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’s DNA synthesis and ‘omics 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), Tanja Woyke, Deputy for User Programs, or Miranda Harmon-Smith (project manager). For questions about the application process, please contact Miranda Harmon-Smith.
Watch the JGI Engagement: Accessing Functional Genomics Capabilities Webinar hosted by the DNA Synthesis Science group. The webinar invites researchers to submit white papers to the Community Science Program’s Functional Genomics call for proposals.
Current Call (OPEN)
The current call for proposals offers multiple capabilities, as described below:
1) Synthesis of genes and pathways for functional characterization. A single proposal can request a total of 100 to 500 kb of DNA synthesis capacity per proposal. A consortium (with co-PIs from at least 3 different institutions) can request up to 1,500 kb. 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. Projects requiring specific nucleotide sequences (such as those required for homology-based recombination) may experience lower successful assemblies due to difficulties in synthesizing precise DNA sequences in the absence of refactoring. Therefore, we may have to adjust the scope of the project depending on the complexity of the sequence constraints. Prospective users are encouraged to contact JGI staff to discuss.
2) Synthesis of combinatorial pathway libraries for fast-track metabolic engineering. Each proposal may request up to 500 kb 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.
3) Synthesis of sgRNA libraries. Each proposal may request up to six libraries comprising up to 12,000 sgRNA sequences per library, or more than six libraries with less degree of variants 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 deliver the libraries to users as glycerol stocks. The subsequent transformation into the targeted microbes and functional screenings will be performed by users. The JGI can further evaluate enriched sgRNA libraries with sequencing-based analysis using MiSeq.
4) Strain Engineering: Genomic Integration of Synthetic Constructs into a Set of Bacterial Strains. JGI is offering a limited capacity of Chassis-independent recombinase-assisted genome engineering (CRAGE) to users. This technology enables integration of large, complex genetic constructs directly into the chromosomes of diverse gamma-proteobacteria with high accuracy and efficiency. Proposals may request up to 96 constructs to be cloned into a CRAGE compatible vector under the control of a T7 promoter and conjugated into a maximum of 5 gamma-proteobacteria hosts. We currently do not offer domestication of new strains to users.
The current list of preferred microbial species offered through this call include:
Pseudomonas putida KT2440
Pantoea agglomerans ATCC 13460 (Eh1087 (ICMP 13301))
Dickeya solani DSM 28711
Yersinia aldovae DSM 18303
Aeromonas piscicola LMG 24783
Photorhabdus luminescens laumondii TTO1
Shewanella oneidensis MR-1
Photobacterium halotolerans DSM 18316
Reference: CRAGE enables rapid activation of biosynthetic gene clusters in undomesticated bacteria https://www.nature.com/articles/s41564-019-0573-8
For additional information (literature citations, video), see this CRAGE blog post.
Applicants are also invited to request one or more other JGI functional genomics capabilities listed below.
5) 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.
6) 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.
7) Mapping of transcription factor binding sites (DAP-seq). 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 between 70-92 transcription factors. DNA/gene synthesis should also be requested for construction of affinity-tagged transcription factor clones used in the assay.
8) 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 between 22 and 54 samples (including replicates) from plants, fungi or microbes for the purpose of testing gene function or elucidating regulatory networks.
9) EcoFAB pilot projects. The JGI can provide up to 50 EcoFAB devices (https://eco-fab.org/) to study plant-microbiome interactions. These devices allow for non-destructive root imaging and sampling of the growth media while maintaining a sterile environment. In addition, the JGI can provide a standardized defined microbial community that colonizes plant roots and Brachypodium germplasm, if desired. Users would conduct experiments using these resources and return samples to the JGI for analysis by existing JGI capabilities e.g. metabolomics and transcriptomics.
Proposal Schedule
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.
Submission deadlines:
- August 1, 2022
- January 30, 2023
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