Generator

Part:BBa_K1486005

Designed by: iGem EPFL 2014   Group: iGEM14_EPF_Lausanne   (2014-08-13)

Arabinose promoter + sfGFP-CpxR [Cterm]

CtermSfgfpcpxr_gradient.jpg

Purpose of the Biobrick

This construct aimed to evaluate the expression of our CpxR construct, and the function of the arabinose promoter in E coli by fusing a superfolder GFP protein to the N terminus of CpxR. The sfGFP was chosen because of its higher intensity compared to GFP. Not knowing if CpxR would react the same way if sfGFP were attached to the N or C terminus, 2 biobricks were built, one with each of the orientations.

An experiment on both possible CpxR - sfGFP orientations was launched to determine whether the proteins were well expressed and folded, and if the arabinose promoter worked well. The basics were the following: increasing concentrations of arabinose as shown below and results of a plate reader with excitation and emission wavelengths of 490 nm and 510 nm respectively.

Screen_Shot_2014-10-13_at_16.20.13_.png

Sfgfpcpxr_C_plot.jpg

The results of this experiment show that CpxR is well expressed, as GFP can be seen. However, the results show that the arabinose promoter doesn't work as expected, although the fluorescence increases with arabinose concentration,.


Associated Biobricks

In the context of the same experiment, we designed another construct with the sfGFP moiety attached to the N terminus of CpxR: http://parts.igem.org/Part:BBa_K1486002.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
    Illegal XhoI site found at 2027
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
    Illegal AgeI site found at 2438
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1235
    Illegal SapI site found at 961


[edit]
Categories
//cds/reporter/gfp
//cds/transcriptionalregulator
//cds/transcriptionalregulator/activator
//chassis/prokaryote/ecoli
//classic/regulatory/other
//direction/forward
//function/regulation/transcriptional
//function/reporter/fluorescence
//promoter
//rbs/prokaryote/constitutive
//regulation/multiple
//terminator/double
regulator
transcriptional
Parameters
biology
chassisEscherichia coli
colorGreen
controlAraC, arabinose
directionForward
emission510
excitation485
functionTransciption regulator
negative_regulatorsCpxA (phosphatase activity)
positive_regulatorsCpxA (Histidine kinase activity when activated)
rbsElowitz
resistanceChloramphenicol