Team:XHD-ShanDong-China/Improvement
Improvement
This part is the improvement of part BBa_K2610001, which is the regulatory region of the degP gene. It contains two CpxR sites, which can bound Phosphorylated DNA-binding transcriptional dual regulator CpxR activates transcription of degP. The degP gene is the target gene in this project. This gene 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. In wild-typeE. coli, there are three CpxR binding sites upstream of degP, which play a positive regulatory role in the expression of degP. In order to follow the original regulation system of E. coli and to study the relationship between distance and regulation, we added a CpxR site on the basis of BBa_K2610001. Compared with the original part, the regulation effect will be more obvious. As shown in Figure 1, the original part BBa_K2610001 only has two sites, CpxR2 and CpxR3, and the new part BBa_K3698003 adds CpxR1, which is an increase of 80bp relative to the original part. For the convenience of characterization, we connected the degP gene fused with chromoprotein after the original parts. The plasmid map is shown in Figure 2.
Figure 1. Schematic diagram of part BBa_K3698003.
Figure 2. Plasmid map of pRdegPLCP.
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.
Figure 3. Gel electrophoresis of rDegP and degP composite fragments.
Figure 4. Gel electrophoresis of linearized pamilCP fragment.
Figure 5. 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.
Figure 6. Growth curve of DH5α_pRdegPLCP.
Figure 7. Enriched bacteria liquid.
Figure 8. Expression level of degP-amilCP.
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.
