Part:BBa_K4509420
HORSERADISH PEROXIDASE with constitutive promoter J23116
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_J23116 constitutive promoter, continues with RBS 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 BBa_J23119 was replaced with BBa_J23116 and transformed into E.coli K12 MG1655. The Transformed colonies were cultured and OD was taken every 30 mins to plot the growth curve of the cells.
The growth curve for the HRP plasmid with J23119, J23116, J23106 and J23100 is plotted in this graph. The burden on cells is inversely proportional to cell viability. Observing the growth curve it may be observed that the growth of J23116 is much more efficient than J23119 in growth regulation. Thus the part has improved cell growth.The promoters BBa_J23100 and BBa_J23106 are used for comparison studies.
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
BBa_J23116 has a burden range of -4.5 ± 2.8%
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