Arabinose promoter + sfGFP CpxR (Nterm)
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. Also, the results show that the arabinose promoter works as expected.
A characterization experiment in a microfluidic chip was also done. The aim of this experiment was to induce CpxR expression with arabinose.
Cells were inoculated and grown overnight in 3 ml LB medium with chloramphenicol. The next morning the cells were loaded on chip and the upper half of the chip had a flow of LB with arabinose and chloramphenicol whereas the lower half of the chip had a flow of LB with only chloramphenicol. The detailed protocol can be found on http://2014.igem.org/Team:EPF_Lausanne/Notebook/Microfluidics
This is a schematic representation of how the bacteria were divided on the chip. M1 and M2 stands for multiplex 1 and 2 and are valves that allow to block the flow of bacteria (or medium). Thanks to these multiplexes up to 16 different types of cells/bacteria can be flown in distinct rows. In this experiment we only used a separation in two region (ara+ and ara-).
Figure 1. Scan of the microfluidic chip at t = 0min. As expected, no signal is detected.
Figure 2. Scan of the microfluidic chip at t = 300min. The upper half of the chip has medium with arabinose and the lower half doesn't. Expression is detected on the upper half.
After GenePix analysis, where we calculated the intensity of each chamber and also the intensity of the area next to the chamber (to subtract as background value) we calculated an average of fluorescence expression. Figure 3 shows the results.
Figure 3. Evolution of CpxR-GFP fluorescence over time
A great GFP signal was detected which confirms the expression of our portein of interest sfGFP-Cpxr.
The increasing standard deviation for the cells with arabinose can be explained as some chambers did not have a lot of cells and so there was a low intensity. As it can be seen in the following picture :
Figure 4. These are chambers with arabinose in the medium, you can see that there are different cell density and thus different intensity in the chambers. Inducing a high standard deviation
In the context of the same experiment, we designed another construct with the sfGFP moiety attached to the C terminus of CpxR: http://parts.igem.org/Part:BBa_K1486005.
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)|