Team:XHD-ShanDong-China/Engineering
Engineering
degP gene expression generates periplasmic serine endoprotease 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°C.
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. The process of degP knockout is 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 flankings was amplified from the plasmid pKD13. 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_pKD46 electrocompetent, 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 was transformed into the strain which degP has been replaced. Finally, we got the degP-deficient strain MG1655_ΔdegP.
Figure 1. Schematic diagram of degP knockout process.
Figure 2. Gel electrophoresis of Kan resistant gene fragment with FRT flanking.
Figure 3. Gel electrophoresis of the degP knockout 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 absorbance of 600nm. The growth curves of the two strains at room temperature and high temperature are shown in the figure 5. It can be seen from the growth curve that the wild-type strain grows faster at 37°C, but at 45°C, the growth rate decreases significantly. The growth rate of degP-defective strains is already very slow at 37°C and even slower at 45°C. Compared to the wild type, the degP-defective strain hardly grows at 45°C. The MG1655_ΔdegP bacterial liquid cultured to 11h is still relatively clear. Therefore, the degP gene is very important to the thermal adaptability of E. coli.
Figure 4. Growth curves of MG1655 and MG1655_ΔdegP at different temperatures.
Dilute the bacterial solution of the two strains which were cultured to the logarithmic phase by 10, 100, and 1000 times respectively, and then spot 100uL on LB-agar plates. Each strain is divided into two groups, one group is cultivated at 37℃, the other group is Cultivate at 45°C. After about 18 hours, the colonies formed by live bacteria are shown in Figure 5. At 37°C, the number of live strains of wild-type strains was significantly greater than that of defective strains, and the colonies of wild-type strains were relatively large. At 45°C, wild-type strains still have colonies alive, while degP defective strains have almost no live bacteria. It can be explained that the lack of degP will bring a fatal threat to E. coli in high temperature environments.
Figure 5. Live bacteria count chart.
The degP gene plays a very important role in E. coli thermal adaptability, and experiments have shown that when E. coli loses the degP gene, it exhibits a significant inability to adapt to 37°C and 45°C temperature conditions, which can even pose a lethal threat. This indicates that it is important for us to study the regulation of degP expression, and there is a high probability that theoretically, by changing the protein expression of this gene, we can achieve changes in E. coli's thermal adaptability.
