Reporter

Part:BBa_K1909013

Designed by: Daniel Wedemeyer   Group: iGEM16_Hamburg   (2016-10-14)


Omp Promoter / GFP

The part BBa_K1909013 consists of the green fluorescent protein GFP and the positively regulated OmpR-controlled promoter (BBa_R0082). The promoter OmpR is taken from the upstream region of ompC. Phosphorylated OmpR binds to the three operator sites and activates transcription.

Usage and Biology

In nature, the promoter OmpR is upstream of the ompC porin gene. The regulation of ompC is determined by the EnvZ-OmpR osmosensing machinery. EnvZ phosphorylates OmpR to OmpR-P. At high osmolarity, EnvZ is more active, creating more OmpR-P. OmpR-P then binds to the low-affinity OmpR operator sites upstream of ompC. Via FACS analysis it was shown in EnvZ deficient cells (JW3367-3), containing the construct BBa_K1909013 ,that the expression of GFP was significantly reduced in comparison to EnvZ containing cells (DH5α).

Functional Parameters

In two approaches, BBa_R0082 (OmpR, positive promoter) was ligated with BBa_E0240 (GFP) into pSB1C3 and transformed the constructs into both ΔEnvZ (JW3367-3) and wt (DH5a) E. coli strains. Positive constructs were validated by sequencing. Negative controls (JW3367-3 and DH5a without our constructs) showed only few to no fluorescence as expected. GFP expression proved to be significantly higher in DH5a (green) cells as compared to EnvZ deficient JW3367-3 cells (pink).

chassisE. coli JW 3367-3

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 779


[edit]
Categories
//cds/reporter
//cds/reporter/gfp
Parameters
chassisE. coli JW 3367-3