HTP-APP_RFP_Attach 1_Performance Specification_Rev. 2.pdf

S814057-SPEC-02 Rev 2

Microbial Molecular Phenotyping Capability

(M2PC)

High-Throughput Automated Phenotyping Platform Performance Specification December 2024

Theva Thevuthasan

DISCLAIMER

This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor Battelle Memorial Institute, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights.

Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof, or Battelle Memorial Institute. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof.

PACIFIC NORTHWEST NATIONAL LABORATORY

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BATTELLE

for the

UNITED STATES DEPARTMENT OF ENERGY

under Contract DE-AC05-76RL01830

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Microbial Molecular Phenotyping Capability (M2PC)

High-Throughput Automated Phenotyping Platform Performance Specification

Theva Thevuthasan

Pacific Northwest National Laboratory Richland, Washington 99354

Signatures ii

Signatures

Submitted by:

Suntharampillai Thevuthasan, M2PC R&D Project Manager Date

Reviewed by:

Samantha Ernst, M2PC Contracts Specialist Date

Darrell De Smet, M2PC R&D/Facility Coordination Project Manager Date

Scott Baker, M2PC R&D Consultant Date

Sam Paulsen, M2PC Project Manager Date

Lanson Oukrop, M2PC Project Director Date

Approval:

Michael Huyck, Associate Lab Director Operational Systems Date

Douglas Mans, M2PC Capability Steward Date

Change Log iii

Change Log

Revision History

Rev. Date Reason 0 July 2024 Original

1 Sept 2024 Address 3rd party review comments, update facility coordination section to align with facility P-Spec and add pressure and fire safety requirements.

2 December 2024 Address PNSO, IRB, and HCA comments. Clarify cybersecurity requirements.

Acronyms and Abbreviations iv

Acronyms and Abbreviations

2-D/3-D two dimensional/three dimensional a.k.a. also known as

ANSI American National Standards Institute

BER Biological and Environmental Research (Office of)

BSL-1 Bio Safety Level 1

BSL-2 Bio Safety Level 2

CDA clean, dry air

CDR Conceptual Design Report

CFM cubic feet per minute

DOE U.S. Department of Energy

EMSL Environmental Molecular Sciences Laboratory

FACP fire alarm control panel

FMCS facility management control system

FWHM full width at half-maximum

GC gas chromatograph/chromatography

HESI heated electrospray ionization

HTP-APP High-throughput Automated Phenotyping Platform

ISO International Organization for Standards

KPPs Key Performance Parameters

LC liquid chromatograph/chromatography

LIMS Laboratory Information Management System

M2PC Microbial Molecular Phenotyping Capability

MESF molecules of equivalent soluble fluorochrome

MS mass spectrometry

N2 nitrogen (gas)

NRTL National Recognized Testing Laboratory

OD optical density

OSHA Occupational Safety and Health Administration

PCR polymerase chain reaction

Acronyms and Abbreviations v

PNNL Pacific Northwest National Laboratory psig pounds per square inch, gauge

RFP Request for Proposal

SBS Society of Biomolecular Screening

SC Office of Science

SDK software development kit

SME(s) subject matter expert(s)

UL Underwriters Laboratories

UV ultraviolet

Contents vi

Contents

Signatures.............................................................................................................................................................................. ii

Change Log ...........................................................................................................................................................................iii

Acronyms and Abbreviations ............................................................................................................................................. iv

1.0 Executive Summary

2.0 Purpose and Scope

2.1 Scope

2.1.1 Schedule of Deliverables

2.1.2 Stretch Goals

2.1.3 Facility Coordination

2.2 Design Approach

2.2.1 Design Requirements

2.2.2 Design Commitment

3.0 Project Management and Execution

3.1 Quality Management

4.0 Performance Requirements

4.1 Key Performance Parameters

4.1.1 Demonstrate HTP Culturing of Microbial Isolates and Consortia

4.1.2 Demonstrate HTP Assaying and Phenotyping of Microbial Isolates and Consortia

4.1.3 Provide Capability for Remote Planning and Operations

4.2 General System Requirements

4.3 Science Core Functions

4.3.1 Media

4.3.2 Cultivation

4.3.3 Sample Preparation

4.3.4 Strain Engineering

4.3.5 Analytical Capabilities

4.4 Remote Planning and Operation

4.5 Automation System Control Software Design

4.4 Safety and Security

4.6.1 Safety by Design

4.6.2 BSL-2 Requirements

4.6.3 Robotics

4.6.4 Electrical Safety

4.6.5 Hearing Safety – Allowable Noise Levels

Contents vii

4.6.6 Cybersecurity

4.6.7 Pressure and Compressed Gas Safety

4.6.8 Fire Safety

4.7 Automation Laboratory Facility Coordination

4.8 System Testing

4.8.1 Factory Testing

4.8.2 On-Site Functional Testing

4.8.3 On-Site Commissioning

4.8.4 Readiness

4.9 Training

4.10 Consumables

4.11 System Backup and Recovery

4.12 Redundancy

4.13 Technical Support

5.0 Stretch Goals

5.1 Exceed Both Threshold KPP#1 and KPP#2 by One Increment above Threshold

5.2 Exceed Both Threshold KPP#1 and KPP#2 by Two Increments above Threshold

5.3 Exceed Both Threshold KPP#1 and KPP#2 by Three Increments above Threshold

5.4 Exceed Both Threshold KPP#1 and KPP#2 by Four Increments above Threshold

APPENDIX A: Deliverables

APPENDIX B: Equipment Specification Examples

APPENDIX C: Definitions

APPENDIX D: Science Core Functions

APPENDIX E: Automation System Control Software HTP-APP

APPENDIX F: Background and Mission Need Statement

Executive Summary 1

1.0 Executive Summary

The mission of the U.S. Department of Energy (DOE) is to ensure America's security and prosperity by addressing its energy, environmental, and nuclear challenges through transformative science and technology solutions. The DOE Office of Science (SC) supports this mission by delivering the scientific discoveries and major scientific tools that transform our understanding of nature and advance the energy, economic, and national security of the United States. The SC Biological and Environmental Research (BER) program’s mission is to advance transformative science and scientific user facilities to achieve a predictive understanding of complex biological, earth, and environmental systems for energy and infrastructure security, independence, and prosperity.

Within BER, basic research to gain a predictive understanding of microbial systems provides the foundation for harnessing and integrating the latest biosystems design techniques with data science, and multi-scale modeling approaches to advance not only a burgeoning bioeconomy but also for predicting the future state of the Earth system. In this regard, the number of microbial systems interrogated is expanding rapidly given the advances in next-generation genome sequencing and engineering capabilities. In contrast, the throughput capabilities to assess the phenotypes (observable traits) of these microbes accurately and expediently is quite low and the range of characterized functions of these microbes in engineered or natural systems is very limited. There is a rapidly growing but unmet need to understand the determinants of microbial function at the molecular and biochemical level at an unprecedented capacity and pace, well beyond those available within BER user facilities. Currently, there is a significant gap in the scientific community’s ability to identify proteins and biochemical pathways of unknown function in microbes at the single-cell to microbial community scales, in part because microbial phenotypes change rapidly due to environmental factors and perturbations.

Without this understanding of the molecular and biochemical determinants of microbial functions complementing existing genomics understanding, the research community is severely limited in their ability to understand, model, and controllably design the range of activities and functions of individual microbes, engineered microbial consortia with bio-industrial potential or ecological relevance, and microbial communities from natural soil environments. The Microbial Molecular Phenotyping Capability (M2PC) project will address this need by providing a new high-throughput, highly automated molecular phenotyping capability for a broad range of microbes and microbial communities. Researchers will use the M2PC to generate data needed to develop a mechanistic and biomolecular understanding of microbial activity and function across a wide range of species in the environment and in engineered consortia at a capacity and pace to match and ideally exceed that of genomics capabilities. Functional understanding at the biomolecular level is critical for scientists to make new discoveries in the use of microbial systems for advancing U.S. leadership in the projected $4T/year global bioeconomy and for providing a more complete and accurate understanding of the Earth system that informs policies and plans for ensuring the security and resilience of the nation’s critical infrastructure and natural resources.

To address these needs, Battelle seeks to acquire a fully functional High-Throughput Automated Phenotyping Platform (HTP-APP) to increase the data throughput, content, quality, and reproducibility of experimental analyses. The platform shall enable researchers to investigate a wide breadth of dynamic parameters to elucidate the relationship between genotype and phenotype with a variety of analytical methods and the ability to integrate future technologies. The platform shall enable researchers to perform experiments efficiently in high throughput with dynamic and interleaved protocol scheduling, optimized resource usage, and require minimal operator intervention.

Executive Summary 2

This document defines the minimum technical performance requirements for a contractor experienced in the field to manage and execute the design, fabrication, assembly, installation, testing, and training for a complete HTP-APP system that will be operated by and housed at the Environmental Molecular Sciences Laboratory (EMSL), located on the Pacific Northwest National Laboratory (PNNL) campus.

The HTP-APP contractor shall coordinate with a yet-to-be-determined design-build facility contractor to ensure the appropriate utilities are available in a newly constructed addition to EMSL where the HTP-APP will reside.

Due to the complexity of the performance requirements for the system modularity, flexibility, software, and equipment necessary to provide a fully functional platform, Battelle recognizes that an HTP-APP contractor may team with other subcontractors for certain activities or lead a group of subcontractors to complete the project.

Final acceptance of the requirements for the HTP-APP contractor will include an on-site functional test that verifies that the project Key Performance Parameters (KPPs), as defined in this document, have been met.

Performance requirements for desired scope, above and beyond the minimum required scope, are also defined.

Purpose and Scope 3

2.0 Purpose and Scope

A key component for one of EMSL’s strategic objectives is to increase the throughput and data content, quality, and reproducibility of its analyses. To create this capability, Battelle seeks to acquire an automated microbial molecular phenotyping platform that enables researchers to investigate a wide breadth of dynamic parameters to elucidate the relationship between genotype and phenotype in microbial systems using a variety of analytical methods with minimal need for operator intervention.

This performance specification contains technical requirements for an HTP-APP that meets the mission need for the M2PC project. The information put forth in this performance specification is the minimum understood and required of the HTP-APP contractor at the time of issue. As an experienced automation contractor, or team, that specializes in the design and installation of high-throughput (HTP) automated phenotyping systems, it is understood that the contractor shall collaborate with EMSL and the facility contractor throughout the design, acquisition, fabrication, factory acceptance testing, installation, functional testing, commissioning, and training processes to successfully deliver a fully operational HTP-APP that meets the performance requirements.

2.1 Scope

The contractor shall provide a fully functional system and demonstrate that the HTP-APP has achieved the project KPPs as given in Table 2.1. The HTP-APP system shall be installed and tested at a newly constructed facility at EMSL.

Table 2.1: Project Key Performance Parameters

2.1.1 Schedule of Deliverables

Providing a fully functional HTP-APP comprises multiple facets and deliverables. Appendix A provides detailed descriptions of each deliverable. At a high level, the deliverables are:

1. System design

a. 30% design package

b. 60% design package (coincides with Design Commitment)

c. 90% design package

d. Final design package

2. Equipment procurement

3. System assembly

KPP# KPP Title KPP Threshold, Minimum Requirement

KPP#1

Demonstrate HTP culturing of microbial isolates and consortia

(i) Capacity to operate M2PC with execution of 500 new strain experiments per week.

(ii) Capacity to operate M2PC with execution of 100 new microbiome experiments per week.

KPP#2

Demonstrate HTP assaying and phenotyping of microbial isolates and consortia

Capacity to operate M2PC to obtain 1,000,000 multimodal analytical measurements per month.

KPP#3

Provide capability for remote planning and operations

Demonstrate that remote users/operators can run pre-defined EMSL protocols to be executed autonomously within M2PC across culturing, assaying, and analyses.

Purpose and Scope 4

4. Factory testing

5. Installation at the M2PC facility

6. Software installation

7. Connection to the EMSL facility Lab Information Management System (LIMS)

8. Startup and commissioning of system components, equipment, and instruments

9. Functional testing and demonstration

10. Training Battelle staff to operate, maintain, and modify the physical system and software

11. KPP verification

2.1.2 Stretch Goals

The primary focus of this performance specification is to establish minimum requirements for the contractor to meet the project threshold KPPs. However, the HTP-APP contractor is encouraged to pursue stretch goals and scope as part of the contract budget to maximize the best value to BER. In order of preference, consider the following stretch goals for inclusion in the offeror’s proposal. The stretch goals are defined in detail in Section 5.0.

1. Exceed both Threshold KPP#1 and KPP#2 by one increment above threshold.

2. Exceed both Threshold KPP#1 and KPP#2 by two increments above threshold.

3. Exceed both Threshold KPP#1 and KPP#2 by three increments above threshold.

4. Exceed both Threshold KPP#1 and KPP#2 by four increments above threshold.

2.1.3 Facility Coordination

The HTP-APP shall be installed and reside in a newly constructed facility that is composed of laboratory and support space in a new expansion to the existing EMSL. EMSL is an SC BER scientific user facility on the PNNL campus in Richland, Washington. The new facility will be designed and built by a construction contractor in parallel with the HTP-APP contract. The facility will include laboratory spaces, functions, systems, and support spaces to house and operate the HTP-APP. The HTP-APP contractor is not responsible for delivering the facility or utilities from the facility; however, the automation contractor shall coordinate their design, utility needs, installation and locations, and delivery schedule with the facility contractor. A partnering attitude and approach with the Battelle M2PC project team and the facility contractor are required to deliver a successful project. The proposal must include a description of the partnering approach.

The HTP-APP contractor shall participate in regular coordination meetings and in facility design reviews (minimum of 4) and respond to facility contractor requests (e.g., RFIs) related to utility and other requirement for the HTP-APP.

2.2 Design Approach

2.2.1 Design Requirements

A Conceptual Design Report (CDR) was previously developed to establish a scope baseline, aligned with a conceptual-level cost estimate, in accordance with DOE Order 413.3B. The CDR is included in the Request for Proposal (RFP) as reference information only and does not indicate or establish requirements or criteria to be executed by the HTP-APP contractor.

Applicable portions of the CDR, experience gained from previous projects, and relevant components of standards have been assembled into this document to identify the performance requirements and criteria for the HTP-APP.

Purpose and Scope 5

The performance requirements have been established with minimal design completion and shall be used by the HTP-APP contractor to complete the appropriate design documentation and test plans. Throughout the design process, the contractor shall perform the design and select the instruments for the HTP-APP in collaboration with Battelle.

The HTP-APP contractor, with its extensive research and high-throughput automated phenotyping equipment knowledge and expertise, shall use the technical requirements defined in this performance specification to develop a complete final design package in collaboration with Battelle staff. This final design package shall include coordination with the facility design-build contractor for the location of and types of utility connection points necessary in the automation laboratory and support spaces. Design packages will require review and approval by Battelle (EMSL/PNNL) and DOE, including the project team, subject matter experts (SMEs), and applicable authorities having jurisdiction.

In the event of conflicting specification content, quantity, or code requirement (see Section 4.6 Safety and Security), the HTP-APP contractor shall use the most stringent criteria and/or the largest/greatest number specified. In case of conflicts, the order of precedence shall be as defined in the RFP.

2.2.2 Design Commitment

The system throughput values included in the offeror’s proposal (minimum base requirements plus stretch goals) are the foundation for the system design and contract award; however, it is understood that the exact equipment types and numbers to achieve the proposed system throughput values could change with further design development. Therefore, to allow for design maturation and flexibility, the project requires design commitment from the HTP-APP contractor coincident with the 60% design package. Design commitment means that the features of work submitted in the 60% design package conform to the proposal throughput values, contractor’s budget, schedule, and the quality requirements of the contract. The contractor commits that the system and associated equipment will be delivered as detailed within the 60% design package. Design Commitment occurs upon the submission, by the HTP-APP contractor, of the 60% design package as detailed in Appendix A, and as approved by the Battelle Contracts Specialist. Reductions in scope, form, fit, finish, or quality after Design Commitment has been achieved shall not be permitted unless expressly approved, in writing, by the Battelle Contracts Specialist.

The 60% design package, including outstanding comments made during the 30% and 60% design package reviews, forms the basis of design.

Project Management and Execution 6

3.0 Project Management and Execution

The contractor shall develop a project management plan detailing the proposed project execution for approval by Battelle. This will include a schedule based on the information below. This schedule is presented as an overview. Refer to the RFP documentation for detailed schedule information.

Proposed Project Schedule

*Presented for information only. These activities are the responsibility of Facility D/B Contractor, not the HTP-APP contractor.

Once the contract is awarded to the HTP-APP contractor, the contractor shall use the above schedule as a framework to develop an integrated schedule with the facility design-build contractor. This schedule shall be indexed on the design-build contractor “ready for equipment” milestone in the automation laboratory. The HTP-APP contractor will not be able to deliver and begin the installation of the equipment in the facility until this facility contractor milestone is accomplished.

The HTP-APP shall be executed in multiple tasks as described in the schedule of deliverables (Appendix A). Modifications to the timing and type of the design, procurement, assembly, testing, and training tasks can be proposed by the HTP-APP contractor to maximize efficient project execution. A proposed modification to the defined timing and deliverable description is subject to Battelle review and approval and there are no guarantees that a modification will be approved.

Project Management and Execution 7

3.1 Quality Management

Refer to S814057-SPEC-01, M2PC HTP-APP General Requirements, Section 5.0, for detailed quality requirements.

Performance Requirements 8

4.0 Performance Requirements

Figure 4.0 diagrammatically depicts the scope of the complete M2PC HTP-APP.

Figure 4.0: HTP-APP Scope

4.1 Key Performance Parameters

The minimum scope requirements of the HTP-APP to meet the BER mission need are defined as the Key Performance Parameters, or KPPs. The contractor shall deliver a fully operational system to meet the KPPs.

The minimum KPPs are defined within Section 4.1. The KPPs that shall be met during acceptance testing are the KPP values defined by the offeror in the proposal, which includes stretch goal commitments. For each KPP, list the factor(s) limiting throughput.

4.1.1 Demonstrate HTP Culturing of Microbial Isolates and Consortia

The HTP-APP shall demonstrate HTP transformation and culturing of microbial isolates and multi-species consortia of diverse physiologies such as heterotrophic, chemotrophic, and phototrophic metabolism in various environments. The complete system shall meet the cultivation KPP during the acceptance testing. The minimum KPP is defined below.

Performance Requirements 9

KPP Threshold (minimum) Demonstrate HTP culturing of microbial isolates and consortia

(i) Capacity to operate M2PC with execution of 500 new strain experiments* per week.

(ii) Capacity to operate M2PC with execution of 100 new microbiome experiments** per week.

*Strain Experiments: A strain is a sub-variety within a species with unique characteristics such as genetic makeup, metabolism, and morphology, and the strain experiments can be designed as the following:

(1) Constructing mutant plasmids to create a test panel

(2) Transforming plasmids to create engineered microbial strains

(3) Cultivating strains across different media, nutrient/metals & carbon sources, temperatures, pH, among others, and analyzing their growth

An example is provided below:

(1) Construct 500 single or multiple gene knockout, up-regulated or down-regulated/”knock-down” constructs.

(2) Use 12 replicates of each.

(3) Grow each in 4 media types with 2 different carbon sources.

(4) Test growth at multiple time points.

(5) Total of 500 “experiments” performed in 500 96-well plates at multiple time points.

**Microbiome Experiments: A microbiome is composed of multiple strains of microorganisms that can usually be found living together in a given habitat, and the microbiome experiments are associated with creating communities of one or more unique microbes in varying proportions and a variety of media/growth conditions to perturb the system and measure how the constituents of the community respond. These perturbations include temperature range, pH range, various metal ion concentrations, addition of various small molecules such as a primary or secondary metabolites and different carbon and/or nitrogen sources.

4.1.2 Demonstrate HTP Assaying and Phenotyping of Microbial Isolates and Consortia

The HTP-APP shall demonstrate HTP assaying of optical, morphological, physiological, biochemical, and/or genetic properties of organisms. In addition, the HTP-APP shall provide HTP cell separation, handling, and sample preparation capability. The complete system shall meet the following assaying/phenotyping KPP during the acceptance testing. The minimum KPP is defined below.

KPP Threshold Demonstrate HTP assaying and phenotyping of microbial isolates and consortia.

Capacity to operate M2PC to obtain 1,000,000 multimodal analytical measurements* per month.

*These measurements are associated with capturing scientifically relevant data during the growth of microorganisms, functional responses associated with experimental perturbations (including but not limited to varying media composition, temperature, nutrient sources, pH, and aeration rate), and after sample processing/preparation. Examples include:

(1) Various spectroscopy and spectrometry measurements can be carried out to characterize the growth and processed samples.

(2) Cell characteristics including cell size, cell count, and cell cycle can be measured using spectral and image cytometry.

(3) The samples can be characterized using gas chromatography (GC) or liquid chromatography (LC) coupled with ultraviolet (UV), fluorescence detection (FLD), mass spectrometry (MS), or refractive index detection (RID) to detect the chemical components.

(4) Automated microscopy measurements can be used to characterize cell morphology and/or fluorescence.

Performance Requirements 10

4.1.3 Provide Capability for Remote Planning and Operations

The HTP-APP shall demonstrate remote planning and operations of the platform by demonstrating that remote users can run pre-defined EMSL protocols to be executed autonomously within M2PC. Protocol settings shall have built-in acceptable safe operating ranges for selection within established instrument specifications from vendors, EMSL protocol best-practices, and Battelle Environmental Safety and Health safe research operating windows. The system shall demonstrate remote user planning and operation prior to the completion of site acceptance testing. The minimum KPP is defined below.

KPP Threshold Provide capability for remote planning and operations.

Demonstrate that remote users/operators can run pre-defined EMSL protocols to be executed autonomously within M2PC across culturing, assaying, and analyses.

4.2 General System Requirements

In addition to the specific requirements for each science core function of the HTP-APP, the contractor shall provide a description of the approach to meet the following requirements:

1. Provide a system to maximize the throughput, efficiency, overall capability uptime, and sample uniformity while incorporating sample tracking and capturing of metadata for the capability.

2. Provide a system that is modular and customizable through the addition or removal of individual components.

a. Time required to add or remove a component shall be within hours to days.

b. Minimize the impact to throughput while an individual component is added or removed

c. Accommodate rapid advances in technology to easily expand the flexible system, integrate emerging technologies, and adopt and/or modify new or existing workflows.

d. Include integrated experimental systems in a modular and flexible manner and shall be able to conduct entire workflows without human intervention. See Appendix D for an example workflow.

e. Provide for integration of new instruments with only minor updates and at minimal expense. In addition, a modular approach to hardware design must allow system architects to quickly retire old instruments and replace them with new ones (with equivalent or different functions), which in turn facilitates easy adoption of emerging technologies.

3. Provide system software that is modular and customizable such that:

a. Protocols can be interleaved and dynamically scheduled.

b. New applications can be created by EMSL staff to integrate new technologies with minimal effort and cost.

c. The system communicates with the EMSL Laboratory Information Management System

(LIMS)

4. The tradeoffs between modularity and longevity as it pertains to system design decisions, shall be described and justified.

Performance Requirements 11

5. Describe the approach towards safety, ergonomics, and serviceability.

6. Provide manufacturer-non-specific equipment and be able to incorporate instruments from multiple manufacturers.

7. The contractor shall develop a list of critical equipment and instruments, in collaboration with Battelle, and provide the listed items in the identified quantities with the system. The critical equipment list shall include the equipment and instruments, and necessary amounts of the either, such that no single point of equipment or instrument failures would stop system operations. This does not mean that every piece or type of equipment requires a spare, only that critical equipment to maintain system operation has adequate redundancy.

8. Describe the capabilities for communicating operational system status and alerts and how they can be customized for our operation.

9. Minimize system components that reduce physical configuration options of the HTP-APP (e.g. building affixed components, non-flexible utility connections, large unadaptable subsystems, specialized expertise or tools required for moves)

10. The system shall communicate with the EMSL building facility management control system (FMCS) and fire alarm control panel (FACP) to coordinate a safe-to-operate status, operational status, general alarm, and accept an external command to initiate orderly shut-down of the HTP-APP operations from FMCS for the FACP. The required FMCS and FACP signals/information will be provided by Battelle.

11. Design the system for microbial phenotyping and do not include workflows, techniques, or equipment specific only to non-microbial systems (e.g., mammalian cells).

12. Provide each core science function to include automated waste accumulation capabilities. Note: This is separate from the waste accumulation space provided with the facility.

13. Provide an overview that defines the experimental diversity in the conceptual design. This list shall include parameters relevant to microbial phenotype and their options or ranges and may include cultivation temperatures, light control, humidity, gas types and concentrations, and shaking speeds.

4.3 Science Core Functions

The contractor shall provide five science core functions: media, cultivation, sample preparation, strain engineering, and analytical capabilities (4.3.1 to 4.3.5). These five science core functions comprise the HTP- APP. Cold storage shall be provided where required to support the science core functions. Provide a sub-set of offline instruments, if necessary, to deliver the science core functions as appropriate to achieve the KPPs.

Contractors are directed to Appendix D for more detailed examples of protocols and workflows.

4.3.1 Media

The ability to explore a wide parameter space of media conditions is a critical enabler for the breadth of experiments that will be performed by the HTP-APP.

1. Provide media dispensing capability in HTP-APP that shall be readily configurable to explore a variety of substrate combinations of growth media.

Performance Requirements 12

2. Provide media dispensing capability that shall be readily configurable to use Society of Biomolecular Screening (SBS) format plates and tube racks.

3. Provide media dispensing capability in HTP-APP that shall be configurable to bulk dispense a single media type into many plates and different media type and composition to every well, every plate, and/or tubes.

4. Provide media dispensing capability in the HTP-APP that shall readily accommodate bottles of fully prepared media, or media components so that the system dispenses media into the correct plates, wells, and/or tubes based on the experimental needs.

5. Provide a media dispensing capability in HTP-APP system that at a minimum:

o blends different media composition from 12 independent media or media component sources into individual wells in the sample plates (different volume of each input).

o affixes 1D and 2-D barcodes/human-readable labels to 4000 consumables/week.

o washes and dries 1000 units of labware/week, including tip boxes and plates.

o stores 600 tip boxes and 600 plates simultaneously.

o dispenses 5-1500 µL media/well into one thousand 96-well plates/week.

o seals plates with gas permeable seals.

o seals plates with solid seals (e.g., foil, plastic).

o sorts system waste by separating liquid and waste streams.

o separates waste according to safety regulations.

o segregates non-contaminated recyclables.

o alerts operators when the waste accumulation capability is nearing capacity so it can be emptied.

o empties the waste shall not interrupt active runs.

o stores solid wastes.

o tips ready for disposal are separated from their recyclable bases.

o waste sorting for BSL-2 workflows shall be separated and stored within a BSL-2 controlled enclosures.

4.3.2 Cultivation

1. Provide incubators in the cultivation capability to grow organisms and equipment to inoculate plates to meet the KPPs. Required incubation capacity and inherent adjustability to create experimental diversity shall be available so that the cultivation conditions and shaking speeds can be achieved.

2. Provide cultivation capability to create experimental diversity so that a wide range of environmentally relevant parameter space can explored, including cultivation temperatures, light exposures, humidity changes, various gas concentrations, and shaking speeds.

3. At least 1/3 of the cultivation incubators shall be fully integrated with the automation system.

4. The cultivation capability shall use SBS format labware.

5. The cultivation capability shall track an individual plate both between and within incubators so that an individual plate can be removed at any given time, based on experimental needs.

Performance Requirements 13

6. The cultivation capability colony picker shall be capable of transferring colonies on solid substrates such as agar plates or biofilms to both liquid media and solid substrates.

7. The cultivation system shall remove a plate from an incubator, perform an analytical/phenotypic measurement such as optical density (OD), and return the plate to an incubator in less than 10 minutes.

Systems capable of real-time ODs, so that growth kinetics can be observed without disturbing the physiology, are acceptable.

8. The cultivation capability in HTP-APP shall:

o simultaneously cultivate 500 medium well SBS format plates and tube racks.

o inoculate 750 plates/week.

o inoculate with volume ranges from 5 to 250 microliters.

o centrifuge the liquid if necessary.

o seal the plates with gas permeable or solid seals after inoculation.

o provide orbital shaking with up to a 3 mm throw and be able to shake up to 1000 rpm.

o provide temperature control between 4°C and 45°C with humidity control.

o provide variable gas concentration control of O2 and CO2 within the incubator(s) o provide controllable illumination, preferably with proper mitigation of heat transfer to the cultivation plates.

4.3.3 Sample Preparation

Provide appropriate sample preparation techniques for the proposed analytical methods and instruments. This requires capabilities common to most sample preparations (e.g., stamping, sealing, cell lysis, bulk reagent dispensing, magnetic bead-based separation, vacuum, centrifugation, and positive pressure-based purification) and capabilities with lower usage but that expand the landscape of possible analytical techniques (single cell isolation, acoustic or other small volume dispensing, affinity purification, protein and/or metabolite fractionation etc.).

1. Sample preparation capability shall accommodate transfer volume ranges from 5 to 1000 microliters.

2. This capability shall have the capacity to seal, peel, and stamp SBS format plates (or the equivalent operations for SBS format tube racks) to meet the KPPs. All plates shall be sealed with gas permeable or solid seals after dispensing.

3. Sample preparation capability shall select from a multiple number of solvents and buffers. There shall be the capability to select at least 6 solvents/buffers.

4. Sample preparation capability shall have heating, cooling, and shaking capabilities.

5. Sample preparation capability shall have bead beaters capable of automatically adding metal beads to a plate, positive pressure device, sample concentrator, fraction collector, and well-based stirring to mix individual wells of a plate.

6. Sample preparation capability shall have the capability to lyophilize plates. The contractor shall determine if this capability is an integrated automation or offline standalone system.

7. The sample preparation capability in HTP-APP system shall:

o seal 3500 plates/week.

Performance Requirements 14 o perform 3500 stamps/week with transfer volumes of 5-250 µL.

o perform hit-picks/cherry-picks (well-to-well transfers, rather than full plate transfers).

o peel 3500 plates/week.

o bulk dispense solvent/buffer into 3500 plates/week selecting from at least 6 solvents/buffer with 50-1000 µL dispense volume.

o centrifuge 2000 plates/week.

o shake plates.

o support enzymatic reactions; there should be capacity to statically incubate plates with at least three temperature options.

o provide (i) bead beater that automatically adds metal beads to a plate, (ii) positive pressure device, (iii) sample concentrator, (iv) fraction collector, (v) temperature controlled plate shaking, (vi) plate-based magnetic bead extraction and (vii) well-based stirring to mix up individual wells of a plate capabilities (provide more than one device if the instrument throughput is low or limits HTP-APP performance).

o ability to perform sample preparation for various offline capabilities including nuclear magnetic resonance, high field Fourier transform infrared spectroscopy MS, and cryo electron microscopy.

4.3.4 Strain Engineering

To support a wide range of genetic diversity, the HTP-APP shall provide strain building capabilities that are integrated into the HTP-APP. Consider BSL-2 operations in the strain building capability.

1. The strain engineering capability shall be able to perform an individual transformation attempt, e.g. a single well of a plate, representing the start of building a new microbial strain with a unique modified genome.

2. The strain engineering capability shall generate a pooled strain library that contains genetic diversity (e.g., RB-Tn insertion libraries).

3. The strain building capability shall accommodate transfer volume ranges from nanoliters to 1000 microliters. At least one device shall include the ability to dispense picoliter volumes.

4. The strain engineering capability in the system shall have the following features:

o seal, peel, and stamp plates with gas permeable or solid seals after dispensing.

o perform heating, cooling, and shaking.

o perform thermocycling using automation-compatible thermocyclers to support strain build and for cell lysis before DNA extraction.

o Perform liquid handling using a special liquid handler to support nanoliter volume hit picking required to set up polymerase chain reactions (PCRs) in addition to the other liquid handlers to meet the requirements.

o some of the additional instruments to be integrated are the following: colony picker, incubator space, -20°C plate storage, plate peelers, plate sealers, plate readers, centrifuges, and liquid handlers.

o additional instruments to be integrated shall be proposed by the contractor, consisting of some of the following instruments, offeror may also provide additional suggestions beyond the instruments listed:

• automation-compatible thermocyclers which support strain build and cell lysis before

DNA extraction.

Performance Requirements 15

• a special liquid handler to support nanoliter volume hit picking required to set up PCRs (in addition to the other liquid handlers to meet the requirements).

4.3.5 Analytical Capabilities

The system shall provide HTP assaying of optical, morphological, physiological, and biochemical properties of organisms. The complete system shall be equipped with enough analytical capabilities from list items 1 and 2 below that are integrated into the HTP-APP to meet the KPPs. Multiple instruments will be required across the five core science functions.

The analytical instruments shall include the following capabilities. The contractor-proposed instruments shall be identified with specific models:

1. Provide analytical capabilities to include automated plate readers which can address a wide range of scientific questions when paired with various dyes/probes/enzymes.

o Throughput that allows dynamic monitoring of microbe growth and/or physiological state.

o Method support for optical density, live/dead assays, glucose/sucrose assays, pH, phosphate abundance, protein binding assays, and DNA quantification.

o Absorbance, luminescence, and fluorescence at controllable wavelengths or with full spectrum scanning shall be able to be measured.

2. Provide the quality, number, and type of instruments needed in each category when required to meet the system throughput quantities defined in the proposed design, such as:

o automated microscopy/imaging capabilities.

o spectral flow-cytometry capabilities.

o imaging flow-cytometry capabilities.

o cell selection capabilities such as fluorescence-activated cell sorting and colony picking.

o DNA sequencing capabilities.

o quantitative PCR (a.k.a., qPCR) capabilities.

o LC-UV handle 1200 bar of pressure or higher, and at least one of these LCs shall be equipped with refractive index (RI) detector.

o GC-FID analysis of volatiles and other molecules.

3. Provide analytical capabilities to include integrated automated MS capabilities, to allow for proteomic, lipidomic, and metabolomic workflows for a diverse set of molecules. The analytical capabilities shall include state-of-the-art MS imaging capabilities. The system shall include a minimum of five distinct capabilities proposed by the contractor from the equipment types below.

o Trapped ion mobility spectroscopy time of flight (a.k.a., TIMS Tof) o Matrix-assisted laser desorption/ionization time of flight (a.k.a., MALDI ToF) o Orbitrap o Triple quadrupole MS o Quadrupole time of flight (a.k.a., Q – ToF) o LC – MS o GC – MS o Inductively coupled plasma mass spectrometry (a.k.a., ICP – MS)

4. The contractor shall provide a list of emerging (leading technological edge) capabilities that directly

Performance Requirements 16 support meeting the assaying and analytical KPPs for consideration in the design beyond those listed above.

4.4 Remote Planning and Operation

1. The architecture of the system shall allow for remote monitoring, operations, and troubleshooting, including (i) error/fault monitoring and recovery; (ii) remote submission of work, monitoring progress, and reviewing data; and (iii) remote troubleshooting.

2. Software shall have security controls in place, such as authentication and authorization via Keycloak to enable single sign-on for remote users to log into the system and execute their work.

3. Remote users shall be able to submit work, monitor progress, and review data.

4. Remote users shall be able to kick-off protocols remotely by specifying which samples/parameters to use with a given protocol. Protocols kicked off by remote users must interleave with protocols running in parallel.

5. Data management, data transfer, and data processes required are outlined in Appendix E.

4.5 Automation System Control Software Design

1. Scheduling software shall have the capability to dynamically route work between the multiple instruments and shall coordinate operation and execution between all subcomponents.

2. Scheduling software shall have interleaved protocols capable of running multiple protocol runs of different types simultaneously, interleaving protocol runs to maximize system throughput.

3. Software shall have the capability for remotely monitoring and troubleshooting highly modular operations. Troubleshooting and remote maintenance shall include remote power cycling of instruments, pulling error logs, and performing basic instrument functions remotely (e.g., on/off, data acquisition, error code).

4. Software shall accommodate maintenance, servicing, upgrades, and expansion of the platform with minimal downtime (on the scale of hours to days) and without interruption of executed protocols on the unaffected portion(s) of the system to ensure maximal operational uptime.

5. Software shall be expandable and scalable with future throughput increases and incorporation of new instruments along with capabilities necessary for scheduling and handling of workflows, integrating data acquisition & analysis, and data storage.

6. Software shall include collaborative configuration management tools.

7. Software shall have the ability to surface fine-grained event and performance data (e.g. executed commands and their timestamps, instrument actuals, real-time sensor output) to stream continuously in real-time to monitoring and metrics tracking applications.

Performance Requirements 17

8. Protocols shall be designed to be fully parameterizable, allowing a user of the system the widest possible design space from which to create an experiment by allowing each parameter of a protocol to be varied. Parameters must include allowed ranges to prevent users from inadvertently creating scripts that won’t run or which will run at a risk of failing due to their selections.

9. The scheduling capability shall support new custom/commercial instrumentation, including simulation and optimization of the processes with the new instrument incorporated into the pre-defined workflows, automatically adjust changes in workflow through instant analysis using analytics packages, and simulate multiple processes in parallel with the new instrumentation incorporated.

10. The software shall integrate with existing relevant EMSL software infrastructure including but not limited to LIMS architecture (enterprise L7 platform) for experiment, workflow, and sample metadata capture, and EMSL’s data storage archive. Integrated experimental systems shall support or manage the capture of instrument performance, sample ID, analytical results in real-time or near real- time, instrument calibration, and quality control information, and associate these data with parallel data and metadata stored on EMSL’s LIMS system.

11. This capability shall run a mix of high-throughput workflows across shared equipment in order to meet the KPPs. The scheduling software shall be capable of receiving and scheduling new protocol run submissions while the system is already executing existing protocol runs.

12. The system shall be capable of dynamically rescheduling operations in response to new protocol run submissions, addition/removal of labware, and errors/slowdowns/accelerations resulting from physical execution of protocol runs. Because the system requires multiple instances of certain devices to accomplish the throughput necessary, it shall dynamically re-route samples to any instance of a given device based on availability at time of scheduling.

13. The software shall be able to continue executing protocols even while one or more individual instruments are offline for service.

14. Reconfiguration of the system shall be feasible, and a reconfigured physical system shall be able to be updated accordingly within the software system within minutes, using a graphical user interface.

15. To facilitate error identification, the software system shall be capable of emitting error messages that are easily consumed by external tools used for monitoring and alerting purposes. In addition, the system shall be designed to facilitate remote error recovery whenever possible, such as remotely restarting and re-initializing devices. Finally, if a protocol run is cancelled by an operator, the system can clean up individual protocol runs without affecting the execution of any concurrent protocol runs.

16. The software shall have a standardized device driver architecture. The contractor shall provide a software development kit (SDK) that provides the details necessary for Battelle/EMSL staff to author customized applications for use with or within the HTP-APP.

17. Software shall include the option to automatically put unused/unscheduled instruments into sleep mode or turn them off to conserve energy without compromising integrity and be able to bring them back online when needed without operator intervention.

Performance Requirements 18

4.4 Safety and Security

4.6.1 Safety by Design

Safety is an important aspect throughout, and after, project delivery. A safety-by-design charrette shall be performed coincident with the 30% and 60% design reviews. The topics of discussion shall include, but not be limited to, biological safety, robotic safety, remote operations safety and security, electrical safety, pressure safety, maintenance, ergonomics, and system clearances.

4.6.2 BSL-2 Requirements

Certain activities associated with HTP-APP have the potential to generate aerosol (e.g., sample preparation, strain engineering science core functions).