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Part:BBa_K514000

Designed by: Arcanum   Group: iGEM11_Sevilla   (2011-09-19)

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>



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