Part:BBa_K4509869
HORSERADISH PEROXIDASE with constitutive promoter J23106
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 and the most studied type is C. The composite part starts with the BBa_J23106 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 445
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 565
Illegal AgeI site found at 719
Illegal AgeI site found at 1022 - 1000COMPATIBLE WITH RFC[1000]
Characterization
The promoter J23119 was replaced with the promoter J23106 which was then transformed into E.coli K12 MG1655. These transformed cells were then cultured and OD was taken at a time interval of 30 mins to plot the growth curve. A graph was plotted between time and OD values of different HRP plasmids with J23119, J23116, J23106, and J23100 to determine the growth curve. From the graph, it is observed that the growth of cells with J23106 is slightly efficient when compared to cells with J23119 and J23100. This is because the cell viability is indirectly proportional to the part burden.
TMB Assay
3,3',5,5'-Tetramethylbenzidine (TMB) is the most commonly used chromogen for horseradish peroxidase (HRP). Upon oxidation, TMB forms a water-soluble blue reaction product that can be measured spectrophotometrically at 605 nm. Upon acidification by stop solution (sulphuric acid), the reaction product becomes yellow with an absorbance peak at 450 nm.
Cell Burden
J23106 promoter has a burden range of 3.5% ± 8.7%.
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