Measurement
sfGFP-DmpR

Part:BBa_K1413001

Designed by: Nandjafot MENDY   Group: iGEM14_Evry   (2014-09-30)
Revision as of 18:11, 1 November 2014 by Nmendy (Talk | contribs)

P0 promoter-RBS B0032-sfGFP- Terminator B0015 - Pr promoter-DmpR

This part consist on a sensor of phenolic compounds based on DmpR, a transcription factor of the Ntrc family. Found in Pseudomonas sp. strain CF 600, DmpR regulates expression from the Po promoter, which drives transcription from one single large operon for phenol degradation (dmpKLMNOPQBCDEFGHI). With GFP attached to P0 promoter, it is then possible to evaluate presence of phenol by fluorescence analysis, if DmpR is expressed. This part is basically composed of [P0 promoter + sfGFP + Pr promoter + DmpR] and allows sensing of phenolic compounds thanks to a constitutively expressed DmpR that will activate expression of GFP. We characterized this biosensor using phenol as effector.


Usage and Biology

We prepared a protocol test to evaluate our Biosensor: E.coli (DH5apha) was grown overnight in M9 medium at 37 °C and then diluted 100-fold to an OD of 0.01 in fresh M9 medium containing Chloramphenicol in 96-well plates. After 6 hours’ of culture at 37 °C, each culture (200 μL) was centrifuged at 2500 r.p.m. for 15 minutes and was suspended in 200 μL of fresh M9 medium containing phenol of different concentrations Then the fluorescence intensity of cultures was measured by microplate reader (TECAN).

IMAGE

Figure 1 Fluorescence intensity per cell of BBa_K1413001 and BBa_K1413002
TECAN measurement of fluorescence during 11h growth, 37 C°, M9 media (0,4% glucose). The values were obtained by substracting raw Fluorescence values of bacteria exposed to phenol by fluorescence of media (M9) then dividing by corresponding OD600.


IMAGE

Figure 2 : Fluorescence induction ratio of BBa_K1413001 and BBa_K1413002
TECAN measurement of fluorescence, 11h growth, 37 C°, M9 media (0,4% glucose). Induction ratio was obtained by dividing the fluorescence intensity of bacteria exposed to phenol by their basal fluorescence intensity (no phenol added)


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1241
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1719
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1404
    Illegal BsaI.rc site found at 1945
    Illegal SapI.rc site found at 211
    Illegal SapI.rc site found at 2602


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