Part:BBa_K3698003
RdegPLCP
A blue DegP expression loop that can be regulated by CpxR.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
RdegPLCP
Introduction
The transcription of degP is regulated by CpxR. As shown in Figure 1, there are three CpxR sites upstream of degP. These three sites can bind to phosphorylated DNA and the transcription dual regulator CpxR to activate transcription. Our project will explore the relationship between the distance between the cpxR and degP genes expressing CpxR and the regulation efficiency, so these three CpxR sites and the degP gene will be amplified from the E. coli MG1655 genome by PCR. The primers obtained contained as shown in Figure 2, and the target fragment was 1757bp.
Aim of experiment
In order to characterize the expression level of degP, we fused a chromoprotein amilCP with it and connected it with a Linker to form a new element named degP_CP. This new element was inserted into the plasmid backbone containing CmR resistance to form the plasmid pdegP_CP. PCR verification showed that the pdegP_CP was successfully constructed. As shown in Figure 3, the target fragment was 1000 bp, and the resulting fragment was in line with expectations. The map of plasmid pRdegPLCP is shown in Figure 4. The plasmid contains a CmR resistance gene.
Figure1. Schematic diagram of degP_CP circuit
Figure2. Gel electrophoresis of degP and CpxR regulatory regions
Figure3. Gel electrophoresis of pdegP_CP recombination verify.
Figure4. Plasmid map of pdegP_CP
Experiment and Results
PdegP_CP was transformed into DH5α competent, and placed in normal temperature (37℃) and high temperature (45℃) respectively. The OD588 and OD600 of the bacterial solution were measured every 1h. After 12h, the DH5α_pdegP_CP at the two temperatures was obtained. The growth curve, as shown in Figure5, the curve in the high temperature environment is significantly lower than the normal temperature. After enriching the bacterial liquid, it can be seen that the bacterial liquid has a very obvious blue color, as shown in Figure 6. Divide OD588 by OD600 to obtain the absorbance of chromin to characterize the expression of degP, as shown in Figure 7，It can be clearly seen that the expression level of degP in a 45℃ environment is much higher than that in a 37℃ environment. This shows that CpxR in our new element can normally regulate the expression of degP and chromin, and can respond to environmental changes.
Figure5. The growth curve of DH5α_pdegP_CP at two temperatures.
Figure6. Photo of the fluid-rich.
Figure7. Expression level of degP_CP at two temperatures
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