Difference between revisions of "Part:BBa K4509569"
(→‎Characterization)
Line 24: Line 24:
 
https://static.igem.wiki/teams/4509/wiki/registry/hrp-100-1.png
 
https://static.igem.wiki/teams/4509/wiki/registry/hrp-100-1.png
  
−
Observing the growth curve it may be observed that the growth of J23100 is much more efficient than J23119 in protein production.
+
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.
 +
 
 +
https://static.igem.wiki/teams/4509/wiki/registry/hrp-expt-1.jpg
 +
 
 +
==Cell Burden==
 +
J23116 has a burden range of -0.6 ± 11.0%

Revision as of 04:24, 12 October 2022


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 J23100 constitutive promoter, continues with RBS and is followed by the Horseradish Peroxidase coding sequence. 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.

hrp-100-1.png

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.

hrp-expt-1.jpg

Cell Burden

J23116 has a burden range of -0.6 ± 11.0%