Part:BBa_K514000
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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:
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