Part:BBa_K5322011
pET-29(a)-pT7-lac operator-(SOD-1+His)-T7
Contents
Usage and Biology
The plasmid pET-29(a)-pT7-lac operator-(SOD-1+His)-T7 enables high-level expression of SOD-1 in Escherichia coli using the pET29a vector. This system controls SOD-1 expression with the weakly inducible lac promoter. The ribosome binding site (RBS) ensures efficient translation of the mRNA, while the T7 terminator provides a clean and efficient end for transcription. This system is designed for the efficient expression of SOD under conditions unaffected by environmental factors, thereby enhancing its antioxidant activity.
Construction of the plasmid
Our team designed the plasmid pET-29(a)-pT7-lac operator-(SOD-1+His)-T7, and the plasmid map is illustrated below.
Figure 2-1 Plasmid pET-29(a)-pT7-lac operator-(SOD-1+His)-T7
Figure 2-2 Colony PCR gel electrophoresis of plasmid pET-29(a)-pT7-lac operator-(SOD-1+His)-T7
Figure 2-3 plasmid pET-29(a)-pT7-lac operator-(SOD-1+His)-T7 sequencing result
Functional Expression
2 mL of overnight induced bacterial culture was used to extract proteins with the Biotech™ bacterial active protein extraction reagent. The SOD enzyme activity was measured using the Beyotime™ SOD enzyme activity assay kit, and the absorbance at 450 nm (A450) was determined using a microplate reader.Definition of SOD Enzyme Activity Units: In the aforementioned xanthine oxidase coupled reaction system, when the inhibition percentage reaches 50%, the enzyme activity in the reaction system is defined as one enzyme activity unit (unit).
Figure 3-1 Inhibition rate and enzyme activity calculation formula
Figure 3-2 The original data of the microplate reader and the calculation results of the inhibition rate
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 577
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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