Part:BBa_K1413002

P0 promoter-RBS SD001-sfGFP- Terminator B0015 - Pr promoter-DmpR

This part consist of 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 of the P0 promoter, which drives transcription of a single phenol degradation large operon (dmpKLMNOPQBCDEFGHI). With GFP attached to the 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 when bound to phenol. We characterized this biosensor using phenol as the 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-wells 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).

Figure 1 describes the 96-wells plate organisation used to evaluate the biosensor. We used three control in this experiment :
Media only : To evaluate the natural fluorescence of the media with different concentrations of phenol.
pSB1C3 : DH5alpha resistant to chloramphenicol used as a growth control.
BBa_J23106 : DH5alpha carrying BBa J23106, allowing constitutive production of GFP. This control was used to evaluate the eventual impact of phenol on GFP expression and/or fluorescence.
Purified GFP : Used to associated fluorescence values to a defined concentration of GFP.

Figure 1 : 96-wells plate organisation scheme

Figure 2 : OD600 values measured over 11h in 96-wells plate(TECAN).

Figure 3 : Fluorescence intensity per cell of BBa_K1413001
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.

These data show an increase in expression of sfGFP in response to increasing concentration of phenol present in wells.They also show that the biosensor is able to sense down to 1µM of phenol. The induction ratio calculated from these data(Figure 4) show a 3-fold increase of fluorescence at 1µM of phenol and up to 37-fold increase at 1000µM. indicate a signal production of sfGFP occurs from a concentration.

Figure 4 : 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)

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.

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 BBa_K1413002 SequenceAndFeatures