Difference between revisions of "Part:BBa K3698001"

Latest revision as of 02:20, 28 October 2020

degP

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.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Introduction

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.

Construction of strain MG1655_ΔdegP

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.

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 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.

Conclusion

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.

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 <partinfo>BBa_K3698001 short</partinfo>
-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.
+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.
 <!-- Add more about the biology of this part here
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 <h2> Introduction </h2>
-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.
+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.
-<h2>Construction of degP-deficient strain</h2>
+<h2>Construction of strain MG1655_ΔdegP</h2>
-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.
+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.
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-   <img src="https://2020.igem.org/wiki/images/9/9a/T--XHD-ShanDong-China--Engineering-Figure1.jpeg" style="width:60%">
+   <img src="https://2020.igem.org/wiki/images/9/9a/T--XHD-ShanDong-China--Engineering-Figure1.jpeg" style="width:80%">
    </body>
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-Figure1. Schematic diagram of DegP knockout process
+Figure 1. Schematic diagram of degP knockout process.
 <html>
    <body>
-   <img src="https://2020.igem.org/wiki/images/e/ed/T--XHD-ShanDong-China--Engineering-Figure2.jpeg" style="width:60%">
+   <img src="https://2020.igem.org/wiki/images/e/ed/T--XHD-ShanDong-China--Engineering-Figure2.jpeg" style="width:40%">
    </body>
 </html>
-Figure2. Gel electrophoresis of Kan resistant gene fragment with FRT flanking
+Figure 2. Gel electrophoresis of Kan resistant gene fragment with FRT flanking.
 <html>
    <body>
-   <img src="https://2020.igem.org/wiki/images/6/64/T--XHD-ShanDong-China--Engineering-Figure3.jpeg" style="width:60%">
+   <img src="https://2020.igem.org/wiki/images/6/64/T--XHD-ShanDong-China--Engineering-Figure3.jpeg" style="width:40%">
    </body>
 </html>
-Figure3.Gel electrophoresis of the kan resistance gene replacement with FRT flanking degP gene verification
+Figure 3. Gel electrophoresis of the degP knockout verification.
 <h2> degP function verification </h2>
-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.
+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.
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-Figure4. Growth curves of MG1655 and MG1655_ΔdegP at different temperatures
+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.
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 </html>
-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.
+Figure 5.  Live bacteria count chart.
+<h2>Conclusion</h2>
+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.