Part:BBa_K3838345

YH-SOD

The overproduction of Superoxide dismutase (SOD) and reactive oxygen species (ROS) is considered to be one of the pathogenesis of IBD. The use of SOD can significantly reduce the peroxidation reaction in the inflamed colon. Studies have shown that the decreased expression of Cu/Zn-SOD has been observed in IBD patients. SOD can not be directly treated, so carrier transport is needed. Therefore, we selected for heteroexpression of superoxide dismutase (SOD) gene from Lactobacillus casei Lc18.We used the P170 promoter to express our protein and constructed a plasmid containing RcfB gene to initiate P170 activity.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 9
  Illegal EcoRI site found at 534
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 9
  Illegal EcoRI site found at 534
  Illegal NheI site found at 147
  Illegal NheI site found at 170
  Illegal NotI site found at 15
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 9
  Illegal EcoRI site found at 534
  Illegal BglII site found at 577
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 9
  Illegal EcoRI site found at 534
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 9
  Illegal EcoRI site found at 534
  Illegal XbaI site found at 24
  Illegal NgoMIV site found at 738
  
• 1000
  COMPATIBLE WITH RFC[1000]

Data:SZU-China 2021 TEAM

We placed the engineered bacteria in media with different pH and cultured them to the same concentration, and then tested their intracellular superoxide dismutase activity.We can verify the difference in the activity of the SOD enzyme expressed between the constitutive and acid-inducible expression systems through this experiment and verify the difference in the expression efficiency of the double-plasmid acid-inducible expression system itself under different pH environments.

1.The DNA level

We selected the DYHSG-RR double plasmid system for verification, transformed PUCYH and PET-RR into DH5α, and extracted the plasmid from the plasmid-transformed DYHSG-RR, as shown in Figure 1. The sample band is between 5000-7500bp, which is in line with the purpose. The band size is around 6700bp. The size of the PCR band is about 1000bp, and the restriction band is about 2300bp, both of which are in line with the current band size.

Fig.1 The transformed DYHSG-RR plasmid, PCR, restriction gel electrophoresis diagram.

2.Functional representation

Fig.2 SOD enzyme activity of constitutive promoter and acid-induced promoter expression system under different PH conditions.