DRAFT_RFQ_ATTACHEMENT_1_-_SOW.pdf

DRAFT

DRAFT RFQ STATEMENT OF WORK

TITLE: Automated Microbial Culture, Sample Preparation, and Analysis System LAB REQUESTING SERVICE: Material Measurement Laboratory, Biosystems and Biomaterials

Division

I. BACKGROUND INFORMATION

The National Institute of Standards and Technology (NIST) Microbial Metrology 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.

In particular, 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 will need 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 will require 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 requirements below; that has the flexibility to perform multiple growth and measurement protocols, including the protocol listed in section VIII, at the end of this document, 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 vendor will provide an automated system for microbial culture sample preparation and analysis. The vendor will also provide software for control of the system and scheduling of processes to be run on the system. All Base components must be fully integrated and automated for software control. Optionally, the vendor will program the system to automate the protocol listed in section VIII at the end of this document. The vendor will perform factory acceptance testing, with the NIST Technical Point of Contact (TPOC) present, if possible. The vendor will install the system at NIST Gaithersburg, perform site acceptance testing (SAT) of the integrated system, and provide training for NIST personnel.

III. SPECIFICATIONS

Responsible quoters shall provide pricing for all of the following line items.

Option Line Items: Contract Options, depending on the availability of funds, may be exercised at award or by contract mod before the end of the period of performance if additional funds become available.

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

The System must have all of the following required capabilities and meet all of the following required specifications. All of the components of the System should be fully integrated and automated:

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

a. Deck capacity for at least 40 SBS-format microplate positions.

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

viii. Capacitance-based liquid level detection for each channel

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 must include hardware for transferring bulk fluid reagents (culture media, water, PBS, etc.) from storage bottles either into SBS-format plates or 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 must 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 at least two (2) of the fluid reagent sources, the fluid transfer must be accomplished with peristaltic pumps, and all components that contact the fluid reagents must be autoclavable.

iii. The system must 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 the plate or trough or for detecting the fill level of the trough

3. “On deck” plate and lid movement

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

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

c. The system must 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 must 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 must 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 must include four (4) 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 must include one heating, cooling, and shaking station with the following capabilities

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 must 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 must include hardware for electroporation of cells in a 96-well format with the following capabilities and specifications:

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

b. Transfection volume between 10 uL and 30 uL

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

a. throughput of at least 100 plates per hour

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

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

d. Sealer must be compatible with foil sealing materials

e. Sealer must 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 must include a plate peeler/de-sealer with the following capabilities and

a. throughput of at least 100 plates per hour

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

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

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

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

f. De-sealer must be compatible with SBS-format plates with a height spanning at least the

12. The system must include storage for consumables (e.g. tips, plates) with the following capabilities

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, e.i. either random access storage or sequential storage with at least 4 stackers.

13. The system must 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 must include a liquid waste collection system with the following capabilities and

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

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

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

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

16. The system must 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 site acceptance test (SAT, Line Item 0014).

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

1. An enclosure around the System of Line Item 0001 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 must be no greater than 10 feet x 12 feet.

h. The maximum height of the enclosure must be no greater than 8 feet 3 inches (251.5 cm).

LINE ITEM 0003 – BASE LINE ITEM: System Design

The Contractor shall provide a finalized System Design for an automated system that meets all the minimum requirements for all Base Line Items and all Option Line Items that have been exercised by

NIST.

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. Must maintain equal pressure across the filter plate

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

d. Located on liquid handler deck

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 following capabilities and specifications:

a. Located on liquid handler deck

b. Compatible with deep-well SBS plates

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: 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

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

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 must be accessible by all pipetting tools

b. Liquid waste collection reservoir must 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.

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 One (1) Temperature Controlled Thermal Block

1. A four-position, temperature-controlled station with the following capabilities and specifications:

a. Located on the liquid handler deck

b. Temperature control spanning at least the range from 0°C to 80°C

c. Temperature control provided by recirculating bath

d. One position for temperature control of SBS format fluid troughs

e. One position with thermal insert for temperature control of 24 reagent tubes

i. size of tubes: 1.5 mL or 2 mL microcentrifuge tubes

f. Two positions open for SBS plate pre-chilling or pre-warming

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 must be compatible with SBS-format plates with a height spanning at least the

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

The Software must 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 must have all of the following required capabilities and meet all of the following required

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

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

4. The software must 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 must include a graphical/visual programming interface.

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

a. for loops and while loops or the equivalent

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

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

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

a. error handling must include programmable error recovery capabilities

9. The software must provide the ability for run-time decision making.

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

LINE ITEM 0012 – OPTION LINE ITEM: Method Programming

Programming the system to automate the FAT/SAT protocol listed in section VIII, below. 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 0013 – BASE LINE ITEM: Factory Acceptance Testing (FAT)

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

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

LINE ITEM 0014 – BASE LINE ITEM: System Installation and Site Acceptance Testing (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 must fit through the lab door, which is 42 inches wide and 92 inches tall. Installation will take place during normal business hours, between 8:30 am and 5:00 pm Eastern Time, Monday through Friday except Federal Holidays, and will be coordinated with the NIST Technical Point of Contact (TPOC). Installation is to occur within 210 days of award.

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

NIST shall 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 installation and SAT.

LINE ITEM 0016 – OPTION LINE ITEM: Quantity One (1) Service Contract Period I

The Service Contract shall include software updates. The Service Contract shall cover all robotic systems and instruments. The Service Contract shall cover all parts, labor and travel. This option shall remain valid for a period of one (1) year from the expiration date of initial warranty period.

LINE ITEM 0017 – OPTION LINE ITEM: Quantity One (1) Service Contract Period II

The Service Contract shall include software updates. The Service Contract shall cover all robotic systems and instruments. The Service Contract shall cover all parts, labor and travel. This option shall remain valid for a period of two (2) years from the expiration date of initial warranty period.

LINE ITEM 0018 – OPTION LINE ITEM: Quantity One (1) Service Contract Period III

The Service Contract shall include software updates. The Service Contract shall cover all robotic systems and instruments. The Service Contract shall cover all parts, labor and travel. This option shall remain valid for a period of three (3) years from the expiration date of initial warranty period.

IV. PERIOD OF PERFORMANCE

The period of performance shall not exceed five (5) years from award of this requirement.

V. PLACE OF PERFORMANCE

Installation of the instrument 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.

VI. DELIVERABLES

Description: Due Date -System Design: 30 days after award of this requirement -Verification of receipt by vendor of major hardware components: 60 days after acceptance of System Design -Factory Acceptance Testing (FAT): 120 days after acceptance of System Design -Automated System: 210 days after award of this requirement.

-Control and Scheduling Software: 210 days after award of this requirement.

-Installation of the Automated System: 210 days after award of this requirement.

-Site Acceptance Testing (SAT): 210 days after award of this requirement.

-Training of NIST personnel at NIST, Gaithersburg, MD: 14 days after SAT.

VII. GENERAL INFORMATION

Safety: The Contractor employee 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.

VIII. PROTOCOL FOR FAT AND SAT

Modified version of Cello RNA-FISH Growth and Sample Prep protocol. References to cultures, buffers, reagents or chemicals are place-holder references only. SAT and/or 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/SAT testing.

1. List of consumables, and reagents needed for 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 IPTG Serial Dilution Plate : Thermo Scientific Nunc 2.0mL DeepWell plate, 95040462

c. 1x Cell Dilution Plate: Thermo Scientific Nunc 2.0mL DeepWell plate, 95040462

d. 1x Reagent Storage Plate: Thermo Scientific Nunc 2.0mL DeepWell plate, 95040462

e. 3x Nuclease-Free Reagent Plate: Agilent Seahorse 24-well irradiated polypropylene storage/reaction microplate, 10ml/square well, pyramid bottoms, 44mm height, 204023-

f. 4x Wash Plates: Corning Axygen P-96-450R-C 500 uL

g. 3x Black Plate: Corning Axygen P-96-450R-BK 500ul, Black

h. 15x SBS Lids

i. 3x Black SBS Lids: (http://www.phenixresearch.com part no. ML-5010; or www.eandkscientific.com part no. EK-2080)

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

k. Filter tips

i. 1 mL: one partial rack/box

ii. 300 uL: 14 racks/boxes

iii. 50 uL: 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: 12 racks/boxes

vii. 150 uL: 1 rack/box

viii. 100 uL 1 rack/box

ix. 50 uL 1 rack/box plus one partial rack/box

l. Starter Cultures: 8x E. coli cultures (strains 1-8) in liquid media:

i. format to be adjusted to match system hardware configuration; could be plates, tubes, etc.

ii. loaded at Start

m. 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. LB agar with appropriate antibiotics manually poured into plates

ii. loaded at Start

n. M9-Kan Media

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

ii. 250 mL

iii. room temperature storage

o. IPTG Stock solution, 50 mmol/L in water

i. In Reagent Storage Plate 1, well A1, with SBS Lid

ii. 1 mL volume {970uL}

iii. loaded onto liquid handler deck at Pause Point 2

p. DI Water

i. In Reagent Storage Plate 1, wells B1-B4, with SBS Lid

ii. 1.5 mL volume per well

iii. loaded onto liquid handler deck at Pause Point 2

q. PBS Buffer

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

ii. stored at room temperature until time of use.

iii. 100 mL

iv. transferred to plate/trough on liquid handler deck at Pause Point 3 and then kept as cold as possible (2°C), and covered with SBS Lid

r. Fixation Buffer

i. In Nuclease-Free Reagent Plate 1, wells A1-A3, with SBS Lid

ii. 20 mL total required

iii. loaded onto liquid handler deck at Pause Point 3

s. Nuclease-Free PBS

i. In Nuclease-Free Reagent Plate 1, wells B1-B6, with SBS Lid

ii. 40 mL total required

iii. loaded onto liquid handler deck at Pause Point 3

t. Nuclease-Free Water

i. In Nuclease-Free Reagent Plate 1, wells C1, with SBS Lid

ii. 3 mL volume

iii. loaded onto liquid handler deck at Pause Point 3

u. 80% Ethanol

i. In Nuclease-Free Reagent Plate 1, wells D1-D3, with SBS Lid

ii. 20 mL volume total

iii. loaded onto liquid handler deck at Pause Point 3

v. Wash Buffer A

i. In Nuclease-Free Reagent Plate 2, wells A1-A3, with SBS Lid

ii. 20 mL volume total

iii. Loaded onto liquid handler deck at Pause Point 4

w. Hybridization Buffer

i. In Black Plate 1, wells A1-A3, with Black SBS Lid

ii. 1 mL volume total

iii. loaded onto liquid handler deck at Pause Point 4

x. Wash Buffer A

i. In Nuclease-Free Reagent Plate 3, wells A1-A5, with SBS Lid

ii. 80 mL volume total

iii. Loaded onto liquid handler deck at Pause Point 5

y. Wash Buffer B

i. In Nuclease-Free Reagent Plate 3, wells B1-B3, with SBS Lid

ii. 20 mL total volume

iii. Loaded onto liquid handler deck at Pause Point 5

z. 2X SSC

i. In Nuclease-Free Reagent Plate 3, wells C1-C2, with SBS Lid

ii. 10 mL total volume

iii. Loaded onto liquid handler deck at Pause Point 5

aa. DAPI Wash Buffer

i. In Black Plate 2, wells A1-D12, with SBS Lid

ii. 20 mL volume total

iii. loaded onto liquid handler deck at Pause Point 5

Start

2. [Optional, if automated streaking is available] Streak or spread each E. coli strain Starter

Culture on an Agar Plate with Lid

3. [Optional automated/manual step] Put Lids on Agar Plates

4. [Manual step] Put Agar Plates with Lids in incubator, 37°C, overnight (16h) or longer Pause Point 1

5. [Optional, if automated colony picking is available] Load 8 Agar Plates with Lids into automated system.

6. Transfer 20 mL M9-Kan Media to bulk reagent plate or trough

7. Prepare Growth Plate 1:

a. Pipette 500 uL per well M9-Kan Media into columns 1-5 (19.2 mL total)

8. [Optional automated/manual step] Pick 3 colonies per Agar Plate and inoculate 1 colony per well into selected wells of 96 well Growth Plate 1

a. strain 1: A2-A4; strain 2: B2-B4; strain 3: C2-C4; strain 4: D2-D4; strain 5: E2-E4;

strain 6: F2-F4; strain 7: G2-G4; strain 8: H2-H4

b. Plate layout on right (strains indicated by color, ‘x’ indicates media blank):

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

10. Incubate Growth Plate 1, 37°C, 16 hours, orbital shaking, OD measurement (in plate reader)

a. OD measurement to be done at both 600 nm and 700 nm Pause Point 2

11. [Manual step] Load Reagent Storage Plate 1 with IPTG stock (1 mL) and DI water (5 mL)

a. Loaded on deck in SBS format plate with Lid

12. Remove Growth Plate 1 from incubator/reader

13. Remove membrane from Growth Plate 1

14. Transfer 16 mL M9-Kan Media to bulk reagent plate or trough

15. Pipette 465 uL M9-Kan Media into each well of columns 6-9 on Growth Plate 1

16. Dilute samples (in 2 steps) from Growth Plate 1 to new wells in Growth Plate 1:

a. For each strain in Growth Plate 1, pick the well with the highest optical density and pipette 35 uL from that well to the corresponding well in column 6 of Growth Plate 1, then mix by pipetting

b. Then pipette 35 uL from each newly mixed destination well to the corresponding well in column 8 of Growth Plate 1

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

18. Incubate Growth Plate 1 at 37°C 3 hours with orbital shaking and OD measurement (in plate reader)

19. Prepare IPTG Serial Dilution Plate,

a. 12 wells with IPTG concentrations ranging from 0 to 50 mmol/L (in DI Water)

b. 11 wells with serial dilution starting from 50 mmol/L ending at 0.25 mmol/L

i. Pipette 970 uL IPTG Stock (50 mmol/L) from Reagent Storage Plate 1, well A1 into IPTG Serial Dilution Plate, well A1

ii. Pipette 400 uL per destination well DI Water from Reagent Storage Plate 1, wells B1-B4 into IPTG Serial Dilution Plate, wells A2-A12

iii. Pipette 570 uL from IPTG Serial Dilution Plate A1 to A2, mix A2 by pipetting

iv. Continue serial dilution in IPTG Serial Dilution Plate by pipetting 570 uL from each well into the next and mixing, stopping with well A11

v. Pipette 570 uL from IPTG Serial Dilution Plate well A11 and send to waste

vi. End result should be IPTG concentrations in wells A1-A12 of: 50, 29.38, 17.27, 10.14, 5.96, 3.50, 2.06, 1.21, 0.71, 0.25, and 0 mmol/L; each well with 400 uL volume

20. Transfer 200 mL M9-Kan Media to bulk reagent plate or trough

a. this transfer may need to be done is steps as the media is used

21. Prepare Growth Plates 2-5: four 96-well clear assay plates with 480 uL M9-Kan Media + IPTG in each well

a. Pipette 470 uL of M9-Kan Media into each well of all 4 plates {180.48 mL M9-kan}

b. Pipette 10 uL from well A1 of IPTG Serial Dilution Plate into each well of column 1

(A1-H1) on each of Growth Plates 2-5

c. Pipette 10 uL from well A2 of IPTG Serial Dilution Plate into each well of column 2

(A2-H2) on each of Growth Plates 2-5

d. Repeat for remaining columns in Growth Plates 2-5

e. End result for Growth Plates 2-5 should be each well with 480 uL volume, with IPTG concentrations in column 1 through column 12 of: 1000, 588, 345, 203, 119, 70, 41, 24, 14, 8.4, 4.9, and 0 umol/L

22. Prepare Cell Dilution Plate 1: 96-well deep-well storage plate

a. Pipette 1152 uL M9-Kan Media into each well of column 1 of Cell Dilution Plate 1

23. Remove Growth Plate 1 from incubator/reader

24. Remove the membrane from Growth Plate 1

25. Pipette 48 uL from each well of column 8 in Growth Plate 1 to the corresponding well of column 1 in Cell Dilution Plate 1, mix by pipetting

26. Distrubute the cell suspensions from column 1 evenly across Cell Dilution Plate 1, 100 uL per well

27. Pipette 20 uL from each well of row A in Growth Plate 1 to each well of rows A-D in Growth

Plate 2

28. Pipette 20 uL from each well of row B in Growth Plate 1 to each well of rows E-H in Growth

Plate 2

29. Apply gas permeable membrane to Growth Plate 2 with adhesive sealer

30. Pipette 20 uL from each well of row C in Growth Plate 1 to each well of rows A-D in Growth

Plate 3

31. Pipette 20 uL from each well of row D in Growth Plate 1 to each well of rows E-H in Growth

Plate 3

32. Apply gas permeable membrane to Growth Plate 3 with adhesive sealer

33. Incubate Growth Plate 2 at 37°C with orbital shaking and OD measurement (in plate readers) for 5 hours

34. Incubate Growth Plate 3 at 37°C with orbital shaking and OD measurement (in plate readers) for 5 hours

35. Pipette 20 uL from each well of row E in Growth Plate 1 to each well of rows A-D in Growth

Plate 4

36. Pipette 20 uL from each well of row F in Growth Plate 1 to each well of rows E-H in Growth

Plate 4

37. Apply gas permeable membrane to Growth Plate 4 with adhesive sealer

38. Pipette 20 uL from each well of row G in Growth Plate 1 to each well of rows A-D in Growth

Plate 5

39. Pipette 20 uL from each well of row H in Growth Plate 1 to each well of rows E-H in Growth

Plate 5

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

41. Incubate Growth Plate 4 at 37°C with orbital shaking and OD measurement (in plate readers) for 5 hours

42. Incubate Growth Plate 5 at 37°C with orbital shaking and OD measurement (in plate readers) for 5 hours Pause Point 3

43. [Manual step] Prepare and load Nuclease-Free Reagent Plate 1 with Fixation Buffer (20 mL), Nuclease-Free PBS (40 mL), Nuclease-Free Water (3 mL), and 80% Ethanol (20 mL)

a. Loaded on deck in SBS format plate with Lid

44. Tansfer 100 mL PBS Buffer to bulk reagent plate or trough

a. Move to cooled reservoir/trough set to 4°C, with SBS Lid

45. Remove Growth Plates 2-3 from incubator/reader

46. Place Growth Plates 2-3 on plate cooling station [if available] set to 4°C, cool plate for 5 minutes

47. Remove membrane from Growth Plates 2-3

48. Centrifuge Growth Plates 2-3 to pellet cells, 5 minutes, 4000g, 4°C

a. Centrifuge with SBS Lids on plates, if possible

49. Remove supernatant from each well of Growth Plates 2-3

50. Resuspend cells in each well of Growth Plates 2-3 in 50 uL per well cold PBS Buffer, mix by pipetting

51. Add 150 uL cold PBS Buffer to each well of Growth Plates 2-3, mix by pipetting

a. Steps 49-50 (and similar steps) done by aspirating 200 uL of cold PBS Buffer 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.

52. Combine rows A-D into row D on Growth Plate 2

53. Combine rows E-H into row E on Growth Plate 2

54. Combine rows A-D into row D on Growth Plate 3

55. Combine rows E-H into row E on Growth Plate 3

56. Centrifuge Growth Plates 2-3 to pellet cells, 5 minutes, 4000g, 4°C

a. Centrifuge with SBS Lids on plates, if possible

57. Remove supernatant from each well of rows D-E of Growth Plates 2-3

58. Resuspend cells in each well of rows D-E of Growth Plates 2-3 with 50 uL per well Fixation

Buffer, mix by pipetting

59. Add 150 uL Fixation Buffer to each well of rows D-E of Growth Plates 2-3, mix by pipetting

60. Put new SBS Lids on Growth Plates 2-3

61. Mix Growth Plates 2-3 at room temperature for 30 minutes

a. Slow shaking speed or intermittent shaking

b. Can alternate plates through a single shaking station, if necessary

62. Repeat last 17 steps for Growth Plates 4-5 (starting with Remove Growth Plates 4-5 from incubator/reader)

63. Centrifuge Growth Plates 2-3 to pellet cells, 8 minutes, 400g, 20°C

a. Centrifuge with SBS Lids on plates, if possible

64. Remove supernatant from rows D-E of Growth Plates 2-3

65. Resuspend cells in each well of rows D-E in Growth Plates 2-3 in 50 ul per well Nuclease-

Free PBS per sample, and mix by pipetting

66. Then add 150 uL Nuclease-Free PBS to each well of rows D-E in Growth Plates 2-3

67. Centrifuge Growth Plates 2-3 to pellet cells, 3.5 minutes, 600g, 20°C

a. Centrifuge with SBS Lids on plates, if possible

68. Remove supernatant from rows D-E of Growth Plates 2-3

69. Repeat last 4 steps

70. Resuspend cells in each well of rows D-E in Growth Plates 2-3 in 25 ul per well Nuclease-

Free Water per sample, and mix by pipetting

71. Then add 175 uL 80% Ethanol to each well of rows D-E in Growth Plates 2-3, mix by pipetting

72. [Optional, if heat sealer is available] Apply foil seals to Growth Plates 2-3 with heat sealer

a. If no heat sealer, then apply SBS Lid to Growth Plates 2-3

73. Mix Growth Plates 2-3 at room temperature for at least 1 hour

a. Slow shaking speed or intermittent shaking

b. Can alternate plates through a single shaking station, if necessary

74. Repeat last 11 steps for Growth Plates 4-5 (starting with Centrifuge Growth Plates 4-5 to pellet cells, 8 minutes, 400g…)

Pause Point 4

75. [Manual step] Prepare and load Nuclease-Free Reagent Plate 2 with Wash Buffer A (20 mL)

a. Loaded on deck in SBS format plate with Lid

76. [Manual step] Prepare and load Black Plate 1 with Hybridization Buffer (1 mL)

77. Prepare Wash Plates 1-2 (96-well polypropylene storage plate)

78. Remove foil seal from Growth Plate 2

79. Pipette row D from Growth Plate 2 to row C of Wash Plate 1

80. Pipette row E from Growth Plate 2 to row D of Wash Plate 1

81. Remove foil seal from Growth Plate 3

82. Pipette row D from Growth Plate 3 to row E of Wash Plate 1

83. Pipette row E from Growth Plate 3 to row F of Wash Plate 1

84. Remove foil seal from Growth Plate 4

85. Pipette row D from Growth Plate 4 to row C of Wash Plate 2

86. Pipette row E from Growth Plate 4 to row D of Wash Plate 2

87. Remove foil seal from Growth Plate 5

88. Pipette row D from Growth Plate 5 to row E of Wash Plate 2

89. Pipette row E from Growth Plate 5 to row F of Wash Plate 2

90. Centrifuge Wash Plates 1-2 to pellet cells, 7 minutes, 600g, 20°C

a. Centrifuge with SBS Lids on plates, if possible

91. Remove supernatant (70% ethanol) from rows C-F of Wash Plates 1-2

92. Resuspend cells in rows C-F of Wash Plates 1-2 in 50 uL per well Wash Buffer A, mix by pipetting

93. Then add 150 uL Wash Buffer A to rows C-F of Wash Plates 1-2, mix by pipetting

94. Centrifuge Wash Plates 1-2 to pellet cells, 7 minutes, 600g, 20°C

a. Centrifuge with SBS Lids on plates, if possible

95. Remove supernatant (40% formamide) from rows C-F of Wash Plates 1-2

96. Resuspend cells in rows C-F of Wash Plates 1-2 in 10 uL per well Hybridization Buffer

a. Viscous solution

97. Apply seals to Wash Plates 1-2 with heat sealer, use foil seal with heat sealer, if available

98. Incubate Wash Plates 1-2 at 30°C for 16 hours in the dark

a. In the incubator/readers Pause Point 5

99. [Manual step] Prepare and load Nuclease-Free Reagent Plate 3 with Wash Buffer A (80 mL), Wash Buffer B (20 mL), and 2X SSC (10 mL)

a. Loaded on deck in SBS format plate with Lid

100. [Manual Step] Prepare and load Black Plate 2 with DAPI Wash Buffer (20 mL)

101. Remove seals from Wash Plates 1-2

102. Add 200 uL per well Wash Buffer A to rows C-F in Wash Plates 1-2, mix by pipetting

103. Pipette samples from Wash Plates 1-2 to new plates (Wash Plates 3-4, same plate type, same wells)

104. Centrifuge Wash Plates 3-4 to pellet cells, 3.5 minutes, 600g, 20°C

a. Centrifuge with SBS Lids on plates, if possible

105. Remove supernatant from rows C-F of Wash Plates 3-4 (40% formamide)

a. Some optimization probably required here; pellet is soft and easy to lose (suck up with the tip) at this point

106. Resuspend cells in 20 uL per well Wash Buffer A to rows C-F in Wash Plates 3-4, mix by pipetting

107. Add 180 uL per well Wash Buffer A to rows C-F in Wash Plates 3-4, mix by pipetting

108. Centrifuge Wash Plates 3-4 to pellet cells, 3.5 minutes, 600g, 20°C

a. Centrifuge with SBS Lids on plates, if possible

109. Remove supernatant from rows C-F of Wash Plates 3-4 (40% formamide)

110. Resuspend cells in 20 uL per well Wash Buffer A to rows C-F in Wash Plates 3-4, mix by pipetting

111. Add 180 uL per well Wash Buffer A to rows C-F in Wash Plates 3-4, mix by pipetting

112. Apply seals to Wash Plates 1-2 with heat sealer, use foil seal with heat sealer, if available

113. Incubate at 30°C for 30 minutes in the dark

a. In the incubator/readers

114. Remove seals from Wash Plates 1-2

115. Repeat last 7 steps (starting with Centrifuge Wash Plates 3-4 to pellet cells…)

116. Centrifuge Wash Plates 3-4 to pellet cells, 3.5 minutes, 600g, 20°C

a. Centrifuge with SBS Lids on plates, if possible

117. Remove supernatant from rows C-F of Wash Plates 3-4 (40% formamide)

118. Resuspend cells in 20 uL per well DAPI Wash Buffer to rows C-F in Wash Plates 3-4, mix by pipetting

119. Add 180 uL per well DAPI Wash Buffer to rows C-F in Wash Plates 3-4, mix by pipetting

120. Apply seals to Wash Plates 1-2 with heat sealer, use foil seal with heat sealer, if available

121. Incubate at 30°C for 30 minutes in the dark

122. In the incubator/readers

123. Remove seals from Wash Plates 1-2

124. Centrifuge Wash Plates 3-4 to pellet cells, 3.5 minutes, 600g, 20°C

a. Centrifuge with SBS Lids on plates, if possible

125. Remove supernatant from rows C-F of Wash Plates 3-4 (40% formamide)

126. Resuspend cells in 20 uL per well Wash Buffer B to rows C-F in Wash Plates 3-4, mix by pipetting

127. Add 180 uL per well Wash Buffer B to rows C-F in Wash Plates 3-4, mix by pipetting

128. Centrifuge Wash Plates 3-4 to pellet cells, 3.5 minutes, 600g, 20°C

a. Centrifuge with SBS Lids on plates, if possible

129. Remove supernatant from rows C-F of Wash Plates 3-4

130. Resuspend cells in 100 uL per well 2X SSC to rows C-F in Wash Plates 3-4, mix by pipetting

131. Prepare Black Plate (96-well round-bottom plate)

132. Pipette row C from Wash Plate 3 to row A of Black Plate

133. Pipette row D from Wash Plate 3 to row B of Black Plate

134. Pipette row E from Wash Plate 3 to row C of Black Plate

135. Pipette row F from Wash Plate 3 to row D of Black Plate

136. Pipette row C from Wash Plate 4 to row E of Black Plate

137. Pipette row D from Wash Plate 4 to row F of Black Plate

138. Pipette row E from Wash Plate 4 to row G of Black Plate

139. Pipette row F from Wash Plate 4 to row H of Black Plate

140. Put Black Lid on Black Plate 2

141. Done with automated protocol, take samples to imaging and/or flow-FISH

IX. PAYMENT TERMS

Subject to FAR 52.232-29, interim commercial payments will be authorized for this procurement under the following notional schedule. All percentages cited do not contemplate the price of CLINs 0016, 0017, and 0018):

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