SOW. Cryptobiotix.docx

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 isolated 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 must be extracted from samples of ex vivo/in vitro, cultured, gut microbiota treated with different foods or food components. The vendor selected must have the capabilities to perform all the steps listed to generate the appropriate end-product samples (DNA). Agricultural Research Service (ARS) scientists will require the following specifications for the samples:

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. This requires the vendor to have the ability to recruit the appropriate donors for the study. 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, the fresh feces must 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. Media used must be degassed and oxygen free.

5. The harvested gut microbiota samples will be cultured for 6, 24 or 48 hours using a computer controlled, automated machine. Automation is required to ensure accuracy. The samples will be maintained anaerobic and at 37 °C during culturing.

6. The media used for culturing must be biorelevant with optimized composition to support colonic microbial growth while maintaining the microbial community structure.

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

8. 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.

9. 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.

10. Experiment must be performed 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. This is a critical component.

11. 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.

PERIOD OF PERFORMANCE

One year. (Based on date of Award)

July 11,2024 to July 11, 2025

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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