Difference between revisions of "Part:BBa K1949050:Design"
 
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===Design Notes===
 
===Design Notes===
−
A point mutation is introduced to <i>amiE</i> to remove the <i>Eco</i>RI recognition site; the 291th nucleotide  was changed from A to a G.
+
A point mutation is introduced to <i>amiE</i> to remove the <i>Eco</i>RI recognition site; the 291th nucleotide  was changed from A to G.
  
  
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-Plasmids
 
-Plasmids
  
−
A. PBAD-rbs-AmiE (pSB6A1)
+
A. PBAD - <i>rbs - amiE</i> (pSB6A1)
  
−
B. Ptet-rbs-luxR-TT-Plux-rbs-gfp (pSB6A1)  
+
B. Ptet - <i>rbs - luxR - tt</i> - Plux - <i>rbs - gfp</i> (pSB6A1)  
  
−
C. Ptet-rbs-rhlR-TT-Prhl-rbs-gfp (pSB6A1)
+
C. Ptet - <i>rbs - rhlR - tt</i> - Prhl - <i>rbs - gfp</i> (pSB6A1)
  
 
====Assay Protocol====
 
====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%.  
+
1. Inoculate A in 3 mL LB medium ampicillin (50 microg / mL), and add glucose so that the final concentration becomes 0.2%.Inoculate B and C in LB medium containing ampicillin (50 microg / mL).
  
−
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%.
+
2. Incubate all samples at 37&deg;C, 180rpm for 12 h.
  
−
3. Dilute the overnight culture of A so that the turbidity becomes about 0.05, and begin fresh culture at 37°C, 180 rpm.
+
3. Dilute the overnight culture of A so that the turbidity becomes about 0.05.
  
−
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.
+
4. Incubate at 37&deg;C, 180rpm so that turbidity becomes about 0.05.
  
−
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.
+
5. Add arabinose into test tube (&#8560;) and (&#8561;) so that the final concentration becomes 0.2%.
  
−
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.
+
6. 2 h after adding arabinose, add 60 microL of 3OC12HSL (500 microM) into test tube (&#8560;) and (&#8562;), and add 200 microL of 3OC12HSL (500 microM) into test tube (&#8561;) and (&#8563;).
  
−
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.)
+
7. 5 h after adding AHLs, dispense the fresh cultures into 1.5 mL tubes, and centrifuge in duplicate for 2 min at 18000G.
  
−
8. Prepare the overnight cultures containing LB medium and 1000 µL antibiotics in a 1.5 mL tube for control.
+
8. Transfer supernatant to another 1.5 mL tube.
  
−
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.
+
9. Add each 10 microL overnight culture for B and C into 800 microL LB medium containing ampicillin (50 microg / mL). Also, add each 10 microL overnight culture for B and C into 1 mL LB medium containing ampicillin (50 microg / mL) for controls. (B: a, c, e, g),(C: b, d, f, h)
  
−
10. Measure the turbiity after incubation at 37°C, 180 rpm for 4 h.
+
10. After 1.5 h, add 200 microL supernatants of A into (&#8560;), (&#8561;), (&#8562;), and (&#8563;), and add 4 microL 3OC12HSL into tube (e), 13.3 microL C4HSL into tube (f), 4 microL DMSO into tube (g), 13.3 microL DMSO into tube (h) for controls.
  
 +
11. incubate at 37&deg;C, 180 rpm for 4 h.
 +
 +
12. Measure RFU of GFP and the terbidity.
  
  
 
===References===
 
===References===
 
Ochiai S, Yasumoto S, Morohoshi T, Ikeda T. AmiE, a Novel <i>N</i>-Acylhomoserine Lactone Acylase Belonging to the Amidase Family, from the Activated-Sludge Isolate <i>Acinetobacter</i> sp. Strain Ooi24. 2014 Nov;80(22):6919-25.
 
Ochiai S, Yasumoto S, Morohoshi T, Ikeda T. AmiE, a Novel <i>N</i>-Acylhomoserine Lactone Acylase Belonging to the Amidase Family, from the Activated-Sludge Isolate <i>Acinetobacter</i> sp. Strain Ooi24. 2014 Nov;80(22):6919-25.

Latest revision as of 07:57, 21 October 2016


amiE


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

A point mutation is introduced to amiE to remove the EcoRI recognition site; the 291th nucleotide was changed from A to 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 in 3 mL LB medium ampicillin (50 microg / mL), and add glucose so that the final concentration becomes 0.2%.Inoculate B and C in LB medium containing ampicillin (50 microg / mL).

2. Incubate all samples at 37°C, 180rpm for 12 h.

3. Dilute the overnight culture of A so that the turbidity becomes about 0.05.

4. Incubate at 37°C, 180rpm so that turbidity becomes about 0.05.

5. Add arabinose into test tube (ⅰ) and (ⅱ) so that the final concentration becomes 0.2%.

6. 2 h after adding arabinose, add 60 microL of 3OC12HSL (500 microM) into test tube (ⅰ) and (ⅲ), and add 200 microL of 3OC12HSL (500 microM) into test tube (ⅱ) and (ⅳ).

7. 5 h after adding AHLs, dispense the fresh cultures into 1.5 mL tubes, and centrifuge in duplicate for 2 min at 18000G.

8. Transfer supernatant to another 1.5 mL tube.

9. Add each 10 microL overnight culture for B and C into 800 microL LB medium containing ampicillin (50 microg / mL). Also, add each 10 microL overnight culture for B and C into 1 mL LB medium containing ampicillin (50 microg / mL) for controls. (B: a, c, e, g),(C: b, d, f, h)

10. After 1.5 h, add 200 microL supernatants of A into (ⅰ), (ⅱ), (ⅲ), and (ⅳ), and add 4 microL 3OC12HSL into tube (e), 13.3 microL C4HSL into tube (f), 4 microL DMSO into tube (g), 13.3 microL DMSO into tube (h) for controls.

11. incubate at 37°C, 180 rpm for 4 h.

12. Measure RFU of GFP and the terbidity.


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.