Part:BBa_K4987003

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.

Usage and Biology

Our team used the low-temperature inducible promoter pCspA to induce the expression of the bacteriolytic proteins T4 holin and T4 lysozyme, with BBa_B0015 as the terminator. We recombined the fragments into the plasmid pET23b and then transformed the constructed plasmid into E. coli DH5α.

Fig 1.genetic circuit diagram

Potential application directions

Firstly, we performed sequence amplification of the low-temperature inducible promoter pCspA, bacteriolytic protein T4 holin, and T4 lysozyme.

Fig 2.Agarose gel electrophoresis to verify the sequence amplification

Secondly, we validated the functionality of the low-temperature inducible promoter. We demonstrated through experiments that the optimal growth temperature for E. coli DH5α is 37°C, and growth slows as the temperature decreases (Fig 3.A). We then introduced the low-temperature inducible promoter CspA, linking it to a red fluorescent protein to test its functionality. The results showed that under low-temperature conditions, the OD600 value of BL21 was only 0.5 after 12 hours, whereas at 37°C, the OD600 of BL21 reached 2.3. However, the Fluorescence/OD600 ratio at low temperatures reached 278, significantly higher than the Fluorescence/OD600 ratio at 37°C (Fig 3.B), indicating that the low-temperature inducible promoter CspA can effectively induce expression under low-temperature conditions.

Finally, after introducing the suicide gene, the engineered bacteria containing the lysozyme sequence remained at a low density at 16°C, with an OD600 value consistently around 0.3 (Fig 3.C), demonstrating the functionality of the suicide system.

Fig 3.Validation of the Low-Temperature Inducible Promoter pCspA Suicide System(A) Effect of Temperature on the Growth of DH5α(B) Expression of Red Fluorescent Protein Induced by the Low-Temperature Inducible Promoter pCspA(C) Growth of E. coli DH5α at 16°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]