Difference between revisions of "Part:BBa K514000"
Line 15: Line 15:
 
Firstly, we seeded the transformed bacteria on a plate with 80ul of X-Gal 1% and 1 ul of C6-3-oxo-HSL 10mg/ml, just to check that it worked.
 
Firstly, we seeded the transformed bacteria on a plate with 80ul of X-Gal 1% and 1 ul of C6-3-oxo-HSL 10mg/ml, just to check that it worked.
 
For the proper charaterization we resorted to an ONPG essay. Ortho-nitrophenyl-betha-galactoside is an artificial substrate that turns the liquid media yellow when it's hydrolized by the betha-galactosidase.  
 
For the proper charaterization we resorted to an ONPG essay. Ortho-nitrophenyl-betha-galactoside is an artificial substrate that turns the liquid media yellow when it's hydrolized by the betha-galactosidase.  
−
We measured
+
 
 +
Protocol:
 +
-Let an inoculum grow until it reaches an O.D(600nm) between 0,1 and 0,3
 +
-Add the inducer signal. In this case we used a final concentration of 0,0001 ug/ml.
 +
 
 +
The next steps should be repeated at different times:
 +
-Take 200 ul of the culture+inducer
 +
-Add 800 ul of Buffer Z (with phosphate)
 +
-Add 20 ul of chloroform to lysate the cells
 +
-Add 10 ul of SDS 1%
 +
-Vortex for 20 seconds
 +
-Add 200 ul of liquid ONPG
 +
Yellow colour should appear soon, in less than 5 minutes (at least in our case)
 +
-After 5 minutes, add 100ul of Na2CO3 to stop the reaction
 +
-Measure O.D (420 nm)
 +
 
 +
These were our results for a concentration of 0,0001 ug/ml, at six different moments:
 +
time(min) Abs(420nm)
 +
5         0,1
 +
12         0,121
 +
20         0,138
 +
25         0,148
 +
32         0,161
 +
40         0,21
 +
 
 +
 
 +
And the corresponding graph and equation:
 +
 
 +
 
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 12:42, 1 October 2011

C6-3OXO-HSL -> β-galactosidase activity

This BioBrick is a composition of F2620 with I732019. It was conceived to characterize the construction F2620: check which concentration of its specific quorum sensing signal (C6-3-oxo-HSL) it's able to detect, and how long does it take the bacterium to produce an answer (in this case, the expression of lacZ).

Sequence and Features


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1004

Characterization

We tested this construction by two different methods. Firstly, we seeded the transformed bacteria on a plate with 80ul of X-Gal 1% and 1 ul of C6-3-oxo-HSL 10mg/ml, just to check that it worked. For the proper charaterization we resorted to an ONPG essay. Ortho-nitrophenyl-betha-galactoside is an artificial substrate that turns the liquid media yellow when it's hydrolized by the betha-galactosidase.

Protocol: -Let an inoculum grow until it reaches an O.D(600nm) between 0,1 and 0,3 -Add the inducer signal. In this case we used a final concentration of 0,0001 ug/ml.

The next steps should be repeated at different times: -Take 200 ul of the culture+inducer -Add 800 ul of Buffer Z (with phosphate) -Add 20 ul of chloroform to lysate the cells -Add 10 ul of SDS 1% -Vortex for 20 seconds -Add 200 ul of liquid ONPG 

Yellow colour should appear soon, in less than 5 minutes (at least in our case)

-After 5 minutes, add 100ul of Na2CO3 to stop the reaction -Measure O.D (420 nm)

These were our results for a concentration of 0,0001 ug/ml, at six different moments: time(min) Abs(420nm) 5 0,1 12 0,121 20 0,138 25 0,148 32 0,161 40 0,21


And the corresponding graph and equation: