(previously DNA Synthesis)
About This Call
The CSP Functional Genomics call is to enable users to perform state-of-the-art functional genomics research and to help them translate genomic information into biological function. The emphasis is on projects leveraging JGI capabilities to enhance understanding of gene and genome function, particularly those not readily achievable without the capabilities and expertise available at the JGI.
Proposals to this call may be submitted to the JGI at any time using a simple web-based form (begin a new proposal document at proposals.jgi.doe.gov to see the proposal format) and are reviewed twice a year. All proposals will undergo an internal pre-review to ensure technical feasibility and alignment with JGI and DOE missions. Screened proposals will be reviewed for scientific merit, DOE relevance, feasibility and impact. Applicants are encouraged to review the Synthetic Biology Internal Review Process guidelines, as insufficient information will delay or potentially defer approval for the proposal.
For questions about whether your project is appropriate, or for program specifics or technical guidance, please contact Yasuo Yoshikuni (DNA synthesis program head), Trent Northen (metabolomics group lead), Susannah Tringe (User Programs Deputy) or Miranda Harmon-Smith (project manager). For questions about the application process, please contact Miranda Harmon-Smith.
Current Call (OPEN)
The current call for proposals offers multiple capabilities, as described below:
- Synthesis of genes and pathways for functional characterization. DNA synthesis technology has advanced to the point that now it allows access to many genes and pathways within diverse, large, and rapidly expanding sequence spaces for heterologous expression. The technology could facilitate discovery and characterization of gene products with novel and/or desired biochemical function at an unprecedented scale. To support this work, the JGI solicits proposals requesting large-scale DNA synthesis and construct assembly of genes and pathways for functional characterization. All constructs are synthesized and assembled into user-defined plasmids, sequence validated, and transformed into an E. coli strain before shipment to users. The products are delivered to users as glycerol stocks.
A single proposal can request a total of 100 to 500 kbp of DNA synthesis capacity per proposal. A consortium (with co-PIs from at least 3 different institutions) can request up to 1,500 kbp per proposal.
- Synthesis of combinatorial pathway libraries for fast-track metabolic engineering. Metabolic engineering traditionally approaches strain development stepwise, requiring cycles of construct design, building, and characterization. However, pathway efficiency may be optimized more efficiently if multiple pathway variations are tested simultaneously. The JGI recently developed the ability to efficiently build a large number of pathway variations comprising promoters, RBSs, gene variations, and terminators with different strengths and/or activities. Each proposal may request up to 500 kbp of DNA de novo synthesis capacity to produce millions of basepairs of combinatorial variants. The JGI will also help identify a panel of each pathway component and design final constructs. All constructs are assembled using type II restriction-enzyme-based technologies (e.g., golden gate assembly) into user-defined plasmids and are transformed into E. coli strains before shipment to users; no sequencing validations will be performed for the constructs. The products are delivered to users as glycerol stocks.
- Synthesis of sgRNA libraries. CRISPR/CAS9 technologies facilitate genome editing (deletion, insertion, and point mutation) and transcriptional regulation (activation and inhibition) of any genes of interest at an unprecedented scale and are therefore useful for high throughput functional genomics studies. Each proposal may request up to six libraries comprising up to 12,000 sgRNA sequences per library. The JGI can help design sgRNA sequences based on the genome sequences of targeted microbes. All sgRNA constructs are synthesized, cloned into user-defined plasmids, and transformed into an E. coli strain as pools. The quality of these libraries is evaluated with sequencing-based analysis using MiSeq before shipment to users. The JGI will create at least 10 replicates of each library to minimize variations in experiments, and will deliver them to users as glycerol stocks. The sgRNA libraries are purified and subsequently transformed into the targeted microbes. These transformants are subjected to user-defined functional screenings. The JGI can further evaluate enriched sgRNA libraries with sequencing-based analysis using MiSeq.
Applicants are also invited to request one or more other JGI functional genomics capabilities listed below.
- Sequence data mining. The JGI’s genome portals IMG, Mycocosm and Phytozome contain a wealth of genomic data from microbes, fungi, plants and microbiomes. Proposals may request assistance with database searches for the selection of target genes and pathways for synthesis. However, capacity for analyzing search results and aiding in target selection is very limited; users needing assistance with these tasks should contact JGI in advance to discuss feasibility.
- Metabolomics based functional analyses. Metabolomic technologies at JGI enable users to examine diverse polar and non-polar metabolites from plants, microbes, and environments. In addition, users may request targeted analysis of stable isotope labeling for specific metabolites. Proposals should clearly indicate how the data obtained will be linked to gene function, and may request up to 50 polar metabolite sample analyses or 150 non-polar metabolite sample analyses.
- Mapping of transcription factor binding sites. High-throughput mapping of putative transcription binding sites enables large-scale characterization of gene regulatory networks in a selected species. Proposals can request in vitro transcription factor binding site mapping by DNA affinity purification sequencing (DAP-seq) for up to 50 transcription factors. DNA/gene synthesis should also be requested for construction of affinity-tagged transcription factor clones used in the assay.
- RNA-seq. Transcriptional profiling can aid in characterizing gene regulatory pathways activated in response to perturbations or environmental stimuli. Proposals may request RNA sequencing of up to 54 samples from plants, fungi or microbes for the purpose of testing gene function or elucidating regulatory networks.
CSP Functional Genomics proposals are accepted on a continuous basis and will be reviewed twice a year. Submission deadline for reach review process is listed below. Letters of intent are not required.
- July 30, 2020
Proposal Review Process
All proposals undergo scientific review as described at https://jgi.doe.gov/user-programs/program-info/csp-review-process-and-contract-documents/. In addition, proposals requesting DNA synthesis that are tentatively approved will undergo an additional review of potential impacts as described below prior to project initiation.
Proposals requesting DNA synthesis are evaluated by at least three external reviewers in a process known as Synthetic Biology Internal Review (SBIR). SBIR encourages investigators to extensively consider broader aspects of their research (e.g., biosafety, biosecurity, bio-containment and environmental issues) to evaluate both positive and negative impacts and to propose strategies to mitigate concerns. If issues are not sufficiently addressed, users will be asked to modify their proposal. If issues are not resolved, the proposal may be rejected. SBIR generally takes three weeks.
Investigators should not merely write “None” or “All research will be conducted in a safe manner according to Federal regulations” in the broader implications statement, as this will lead to requests for proposal modifications, incurring delays of three weeks or longer.
Investigators must explicitly state whether their proposed research would:
- Demonstrate how to make a vaccine ineffective
- Confer resistance to antibiotics or antiviral agents
- Enhance a pathogen’s virulence or make a non-virulent microbe virulent
- Increase transmissibility of a pathogen
- Alter the host range of a pathogen
- Enable a pathogen’s ability to evade diagnostic or detection modalities
- Enable the weaponization of a biological agent or toxin