Difference between revisions of "Part:BBa K514000"
 
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===Characterization===
 
===Characterization===
 
We tested this construction by two different methods.
 
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.
+
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.</br>
−
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. </br>
  
−
Protocol:
+
Protocol:</br>
−
-Let an inoculum grow until it reaches an O.D(600nm) between 0,1 and 0,3
+
-Let an inoculum grow until it reaches an O.D(600nm) between 0,1 and 0,3</br>
−
-Add the inducer signal. In this case we used a final concentration of 0,0001 ug/ml.  
+
-Add the inducer signal. In this case we used a final concentration of 0,0001 ug/ml. </br>
  
−
The next steps should be repeated at different times:
+
The next steps should be repeated at different times:</br>
−
-Take 200 ul of the culture+inducer
+
-Take 200 ul of the culture+inducer</br>
−
-Add 800 ul of Buffer Z (with phosphate)
+
-Add 800 ul of Buffer Z (with phosphate)</br>
−
-Add 20 ul of chloroform to lysate the cells
+
-Add 20 ul of chloroform to lysate the cells</br>
−
-Add 10 ul of SDS 1%
+
-Add 10 ul of SDS 1%</br>
−
-Vortex for 20 seconds
+
-Vortex for 20 seconds</br>
−
-Add 200 ul of liquid ONPG
+
-Add 200 ul of liquid ONPG. Yellow colour should appear soon, in less than 5 minutes (at least in our case)</br>
−
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</br>
−
-After 5 minutes, add 100ul of Na2CO3 to stop the reaction
+
-Measure O.D (420 nm)</br>
−
-Measure O.D (420 nm)
+
  
−
These were our results for a concentration of 0,0001 ug/ml, at six different moments:
+
These were our results for a concentration of 0,0001 ug/ml, at six different moments:</br>
−
time(min) Abs(420nm)
+
time(min) Abs(420nm)</br>
−
5         0,1
+
5         0,1</br>
−
12         0,121
+
12         0,121</br>
−
20         0,138
+
20         0,138</br>
−
25         0,148
+
25         0,148</br>
−
32         0,161
+
32         0,161</br>
−
40         0,21
+
40         0,21</br>
  
  
−
And the corresponding graph and equation:
+
And the corresponding graph and equation:</br>
  
  

Latest revision as of 12:45, 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.</br> 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. </br>

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

The next steps should be repeated at different times:</br> -Take 200 ul of the culture+inducer</br> -Add 800 ul of Buffer Z (with phosphate)</br> -Add 20 ul of chloroform to lysate the cells</br> -Add 10 ul of SDS 1%</br> -Vortex for 20 seconds</br> -Add 200 ul of liquid ONPG. Yellow colour should appear soon, in less than 5 minutes (at least in our case)</br> -After 5 minutes, add 100ul of Na2CO3 to stop the reaction</br> -Measure O.D (420 nm)</br>

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


And the corresponding graph and equation:</br>