HR001123S0012-Amendment-01.pdf

The purpose of Amendment 1 to HR001123S0012, the Ceres Broad Agency Announcement, is to extend the full proposal deadline, clarify references to “synthetic community”, update the Phase II TA1/TA2 metrics for the Cleaner and Combined Tracks, and clarify University funding guidelines. Changes bolded/highlighted/underlined below.

Part I Overview Information is updated as follows:

o Full Proposal Due Date and Time: January 11 2023, 4:00 PM ET o BAA Closing Date: January 11, 2023

Section 1.5.2 Technical Area 2 (TA2): Engineer Plants is updated as follows:

TA2 will focus on generating plant scaffolds that successfully support the engineered microbial communities (TA1) in their track-specific function. The many, rich pathways where plants support the rhizosphere and the diverse communication pathways that control that support are finally being revealed. Proposers will modify plants to specifically enhance the target rhizosphere community developed in in TA1. Example strategies include the manipulation of root exudates, the release of signaling molecules, and the creation of a niche. Given the challenges associated with engineering plants, proposals should identify and justify plant selection, briefly delineate the extent to which that plant has been engineered to date, and describe the proposer’s specific engineering strategy with clear risk mitigation. Additionally, proposals should outline which tools (e.g., transcriptomics, metabolomics, etc.) will be used to identify key metabolites to communicate directly with and enhance the rhizosphere community. For the Cleaner Track, the plant supports the TA1 generated community’s metabolic activity to increase contaminant degradation rates. For the Beacon Track, the plant should support and stabilize the TA1 generated community with specific emphasis on receiving engineered orthogonal molecular signals from the rhizosphere and converting the relayed signals into an overt change in aboveground plant phenotype. By the end of Phase I, the plant–microbe synthetic communities should be fully integrated. By the end of Phase II, synthetic communities will either detect and signal contaminant presence at the EPA approved low RSL concentration (Beacon) or fully degrade the contaminant to EPA approved concentrations (Cleaner).

Section 1.7 (CLEANER TRACK) is updated as follows:

TA1 and TA2 METRICS and MILESTONES BY TRACK AND BY PHASE

►CLEANER TRACK

Phase I (Months 1 through 24) Goal: Phase I, 24 months, will require proposers to obtain and engineer intact rhizosphere communities (TA1) and engineer the plant host (TA2) as integrated contaminant-specific Cleaner synthetic communities. Synthetic communities will be optimized to sense and respond to the soil contaminant(s) with engineered plants supporting and enhancing rhizosphere community contaminant degradation. By the end of Phase I, proposers must demonstrate integrated TA1/TA2 synthetic community retains degradation function for ≥ 15 days in the simplified testbeds.

Proposals must concisely describe known metabolic pathways pertinent to the proposed synthetic community function, by-products produced, and potential environmental or health impacts of biodegradation of the contaminant and how the proposers plan to mitigate these risks.

A principal goal of Ceres is achieving rates of bioremediation comparable to conventional bioremediation, such as biopiling or windrowing, but with significantly less effort or energy.

Consequently, proposals should outline strategies for referencing the rate of synthetic community degradation to that achieved via biopiling to serve as the final target degradation rate for the synthetic community. Biopiling, where excavated contaminated soil is supplemented with specific microbes, nutrients and environmental conditions in order to optimize the targeted microbial degradation activity, can accelerate soil bioremediation ≈10-fold versus native rates. The rate of biopiling varies substantially by specific rate limitation(s), e.g. temperature in cold biomes, water availability in dry climates, or nutrient availability in oligotrophic soil. The goal for the Cleaner synthetic community is to match the decontamination rate achievable locally with biopiling but without the added human effort and energetic inputs associated with biopiling.

TA 1

Demonstrate the rhizosphere community can be engineered either through exogenous gene circuit expression or by a ≥5-fold increase in a Track-specific metabolic pathway. (12 months)

Demonstrate rhizosphere community doubles the degradation rate of the target contaminant compared to matched native soils in simplified testbeds. At the end of the remediation period, no side products of the bioremediation should exceed the Industrial Screening Level as defined by the EPA. (12 months)

Demonstrate rhizosphere community degrades the soil contaminant from the high (Table

1) concentration 2x faster than matched native soil, i.e., matched soil without any biopiling.

(15 months)

TA 2

Demonstrate the plant supports the engineered microbiome community and its track-specific function in non-sterile soil. By either increasing the functional population or enhancing the target metabolism, the overall targeted metabolic activity should increase by ≥2x. (15 months)

The plant support, such as via root exudates, must be engineered to selectively enhance the targeted rhizosphere community’s metabolic activity in vitro, doubling contaminant-specific degradation from high concentration compared to matched native soil. (12 months)

TA1/2 Integrated synthetic community Demonstrate the synthetic community can survive together in non-sterile soil with native microbes for at least 30 days. (12 months)

Demonstrate the plant-microbe synthetic community degrades the soil contaminant from high starting concentration (Table 1) in non-sterile soil 2x faster than matched native soil.

(18 months)

Demonstrate the synthetic community degrades the soil contaminant from high concentration (Table 1) to ISL for the marker compound 4x faster than matched native soil.

The Cleaner synthetic community is functional ≥15 days in the simplified testbeds (24 months)

Phase II Option (Months 25 through 48) Goal: The Phase II Option, 24 months, will focus on further synthetic community refinement under increasingly stringent conditions. Proposers will optimize the integrated synthetic community (TA1/TA2) function for the following Cleaner-specific metrics. By the end of Phase II, the Cleaner synthetic community must be functionally operational for ≥60 days in greenhouse testbeds.

TA1/2 Integrated synthetic community

Delivery of the Cleaner synthetic community to the IV&V partners for testing in one DoD target biome soil capable of degrading to ISL. (26 months)

Demonstrate the plant host supports the rhizosphere community function, for instance via modulated and orthogonal root exudation under the track-specific EPA thresholds. (36 months)

Demonstrate synthetic community degrades target soil contaminant from both high and low concentrations (Table 1) down to RSL for the marker compound. Remediation must occur 4x faster than matched native soil. (36 months)

Demonstrate that the synthetic community functions to the track-specific EPA threshold for at least 60 days in a greenhouse testbed. (48 months)

Demonstrate synthetic community degrades target soil contaminant down to RSL for the marker compound(s) 10x faster than matched native soil, is functionally operational for at least 60 days, and scales to a 1 m3 greenhouse plot. (48 months)

Delivery of the Cleaner synthetic community to the IV&V partners for final Phase III demonstrations. (48 months)

Phase III Demonstration (Months 49 through 54) IV&V teams will demonstrate that the Cleaner synthetic community can biodegrade the chosen contaminant to RSL 10x faster than matched native soil in the three distinct U.S. biomes— Eastern Woodlands, Great Plains, and Mediterranean Climate. The biome is defined as the soil, the associated microbiota, and the local abiotic conditions. The synthetic community must be functionally operational to biodegrade the target contaminant for at least 60 days and scale to a 1 m3 greenhouse plot. (54 months)

Section 1.7 (COMBINED TRACK) is updated as follows:

►COMBINED TRACK

Phase I (Months 1 through 24) Goal: Phase I, 24 months, will require proposers to obtain and engineer intact rhizosphere communities (TA1) and engineer the plant host (TA2) as integrated track-specific synthetic communities outlined above. synthetic communities will be optimized to sense and respond to the soil contaminant(s) with engineered host plants that support and enhance the track-specific function of the rhizosphere community. By the end of Phase I, proposers must demonstrate integrated TA1/TA2 synthetic communities retain track-specific function for ≥ 15 days in the Phase I testbeds.

TA 1

Demonstrate the rhizosphere community can be engineered either through exogenous gene circuit expression or by a ≥ 5-fold increase in a Track-specific metabolic pathway. (12 months)

Demonstrate the Cleaner rhizosphere community doubles the degradation of the target contaminant compared to matched native soils in Phase I testbeds. Degradation products must not be contaminants. (12 months)

Demonstrate the Beacon rhizosphere community can generate a signal in response to the target contaminant at the marker-specific ISL in vitro. (12 months)

Demonstrate the Cleaner rhizosphere community degrades the soil contaminant from the high (and from low) concentration 2x faster than matched native soil. (18 months)

Demonstrate the Beacon rhizosphere community can generate the signal molecule(s) for orthogonal microbe-to-plant communication. (18 months)

TA 2

Demonstrate the plant supports the engineered microbiome community and its track-specific function in non-sterile soil. By either increasing the functional population or enhancing the target metabolism, the overall targeted metabolic activity should increase by ≥2x. (15 months)

The Cleaner plant support, such as via root exudates, must be engineered to selectively enhance the targeted rhizosphere community’s metabolic activity in vitro, doubling contaminant-specific degradation from high concentration compared to matched native soil. (12 months)

Demonstrate Beacon plant can detect and respond to the target signal molecule(s) from the rhizosphere community for microbe-to-plant communication. (12 months)

Demonstrate Beacon plant phenotypically responds aboveground to the rhizosphere community’s metabolic signal(s). (18 months)

TA1/2

Demonstrate each synthetic community can survive together in non-sterile soil with native microbes for at least 30 days. (12 months) Demonstrate functionality of the synthetic community to the track-specific EPA threshold.

(24 months) Demonstrate Cleaner synthetic community degrades the soil contaminant from the high

(and separately from low) concentration in non-sterile soil 2x faster than matched native soil. (18 months)

Demonstrate Cleaner synthetic community degrades the soil contaminant from the high (and separately from low) concentration down to ISL for the marker compound 4x faster than matched native soil. The Cleaner synthetic community is functional ≥15 days in the simplified testbeds (24 months)

Demonstrate Beacon synthetic community can communicate with microbe-to-plant metabolic signals in non-sterile soil. (18 months) Demonstrate Beacon synthetic community can detect and report on a contaminant status at or below ISL for the marker after at least 15 days of living in Phase I testbeds. (24 months)

Phase II Option (Months 25 through 48) In Phase II, teams will focus on further synthetic community refinement under increasingly stringent conditions. Proposers will optimize both integrated synthetic communities (TA1/TA2) function for the specific application-track metrics outlined above. By the end of Phase II, both synthetic communities must be functionally operational for ≥ 60 days in greenhouse testbeds.

TA1/2

Demonstrate the plant host supports the rhizosphere community function, for instance via modulated and orthogonal root exudation under the track-specific EPA thresholds. (36 months)

Demonstrate that the synthetic community functions to the track-specific EPA threshold for at least 60 days in a greenhouse testbed. (48 months)

Demonstrate the plant host supports track-specific rhizosphere community function EPA screening levels, relative to the rhizosphere community function without the plant host, for at least 60 days in a greenhouse testbed. (48 months)

Demonstrate Cleaner synthetic community degrades target soil contaminant from high (and separately from low) concentration down to RSL for the marker compound 4x faster than matched native soil. (36 months)

Demonstrate Cleaner synthetic community degrades target soil contaminant down to RSL for the marker compound(s) 10x faster than matched native soil and is functionally operational to biodegrade the target contaminant for at least 60 days and scale to a 1 m3 greenhouse plot. (48 months)

Demonstrate Beacon synthetic community limit of detection occurs at or below RSL for the target contaminant marker(s). (36 months)

Demonstrate Beacon synthetic community limit of detection and signal generation occurs at or below RSL for the target contaminant marker(s). The signal must be overt (detectable by an unaided human at 2m), and the synthetic community must detect and report soil contaminant status and be functionally operational for at least 60 days in the simplified testbeds with native soils. (48 months)

By the end of Phase II, teams will have worked with IV&V partners to test both synthetic communities each in one biome soil. (48 months)

Phase III Demonstration (Months 49 through 54) IV&V teams will demonstrate that the Cleaner and Beacon synthetic communities can function in the three distinct U.S. biomes—Eastern Woodlands, Great Plains, and Mediterranean Climate.

The biome is defined as the soil, the associated microbiota, and the local abiotic conditions. The Cleaner biodegrades the target contaminant to RSL 10x faster than matched native soil. The Beacon synthetic community generates an overt signal of target contamination status with a limit of detection at or below RSL. Both synthetic communities must be functionally operational for at least 60 days and scale to a 1 m3 greenhouse plot. (54 months)

Section 8 is updated as follows:

8.1 UNIVERSITY FUNDING

In order to ensure that U.S. scientific and engineering students will be able to continue to make strategic technological advances, DARPA is committed to supporting the work and study of Ph.D students and post-doctoral researchers that began work under a DARPA-funded program awarded through an assistance instrument. Stable and predictable federal funding enables these students to continue their scientific and engineering careers.

To that end, should a DARPA funded program (awarded through a grant or cooperative agreement with a university or a Research Other Transaction pursuant to 10 U.S.C. § 4021 where the university is a participant) end before the negotiated period of performance, DARPA will continue to fund, for no more than two semesters (or equivalent), stipend costs to Ph.D students and/or post-doctoral researchers. The stipend amount will be determined at the time of award based on the costs included for such participants in the University’s original proposal. Universities are expected to make reasonable efforts to find alternative research opportunities for these participants before stipend funding is provided in this situation. This additional funding will not be provided in cases where an assistance award option is not exercised or any other scenario in which the University was aware at the time of award that the period of performance might not continue after a designated programmatic decision (i.e. a down-selection or inclusion of a subsequent programmatic phase).