Difference between revisions of "Part:BBa K4509869"
 
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<partinfo>BBa_K4509869 short</partinfo>
 
<partinfo>BBa_K4509869 short</partinfo>
  
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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 J23116 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.
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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 [https://parts.igem.org/Part:BBa_J23106 BBa_J23106] constitutive promoter, continues with RBS [https://parts.igem.org/Part:BBa_B0034 BBa_B0034] and is followed by the Horseradish Peroxidase coding sequence [https://parts.igem.org/Part:BBa_K1291571 BBa_K1291571]. The sequence ends with a terminator and a BamH1 restriction site.
  
 
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=Characterization=
 
=Characterization=
  
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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.  
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The promoter J23119 was replaced with the promoter [https://parts.igem.org/Part:BBa_J23106 BBa_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.  
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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.
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A graph was plotted between time and OD values of different HRP plasmids with [https://parts.igem.org/Part:BBa_J23119 BBa_J23119], [https://parts.igem.org/Part:BBa_J23116 BBa_J23116], [https://parts.igem.org/Part:BBa_J23106 BBa_J23106], and [https://parts.igem.org/Part:BBa_J23100 BBa_J23100] to determine the growth curve. Absorbance was taken at 600nm. From the graph, it is observed that the growth of cells with [https://parts.igem.org/Part:BBa_J23106 BBa_J23106] are slightly more efficient when compared to cells with [https://parts.igem.org/Part:BBa_J23119 BBa_J23119] and [https://parts.igem.org/Part:BBa_J23100 BBa_J23100]. This is because the cell viability is indirectly proportional to the part burden.
  
 
https://static.igem.wiki/teams/4509/wiki/registry/hrp-106-1.png
 
https://static.igem.wiki/teams/4509/wiki/registry/hrp-106-1.png
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https://static.igem.wiki/teams/4509/wiki/registry/hrp-expt-1.jpg
 
https://static.igem.wiki/teams/4509/wiki/registry/hrp-expt-1.jpg
 
   
 
   
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Cell Burden:
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==Cell Burden==
 
J23106 promoter has a burden range of 3.5% ± 8.7%.
 
J23106 promoter has a burden range of 3.5% ± 8.7%.

Latest revision as of 06:07, 13 October 2022


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


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 was replaced with the promoter BBa_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 BBa_J23119, BBa_J23116, BBa_J23106, and BBa_J23100 to determine the growth curve. Absorbance was taken at 600nm. From the graph, it is observed that the growth of cells with BBa_J23106 are slightly more efficient when compared to cells with BBa_J23119 and BBa_J23100. This is because the cell viability is indirectly proportional to the part burden.

hrp-106-1.png


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.

hrp-expt-1.jpg

Cell Burden

J23106 promoter has a burden range of 3.5% ± 8.7%.