Difference between revisions of "Part:BBa K3037000"
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<partinfo>BBa_K3037000 short</partinfo>
 
<partinfo>BBa_K3037000 short</partinfo>
 
{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
 
{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
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! colspan="2" style="background:#FFBF00;"|Horseradish Peroxidase
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! colspan="2" style="background:#FFBF00;"|pOCC97
 
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|-
 
|'''Function'''
 
|'''Function'''
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|-
 
|-
 
|'''Use in'''
 
|'''Use in'''
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|Escherichia coli
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|Escherichia coli T7
 
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|-
 
|'''RFC standard'''
 
|'''RFC standard'''
 
|RFC 25 compatible  
 
|RFC 25 compatible  
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|-
 
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|'''Backbone'''
 
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|pSB1C3<br>
 
 
|-
 
|-
 
|'''Submitted by'''
 
|'''Submitted by'''
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=== Overview ===
 
=== Overview ===
  
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The TU Dresden 2019 team design this biobrick in order to make a fusion protein with dCas9 in accordance to the RFC 25 standard. [https://2019.igem.org/Team:TU_Dresden (more information)]
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The TU Dresden 2019 team design this biobrick in order to express its fusion protein [https://2019.igem.org/Team:TU_Dresden (more information)]
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In the experiment, HRP was used as a color proof reading for detection of specific DNA sequences.
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HRP was inserted into the pSB1C3 vector for transformation and expressed in E. coli .
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===Biology===
 
===Biology===
  
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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).
 
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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)
 
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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)
 
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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).
 
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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)
 
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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)
 
  
  
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This Biobrick was used by the TU Dresden iGEM team 2019 using the RFC 25 standard
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This BioBrick was site mutated to take out a XbaI restriction site
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=== References ===
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<small>
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Veitch, N. C. (2004). Horseradish peroxidase: a modern view of a classic enzyme. Phytochemistry, 65(3), 249-259.
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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.
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−
 
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Humer, D., & Spadiut, O. (2019). Improving the Performance of Horseradish Peroxidase by Site-Directed Mutagenesis. International journal of molecular sciences, 20(4), 916.
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Revision as of 18:28, 12 October 2019

pOCC97 plasmid backbone for expression (optimized)

pOCC97
Function Expression
Use in Escherichia coli T7
RFC standard RFC 25 compatible
Submitted by Team:TU_Dresden 2019[1]

Overview

The TU Dresden 2019 team design this biobrick in order to express its fusion protein (more information)


Biology

Sequence


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

This BioBrick was site mutated to take out a XbaI restriction site