Difference between revisions of "Part:BBa K5322011"

 
 
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===Usage and Biology===
 
  
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<span class='h3bb'>Sequence and Features</span>
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==Usage and Biology==
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The plasmid pET-29(a)-pT7-lac operator-(SOD-1+His)-T7 enables high-level expression of SOD-1 in <i>Escherichia coli</i> 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.
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==Construction of the plasmid==
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Our team designed the plasmid pET-29(a)-pT7-lac operator-(SOD-1+His)-T7, and the plasmid map is illustrated below.
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<img src="https://static.igem.wiki/teams/5322/t-imgs/3-pet-29-a-pt7-lac-operator-sod-1-his-t7.png" alt="pET-29(a)-pT7-lac operator-(SOD-1+His)-T7 " width="600">
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<p align="center"><b>Figure 2-1</b>  Plasmid pET-29(a)-pT7-lac operator-(SOD-1+His)-T7</p>
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<img src="https://static.igem.wiki/teams/5322/wet-lab/sod-jun-pcr.png" alt="Colony PCR gel electrophoresis of pET-29(a)-pT7-lac operator-(SOD-1+His)-T7" width="600">
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<p align="center"><b>Figure 2-2</b>  Colony PCR gel electrophoresis of plasmid pET-29(a)-pT7-lac operator-(SOD-1+His)-T7</p>
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<img src="https://static.igem.wiki/teams/5322/wet-lab/pet-29-a-lac-operator-sod-1-his-t72-map.png" alt="cexu" width="600">
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<p align="center"><b>Figure 2-3</b>  plasmid pET-29(a)-pT7-lac operator-(SOD-1+His)-T7 sequencing result</p>
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==Functional Expression==
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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).
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<img src="https://static.igem.wiki/teams/5322/wet-lab/part1meihuogongshi.png" alt="Inhibition rate and enzyme activity calculation formula" width="600">
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<p align="center"><b>Figure 3-1</b> Inhibition rate and enzyme activity calculation formula
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The original data is shown in the table below. The calculated inhibition rate for the inducible SOD enzyme is approximately 46%, while the inhibition rate for DH5α is around 38%. This indicates successful expression of the SOD protein.
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<img src="https://static.igem.wiki/teams/5322/wet-lab/part1meihuojieguo.png" alt="The original data of the microplate reader and the calculation results of the inhibition rate" width="600">
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<p align="center"><b>Figure 3-2</b> The original data of the microplate reader and the calculation results of the inhibition rate
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The inhibition percentage for DH5α is 38.4%, with an enzyme activity of 0.6245 U. The inhibition percentage for SOD is 46.1%, with an enzyme activity of 0.8537 U.
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==Sequence and Features==
 
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==Functional Parameters==
 
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Latest revision as of 11:56, 2 October 2024

pET-29(a)-pT7-lac operator-(SOD-1+His)-T7


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.

pET-29(a)-pT7-lac operator-(SOD-1+His)-T7

Figure 2-1 Plasmid pET-29(a)-pT7-lac operator-(SOD-1+His)-T7

Colony PCR gel electrophoresis of 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

cexu

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).

Inhibition rate and enzyme activity calculation formula

Figure 3-1 Inhibition rate and enzyme activity calculation formula

The original data is shown in the table below. The calculated inhibition rate for the inducible SOD enzyme is approximately 46%, while the inhibition rate for DH5α is around 38%. This indicates successful expression of the SOD protein.
The original data of the microplate reader and the calculation results of the inhibition rate

Figure 3-2 The original data of the microplate reader and the calculation results of the inhibition rate

The inhibition percentage for DH5α is 38.4%, with an enzyme activity of 0.6245 U. The inhibition percentage for SOD is 46.1%, with an enzyme activity of 0.8537 U.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 577
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]