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]
Introduction
This part is a composite part of BBa_K3698002, BBa_K3698001, BBa_K3698004 and BBa_K592009, As shown in Figure 1. The BBa_K3698002 is regulatory region of degP, it contains three CpxR sites, which can bound Phosphorylated DNA-binding transcriptional dual regulator CpxR activates transcription of degP. The BBa_K3698002 is degP gene which expresses periplasmic serine endoprotease. This protease is responsible for digesting abnormally folded proteins in E. coli in a high temperature environment to protect E. coli. There is also experimental evidence that DegP is beneficial to E. coli to resist acid stress. For the convenience of characterization, we connected the degP gene fused with chromoprotein after the rDegP. This competent part was inserted into a plasmid containing the chloramphenicol resistance gene for characterization, as shown in Figure 2.
Figure1. Schematic diagram of degP_CP circuit
Figure2. Plasmid map of pRdegPLCP
Experiment and Results
We first constructed a composite fragment of rDegP and degP. The result is shown in Figure 3. The fragment size is 1757bp. Then the pre-constructed pamilCP was linearized by PCR. The result is shown in Figure 4, and the fragment size is 2876bp. The fragments recovered and purified in the first two steps are transformed into DH5α competent after homologous recombination, and spread on a chloramphenicol-resistant plate, and a single colony is picked for culture. The obtained culture fluid was used for PCR verification, and the primers used were Test-Top and Test-Bottom. After the PCR was completed, gel electrophoresis was performed. The result is shown in Figure 5. The target fragment should be 1000 bp, and the fragment obtained is in line with the expected size, confirming the successful recombination of our pRdegPLCP plasmid.
Figure3. Gel electrophoresis of rDegP and degP composite fragments
Figure4. Gel electrophoresis of linearized pamilCP fragment
Figure5. Gel electrophoresis of pRdegPLCP recombination verifies
The pRdegPLCP was transformed into DH5α, and placed in normal temperature (37°C) and high temperature (45°C) respectively. The OD588 and OD600 of the bacterial solution were measured every 1h. After 11h, the DH5α_pRdegPLCP at the two temperatures was obtained. The growth curve, as shown in Figure 6, 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 7. Divide OD588 by OD600 to obtain the absorbance of chromprotein amilCP to characterize the expression of degP, as shown in Figure 7. The expression level of degP at 45°C is obviously higher than that at 37°C. This shows that CpxR in our new part can normally regulate the expression of degP, and can respond to environmental changes.
Figure6. Growth curve of DH5α_pRdegPLCP
Figure7. Enriched bacteria liquid
Figure8. Expression level of degP-amilCP
Conclusion
Adding a CpxR site can follow the original regulatory relationship of E. coli, and the two sites of CpxR2 and CpxR3 have a overlap, so that only one phosphorylated CpxR can be bound at the same time. The increased CpxR site is located in a more upstream position and can be bound with phosphorylated CpxR alone, which is more beneficial for the regulation of degP.
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