Team:Caltech/Protocols/Folate assay

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Folate Microbiological Assay Protocol

Purpose

To quantify and measure levels of folate production in the cell lysate and supernatant of 'E.coli' transformed with high copy folate biosynthesis genes 'folB,' 'folKE,' 'folBKE'.

Materials

• Folic Acid Assay media
• Lactobacilli Broth AOAC
• Folic Acid (for standard curve)
• 0.1M sodium acetate buffer (pH4.8) -1% ascorbic acid
  • Ascorbic acid (Sigma, A4544-25G)
• 0.1M 2-mercaptoethanol-0.5% sodium ascorbate (We used the above 0.1M sodium acetate - 1% ascorbic acid buffer in place of the sodium ascorbate)(for deconjugation mix)
• Human plasma (Sigma, P9523-1ML)(for deconjugation mix)
• Lactobacillus rhamnosus (ATCC 7469)

Deconjugation Mixture Sybesma

1. Dilute 1 g of human plasma in 5ml of 0.1M 2-mercaptoethanol-0.5% sodium ascorbate

2. Clear from precipitates by centrifugation (10,000xg,2 min)

3. Add 2.5% (vol/vol) concentration of the clarified human plasma solution to the folate samples.

Sample Preparation

1. Centrifuge the full-grown cell culture (5ml) after centrifugation (12,000x g, 10 min, 20 C).

2. Recover both cells and supernatant.

3. Dilute the supernatant 1:1 with 0.1M sodium acetate buffer (pH4.8) -1% ascorbic acid.

4. Wash with the 0.1M sodium acetate-1% ascorbic acid and resuspend in 5 mL of the same buffer.

5. Release folate from the cells by incubating the samples at 100C for 5 min (determined to be optimal for folate release + the heat inactivates the bacteria)

6. Add the deconjugation reaction mixture (2.5% vol/vol)

7. Incubate for 4h at 37C, you should see obvious cell debris at the bottom of the cell lysate tubes.

8. Add 50uL of prepared L.rhamnosus inoculum to each assay tube.

Standard Curve

Notes

• Previously, we had been using this protocol with the other folate-dependent strain Entercoccus hirae ATCC 8043, and were able to generate a linear standard curve from 0.1-1ng. However, this strain did not grow in our samples, and so we switched to L. rhamnosus.
• This is why we used Lactobacilli Broth AOAC instead of the recommended Lactobacilli MRS broth.