Difference between revisions of "Part:BBa K1949050:Design"

Revision as of 05:04, 16 October 2016

amiE

Design Notes

A point mutation is introduced to amiE to remove the EcoRI recognition site; the 291th nucleotide was changed from A to a G.

Source

The DNA fragment of amiE was artificially synthesized.

Materials and Methods

Construction

-strain

All the sample were XL1-Blue strain.

-Plasmids

A. PBAD-rbs-AmiE (pSB6A1)

B. Ptet-rbs-luxR-TT-Plux-rbs-gfp (pSB6A1)

C. Ptet-rbs-rhlR-TT-Prhl-rbs-gfp (pSB6A1)

Assay Protocol

1. Inoculate A into 2997 µL LB culture containing 3 µL ampicillin, and incubate at 37°C, 180 rpm for 12 h, so that the final concentration of glucose becomes 0.2%.

2. Inoculate B and C into 2997 µL LB culture containing 3 µL ampicillin, and incubate at 37°C, 180 rpm for 12 h, so that the final concentration of glucose becomes 0.2%.

3. Dilute the overnight culture of A so that the turbidity becomes about 0.05, and begin fresh culture at 37°C, 180 rpm.

4. Add arabinose into test tube ① and ② so that the final concentration becomes 0.2% when the turbidity reaches 0.6 to 0.7.

5. 2 h after addition arabinose, add 60 µL of 500 µM C12AHL into test tube ① and ③, add 200 µL of 500 µM C12AHL into test tube ② and ④.

6. 5 h after adding AHL, dispense the fresh cultures into 1.5 mL tubes, and centrifuge in duplicate for 2 min at 18000x g. Transfer it to other 1.5 mL tube.

7. Dilute the overnight cultures of B and C, and begin fresh culture at 37°C, 180 rpm. (Add LB + antibiotics 800 µL into 10 µL overnight culture.)

8. Prepare the overnight cultures containing LB medium and 1000 µL antibiotics in a 1.5 mL tube for control.

9. After 1.5 h, add 200 µL supernatants of A ①, ②, ③ and ④, and add 4 µL C12AHL into tube e, 13.3 µL C4AHL into tube f, 4 µL DMSO into tube g, 13.3 µL DMSO into tube h for control.

10. Measure the turbiity after incubation at 37°C, 180 rpm for 4 h.

References

Ochiai S, Yasumoto S, Morohoshi T, Ikeda T. AmiE, a Novel N-Acylhomoserine Lactone Acylase Belonging to the Amidase Family, from the Activated-Sludge Isolate Acinetobacter sp. Strain Ooi24. 2014 Nov;80(22):6919-25.