substrate specificity towards azidonorleucine (Anl), Anl metabolic labeling was significantly enhanced in growth medium containing rapamycin, which stabilizes the FKBP12-mTOR complex. In addition, fusion of MetRS fragments to the termini of the ligand-binding domain of the estrogen receptor yielded proteins whose Anl metabolic labeling was significantly enhanced when 4-hydroxytamoxifen was added to the growth medium. These findings suggest that split MetRS can be fused to a range of ligand-binding proteins to create aaRS whose metabolic labeling activities depend upon post-translational interactions with ligands.
The gut microbiota is implicated in numerous aspects of health and disease, but dissecting these connections is challenging because genetic tools for gut anaerobes are limited. Inducible promoters are particularly valuable tools because these platforms allow real-time analysis of the contribution of microbiome gene products to community assembly, host physiology, and disease. We developed a panel of tunable expression platforms for the prominent genus Bacteroides in which gene expression is controlled by a synthetic inducer. In the absence of inducer, promoter activity is fully repressed; addition of inducer rapidly increases gene expression by four to five orders of magnitude. Because the inducer is absent in mice and their diets, Bacteroides gene expression inside the gut can be modulated by providing the inducer in drinking water. We use this system to measure the dynamic relationship between commensal sialidase activity and liberation of mucosal sialic acid, a receptor and nutrient for pathogens.
Chronic inflammatory diseases such as arthritis are characterized by dysregulated responses to pro-inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α). Pharmacologic anti-cytokine therapies are often effective at diminishing this inflammatory response but have significant side effects and are used at high, constant doses that do not reflect the dynamic nature of disease activity. Using the CRISPR/Cas9 genome-engineering system, we created stem cells that antagonize IL-1- or TNF-α-mediated inflammation in an autoregulated, feedback-controlled manner. Our results show that genome engineering can be used successfully to rewire endogenous cell circuits to allow for prescribed input/output relationships between inflammatory mediators and their antagonists, providing a foundation for cell-based drug delivery or cell-based vaccines via a rapidly responsive, autoregulated system. The customization of intrinsic cellular signaling pathways in stem cells, as demonstrated here, opens innovative possibilities for safer and more effective therapeutic approaches for a wide variety of diseases.
To get content containing either thought or leadership enter:
To get content containing both thought and leadership enter:
To get content containing the expression thought leadership enter:
You can enter several keywords and you can refine them whenever you want. Our suggestion engine uses more signals but entering a few keywords here will rapidly give you great content to curate.
Your new post is loading...
