RFQ_ATTACHMENT_1_-_SOW.pdf

STATEMENT OF WORK

Automated Microbial Culture, Sample Preparation, and Analysis System

I. BACKGROUND INFORMATION

The National Institute of Standards and Technology (NIST) Microbial Metrology Group (the Group) provides robust protocols and reference materials to establish confidence in measurements of complex microbial systems; develops measurements and other tools to enable improved predictability and scalability in the engineering of biological systems; and provides measurement science and standards to enable exchange and operation of engineered organisms within and between organizations. The Group is working to develop and disseminate best practices for specifying and measuring the operating conditions, functional characterizations, and performance metrics for engineered organisms.

For this work, the Group needs to perform a large number of measurements of engineered organisms.

This will include replicates of nominally identical measurements to test reproducibility. It will also include multiple measurements with variation in the organism operating conditions to test the sensitivity/robustness of functional characterization and organism performance. This work requires precise control and reproducibility of both the organism operating conditions (i.e. culture conditions) and the measurement processes. In addition, the protocols for growth, operation, and measurement of engineered organisms typically span multiple days, with requirements for unit operations spread across two or three nominal eight-hour work shifts.

The Group has a requirement for an automated system for culture, sample preparation, and analysis of microbial cells, including bacteria. Specifically, the Group requires a system that has the ability to perform multiple laboratory unit operations, as detailed in the system specifications below; that has the flexibility to perform multiple growth and measurement protocols, including the protocol listed in Section VII below as well as protocols to be developed in the future using similar unit operations; that can maximize “walk away” time for those protocols; and that is expandable/upgradable for potential future integration of new hardware.

II. SCOPE OF WORK

The Contractor shall provide an automated system for microbial culture sample preparation and analysis, software for control of the system and scheduling of processes to be run on the system, factory acceptance testing (FAT) in the presence of the NIST Technical Point of Contact (TPOC), installation of the system, site acceptance testing (SAT) of the integrated system and training of NIST personnel at NIST, Gaithersburg. All Base Line Item components shall be fully integrated and automated for software control. Optionally, the Contractor will program the system to automate the protocol listed in Section VII below.

III. SPECIFICATIONS

Note Re: Option Line Items: Option Line Items, depending on the availability of funds, may be exercised at award or by way of purchase order modification before the end of the delivery schedule if additional funds become available. For Option Line Items 0004, 0005, 0006, 0007, 0008, 0009, 0010, and 0012, the Contractor shall specify the maximum period (i.e., latest possible date), not to exceed six (6) months by which the Government may exercise each option without impacting the delivery schedule for the system.

Quoted prices for all Option Line Items shall include the additional cost, if applicable, for hardware and software required to fully integrate the optional components into the automated system.

LINE ITEM 0001 – BASE LINE ITEM: Quantity One (1) Automated Microbial Culture, Sample Preparation, and Analysis System

The Contractor shall deliver a system meeting all of the following specifications. The Contractor shall fully integrate and automate all the components of the system.

1. The system shall include hardware for automated liquid handling with the following components and specifications:

a. Deck capacity for at least 40 SBS-format microplate positions. For flexibility of method development, NIST requires at least a 40-position deck inside the envelope of the liquid handler.

b. Eight-channel air displacement pipetting tool

i. For use with disposable tips

ii. Pipetting volume spanning at least the range from 1 uL to 1000 uL

iii. Independent pipetting volume for each channel

iv. Pipetting precision (with optimal tip size) of better than:

1. 6% CV at 1 uL

2. 1.5% CV at 10 uL

3. 1.5% CV at 100 uL

4. 1% CV at 1000 uL

v. Pipetting accuracy (with optimal tip size) of better than:

1. 8% at 1 uL

2. 2% at 10 uL

3. 2% at 100 uL

4. 1% at 1000 uL

vi. Variable spacing and height (i.e. independent ‘Y’ and ‘Z’) for each channel

vii. Pressure-based liquid level detection for each channel to detect the level of liquid in SBS plates, tubes, or other labware used with the system

viii. Capacitance-based liquid level detection for each channel to detect the level of liquid in SBS plates, tubes, or other labware used with the system

ix. Capability to monitor pressure inside each pipette channel during aspiration and dispensing for verification of sample transfer

c. 96-channel air displacement pipetting tool

i. For use with disposable tips

ii. Pipetting volume spanning at least the range from 1 uL to 500 uL

iii. Pipetting precision (with optimal tip size) of better than:

1. 6% CV at 1 uL

2. 5% CV at 10 uL

3. 2% CV at 100 uL

iv. Pipetting accuracy (with optimal tip size) of better than:

1. 8% at 1 uL

2. 5% at 10 uL

3. 3% at 100 uL

v. Capability to pick up and use a single row or a single column of tips

2. The system shall include hardware for transferring bulk fluid reagents (culture media, water, PBS, etc.) from storage bottles into SBS-format fluid troughs on the liquid handler deck, with the following capabilities and specifications:

a. Capability for at least four (4) independent fluid reagent sources

i. For all four (4) reagent sources, the system shall be capable of transferring the fluid reagents from a storage bottle located outside the envelope of the liquid handler deck (e.g., from off deck or below the deck).

ii. For all four (4) reagent sources, the fluid transfer shall be accomplished with peristaltic pumps, and all components that contact the fluid reagents shall be autoclavable.

iii. The system shall have storage capacity (off the liquid handler deck) for at least two media bottles, each up to 5 L in size (7.5 inches diameter, 14 inches tall), in a location that is accessible as a source for the bulk fluid reagent transfer.

1. Note that 5 L media bottles are NOT required as part of the system; the requirement is just for sufficient space to put two 5 L media bottles.

b. Capability either for measuring the volume of fluid reagents as they are transferred into each trough or for detecting the fill level of each trough

3. “On deck” plate and lid movement

a. The system shall be capable of moving SBS plates and pipette tip racks/boxes between positions on the liquid handler deck.

b. The system shall be capable of moving SBS plates between all integrated instruments in the system.

c. The system shall be capable of removing the lid from an SBS plate on the deck, pipetting to/from the plate, and replacing the lid on the plate, all while keeping the plate and lid inside the envelope of the liquid handler.

4. The system shall include hardware for bar code reading with the following specifications:

a. Capability to track bar-coded SBS-format plates as they are moved between all devices on the system

5. The system shall include a multi-mode plate reader with the following capabilities and specifications:

a. Xenon flash lamp light source

b. Absorbance detection mode with wavelengths spanning at least the range from 230 nm to

999 nm.

c. Absorbance linearity up to OD 4.

d. Automatic pathlength correction

e. Fluorescence detection mode with wavelengths spanning at least the range from 280 nm to 850 nm

f. Variable fluorescence bandwidth spanning at least the range from 9 nm to 50 nm

g. Capability for performing fluorescence and absorbance spectral scans

h. Hybrid optical system combining both monochromator fluorescence optical system and filter-based fluorescence optical system.

i. Quadruple grating fluorescence detection (two gratings per monochromator)

j. Incubation temperature spanning at least the range from 7°C above ambient to 60°C

k. Temperature uniformity ±0.5°C or better at 37°C

l. Capability for warmer temperature above the plate vs. below the plate to reduce condensation on lidded plates

m. Compatibility with SBS format plates from 6-well to 384-well plates

n. Compatibility with SBS format plates with height up to 22.5 mm.

o. Capability to define and use plates with non-standard formats with wells in non-standard positions.

p. Capability for linear, orbital, and double orbital shaking

q. Compatible with micro-volume plate format for absorbance and fluorescence measurements of samples with approximately 2 uL volume

6. The system shall include two (2) identical absorbance plate readers each with the following capabilities and specifications:

a. Xenon flash lamp light source

b. Absorbance detection mode with wavelengths spanning at least the range from 200 nm to

999 nm.

c. Absorbance linearity up to OD 4.

d. Capability for performing absorbance spectral scans

e. Incubation temperature spanning at least the range from 7°C above ambient to 60°C

f. Temperature uniformity ±0.5°C or better at 37°C

g. Capability for warmer temperature above the plate vs. below the plate to reduce condensation on lidded plates

h. Compatibility with SBS format plates from 6-well to 384-well plates

i. Compatibility with SBS format plates with height up to 22.5 mm.

j. Capability to define and use plates with non-standard formats with wells in non-standard positions.

k. Capability for linear, orbital, and double orbital shaking

l. Compatible with micro-volume plate format for absorbance measurements of samples with approximately 2 uL volume

7. The system shall include one (1) heating, cooling, and shaking station with the following

a. Temperature control spanning at least the range from 4°C to 70°C

b. Capability for linear and orbital shaking

c. Temperature accuracy of ±0.8°C or better at 37°C

d. Temperature uniformity of ±0.6°C or better at 37°C

e. Adapter plates for deep-well 96-well SBS plates.

8. The system shall include a centrifuge with the following capabilities and specifications:

a. Maximum g-force of at least 4000g

b. Temperature control spanning at least the range from -20°C to 40°C

c. Rotor with 4 buckets for SBS plates

d. Compatibility with standard depth and deep-well SBS plates

9. The system shall include hardware for electroporation of cells in a 96-well format with the

a. Capability for up to 96 different electroporation programs per plate

b. Transfection volume between 10 uL and 30 uL

10. The system shall include a plate sealer with the following capabilities and specifications:

a. throughput of at least 100 plates per hour

b. Sealer shall be compatible with adhesive-based sealing materials

c. Sealer shall be compatible with gas-permeable sealing materials

d. Sealer shall be compatible with foil sealing materials

e. Sealer shall be compatible with transparent perforated gas permeable film such as

QuickSeal Gas Perm™ or similar.

f. Sealer shall be compatible with SBS-format plates with a height spanning at least the range from 14.2 mm (standard) to 44 mm (deep-well)

11. The system shall include a plate peeler/de-sealer with the following capabilities and

a. throughput of at least 100 plates per hour

b. De-sealer shall be compatible with adhesive-based sealing materials

c. De-sealer shall be compatible with heat-based sealing materials

d. De-sealer shall be compatible with gas-permeable sealing materials

e. De-sealer shall be compatible with foil sealing materials

f. De-sealer shall be compatible with SBS-format plates with a height spanning at least the range from 14.2 mm (standard) to 44 mm (deep-well)

12. The system shall include storage for consumables (e.g. tips, plates) with the following capabilities and specifications:

a. automated access

b. capacity for at least 15 racks/boxes each with 96 tips (300 uL filter tips or 500 uL filter tips compatible with the 96-channel pipetting tool) and 36 SBS-format plates that are each 22 mm in height

c. capability for storage and access of at least 4 different kinds of consumable, i.e., either random access storage or sequential storage with at least 4 stackers.

13. The system shall include a solid waste collection system with the following capabilities and

a. off-deck waste collection compartment with capacity for disposal of at least 1440 (20 x

96) 300 uL or 500 uL pipette tips and 40 SBS-format plates that are each 22 mm in height

14. The system shall include a liquid waste collection system with the following capabilities and

a. Liquid waste collection system shall be accessible by all pipetting tools

b. liquid waste collection reservoir shall be off the liquid handler deck.

c. location of liquid collection reservoir shall be large enough to fit a 2 ½ gallon square carboy (8” x 8” x 14” tall)

15. The system shall include a computer for control of the hardware. The computer shall have the Windows 7 or Windows 10 operating system installed.

16. The system shall include other hardware components necessary for integration and automated operation of all listed components.

17. The Contractor shall provide, at a minimum, a one-year warranty for the system which covers the base system and any optional line items that may be exercised. The warranty shall include software updates. The warranty shall cover all robotic systems and instruments. The warranty shall cover all parts, labor and travel. The warranty shall commence upon successful completion of the SAT.

LINE ITEM 0002 – BASE LINE ITEM: Quantity One (1) Enclosure

1. An enclosure around the Line Item 0001 system with the following capabilities and

a. Interlocked or otherwise guarded for mechanical safety

b. Doors to allow access to equipment for loading of reagents and disposables and servicing of system hardware

c. Status lights that provide visible indications of system state (running, error conditions, etc.)

d. Positive-pressure enclosure fed by HEPA-filtered air

e. Control panel to control and monitor lighting inside enclosure, airflow, and filter pressure

f. Enclosure contains all operations of the system that involve open sample plates or reagents (e.g. pipetting, plate sealing, plate de-sealing)

g. The outside dimensions of the enclosure shall be no greater than 12 feet x 12 feet.

h. The maximum height of the enclosure shall be no greater than 9 feet 0 inches.

i. The enclosure shall have sufficient internal space to enclose the Line Item 0001 system and the optional system components of Line Items 0004, 0007, 0009, and 0010.

LINE ITEM 0003 – BASE LINE ITEM: Quantity One (1) System Design

The Contractor shall provide a finalized system design for an automated system that meets all the minimum requirements for all other Base Line Items and all Option Line Items that have been exercised by NIST at the time of purchase order award.

LINE ITEM 0004 – OPTION LINE ITEM: Quantity One (1) Positive-Pressure Solid Phase Extraction

1. Hardware to apply a controlled, positive pressure to the top of the wells of an SBS-format solid phase extraction plate or filter plate with the following capabilities and specifications:

a. Pressure control up to 100 psi

b. Shall maintain equal pressure across the filter plate

c. Compatible with filter collection plates up to 110 mm in height

d. 96-well plate only

e. NIST's initial plan is to use Agilent filter plates, part number 203980-100. However, NIST is willing to change the disposable labware that it will use if it will better suit the automated hardware system. NIST's applications will require filter membranes with 0.2 um pores and filter plates that are available in sterile packaging.

2. The Contractor shall integrate the Positive-Pressure Solid Phase Extraction hardware into the automated system of Line Item 0001 so that its operation can be controlled by the software of Line Item 0011.

This option shall remain valid for a period specified by the Contractor, not to exceed six (6) months from the date of award.

LINE ITEM 0005 – OPTION LINE ITEM: Quantity One (1) Active SBS Plate Position

1. Active SBS plate position to securely hold SBS-format deep-well plates during piercing with the

a. Located on liquid handler deck

b. Compatible with deep-well SBS plates

2. The Contractor shall integrate the Active SBS Plate Position into the automated system of Line

Item 0001 so that its operation can be controlled by the software of Line Item 0011.

This option shall remain valid for a period specified by the Contractor, not to exceed six (6) months from

LINE ITEM 0006 – OPTION LINE ITEM: Quantity One (1) Automated Bacterial Colony Picking

1. Capability for automated bacterial colony picking from an agar plate with the following

a. Colony picking based on brightfield imaging

b. Colony picking rate of at least three (3) colonies per minute

c. Integrated into Liquid Handler deck

d. Colony picking from SBS format plates (e.g., Omnitray) will meet the requirements.

Also, colony picking that requires a round petri-dish labware format would also meet the requirements if the automated system is capable of lidding/de-lidding and moving the round petri dishes within the system.

2. The Contractor shall integrate the Automated Bacterial Colony Picking into the automated system of Line Item 0001 so that its operation can be controlled by the software of Line Item 0011.

This option shall remain valid for a period specified by the Contractor, not to exceed six (6) months from

LINE ITEM 0007 – OPTION LINE ITEM: Quantity One (1) Thermocycler

1. A thermocycler suitable for polymerase chain reaction in 96-well plate format with the following

a. Temperature control spanning at least the range from 4°C to 99°C

b. Temperature accuracy of ±0.3°C or better at 55°C

c. Temperature uniformity of ±0.2°C or better from 55°C to 95°C

d. Heating rate adjustable from 0.1 to 4.4 K/s

e. Cooling rate adjustable from 0.1 to 2.2 K/s

f. Lid heating with temperature control spanning at least the range from 5°C above ambient to 115°C

g. Compatible with 96-well skirted plates

2. The Contractor shall integrate the Thermocycler into the automated system of Line Item 0001 so that its operation can be controlled by the software of Line Item 0011.

This option shall remain valid for a period specified by the Contractor, not to exceed six (6) months from

LINE ITEM 0008 – OPTION LINE ITEM: Quantity One (1) Extra Liquid Waste Collection System

1. A second liquid waste collection system with the following capabilities and specifications:

a. Liquid waste collection system shall be accessible by all pipetting tools

b. Liquid waste collection reservoir shall be off the liquid handler deck.

c. In combination with the liquid waste collection system listed under Line Item 0001, this will provide the capability to segregate two types of liquid waste for separate disposal.

d. Capability to discard Formamide (up to 50% in water) and formaldehyde (up to 10% in water).

This option shall remain valid for a period specified by the Contractor, not to exceed six (6) months from

LINE ITEM 0009 – OPTION LINE ITEM: Quantity Two (2) Additional Absorbance Plate Readers (identical to the absorbance plate readers specified in Line Item 0001.6)

1. The Contractor shall integrate the Two Additional Absorbance Plate Readers into the automated system of Line Item 0001 so that its operation can be controlled by the software of Line Item 0011.

This option shall remain valid for a period specified by the Contractor, not to exceed six (6) months from

LINE ITEM 0010 – OPTION LINE ITEM: Quantity One (1) Additional Plate Sealer

1. A plate sealer with the following capabilities and specifications:

a. Heat-based sealer

b. Compatible with both foil and gas permeable sealing materials

c. Sealer shall be compatible with SBS-format plates with a height spanning at least the range from 14.2 mm (standard) to 44 mm (deep-well).

2. The Contractor shall integrate the Additional Plate Sealer into the automated system of Line Item 0001 so that its operation can be controlled by the software of Line Item 0011

This option shall remain valid for a period specified by the Contractor, not to exceed six (6) months from

LINE ITEM 0011 – BASE LINE ITEM: Quantity One (1) Software for Control of Automated System and Scheduling of Automated Processes

The software shall include drivers for all of the system hardware components listed in Line Item 0001 and a scheduler for programing, simulating, and scheduling processes for the automated system. The software shall have the flexibility to automate new protocols and integrate new components into system. The software shall meet all of the following required specifications:

1. The software shall be compatible with the Windows 7 operating system.

2. The software shall provide the ability to program, develop, simulate, schedule, and control processes on the automated system using the computer listed under Line Item 0001.

3. The software shall provide the ability to program, develop, and simulate processes on at least one additional computer (other than the computer running the automated system) while the automated system is running.

4. The software shall have multi-level user capability with:

a. A user/operator mode for graphical process setup and to run existing processes; and

b. A developer/programmer/integrator mode for more detailed programming.

5. The software shall include a graphical/visual programming interface.

6. The software shall include capability for programming flow control including:

a. for loops and while loops or the equivalent; and

b. if-then and if-then-else statements or the equivalent.

7. The software shall provide the ability for remote monitoring of the System and scheduling via a network connection.

8. The software shall have the capability for error handling and error notification:

a. Error handling shall include programmable error recovery capabilities.

9. The software shall provide the ability for run-time decision making using data collected by the instruments in the automated system.

a. e.g., incubate bacteria in a plate reader until the optical density reaches a threshold value, then remove plate from reader.

10. The software shall include capability for programming the unit operations listed in Section VII of this document, “PROTOCOL FOR FAT AND SAT.”

LINE ITEM 0012 – OPTION LINE ITEM: Quantity One (1) Method Programming

Programming the system to automate the FAT/SAT protocol listed in section VII, below.

This option shall remain valid for a period specified by the Contractor, not to exceed six (6) months from

LINE ITEM 0013 – BASE LINE ITEM: Quantity One (1) Factory Acceptance Test (FAT)

Factory acceptance testing to include testing for basic functionality of all integrated system components, and the protocol listed in Section VII, below. Factory acceptance testing not to exceed two (2) days.

NIST will supply all disposables (pipette tips, SBS plates, etc.) for use during the FAT.

LINE ITEM 0014 – BASE LINE ITEM: Quantity One (1) System Installation and One (1) Site Acceptance Test (SAT)

The Contractor shall install the instrument in Building 227, Room A338, at NIST Gaithersburg, MD. It will be the Contractor’s responsibility to see that the system is delivered to the installation site including rigging from the dock to the lab if necessary. A freight elevator is available with internal dimensions of 14 ft. x 7 ft. Upon delivery, all components of the system shall fit through the lab door, which is 42 inches wide and 92 inches tall. Installation shall take place during normal business hours, between 8:30 am and 5:00 pm Eastern Time, Monday through Friday except Federal Holidays, and shall be coordinated with the NIST TPOC. Installation shall be completed within 210 days of award. The SAT shall include testing for basic functionality of all integrated system components, and the protocol listed in Section VII below. The SAT shall not exceed two (2) days in duration. NIST will supply all disposables (pipette tips, SBS plates, etc.) for use during the SAT.

LINE ITEM 0015 BASE LINE ITEM: Quantity One (1) Training

The Contractor shall schedule and facilitate training for three (3) NIST personnel, on-site at NIST Gaithersburg. The training shall provide a thorough demonstration of all equipment functions, equipment operation, methods/instrument modes, and basic troubleshooting. The training may be completed on-site at NIST immediately after completion of installation and the SAT.

IV. PLACE OF PERFORMANCE

Installation of the instrument, the SAT, and training shall be accomplished at NIST, Gaithersburg, Maryland. Normal duty hours are Monday through Friday, 8:30 a.m. to 5:00 p.m. except for Federal holidays.

V. DELIVERABLES AND PERIOD OF PERFORMANCE

All deliverable time requirements are set forth in the attached LINE ITEM SCHEDULE. The period of performance shall not exceed one (1) year after receipt of this purchase order (ARO).

VI. GENERAL INFORMATION

Safety: The Contractor shall be responsible for knowing and complying with NIST installation safety prevention regulations (http://www-i.nist.gov/mml/safety/policies/index.htm.) Such regulations include, but are not limited to, general safety, fire prevention, and waste disposal.

Security: NIST is a restricted campus. An identification badge is required for access for entry into buildings and is shown to the armed Security Police when entering the campus.

VII. PROTOCOL FOR FAT AND SAT

This section provides a detailed listing of the protocol that the Contractor is required to program to satisfy the requirements of Option Line Item 0012. References to cultures, buffers, reagents or chemicals are place-holder references only. SAT and FAT to be run either dry or with DI water only (no chemicals or biologicals in the system). Also, all incubation steps should be shortened as appropriate for FAT and SAT testing.

List of consumables, and reagents needed for the protocol. As much as possible, the consumables should be loaded into the storage hotel/carousel of Line Item 0001.12 at the start of the protocol.

a. 5x Growth Plates: 4titude 96-well clear assay plates, 4titude part number: 4ti-0255 (http://www.4ti.co.uk/microplates/clear-assay-microplates/96-well-clear-assay-pyramid)

b. 1x Skirted PCR Plate: 4titude skirted PCR plate, part number: 4ti-0960

c. 1x Electroporation Plate: 96-well electroporation plate compatible with Line Item 0001.9

d. 1x Filter Plate: 96-well filter plate, Agilent part number 203980-100 or similar

e. 1x Receiver Plate: 96-well plate, Agilent part number 203942-100 or similar

f. 7x SBS Lids

g. disposable reagent troughs or plates for bulk reagent transfers, format depends on system hardware configuration.

h. Filter tips

i. 1 mL: 2 racks/boxes plus one partial rack/box

ii. 300 uL: 1 rack/box

iii. 50 uL: 1 rack/box plus one partial rack/box

iv. 10 uL: 1 rack/box plus one partial rack/box

OR:

v. 1 mL: one partial rack/box

vi. 500 uL: 3 racks/boxes plus one partial rack/box

vii. 50 uL: 1 rack/box plus one partial rack/box

viii. 10 uL: 1 rack/box plus one partial rack/box

i. Starter Cultures: 4x E. coli cultures (strains 1-4) on Agar Plates with Lids: Format as appropriate for system hardware configuration (e.g. Thermo Scientific Nunc OmniTray

242811, or round polystyrene petri dishes with lids)

i. loaded at Start

j. M9-Kan Media

i. in off-deck media bottle connected to Bulk Reagent Source no. 1

ii. room temperature storage

k. DI Water

i. in off-deck media bottle connected to Bulk Reagent Source no. 2

ii. room temperature storage.

Start

1. [Optional, if automated colony picking is available] Load 4 Starter Cultures on Agar Plates with Lids into automated system.

2. Transfer 50 mL M9-Kan Media to bulk reagent trough

3. Prepare Growth Plate 1:

a. Pipette 500 uL M9-Kan Media into each well

4. [Optional automated/manual step] Pick 12 colonies per Agar Plate and inoculate 1 colony per well into selected wells of 96 well Growth Plate 1

a. strain 1: A1-A12; strain 2: C1-C12; strain 3: E1-E12; strain 4: G1-G12

5. Apply gas permeable membrane to Growth Plate 1 with adhesive sealer

6. Incubate Growth Plate 1, 37°C, 5 minutes, orbital shaking, OD measurement (in multi-mode plate reader)

a. OD measurement to be taken every 30 seconds

7. Transfer 200 mL M9-Kan Media to bulk reagent trough (possibly in multiple steps)

8. Prepare Growth Plates 2-5:

a. Pipette 496 uL M9-Kan Media into each well of all four plates

9. Remove Growth Plate 1 from incubator/reader

10. Remove membrane from Growth Plate 1

11. Dilute samples from Growth Plate 1 to Growth Plates 2-5:

a. For each strain in Growth Plate 1, pick the well with the highest optical density and pipette 4 uL from that well into each well on one of the new Growth Plates: strain 1 into Growth Plate 2, strain 2 into Growth Plate 3, strain 3 into Growth Plate 4, strain 4 into Growth Plate 5.

12. Apply gas permeable membrane to Growth Plates 2-5 with adhesive sealer

13. Incubate Growth Plates 2-5 at 37°C, with orbital shaking and OD measurement (in absorbance plate readers)

a. OD measurement to be taken every 30 seconds

b. Incubate until average OD value reaches a threshold value (0.2 or adjusted for FAT/SAT) or until max time of 5 minutes.

14. Transfer 100 mL DI Water to bulk reagent plate or trough

a. Move to cooled reservoir/trough on heating, cooling, and shaking station set to 4°C, with SBS Lid

15. Remove Growth Plates 2-5 from incubators/readers

16. Remove membrane from Growth Plates 2-5

17. Centrifuge Growth Plates 2-5 to pellet cells, 5 minutes, 4000g, 4°C

a. Centrifuge with SBS Lids on plates, if possible

18. Remove supernatant from each well of Growth Plate 2

19. Resuspend cells in each well of Growth Plate 2 in 50 uL per well cold DI Water, mix by pipetting

20. Add 150 uL cold DI Water to each well of Growth Plate 2, mix by pipetting

a. Steps 19-20 (and similar steps) done by aspirating 200 uL of cold DI Water into each pipette tip, then dispensing 50 uL into each destination well, mixing 50 uL by 3x repeated aspiration and dispensing, then dispensing the entire 200 uL, and mixing.

21. Put SBS Lids on Growth Plates 2-5

22. Incubate Growth Plates 2-5 at room temperature for 5 minutes (In absorbance plate readers)

a. Slow shaking speed or intermittent shaking

b. In incubators with temperature control turned off.

23. [Optional, if thermocycler is available] Prepare Skirted PCR Plate

a. Pipette 50 uL DI Water into each well of the plate

24. [Optional, if thermocycler and heat sealer are available] Apply foil seal to Skirted PCR Plate with heat sealer

25. [Optional, if thermocycler is available] Thermocycle Skirted PCR Plate

a. 95° for 30 s

b. 45° for 30 s

c. 68° for 30 s

d. Repeat 2 times

26. Remove Growth Plates 2-5 from incubators/readers

27. Remove lid from Growth Plate 2

28. Remove Skirted PCR Plate from thermocycler

29. Remove foil seal from Skirted PCR Plate

30. Combine rows A-D of Growth Plate 2 into row D of Growth Plate 2 and mix by pipetting

a. Use 96-channel head with a single row of 12 tips

31. Pipette 5 uL from each well in Skirted PCR Plate into the corresponding well in

Electroporation Plate

32. Pipette 15 uL from each well in row D of Growth Plate 2 into the corresponding well in each row in Electroporation Plate, mix by pipetting

33. Electroporate 96 samples in Electroporation Plate

34. Remove lid from Growth Plates 3-4

35. Pipette 10 uL from well A1 of Growth Plate 3 to well D7 of Growth Plate 4

36. Pipette 10 uL from well B1 of Growth Plate 3 to well E5 of Growth Plate 3

37. Pipette 30 uL from each well of column 2 of Growth Plate 3 to the corresponding well in column 3 of Growth Plate 3

a. Use 96-channel head with a single column of 8 tips

38. Pipette 30 uL from each well of column 4 of Growth Plate 3 to the corresponding well in column 7 of Growth Plate 4

a. Use 96-channel head with a single column of 8 tips

39. [Optional, if positive-pressure solid phase extraction is available] Pipette samples from each well of Electroporation Plate into corresponding well in Filter Plate

40. [Optional, if positive-pressure solid phase extraction is available] Pipette 480 uL M9-Kan Media into each well of Filter Plate, mix by pipetting

41. [Optional, if positive-pressure solid phase extraction is available] Move Filter Plate to solid-phase extraction and filter samples, depositing filtrate into Receiver Plate.

VIII. PAYMENT TERMS

Subject to FAR 52.232-29, interim commercial payments will be authorized for this procurement under the following notional schedule.

1. NIST’s acceptance of System Design: 40% of FFP

2. NIST’s acceptance of FAT: 30% of FFP

3. NIST’s final acceptance of the system, installation, and training: 30% of FFP