Part:BBa_K4509569

HORSERADISH PEROXIDASE with constitutive promoter J23100

The enzyme horseradish peroxidase, found in the roots of horseradish catalyzes the oxidation of various organic substrates by hydrogen peroxide. It is a metalloenzyme with many isoforms; the most studied type is C. The composite part starts with the BBa_J23100 constitutive promoter, continues with RBS BBa_B0034 and is followed by the Horseradish Peroxidase coding sequence BBa_K1291571. The sequence ends with a terminator and a BamH1 restriction site.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 445
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 565
  Illegal AgeI site found at 719
  Illegal AgeI site found at 1022
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

The promoter J23119 is replaced with the promoter J23100 to increase the expression of the enzyme. Absorbance was taken at 600 nm.

The increased expression of HRP was reviewed by performing the TMB assay.

TMB assay

3,3',5,5'-Tetramethylbenzidine (TMB) is the most commonly used chromogen for horseradish peroxidase (HRP). TMB produces a yellow-orange colour with Horseradish Peroxidase and a stop solution (Sulfuric acid), that absorbs at 450nm.

Cell Burden

J23116 has a burden range of -0.6 ± 11.0%