Part:BBa_K817000

Method

To check whether our bacteria successfully secrete GLP-1 to the environment, we collected the bacterial media and measure its GLP-1 concentration. After centrifugation of bacterial culture, we collected the supernatant and passed it through 0.22 μm filter to exclude bacteria. Since the secreted GLP-1 is too diluted by the large volume of media, we concentrated the solution by 5 kDa ultra centrifugal filter tubes. We also collected the bacterial pellet and add lyze it by E. coli lysis buffer and freeze-thaw to measure GLP-1 inside bacteria. Both Immunoblotting and ELISA are used to detect GLP-1 concentration inside and outside bacteria.

Protocol

A. Sample Preparation

1. 310 μL Bacterial culture was cultured in 5mL LB at 37℃, with suitable concentration of antibiotics shaking till 600 nm absorbance in cuvette is 1.5 (usually it takes about 18 hr).
2. Centrifuge the bacterial culture at 12000 rpm for 3 min.
3. Pass the supernatant through 0.22 μm filter. Transfer the supernatant to 5 kDa ultra centrifugal filter tubes and centrifuge at 10000 rpm for 40 min.
4. Add 250 μL of E. coli lysis buffer to the pellet and use liquid Nitrogen to freeze-thaw for three cycles.
5. Heat the samples at 95。C for 10 min before immune-blotting.

B. Dot-Blotting

1. Soak the PVDF membrane in ethanol for 5 min to activate the membrane.
2. Drop every 2 μL sample in a very small area on the membrane.
3. Blocking with 5% de-fat milk (in PBST) for 1 hr.
4. Wash with PBST for 10 min, repeat 3 times.
5. Add primary antibody (1:1000 dilution in PBST) and incubate at 4℃ overnight.
6. Wash with PBST for 10 min, repeat 3 times.
7. Add secondary antibody (1:5000 dilution in PBST) and incubate at 25℃ 2 hr.
8. Wash with PBST for 10 min, repeat 3 times.
9. Mix 2 mL ECL with 2mL substrate, add to the membrane and let stand for 5 min.
10. Expose the membrane to the film in dark room.

C. ELISA

1. Wash the wells with 250 μL Wash Buffer.
2. Add 100 μL Assay Buffer to each well, and add 100 μL standards in ascending orders to wells, and add samples in the remaining wells.
3. Incubate at 4℃ overnight.
4. Decant liquid from plate.
5. Wash 5 times with 250 μL Wash Buffer.
6. Add 200 μL Detection Conjugate in each well. Incubate 2 hr at room temperature.
7. Wash 3 times with 250 μL Wash Buffer.
8. Add 200 μL diluted substrate and incubate for 20 min.
9. Read plate on fluorescence plate reader with excitation/emission wavelength of 355 nm/460 nm.

Data

A. Dot-Blotting

The upper dot is from the lyzed bacterial pellet, which represents the GLP-1 inside the bacteria. The middle part is from bacterial culture supernatant, which means the secreted GLP-1, and the left dot is the media we did 10X concentration, the right dot is the one we did 100X concentration. The lower dot is from pure GLP-1 solution as positive control.

B. ELISA

The left bar is from the lyzed bacterial pellet, which represents the GLP-1 inside the bacteria. The right bar is from 10X concentrated bacterial culture supernatant, which means the secreted GLP-1.

Conclusion

Our data indicates that although there is much GLP-1 inside the bacteria, some of GLP-1 is secreted by our signal peptide design. The secreted GLP-1 can be delivered to intestinal lumen and carry out its physiological function once absorbed.