Part:BBa_K3376012

ldhp-AQP-RBS-SpxB-Tr-tpxp-KatG-Tr/pSB1C3

The device contains pyruvate oxidase (SpxB) gene from S. sanguinis to generate H2O2, aquaporin (AQP) gene from S. cristatus to facilitate H2O2 transport, and catalase (KatG) gene from E. coli to revive from oxidative stress by decomposing H2O2 to oxygen and water. The SpxB gene is driven under the constitutive and strong promoter of lactate dehydrogenase (ldhp) of S. mutans. The KatG gene is regulated under the promoter of thiol peroxidase (tpxp) of S. mutans, which is induced by H2O2. Therefore, catalase is expressed in the presence of H2O2 and detoxify the H2O2. Furthermore, the endogenous gene of lactate dehydrogenase is broken to eliminate the production of lactic acid.

THE PROMOTERS

First of all, two kinds of promoters from S. mutans were demonstrated. Lactate dehydrogenase promoter (ldhp) is a strong and constitutive promoter, and thiol peroxidase promoter (tpxp) is induced by the presence of H2O2 in the response to an environmental stress. The gene expression in E. coli Nissle was measured with GFP reporter by the fluorescence intensity at Ex/Em = 483/513 nm. The data showed a strong activity of ldhp and a limited expression of tpxp.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3042
  Illegal BglII site found at 3977
  Illegal BglII site found at 4104
  Illegal BamHI site found at 3894
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1352
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1271
  Illegal SapI site found at 1949
  Illegal SapI.rc site found at 2405