DARPA-BAA-16-50.pdf

Broad Agency Announcement Engineered Living Materials

BIOLOGICAL TECHNOLOGIES OFFICE

DARPA-BAA-16-50

August 5, 2016

DARPA-BAA-16-50, Engineered Living Materials

TABLE OF CONTENTS

PART I: OVERVIEW INFORMATION

PART II: FULL TEXT OF ANNOUNCEMENT

1. Funding Opportunity Description

1.1. PROGRAM OVERVIEW

1.2. Program Introduction and Scope

1.3. ELM Program Structure

2. Award Information

3. Eligibility Information

3.1 ELIGIBLE APPLICANTS

3.2. COST SHARING/MATCHING

3.3. OTHER ELIGIBILITY REQUIREMENTs

4. Application and Submission Information

4.1. ADDRESS TO REQUEST APPLICATION PACKAGE

4.2. CONTENT AND FORM OF APPLICATION SUBMISSION

4.3. FORMATTING CHARACTERISTICS

4.4. SUBMISSION DATES AND TIMES

4.5. FUNDING RESTRICTIONS

4.6. OTHER SUBMISSION REQUIREMENTS

5. Application Review Information

5.1. EVALUATION CRITERIA

5.2. REVIEW AND SELECTION PROCESS

6. Award Administration Information

6.1. SELECTION NOTICES

6.2. ADMINISTRATIVE AND NATIONAL POLICY REQUIREMENTS

6.3. REPORTING

6.4. ELECTRONIC SYSTEMS

7. Agency Contacts

8. Other Information

8.1. INTELLECTUAL PROPERTY

9. APPENDIX 1 – Volume II checklist

PART I: OVERVIEW INFORMATION

Federal Agency Name – Defense Advanced Research Projects Agency (DARPA), Biological Technologies Office

Funding Opportunity Title – Engineered Living Materials Announcement Type – initial announcement Funding Opportunity Number – DARPA-BAA-16-50 Catalog of Federal Domestic Assistance Numbers (CFDA)- Not applicable Dates o Posting Date – August 5, 2016 o Proposal Abstract Due Date – September 15, 2016 (Track 2 only) o Full Proposal Due Dates – September 27, 2016 (Track 1), November 10, 2016 (Track 2) o BAA Closing Date – November 10, 2016 o Any other relevant date(s) – Proposers Day, August 26, 2016 https://www.fbo.gov/spg/ODA/DARPA/CMO/DARPA-SN-16-56/listing.html

Optional Additional Overview Content:

Concise description of the funding opportunity: The Engineered Living Materials

(ELM) program will develop tools and methods to enable the engineering of structural features into cellular systems that function as living materials, thereby opening a new design space for construction technology.

Total amount of money to be awarded:

Anticipated individual awards - Multiple awards are anticipated.

Types of instruments that may be awarded - Procurement contract, cooperative agreement, or other transaction.

Agency contact o Points of Contact The BAA Coordinator for this effort may be reached at:

DARPA-BAA-16-50@darpa.mil

DARPA/BTO

ATTN: DARPA-BAA-16-50

675 North Randolph Street Arlington, VA 22203-2114 https://www.fbo.gov/spg/ODA/DARPA/CMO/DARPA-SN-16-56/listing.html mailto:DARPA-BAA-16-50@darpa.mil

PART II: FULL TEXT OF ANNOUNCEMENT

1. Funding Opportunity Description

The Defense Advanced Research Projects Agency often selects its research efforts through the Broad Agency Announcement (BAA) process. This BAA is being issued, and any resultant selection will be made, using procedures under Federal Acquisition Regulation (FAR) 35.016 and the Department of Defense Grant and Agreement Regulatory System (DoDGARS) Part 22 for Grants and Cooperative Agreements. Any negotiations and/or awards will use procedures under FAR 15.4, Contract Pricing, as specified in the BAA (including DoDGARS Part 22 for Grants and Cooperative Agreements). Proposals received as a result of this BAA shall be evaluated in accordance with evaluation criteria specified herein through a scientific review process.

DARPA BAAs are posted on the Federal Business Opportunities (FedBizOpps) website, https://www.fbo.gov, and, as applicable, the Grants.gov website at http://www.grants.gov. The following information is for those wishing to respond to the BAA.

DARPA is soliciting innovative research proposals to develop materials that combine the structural properties of traditional building materials with attributes of living systems, including the ability to rapidly grow, self-organize, self-repair, and adapt to the environment.

1.1. PROGRAM OVERVIEW

The Engineered Living Materials (ELM) program will develop design tools and methods that enable the engineering of structural features into cellular systems that function as living materials, thereby opening up a new design space for construction technology. These methods will be validated through the production of living materials that display hallmarks of biological systems, such as the ability to actively sense and respond to the environment, or to heal after damage. Successful completion of ELM program objectives will require innovations in the ability to functionally unite living components with inert structural materials, to program structural features into living systems, and to extend the scale of synthetic biology building blocks from the molecular to the cellular. The deliverables from this program will comprise a suite of technologies that enable the production of living structural materials tailored to design specifications, such as those provided by architects and builders.

1.2. PROGRAM INTRODUCTION AND SCOPE

The structural materials that are used to construct our homes, buildings, and infrastructure are expensive to produce and transport, are subject to damage due to environmental insults and aging, and have limited ability to respond to changes in the immediate surroundings. As a result, the energy and financial costs of building and infrastructure construction and repair, to both the DoD and the nation, are enormous. Living biological materials may have advantages over inert materials, in that they might be grown on-site from simple feed stocks under ambient conditions, self-repair when damaged, or respond to changes in their surroundings. The inclusion of living components in our built environments has obvious benefits; however, today we are unable to control the structural aspects (shapes and sizes) of living materials so that they can be useful for construction. The ELM program seeks to deliver technologies that will enable the addition of living structural materials into our built environments. Such novel materials would reduce the energy and https://www.fbo.gov/ http://www.grants.gov/ financial burden associated with the manufacture and transport of materials to construction sites, since they will be able to grow on-site from natural feedstocks. Furthermore, as they will contain elements that are alive, the resulting structures will be endowed with the ability to self-repair and respond appropriately to changes in the environment.

A major inspiration for the ELM program is the recent development of biologically-sourced structural materials that are grown to specified size and shape from inexpensive feedstocks. For example, mycelia can be grown on agricultural byproducts to produce materials that are drop-in replacements for polystyrene. Similarly, bacteria can be used to bind sand to produce drop-in replacements for bricks. That factory-scale production of grown materials can be economically competitive with materials as common as polystyrene and brick, demonstrates the feasibility of using biological approaches to reduce the energy and waste associated with the manufacture of structural materials. However, as the final products are rendered inert during the manufacturing process, these early examples of grown materials retain few of the benefits of the biological components they contain; for example, the ability to respond to environmental cues or to self-repair.

DARPA is seeking technologies that enable the engineering of hybrid materials composed of structural scaffolds that support the rapid growth and long-term viability of living cells that endow the final products with biological functions. These materials should exhibit aspects of both the inert grown materials that are being produced today at the factory scale, such as structural integrity, as well as those of living systems, such as self-repair. The platform technologies developed in the ELM program are intended to be scalable and generalizable, so as to be transitioned from the lab to industry in the near-term.

In addition, DARPA seeks the ability to engineer structural properties directly into the genomes of biological systems, so that living materials can be grown from progenitor cells (e.g., seeds), without the need of non-living scaffolds or external developmental cues. To address this goal, it will be necessary to program developmental pathways that result in multicellular systems with defined patterns and 3D shapes. The ability of multicellular organisms to develop and maintain defined body plans is evidence of the inherent potential of genetically-programmed biological structures. However, it is not yet possible to engineer these properties de novo. To enable genetic programming of multicellular morphology, synthetic biology will need to advance toward the engineering of multicellular systems derived from a single genotype. It is expected that successful proposers will not only create new advances in synthetic biology, but also leverage the state-of-the-art in experimental and/or theoretical developmental biology.

Through ELM, DARPA seeks to cultivate foundational principles, as well as novel approaches and methods that will ultimately enable living structural materials with advanced capabilities to be rationally designed, and implemented through genetic engineering. DARPA has identified five fundamental capabilities that can conceivably be used in combination to enable the invention of a wide range of living materials of arbitrary form and function. The demonstration of these fundamental capabilities will form the major deliverables of the program, and are: (1) on-site growth, maintenance, and reproduction of a living structural material on inexpensive feedstock;

(2) the precise coordination of cells and inert particles to form tunable multi-scale patterns; (3) the ability to self-repair in response to damage; (4) genetically programmed multicellular patterns; and

(5) genetically programmed multicellular 3D shapes.

1.3. ELM PROGRAM STRUCTURE

The ELM program seeks to enable a new class of building materials that can be combined with traditional construction methods to ensure advanced capabilities in the near term, as well as lay the foundation for entirely new technologies that will revolutionize the way we build far into the future. The ELM program consists of two tracks: (1) Hybrid Engineered Living Materials (Hybrid- ELM), and (2) Programmable Engineered Living Materials (Programmable-ELM). Each track is divided into specific task areas that independently address five fundamental capabilities needed to creating living structural materials. An individual proposal may address multiple task areas within a program track, but may not address both program tracks. Together, the ELM task areas define a comprehensive set of capabilities that could hypothetically be combined in the rational design of living structural materials of arbitrary function.

Track 1: Hybrid-ELM (48-month Period of Performance)

The major objective of ELM Track 1 is to develop methods for adding living cells to scaffolding materials such that the resulting hybrid material is both structurally sound, and exhibits desirable characteristics of living things. Just as is the case with natural living structural materials, such as wood or bone, the engineered living hybrid materials need not contain a large percentage of living cells; however, it is essential that the viability of the living components be maintained within the environment for which the final product is designed to be used, and that the cells contribute significantly to the function of the final product. DARPA is not interested in approaches that result in drop-in replacements for existing building materials (i.e., materials that do not have capabilities beyond today’s building materials). Moreover, DARPA is not interested in funding the development of living materials for medical applications, such as tissue engineering. Proposals that address drop-in replacements for existing materials, or materials for medical applications will be deemed nonresponsive.

A challenge of Track 1 is identifying material(s) with appropriate strength and density to serve the structural role that can also support the long-term viability of living cells. To permit future scale-up and eventual commercial potential, the scaffold material should be readily obtainable at a reasonable cost. Furthermore, as discussed in detail below, the living cellular components of the hybrid material will need to be engineered to perform a relevant and practical biological function in the final product, which the performer must specify. Performer teams will be required to develop materials, engineer the genomes of organisms, and design production practices that result in living structural materials. Successful completion of these goals will likely require the coordinated efforts of researchers from the fields of materials science and engineering biology, as well as industrial partners. Proposals should specifically address teaming strategies and should involve performers with expertise in the relevant fields. Although demonstrations will be performed at lab-scale (on the order of centimeter-sized products), the technologies created through ELM Track 1 are intended to be scalable and generalizable, so as to be transitioned from the lab to industry in the near-term. Performers must include justification for how their approach is scalable. One-off demonstrations or methods that are not compatible with large-scale production are specifically discouraged.

As described in the following sections, DARPA has identified three classes of living hybrid materials, each of which takes advantage of distinct attributes of biological systems, to be individually addressed by the three ELM Track 1 Task Areas. Proposers may choose to address one or multiple Task Areas in Track 1. Each Task Area is designed to occur in three Phases over 48 months. In the first Phase, performing teams will explore and validate approaches to joining the inert scaffold with the living cells that will compose the hybrid material. In Phase II, the biological component(s) of the hybrid material will be engineered to perform a relevant practical function in the final product. Phase III will involve optimization, testing, and validation of the hybrid material to a set of standards specified in each Task Area. The final deliverable for each Task Area will be a living hybrid material with the following minimum characteristics:

Composed of a coordinated system of living tissue/cells and structural particles/materials.

Produced through methods that are generalizable and scalable to building industry standards.

Maintains viability of the living component in environmental conditions representative of a built environment.

Exhibits relevant attribute(s) of living systems.

Track 1 - Task Area A: Rapid On-Site Growth & Reproduction

Through Task Area A, DARPA seeks innovative approaches to leveraging biology’s ability to grow and reproduce rapidly on inexpensive resources for the on-demand production of structural materials. Specifically, Task Area A seeks to prototype living hybrid materials that can be rapidly grown outside of a factory environment, and that sustain a cadre of living cells that can reproducibly generate more of the same material when needed. Proposals should describe an original and innovative approach that conforms to the following minimum criteria:

Proposals should describe in detail the relevant environment in which the proposed hybrid living material will operate.

Identify or engineer an organism, or community of organisms, with the ability to grow and persist on inexpensive and locally available feedstock at ambient temperature.

The feedstock and scaffold may be derived from the same or separate substrates; however, it is essential that both the structural integrity and the long-term viability of cellular components of the hybrid material be considered in the design of the final product.

The taxonomic identity of the cellular component(s) of the hybrid materials is left to the discretion of the proposer, and may derive from any kingdom of life.

Successful approaches to Task Area A may build on the state-of-the-art for biologically produced materials, such as mycelium-based materials, but must alter manufacturing practices to allow the living components to survive production.

The size and shape of the hybrid material may be externally controlled, for example using molds or patterns; however, the shape must be stabilized after production to facilitate use as a construction material. Proposals should specifically address methods for forming arbitrary shapes, as well as stabilizing the shape of the hybrid material.

The percentage of living matter in the final composition is left to the proposer, but proposals should describe an approach that results in a stable structural material with the ability to reproduce itself within 3 days when starting with a maximum of 50% existing material plus additional scaffold (i.e., the hybrid material should have a doubling time of ≤ 3 days, when required). It is assumed that this will require the ability to switch living components from a quiescent or non-dividing state to a growth state, although alternative solutions are acceptable.

As described in more detail in Section 1.4, the final demonstration will be performed at lab scale; however, proposals must describe potential approaches to scaling-up production to industrially relevant scales.

The exact utility of the resulting hybrid material is left to the discretion of the proposer;

however, for this task area the final deliverable should be a structural material that could be used in the construction of shelters or barriers. Proposals should include detailed target metrics for physical properties of the hybrid material that are appropriate for the intended application, such as strength, resiliency, and density.

Track 1 - Task Area B: Self-Organizing Structures

Through Task Area B, DARPA seeks innovative approaches to leveraging biology’s ability to self-organize into arrays at multiple length scales for the production of precisely patterned composite materials. There are many examples of living systems that organize non-living components, such as insect exoskeletons with high strength-to-weight ratios comprised of patterned chitin, sea sponges that grow biological glass from ordered arrays of microscopic silica rods, and butterfly wings with structural color derived from precisely organized 3D layers of cuticle proteins. Task Area B is aimed at developing methods to leverage the innate ability of biology to produce highly ordered structures with desirable emergent physical properties. Proposals should describe an original and innovative approach that conforms to the following minimum criteria:

The proposed hybrid living material must be composed of a living component and a scaffold component that can be organized into multi-scaled patterns. The patterning may, for example, arise through subcellular interactions (e.g., cell-surface proteins binding to the scaffold component), that are further organized through intercellular interactions.

Therefore, the final product could exhibit a pattern at the nano- or micro-scale, which itself is arranged to produce higher-order patterns at the millimeter- or greater-scale.

The specific identities of the living and scaffold components of the hybrid material are at the discretion of the proposer; however, it is essential that the living component be a genetically tractable organism to facilitate further engineering. Proposals should describe in detail the choice of components and justify their relevant attributes.

The ordered pattern of the hybrid material must arise due to the specific interactions of the cells with the scaffold material. It is expected that the living component will be engineered to enable the resulting material to be tuned in various ways, including but not limited to scaffold spacing, aspect ratio, and density. Proposals should include detailed plans for how relevant physical parameters will be addressed, and how they will be made to be tunable within appropriate ranges.

The resulting material must be self-organizing, such that external cues are not needed to template the pattern during the growth of the material.

The final material should include features that are amenable to regulation by changes in the environment.

As described in more detail in Section 1.4, the final demonstration will be performed at lab scale (final products must be > 1 cm3); however, proposals must describe potential approaches to scaling-up production to industrially relevant scales.

The specific function of the resulting material is left to the discretion of the proposer;

however, the multi-scale pattern created by the interaction of living and scaffold components must contribute directly to the function and form of the final product.

Track 1 - Task Area C: Responsive Surfaces

Through Task Area C, DARPA seeks innovative approaches to leveraging biology’s ability to form environmentally responsive protective outer layers for the production of construction materials with self-healing surfaces. Multicellular organisms are typically wrapped in a tough external layer of cells, such as skin or bark. This layer serves to protect the internal tissues and organs, provide a security barrier, and act as a sensing interface with the external environment.

Environmental sensing can often induce appropriate changes in the physical features of the surface to, for example, increase porosity, change appearance or color, or vary tensile strength to achieve a functional response to a specific stimulus. Importantly, this outer sheath also comes with the ability to heal after damage, to preserve the overall function of the barrier and the organism. Task Area C is aimed at developing methods to produce hybrid materials with living surfaces that serve as sensitive protective barriers capable of responding to environmental cues, and self-healing in response to damage. Proposals should describe an original and innovative approach that conforms to the following minimum criteria:

The proposed hybrid living material must be composed of a core structure, or scaffold, that supports the growth and viability of a living protective surface. The core material can take the form of a solid or skeletal frame, but must provide structural integrity to the final hybrid material.

The specific identities of the living and scaffold components of the hybrid material are at the discretion of the proposer; however, it is essential that the living component be a genetically tractable organism to facilitate engineering. Proposals should describe in detail the choice of components and justify their relevant attributes.

The cells that comprise the living component of the hybrid material must form a cohesive tissue, such that they can be engineered to perform coordinated functions in response to an environmental stimulus sensed anywhere on the surface.

The growth of the living component should be constrained to 2.5-dimensions (i.e., a thin layer with potentially large surface area (> 100 cm2)) along the surface of the core material.

The living component must be engineered with the ability to sense a relevant environmental stimulus, and to respond with a tissue-level change in a physical property of the entire material. For example, response to an increase in ambient temperature by increasing the porosity of, and the airflow through the hybrid material. The specific sensing and response capabilities are at the discretion of proposers and should be chosen to align with the intended function of the hybrid material.

The living component of the hybrid material must be engineered with the ability to self-heal following at least three types of damage (e.g., tearing, erosion, and infection by other organisms). The type and extent of damage that will be repaired during demonstrations should be described in the proposal and should align with the intended application of the hybrid material. It is not necessary that the healing process be scarless; however, the overall function of the responsive skin must be retained following damage and healing.

As described in more detail in Section 1.4, the final demonstration will be performed at lab scale; however, proposals must describe potential approaches to scaling-up production to industrially relevant scales.

The exact utility of the resulting responsive surface is left to the discretion of the proposer;

however, materials that serve as a protective outer layer for a shelter or barrier are particularly encouraged. Proposals should include detailed metrics for physical properties of the hybrid material that are appropriate for the intended application, such as tensile strength, porosity, and hydrophobicity.

Track 2: Programmable-ELM (24-month Period of Performance)

The major objective of ELM Track 2 is to explore methods for programming structural features into multicellular biological systems through genetic engineering. If successful, the technologies developed in Track 2 of the ELM program will lead to the ability to grow living structural materials to design specifications from a single progenitor cell, such as a seed.

Unlike Track 1 efforts, which are directed at specific applications, Track 2 is meant to support basic research projects that answer fundamental questions about how assemblies of cells form higher-order structures, and to explore new methodology for encoding developmental programs at the genetic level. The foundational knowledge being sought through this program track will likely require collaborative efforts from members of the synthetic biology, computer science, and developmental biology communities. Proposers are strongly encouraged to form teams that include experts in each of these areas, and teaming strategies should be specifically addressed in the proposal.

DARPA has identified two key objectives for controlling structural features of multicellular systems, which are individually addressed by the Track 2 Task Areas described below. Proposals can address one or both of the task areas in Track 2. Each task area is designed to occur in two phases over 24 months. In the first phase, performing teams will design and prototype genetic circuits to enable differentiation in a clonal population, according to the specifics of the individual Task Area. In Phase II, teams will engineer and demonstrate control over the final form of the model system by programming a range of arbitrary patterns or shapes. Approaches that represent incremental improvements on existing technology are specifically discouraged. The final deliverable for both task areas is the demonstration of a set of design principles for a multicellular system with the following minimum characteristics:

A single genome that is engineered to include a developmental pathway to produce a multicellular structure or pattern with specified properties from a single progenitor cell.

The method of development must not require active cues from the environment or experimenter.

Track 2 - Task Area A: Programmable Patterns

Track 2 - Task Area A is aimed at exploring design approaches to create artificial biological multicellular systems that can be programmed to form precise patterns of differentiated cells from a single progenitor cell with a defined genotype. The particular functions that cells can be programmed to perform within a multicellular system are beyond the scope of the ELM program.

Proposals should describe an original and innovative approach that conforms to the following minimum criteria:

The taxonomic identity of the progenitor strain is at the discretion of the proposer and can be derived from prokaryotic or eukaryotic organisms. Proposals should describe in detail the choice of progenitor species and justify its relevant attributes.

Pattern formation must be autonomous and genetically programmed. Neither the pattern, nor the tuning of the pattern should rely on external stimuli provided by the experimenter.

The approach must involve the engineering of a synthetic genetic pathway(s), as opposed to the triggering of developmental pathways of a naturally pluripotent cell.

The pattern must be formed by the spatial distribution of at least two differentiated cell types derived from the same progenitor cell line. The two cell types must be readily and persistently discernable.

The programmed pattern must be stable and irreversible for at least one week.

The desired pattern must be tunable, such that the system can be programmed to produce one of multiple potential patterns prior to the start of development, at the user’s discretion.

The genetic circuits that control pattern formation can leverage mechanisms of natural biological tissue patterning or be entirely synthetic. Proposals should describe in detail the components of the patterning circuit(s), including control points, logic or information processing mechanisms, and underlying theory.

Design approaches should be amenable to modeling and computational simulations.

Proposals should include both experimental and theoretical analysis of patterning control.

Track 2 - Task Area B: Programmable Shapes

Through Track 2 - Task Area B, DARPA seeks innovative approaches to genetically program the development of multicellular systems with defined three-dimensional forms from a single progenitor cell. An obvious but important feature of building materials is that they can be formed into arbitrary shapes to fit a specific space or function. To realize the long-term vision of living matter as a programmable source of structural materials, the growth of the component cells must be coordinated to produce a material of desired shape, and be self-limiting to maintain the desired size and shape while in the built environment.

Track 2 - Task Area B aims to explore design approaches and methods to genetically program structural information, such that an engineered progenitor cell can give rise to a multicellular system with a predetermined three-dimensional form. Proposals should describe an original and innovative approach that conforms to the following minimum criteria:

The taxonomic identity of the progenitor strain is at the discretion of the proposer and can be derived from prokaryotic or eukaryotic organisms. Proposals should describe in detail the choice of progenitor species and justify its relevant attributes.

The approach must involve the engineering of a synthetic genetic pathway(s), as opposed to solely triggering of developmental pathways of a naturally pluripotent cell.

Multicellular shape formation must be autonomous and genetically programmed. Neither the shape, nor the tuning of the shape should rely on external stimuli provided by the experimenter.

Cells on the surface of the developed system must be distinct from those in the interior. The difference between interior and exterior cells can be a simple as the differential expression of a reporter, such as a fluorescent protein. The two cell types must be readily and persistently discernable and derive from the same progenitor cell line.

The programmed size and shape must be stable in the developed system for at least one week.

The desired shape must be tunable, such that the system can be programmed to produce one of multiple potential shapes prior to the start of development, at the user’s discretion.

The genetic circuits that control the engineered morphology can leverage mechanisms of natural biological body plan development or be entirely synthetic. Likewise, the design of morphology circuits can be based on new or existing theory. Proposal should describe in detail the components of the morphology circuit, including control points, logic or information processing mechanisms, and underlying theory.

Design approaches should be amenable to modeling and computational simulations.

Proposals should include both experimental and theoretical analysis of morphology control.

1.4 ELM Program Metrics & Milestones:

Proposals should be written to address either Track 1 or Track 2 – proposals that address both Tracks will be considered non-conforming and rejected without review. A proposal may address multiple task areas within the chosen program track; however, the approach to each task area must be described independently, with a separate statement of work and budget plan, and proceed in parallel to the other(s). The metrics and major milestones for each phase of each task area are outlined below. Proposals should include a statement of work that meets these minimum requirements on the provided schedule. Proposals should suggest relevant metrics and minor milestones that are consistent with the proposed approach.

0 10 20 30 40 50 60

Phase II

Track 2 Phase I

Phase III

Phase II

Track 1 Phase I

Months

ELM Program Structure

Track 1: Hybrid Engineered Living Materials

Task Area A: Phase I (Months 1 through 12)

Goal: design and prototype a hybrid living material with short doubling time under ambient conditions.

Milestone 1: demonstrate growth of the proposed living components on the proposed support substrate(s), with a doubling time of ≤ 7 days.

Milestone 2: demonstrate viability of the living component on the support substrate under relevant environmental conditions for a minimum of 1 month. Viability is defined as the ability of the cells to reproduce, as measured by an assay that is appropriate for the chosen organism (e.g., colony forming assay). The environmental conditions to be tested must be specified by the proposal and must be relevant to the intended application of the final material.

Milestone 3: demonstrate the ability to form arbitrary shapes from the hybrid living material. The method should produce a piece of living material of approximately 500 cm3 within 7 days of growth.

Task Area A: Phase II (Months 13 through 30)

Goals: expand on work from Phase I by engineering growth control into the hybrid living material such that arbitrary shapes are stable (i.e., do not overgrow or deteriorate over time). Optimize growth rate during material production.

Milestone 4: demonstrate the stability of arbitrary shapes formed from the hybrid living material in the relevant growth environment (as for milestone 2) over a period of 1 month.

Relevant physical properties (e.g., strength, density, porosity) must also be stable over this time period. The target ranges of these measurements must be included in the proposal and be consistent with the intended function of the final product.

Milestone 5: demonstrate improved growth of the living component on the support material with doubling time of ≤ 3 days under the relevant environmental conditions.

Task Area A: Phase III (Months 31 through 48)

Goal: expand on work from Phase II by engineering the hybrid living material with the ability to reproduce itself from pre-existing material.

Milestone 6: demonstrate the ability to seed the growth of new batches of hybrid living material from at most 50% pre-existing material that had been formed into stable arbitrary shapes. Proposals should include target metrics for minimum seeding requirements, as well as material doubling time. The doubling time for the final product must be ≤ 3 days.

Milestone 7: demonstrate the ability to produce at least five consecutive generations of hybrid living material using the methods demonstrated for milestone 6. Demonstrate that the relevant physical properties, as described for milestone 4, are identical across successive generations.

Task Area B: Phase I (Months 1 through 12)

Goal: design and prototype a hybrid living material composed of living cells that organize an inert material into a patterned array at a sub-cellular scale.

Milestone 1: demonstrate the functional interaction of living and structural components of the material, such that the structural components are organized by the living components into a precise pattern through sub-cellular interactions. Proposals must include target metrics for the distribution and regularity of the organized structural material, and target measurements for the expected emergent physical properties produced by the pattern.

Milestone 2: demonstrate the viability of the living component in the hybrid material under relevant environmental conditions for a minimum of 1 month. Viability is defined as the ability of the cells to reproduce when transferred to optimal growth conditions, as measured by an assay that is appropriate for the chosen organism (e.g., colony forming assay). The environmental conditions to be tested must be specified by the proposal and must be relevant to the intended application of the final material.

Task Area B: Phase II (Months 13 through 30)

Goal: expand on work from Phase I to develop a living material that self-organizes into a pattern at a multi-cellular scale.

Milestone 3: engineer the living components of the hybrid material such that they organize the sub-cellular pattern developed in Phase I into a higher-order multi-cellular pattern (i.e., a pattern of patterns). Proposals must include target metrics for the distribution and regularity of the organized structural material at each relevant scale.

Milestone 4: demonstrate production of the self-organizing living hybrid material with multi-scale patterning. Proposals must provide target measurements for the expected emergent physical properties produced by the multi-scale pattern. Demonstration product must be at least 1 cm3 in size.

Task Area B: Phase III (Months 31 through 48)

Goal: expand on work from Phase II to develop a living material that self-organizes into a multi-scale pattern with tunable physical properties.

Milestone 5: engineer the living components of the hybrid material developed in Phase II to allow the multi-scale pattern to be controlled across a range of relevant parameters, including but not limited to particle spacing, density, and aspect ratio. Proposals must include target metrics for the distribution and regularity of the organized structural material at each relevant scale.

Milestone 6: demonstrate production of at least three variations of the self-organizing living hybrid material, as in milestone 4, with a range of emergent physical properties, specified by the various patterns produced through implementation of the control mechanisms developed for milestone 5. Proposals must provide target measurements for the expected emergent physical properties produced by each multi-scale pattern.

Demonstration products must be at least 1 cm3 in size.

Task Area C: Phase I (Months 1 through 12)

Goal: design and prototype a hybrid living material composed of a structural core and a living protective surface.

Milestone 1: demonstrate that the growth of the living component is constrained to 2.5-dimensions along the surface of the core material. The demonstration product must have a surface area of at least 100 cm2.

Milestone 2: demonstrate the viability of the living component in the hybrid material under relevant environmental conditions for a minimum of 1 month. Viability is defined as the ability of the cells to reproduce when transferred to optimal growth conditions, as measured by an assay that is appropriate for the chosen organism (e.g., colony forming assay). The environmental conditions to be tested must be specified by the proposal and must be relevant to the intended application of the final material.

Milestone 3: demonstrate that the cells of the living surface of the hybrid material form a cohesive tissue of interconnected cells. Proposals must describe methods for examining the connections between cells according to the material design, and the minimal metrics of connectivity for establishing a functional tissue. Proposals must include target metrics for physical properties of the living hybrid material (e.g., tensile strength, porosity, and hydrophobicity).

Task Area C: Phase II (Months 13 through 30)

Goals: expand on work from Phase I to engineer the hybrid living material with the ability to self-repair in response to damage. Engineer the hybrid living material with the ability to sense a relevant environmental stimulus.

Milestone 4: demonstrate the self-repair of the hybrid living material in response to a single type of damage (e.g., tearing, erosion, or infection) using methods and metrics developed for milestone 3 to measure the formation of regenerated material at the site of damage. The target self-repair rate must be at least 1 cm2 per day.

Milestone 5: demonstrate the ability of the hybrid living material to sense a relevant environmental stimulus. Proposals must include methods and target metrics for measuring sensing that are appropriate for the proposed design. The cells of the hybrid living material must be able to sense the environmental stimulus uniformly, even when the stimulus is presented to only a portion of the surface. Methods for measuring environmental sensing must reveal the dynamics of signal propagation throughout the entire material.

Task Area C: Phase III (Months 31 through 48)

Goals: expand on work from Phase II to engineer the hybrid living material with the ability to self-repair in response to two additional types of damage. Engineer the hybrid living material with the ability to respond to an environmental stimulus with a tissue-level change in a physical property of the entire living material.

Milestone 6: demonstrate the self-repair of the hybrid living material in response to additional types of damage (e.g., tearing, erosion, or infection) using methods and metrics developed for milestones 3 and 4. The target self-repair rate must be at least 1 cm2 per day.

Milestone 7: demonstrate the ability of the hybrid living material to respond to the environmental stimulus targeted in milestone 5 with a coordinated tissue-level response in the hybrid living material. The cells of the hybrid living material must be able to respond the environmental stimulus uniformly, even when the stimulus is presented to only a portion of the surface. The engineered tissue-level response must be a change in some physical property of the hybrid living material. Proposals must include methods and target metrics for measuring the physical change and the change dynamics.

Milestone 8: demonstrate that the engineered sense-and-response functions developed for milestone 7 are retained following self-repair in response to the types of damage tested in milestone 6.

Track 2: Programmable Engineered Living Materials

Task Area A: Phase I (Months 1 through 12)

Goals: explore design approaches for genetically programming a single cell line to differentiate into multiple cell types, and to autonomously form regular patterns of differentiation in an expanding clonal population.

Milestone 1: develop theoretical models for programmed cellular differentiation patterning circuits. Perform computer simulations to predict the range of possible differentiation patterns, as well as the stability and reproducibility of the patterns that arise for a chosen genetic design.

Milestone 2: design and build genetically programmed circuits that control the autonomous differentiation of cells in a clonal population to form at least two distinct cell types. The cell fates must be irreversible; however, differentiated cells can be programmed to give rise to daughter cells of the same or different cell type, or to be non-dividing, as needed for the approach to milestone 3. Methods and target metrics for monitoring the differentiation of cells and distinguishing the two (or more) populations within the colony must be included in the proposal.

Milestone 3: design and build genetically programmed circuits that control the pattern of differentiation of the cells engineered for milestone 1. Methods and target metrics for monitoring the pattern formation within the clonal population must be included in the proposal.

Milestone 4: demonstrate that the differentiation pattern engineered for milestone 3 is stable for at least 1 week under optimal growth/differentiation conditions.

Task Area A: Phase II (Months 13 through 24)

Goals: expand on work from Phase I to design and build tunable control of differentiation patterns in a clonal cell population.

Milestone 5: use theoretical models developed for milestone 1 to predict points and potential mechanisms of control of differentiation patterns. Perform computer simulations that illustrate the range of possible patterns, as well as the stability and reproducibility of pattern formation.

Milestone 6: design and build genetically programmed control elements that allow differentiation patterns to be tuned in predictable ways to produce a variety of potential patterns. Proposals must include methods for programming potential patterns into the progenitor cell line, and metrics for identifying individual patterns. Programming methods may be reversible or irreversible, provided that the final differentiation pattern is stable.

Programming methods may include external stimuli; however, once development of the pattern has started it must proceed autonomously without external cues provided by the experimenter.

Milestone 7: demonstrate the production of at least three distinct patterns from a single engineered progenitor cell line based on methods developed for milestones 5 and 6.

Demonstrate that each programmed pattern is stable over a minimum period of 1 week under optimal growth/differentiation conditions.

Task Area B: Phase I (Months 1 through 12)

Goals: explore design approaches for genetically programming a single cell line to give rise to a multicellular system with a predetermined three-dimensional form (i.e., morphology).

Milestone 1: develop theoretical models for programmed development of morphology in an expanding clonal cell population. Perform computer simulations to predict the range of possible forms, as well as the stability and reproducibility of the morphology that arises for a chosen genetic design.

Milestone 2: design and build genetically programmed circuits that control the autonomous differentiation of cells according to whether they occupy an interior or exterior position in the three-dimensional cell mass. Methods and target metrics for monitoring the differentiation of cells and distinguishing the phenotypes of interior and exterior cells must be included in the proposal.

Milestone 3: design and build genetically programmed circuits that control the development of a defined morphology (e.g., a platonic solid) in an expanding population of cells engineered for milestone 1. Methods and target metrics for monitoring morphology development within the clonal population must be included in the proposal.

Milestone 4: demonstrate that the final three-dimensional size and shape of the clonal population engineered for milestone 3 is stable for at least 1 week under optimal growth/differentiation conditions.

Task Area B: Phase II (Months 12 through 24)

Goals: expand on work from Phase I to design and build tunable control of three-dimensional form in a clonal cell population, with differentiated surfaces.

Milestone 5: use theoretical models developed for milestone 1 to predict points and potential mechanisms of control of morphology development. Perform computer simulations that illustrate the range of possible forms, as well as the stability and reproducibility of morphology development.

Milestone 6: design and build genetically programmed control elements that allow development to be tuned in predictable ways to produce a variety of potential forms (e.g., a series of platonic solids). Proposals must include methods for programming potential morphologies into the progenitor cell line, and metrics for identifying individual forms.

Programming methods may be reversible or irreversible, provided that the final size and shape is stable. Programming methods may include external stimuli; however, once development of the system has started it must proceed autonomously without external cues provided by the experimenter.

Milestone 7: design and build differentiation pathways that distinguish between exterior cells on various faces of the final three-dimensional form (analogous to the different faces of gaming dice).

Milestone 8: demonstrate the production of at least three distinct three-dimensional forms with discernible faces from a single engineered progenitor cell line based on methods developed for milestones 5, 6, and 7. Demonstrate that each programmed form is stable in size and shape over a minimum period of 1 week under optimal growth/differentiation conditions.

2. Award Information

Multiple awards are possible. The amount of resources made available under this BAA will depend on the quality of the proposals received and the availability of funds.

The Government reserves the right to select for negotiation all, some, one, or none of the proposals received in response to this solicitation, and to make awards without discussions with proposers.

The Government also reserves the right to conduct discussions if it is later determined to be necessary. If warranted, portions of resulting awards may be segregated into pre-priced options.

Additionally, DARPA reserves the right to accept proposals in their entirety or to select only portions of proposals for award. In the event that DARPA desires to award only portions of a proposal, negotiations may be opened with that proposer. The Government reserves the right to fund proposals in phases with options for continued work at the end of one or more of the phases.

Awards under this BAA will be made to proposers on the basis of the evaluation criteria listed below (see section labeled “Application Review Information”, Sec. 5.), and program balance to provide overall value to the Government. The Government reserves the right to request any additional, necessary documentation once it makes the award instrument determination. Such additional information may include, but is not limited to, Representations and Certifications.