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degP

degP expression generates DegP. DegP acts as a chaperone at low temperatures but switches to a peptidase (heat shock protein) at higher temperatures. Degrades transiently denatured and unfolded or misfolded proteins which accumulate in the periplasm following heat shock or other stress conditions. DegP is indispensable for bacterial survival at temperatures above 42 degrees Celsius.

Sequence and Features

Introduction

degP expression generates DegP. DegP acts as a chaperone at low temperatures but switches to a peptidase (heat shock protein) at higher temperatures. Degrades transiently denatured and unfolded or misfolded proteins which accumulate in the periplasm following heat shock or other stress conditions. DegP is indispensable for bacterial survival at temperatures above 42 degrees Celsius.

Construction of degP-deficient strain

In order to explore whether degP plays a decisive role in the thermal adaptation of E. coli, we need degP-deficient strains as a control. As shown in Figure 1, the plasmid pKD46 was first transformed into the MG1655 wild-type strain and prepared as electrocompetent. Then, the kan resistance gene fragment containing the FRT flanks was amplified from the pKD13 plasmid, and the 5'end of the primer used in PCR Containing the upstream and downstream homology arms of the degP gene, the PCR fragment size is 1395 bp, as shown in Figure 2, the fragment meets expectations, and the gel is cut and recovered for use. The recovered fragments were electrotransformed into the MG1655 strain containing pKD46 plasmid, the recombinase expressed by plasmid pKD46 would replace degP with the kan resistance gene flanking FRT through homologous recombination. As shown in Figure 3, PCR verification showed that the fragment replacement was successful . Finally, the plasmid pCP20 is transformed into the strain where degP has been replaced. The recombinase expressed by pCP20 will overlap the two FRTs and knock out the kan resistance gene in the middle. As shown in Figure 4, PCR verification shows that the kan resistance gene Knockout is successful. Finally, we got the degP-deficient strain MG1655_ΔdegP.

Figure1. Schematic diagram of DegP knockout process

Figure2. Gel electrophoresis of Kan resistant gene fragment with FRT flanking

Figure3.Gel electrophoresis of the kan resistance gene replacement with FRT flanking degP gene verification

degP function verification

We cultured both the MG1655_ΔdegP strain and the wild-type MG1655 strain at room temperature (37°C) and high temperature (45°C), and took samples every 1 hour to identify their OD600. The growth curves of the two strains at room temperature and high temperature are shown in the figure. 5 shown. It can be clearly seen from the figure that the growth state of the degP-deficient strain at room temperature is not very different from that of the wild-type, but in a high temperature environment, the OD600 of the degP-deficient strain is significantly lower than that of the wild-type strain. There is no growth on it. Then, we diluted the bacteria liquids of the two strains grown to the logarithmic phase at 37°C by 10, 100, and 1000 times and spread them on a non-resistant LB-agar medium. Each strain was coated with two groups, one group Incubate at 37°C and the other group at 45°C. After about 18 hours, the observed colonies are shown in Figure 6. The number of colonies formed by the two strains cultured at 37°C is similar, while at 45°C, there are almost no colonies of the defective strains, while the wild type still has more colonies. . It can be explained that the lack of degP does not have a significant impact on the normal growth of E. coli at room temperature, but in high temperature environments, degP is very important, and the lack of degP will bring a fatal threat to E. coli in high temperature environments.

Figure4. Growth curves of MG1655 and MG1655_ΔdegP at different temperatures

Figure5. were grown to log phase in LB broth, serially diluted, spotted on LB agar plates, and grown overnight at 37°C or 45°C.