Part:BBa_K4987005
low-temperature inducible promoter (pCspA)+GalU
Due to the lower outdoor temperature compared to the ideal laboratory temperature, the UDP-glucose pyrophosphorylase encoded by the GalU gene cannot efficiently catalyze the EPS substrate (glucose-6-phosphate). In order to enhance the enzyme activity and ensure the production of EPS, we plan to use a low-temperature inducible promoter (pCspA) to ensure normal EPS production in a low-temperature environment.
Usage and Biology
Figure1 Design of the pCspA and galU .
We utilized pET28a as the vector and incorporated the low-temperature inducible promoter (pCspA), ureA, B, C structural genes, GalU gene, and B0015 terminator into E. coli Rosetta strain (host cells). Subsequently, the constructed plasmid was introduced into E. coli Rosetta strain via transformation.
Characterization
Figure2 Gel image of pCspA and galU.
Figure3 Schematic diagram illustrating the principle of the GalU gene.
The UDP-glucose pyrophosphorylase encoded by the GalU gene catalyzes the production reaction of the EPS substrate, UDP-glucose pyrophosphate. UDP-glucose pyrophosphorylase can catalyze the conversion of UDP-glucose to UDP-glucose pyrophosphate, and a large amount of UDP-glucose pyrophosphate can further generate EPS through a series of intracellular reactions (as shown in Figure 3), achieving high production of EPS.
As shown in Figure 4a, the growth of urease at 25°C and 37°C was observed. It can be seen that the OD600 (1.7) at 37°C was significantly higher than the OD600 (0.9) at 25°C. The optical density and mRFP fluorescence intensity were measured using a plate reader, and the relative fluorescence unit was calculated by dividing the raw mRFP fluorescence intensity by the OD600 value. As depicted in Figure 4b and 4c, the fluorescence intensity of mRFP gene expression coupling at different temperatures was analyzed. It can be observed that the fluorescence intensity of urease at 25°C was higher than that at 37°C. Inserting the CspA promoter upstream of the target gene for urease expression indeed enhanced urease expression and improved the adaptability of the bacteria to lower temperature conditions. Furthermore, it can also increase the production of EPS in subsequent operations. The seed solution was also cultured at 37°C, 180 rpm until the cell density reached OD600=0.4. Then, 0.5 mM IPTG was added for induction for 16 hours. The cell density of the bacterial solution was adjusted to OD600=1. A 10 mL culture medium was taken to detect the EPS yield using the anthrone-sulfuric acid method, as shown in Figure 5. Comparing the wild-type strain, which had an EPS yield of 46 mM, to the engineered strain induced by 0.5 mM IPTG for 16 hours, which had an EPS yield of 105 mM, it is evident that the engineered strain exhibited approximately twice the EPS yield of the control group. Hence, it can be concluded from this study that overexpressing the GalU gene can significantly increase EPS production.
Figure4 Growth status and expression of fluorescent protein in strains induced by low-temperature promoter.
Figure5 Comparison of EPS production data between the wild-type control bacterial group and the pT7-galU engineered bacterial group.
Potential application directions
Experimental results have demonstrated that the low-temperature inducible promoter and GalU gene effectively enhance EPS production, which is expected to have potential improvements on the surrounding soil. EPS possesses gel-like properties that can combine with soil particles and organic substances, forming colloidal aggregates that increase soil aggregation and structural stability. This helps improve soil permeability, erosion resistance, and wind erosion resistance. Overall, the application of the GalU gene and EPS can improve soil structural stability and water retention capacity, which are significant for enhancing soil quality, increasing agricultural production, and protecting the environment. The use of the low-temperature inducible promoter can effectively increase enzyme activity and expression efficiency, improving the effectiveness of our biocement. It also addresses temperature limitations in future project applications, making laboratory operations more practical and contributing to the creation of a better living environment.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 135
Illegal AgeI site found at 453
Illegal AgeI site found at 576 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 849
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