Part:BBa_K5013003
This is a phenylalanine degrading enzyme controlled by a hypoxic promoter
Considering the functionality of phenylalanine lyase (PAL) in an anaerobic environment, we decided to position PAL downstream of the hypoxia promoter (pPepT), as this part allows for the regulation of PAL expression in such conditions. Ultimately, this composite part facilitates the transformation of phenylalanine into trans-cinnamic acid.
Usage and Biology
We used a hypoxia-sensing promoter, constructed phenylalanine lyase (PAL) gene downstream of it, and used B0015 terminator. The constructed plasmid was introduced into E. coli BL21.
Figure 1 Design of the pPepT promoter for pal.
Characterization
We used the pPepT promoter to create engineered bacteria that express PAL. These bacteria were then cultured in a CO2 incubator with varying levels of O2 (0%, 10%, and 20%). The bacterial solution was suspended in 1 mL of Phe experimental buffer, which consisted of M9 medium with 0.5% dextrose and 1 mM Phe. The suspension was adjusted to an optical density (OD600) of 0.1 and placed in microtubes, which were then incubated at 37°C for 1 hour.After incubation, we determined the concentration of TCA using an enzyme labeling method and measuring the absorbance at 290 nm (OD290). The content of Phe was measured using an Elisa kit.
Figure 2 Validation of whether the anaerobic promoter can effectively activate PAL expression (x-axis represents oxygen concentration)
As depicted in the figure2, when the oxygen concentration is 0%, the TCA concentration is approximately 0.4 mM, while the Phe concentration is above 0.5 mM and below 1 mM. When the oxygen concentration is 10%, the TCA concentration decreases slightly, and there is a slight increase in Phe concentration. Under an oxygen concentration of 20%, the TCA concentration further decreases to around 0 mM, while the Phe concentration continues to rise. This experiment provides evidence that the hypoxia promoter significantly activates the PAL enzyme, effectively enhancing the conversion of Phe to TCA.
Potential application directions
This experiment demonstrated the efficacy of pPepT-PAL in regulating PAL expression in an anaerobic environment. This finding has potential applications in future probiotic production, as it addresses the issue of nutritional depletion caused by protein expression during bacterial cultivation and storage. By extending the growth cycle and reducing shelf-life, probiotic bacteria can now effectively express relevant proteins in the anaerobic intestinal environment. This not only lowers production costs but also enhances patient efficacy. Therefore, this research holds promising development prospects.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2346
Illegal BamHI site found at 608
Illegal XhoI site found at 659
Illegal XhoI site found at 722
Illegal XhoI site found at 740
Illegal XhoI site found at 818
Illegal XhoI site found at 1019
Illegal XhoI site found at 1262
Illegal XhoI site found at 2009 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1178
Illegal NgoMIV site found at 1424
Illegal NgoMIV site found at 1592
Illegal NgoMIV site found at 1735
Illegal NgoMIV site found at 2069 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 719
Illegal BsaI site found at 1049
Illegal BsaI site found at 1055
Illegal BsaI.rc site found at 2180
Illegal SapI site found at 1009
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