Difference between revisions of "Part:BBa K4987003"

 
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Because the outdoor environment is much lower than 37 degrees Celsius, low temperature conditions may have a detrimental effect on enzyme activity, leading to low expression efficiency. To address this issue in the low-temperature expression of urease, we have designed a solution using a cold-inducible promoter (CspA). This promoter ensures efficient expression of urease even under low-temperature conditions.
 
Because the outdoor environment is much lower than 37 degrees Celsius, low temperature conditions may have a detrimental effect on enzyme activity, leading to low expression efficiency. To address this issue in the low-temperature expression of urease, we have designed a solution using a cold-inducible promoter (CspA). This promoter ensures efficient expression of urease even under low-temperature conditions.
  
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===Usage and Biology===
 
===Usage and Biology===
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The Gene pCspA was cloned from Escherichia coli and inserted upstream of the target gene of urease. Then, it was transformed into E. coli.
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<div style="display:flex; flex-direction: column; align-items: center;">
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<img src="https://static.igem.wiki/teams/4987/wiki/part/emphasis-basic-parts-2-low-temperature-induced-promoter-pcspa-new-part-successful-project/image-9.png" style="width: 500px;margin: 0 auto" />
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<p style="font-size: 98%; line-height: 1.4em;">Figure1 Design of the pCspA.</p >
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<div style="display:flex; flex-direction: column; align-items: center;">
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<img src="https://static.igem.wiki/teams/4987/wiki/part/emphasis-basic-parts-2-low-temperature-induced-promoter-pcspa-new-part-successful-project/image-10.png" style="width: 300px;margin: 0 auto" />
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<p style="font-size: 98%; line-height: 1.4em;">Figure2 Design of the pCspA</p >
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</div>
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===Characterization===
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====1.Temperature effect on E. coli Rosetta ====
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Escherichia coli was inoculated in LB medium and cultured at different temperatures (180 rpm) for 6 hours. The environmental temperature was controlled using an oscillating incubator, and the optical density at 600 nm (OD600) was measured at each temperature. As shown in the figure, the results indicate that E. coli exhibits robust growth at 37°C, mild cold-shock inhibition at 25°C, but minimal growth at 10°C. These research findings confirm that temperature significantly impacts the activity of E. coli and demonstrates its sensitivity to temperature changes.
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<img src="https://static.igem.wiki/teams/4987/wiki/part/emphasis-basic-parts-2-low-temperature-induced-promoter-pcspa-new-part-successful-project/image-11.png" style="width: 400px;margin: 0 auto" />
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<p style="font-size: 98%; line-height: 1.4em;">Figure3 The effect of different temperatures on the engineered bacterial strain.</p >
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====2.Test the pCspA====
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In this study, the mRFP gene was cloned downstream of a cold-inducible promoter. To analyze the expression of mRFP, cells were inoculated into LB medium and grown at two different temperatures, 37°C and 25°C, with an oscillation of 180 rpm for 16 hours. The growth of cells was quantified by measuring the optical density at 600 nm (OD600) using a spectrophotometer. Additionally, the fluorescence intensity of mRFP was measured using the same instrument. Figure A shows the impact of different temperatures on E. coli when the cold-inducible promoter is added. It is noteworthy that the fluorescence of mRFP is significantly enhanced at 37°C compared to 25°C. The results indicate that temperature has a significant effect on the activity of mRFP, with 37°C being the optimal temperature for mRFP. Figures B and C represent the fluorescence of mRFP at different temperatures.​​​​​​​​​
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<img src="https://static.igem.wiki/teams/4987/wiki/part/emphasis-basic-parts-2-low-temperature-induced-promoter-pcspa-new-part-successful-project/2023-09-11-22-30-43.png" style="width: 800px;margin: 0 auto" />
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<p style="font-size: 98%; line-height: 1.4em;">Figure4  Test the pCspA.​</p >
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===Potential application directions===
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The application of pCspA on the pSB1A3 vector provides a promising solution for temperature-restricted enzyme expression. This promoter is able to independently regulate gene expression without being influenced by environmental temperature. These findings open up possibilities for improving enzyme expression at low temperatures, which has practical significance in various fields such as biotechnology and industrial processes.
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Revision as of 14:15, 10 October 2023


a cold-inducible promoter (CspA)

Because the outdoor environment is much lower than 37 degrees Celsius, low temperature conditions may have a detrimental effect on enzyme activity, leading to low expression efficiency. To address this issue in the low-temperature expression of urease, we have designed a solution using a cold-inducible promoter (CspA). This promoter ensures efficient expression of urease even under low-temperature conditions.

Usage and Biology

The Gene pCspA was cloned from Escherichia coli and inserted upstream of the target gene of urease. Then, it was transformed into E. coli.

Figure1 Design of the pCspA.

Figure2 Design of the pCspA

Characterization

1.Temperature effect on E. coli Rosetta

Escherichia coli was inoculated in LB medium and cultured at different temperatures (180 rpm) for 6 hours. The environmental temperature was controlled using an oscillating incubator, and the optical density at 600 nm (OD600) was measured at each temperature. As shown in the figure, the results indicate that E. coli exhibits robust growth at 37°C, mild cold-shock inhibition at 25°C, but minimal growth at 10°C. These research findings confirm that temperature significantly impacts the activity of E. coli and demonstrates its sensitivity to temperature changes.

Figure3 The effect of different temperatures on the engineered bacterial strain.

2.Test the pCspA

In this study, the mRFP gene was cloned downstream of a cold-inducible promoter. To analyze the expression of mRFP, cells were inoculated into LB medium and grown at two different temperatures, 37°C and 25°C, with an oscillation of 180 rpm for 16 hours. The growth of cells was quantified by measuring the optical density at 600 nm (OD600) using a spectrophotometer. Additionally, the fluorescence intensity of mRFP was measured using the same instrument. Figure A shows the impact of different temperatures on E. coli when the cold-inducible promoter is added. It is noteworthy that the fluorescence of mRFP is significantly enhanced at 37°C compared to 25°C. The results indicate that temperature has a significant effect on the activity of mRFP, with 37°C being the optimal temperature for mRFP. Figures B and C represent the fluorescence of mRFP at different temperatures.​​​​​​​​​

Figure4 Test the pCspA.​

Potential application directions

The application of pCspA on the pSB1A3 vector provides a promising solution for temperature-restricted enzyme expression. This promoter is able to independently regulate gene expression without being influenced by environmental temperature. These findings open up possibilities for improving enzyme expression at low temperatures, which has practical significance in various fields such as biotechnology and industrial processes.


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]