Difference between revisions of "Part:BBa K3037000"
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<partinfo>BBa_K3037000 short</partinfo>
 
<partinfo>BBa_K3037000 short</partinfo>
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{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
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! colspan="2" style="background:#FFBF00;"|CMV:Ha-NLS-dCas9-Linker-VP16-NLS:BGH
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|-
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|'''Function'''
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|Expression
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|-
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|'''Use in'''
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|Prokaryotic cells
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|-
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|'''RFC standard'''
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|[https://parts.igem.org/Help:Standards/Assembly/RFC25], RFC 25 compatible
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|-
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|'''Backbone'''
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|pSB1C3<br>
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|-
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|'''Submitted by'''
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|[http://2019.igem.org/Team:TU-Dresden]
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|}
  
 
===Horseradish Peroxidase (HRP)===
 
===Horseradish Peroxidase (HRP)===

Revision as of 16:23, 2 October 2019

pOCC97 plasmid backbone for expression (optimized)

CMV:Ha-NLS-dCas9-Linker-VP16-NLS:BGH
Function Expression
Use in Prokaryotic cells
RFC standard [1], RFC 25 compatible
Backbone pSB1C3
Submitted by [http://2019.igem.org/Team:TU-Dresden]

Horseradish Peroxidase (HRP)

In our lab, we designed an HRP part in accordance to RFC 25.

HRP in our experiment will serve as, in combination with Cas9, a color proof reading for detection of specific DNA sequences.

HRP will be inserted into the pOCC97 vector for transformation and expression in E. coli .

Horseradish peroxidase (HRP) is an extensively studied and one of the most important enzymes obtained from plants. The reason because of the interest in the enzyme is because it has a lot of commercial and practical applications (Veitch, N. C. 2004).

HRP is a peroxidase which oxidizes different substrates (e.g. aromatic phenols) using commonly H2O2, as initial electron acceptors. Physiologically, HRP is involved in many reactions, such as the regulation of the levels of H2O2 and the crosslinking of phenolic molecules. Because of its large amount of different functions, HRP has many isoenzymes. (Krainer, F. W. et al 2014)

The oxidative properties of the HRP allow them to produce color changes in specific substrates. Therefore, in the industry HRP has many applications, especially biosensors and diagnostic kits (e.g., immunoassays, ELISA, EMSA…). (Krainer, F. W. et al 2014)

Also, HRP has many characteristics that make it suitable for therapeutic use as it is stable at 37 °C, shows high activity at physiological pH and can be conjugated to antibodies or lectins. (Humer, D., & Spadiut, O. 2019) In addition, site-directed mutagenesis and directed evolution techniques are beeing used to improve the properties of the HRP. (Veitch, N. C. 2004). The commercially available HRP is extracted from Armoracia rusticana roots. However, Armoracia rustica require long cultivation times and produce low yields which make the classical production method quite inefficient. (Humer, D., & Spadiut, O. 2019)

As a consequence, many studies have addressed Saccharomyces cerevisiae or Pichia pastoris as host organisms. However, these organisms have problems to produce glycoproteins with disulphide bridges. In contrast, E. coli has shown to have no obstacles due to hyper-glycosylation and it is also a suitable organism because of its cheap and easy cultivation. (Humer, D., & Spadiut, O. 2019)


Sequence

pOCC97 plasmid backbone for expression (optimized)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 5283
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 5289
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 5283
    Illegal BglII site found at 5169
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 5283
    Illegal suffix found at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 5283
    Plasmid lacks a suffix.
    Illegal XbaI site found at 5298
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NgoMIV site found at 342
    Illegal NgoMIV site found at 3389
    Illegal NgoMIV site found at 3549
    Illegal NgoMIV site found at 5137
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal SapI.rc site found at 2468


Design Notes

Followed by the biobrick prefix there is an RBS with an NgoIV restriction site, which is used for the RFC 25 standard when trying to make fusion proteins.


References

Veitch, N. C. (2004). Horseradish peroxidase: a modern view of a classic enzyme. Phytochemistry, 65(3), 249-259.

Krainer, F. W., Pletzenauer, R., Rossetti, L., Herwig, C., Glieder, A., & Spadiut, O. (2014). Purification and basic biochemical characterization of 19 recombinant plant peroxidase isoenzymes produced in Pichia pastoris. Protein expression and purification, 95, 104-112.

Humer, D., & Spadiut, O. (2019). Improving the Performance of Horseradish Peroxidase by Site-Directed Mutagenesis. International journal of molecular sciences, 20(4), 916.