A1. Updated SOW. DNA Production Gut Microbiota.pdf

Statement of work DNA Production Set-up, Generation: Gut Microbiota.

Objective:

Scientists at the Eastern Regional Research Center (ERRC) has a requirement for a laboratory / contractor to provide samples of DNA from cultures inoculated with fresh fecal samples that are subjected to food or food components.

BACKGROUND

The Gut Microbiome Group within the Dairy and Functional Foods Research Unit (DFFRU) at the EERC performs research on the effect of food and food ingredients on the gut microbiota of humans. This project requires testing of food components on the gut microbiota and subsequent DNA extraction for community level analysis and becomes part of our DNA library. Sequencing of the DNA then allows for the evaluation of how these foods may impact the gut microbiota of different individuals.

GENERAL SCOPE

The gut microbiome group is looking to purchase DNA for our DNA library on the gut microbiome. The DNA is extracted from samples of in vitro, cultured, gut microbiota treated with different foods or food components.

Agricultural Research Service (ARS) scientists will require the following specifications:

1. The gut microbiota used for culturing is required to be feces harvested fresh (not frozen) from subjects that are healthy humans. ARS scientists will provide a list of requirements that will be used to define the term healthy humans. Further demographic data on the subjects will be required on demand, but only if deemed necessary by ARS scientists.

2. The feces used for culturing must be within specific age groups as determined by ARS scientists.

3. After harvesting fresh fecal samples, they will be processed using a phosphate buffer following industry standards and used to inoculate miniaturized anaerobic bioreactors.

4. The bioreactors used must be anaerobic, and capable of monitoring pH and gas production during the incubation period.

5. The harvested gut microbiota samples will be cultured for 6, 24 or 48 hours. The samples will be maintained anaerobic and at 37 °C during culturing.

6. Samples will be taken at set time points during culturing for DNA extraction. Scientists will determine what time points require sampling.

7. The DNA extraction must be performed following collection using current industry standards, for example, a DNA extraction kit. The type of extraction kit will be verified by ARS scientists to ensure suitability.

8. The DNA samples are required to be ≥ 5 ng/µL in a total volume of > 49 µL each. Samples will be frozen and shipped on dry ice to maintain integrity.

The gut microbiota of subjects will be cultured using ex vivo technology. We require the ability to perform these gut microbiome studies in a high throughput fashion with a volume higher than 2mL but less than 15 mL. We require a miniaturized platform because it will need less resources and is more cost efficient for ARS. We require an automated system that can culture the gut microbiota under sealed, anaerobic conditions, while simultaneously measuring gas output under controlled temperature conditions. We require the use of a medium for colonic microbiota that provides a biorelevant output with optimized composition to support colonic microbial growth while maintaining the microbial community structure. We require the ability to perform 6-36 culturing reactions simultaneously, using fresh feces harvested from donors that are healthy, and fall within a specific age group.

Example of Objectives/Results:

Type “A” DNA production set-up of approximately 70 (or greater) QC DNA extracts

Additional Type “A” DNA extracts of approximately 36 (or greater)

Dry Ice Shipment of DNA Products

Shipping of any additional study materials

PERIOD OF PERFORMANCE

One year. (Based on date of Award)

February 15, 2023 to February 14, 2023.

PLACE OF PERFORMANCE

DEPT OF AGRICULTURE

ARS EERC

600 EAST MERMAID LANE

WYNDMOOR PA 19038

GOVERNMENT FURNSIHED PROPERTY

No physical property furnished by the USDA

DELIVERY ORDER REQUIREMENTS

1. Scientists will provide a list of food/ food components to be tested including concentrations.

2. The contractor will collect fresh fecal samples from human adults and use these to cultivate the gut microbiota. Donors must be healthy and fall within a specific age group. ARS scientists will determine the criterion for the healthy donor and specify which age groups should be tested.

3. The number of donors tested at one time will be between 6-36.

4. The gut microbiota will be cultivated anaerobically at 37C, and samples harvested at pre-indicated time points and frozen immediately.

5. Cultivation must be high throughput and small volume- i.e. at least 6 donors and only 2-15mL culture volume.

6. During the cultivation, there must be automated recording of the pH of the cultures, and gas measurements.

7. DNA will be extracted from frozen samples using a method approved by ARS scientists and quantified and frozen.

8. DNA will be shipped to the selected sequencing facility for analysis.

9. All data will be transferred to the ARS research scientists.

DELIVERY REQUIREMENTS

Delivery of DNA should be within 30-Days of running the experiment.

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