Part:BBa_K1413001
P0 promoter-RBS B0032-sfGFP- Terminator B0015 - Pr promoter-DmpR
This part consists 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 of the Po promoter, which drives transcription of one single large operon for phenol degradation (dmpKLMNOPQBCDEFGHI).
With GFP attached to P0 promoter, it is then possible to evaluate the presence of phenol by fluorescence analysis, if DmpR is expressed.
This part is basically composed of
- P0 promoter carrying two DmpR binding sites, a IHF binding site and a sigma factor 54 binding site.
- RBS BBa_B0032
- The super folded GFP (sfGFP)
- Pr promoter, a constitutive promoter
- DmpR the transcription factor that enable sensing of phenol.
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 at different concentrations. Then the fluorescence intensity of cultures was measured by microplate reader (TECAN).
Figure 2 describes the 96-wells plate organisation used to evaluate the biosensor.
We used three controls 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.
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 an 8-fold increase of fluorescence at 1µM of phenol and up to 45-fold increase at 1000µM.
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)
Comparing these data with those obtained by iGEM Peking 2013 indicates an improvement in the ability to produce a distinctive signal in response to phenol.
(Figure 5)
Left : Fluorescence induction ratio of Peking 2013 biosensor. Green curve corresponding phenol sensing.
Right : Fluorescence induction ratio of BBak1413001 in response to phenol.
Improvement of BBa_K1413001 response to phenol.
We decided to strenghten the signal produced by our biosensor by mutating the ribosome binding site of sfGFP. This mutation is processed in a way that it reproduces the consensus sequence of Shine Dalgarno (AGGAGGUAA)allowing mRNA to bind more specifically to 16s rRNA. This in turn increases the translation rate of the mRNA.
See BBa_K1413002 part.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1241
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1719
- 1000INCOMPATIBLE 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
| None |