Arabinose promoter + sfGFP-CpxR [Cterm]
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.
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,.
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
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 1205
- 21Illegal BamHI site found at 1144
Illegal XhoI site found at 2027
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 979
Illegal AgeI site found at 2438
- 1000Illegal SapI.rc site found at 1235
Illegal SapI site found at 961
|negative_regulators||CpxA (phosphatase activity)|
|positive_regulators||CpxA (Histidine kinase activity when activated)|