Difference between revisions of "Part:BBa K3037008"

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|'''Backbone'''
 
|'''Backbone'''
 
|[https://parts.igem.org/Help:2019_DNA_Distribution pSB1C3<br>]
 
|[https://parts.igem.org/Help:2019_DNA_Distribution pSB1C3<br>]
 +
|-
 +
|'''Experimental Backbone'''
 +
|[https://parts.igem.org/Part:BBa_K3037000 pOCC97<br>]
 
|-
 
|-
 
|'''Submitted by'''
 
|'''Submitted by'''
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== Overview ==
 
== Overview ==
  
−
The TU Dresden 2019 team design this fusion protein in order to increase the expression of HRP. In order to make it the RFC 25 standard was used [https://2019.igem.org/Team:TU_Dresden/Parts (more information).]
+
The TU Dresden 2019 team designed this fusion protein in order to increase the expression of HRP. For creating this fusion protein, the RFC 25 standard was used [https://2019.igem.org/Team:TU_Dresden/Parts (more information).]
  
−
HRP was inserted into the pSB1C3 vector for transformation and expressed in <span style="font-style: italic;">Escherichia coli</span>.
+
HRP was inserted into the pSB1C3 vector for transformation and expressed with pOCC97 [https://parts.igem.org/Part:BBa_K3037000 (BBa_K3037000)] in <span style="font-style: italic;">Escherichia coli</span>.
  
−
The HRP was made from [https://parts.igem.org/Part:BBa_K1800002 BBa_K1800002] but adapted to the RFC 25 standard
+
The HRP was made from [https://parts.igem.org/Part:BBa_K1800002 BBa_K1800002]and adapted to the [[Help:Assembly_standard_25|Freiburg RFC25 standard.]]
 +
 
 +
=== Biology ===
 +
 
 +
The metalloenzyme horseradishperoxidase (HRP) is widely used in many biochemical and immunological applications. This enzyme on its own does not give any visual readout, but however, upon addition of a suitable substrate, HRP oxidizes this substrate and yields a colour change and this can be spectrophotometrically analysed. One such common example for chromogenic substrate is TMB (3,3',5,5'-Tetramethylbenzidine) which is oxidized by HRP. Additionally, an advantage of using HRP includes its stability and small size, which results in the reduced interference during the conjugation reactions such as for secondary antibody-HRP conjugation. Moreover, it is economical in comparison with other alternative enzymes like the alkaline phosphatase.
 +
 
 +
MBP is a well-established, reliable protein-tag that is meant to increase the solubility of the proteins it is fused to. Therefore it limits the risk of accumulation of over expressed recombinant protein in inclusion bodies and increases the total protein yield. It can also improve expression of difficult enzymes like Cas9. It can further be used to purify proteins via affinity chromatography in amylose resin.[1]
 +
 
 +
You can find more information in the pages of the individual parts of this composite: [https://parts.igem.org/Part:BBa_K3037007 BBa_K3037007] [https://parts.igem.org/Part:BBa_K3037001 BBa_K3037001]
  
 
== Characterization ==
 
== Characterization ==
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=== Outline ===
 
=== Outline ===
  
−
<b>1)</b> Expression in pOCC97, growing curve [https://parts.igem.org/Part:BBa_K3037000 (BBa_3037000)]
+
<b>1)</b> Expression in pOCC97, growth curve [https://parts.igem.org/Part:BBa_K3037000 (BBa_3037000)]
  
 
<b>2)</b> Expression in pOCC97, SDS-PAGE [https://parts.igem.org/Part:BBa_K3037000 (BBa_3037000)]
 
<b>2)</b> Expression in pOCC97, SDS-PAGE [https://parts.igem.org/Part:BBa_K3037000 (BBa_3037000)]
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=== Experiments in Detail ===
 
=== Experiments in Detail ===
  
−
==== Expression in pOCC97, growing curve [https://parts.igem.org/Part:BBa_K3037000 (BBa_3037000)] ====
+
==== 1) Expression in pOCC97, growth curve [https://parts.igem.org/Part:BBa_K3037000 (BBa_3037000)] ====
  
 
<gallery mode="packed", caption="Growing curve", widths=400px, heights=200px>
 
<gallery mode="packed", caption="Growing curve", widths=400px, heights=200px>
−
File:T--TU_Dresden--Expression_MBP_HRP_in_pOCC97.png|<span style="color:#0000ff"> ''Growing curve''  </span> (Check that the growing curve of the bacteria is not affected by the expression of the protein)
+
File:T--TU_Dresden--Expression_MBP_HRP_in_pOCC97.png|<span style="color:#0000ff"> ''Growth curve''  </span> (Normal bacterial Growth curve is seen during the expression of MBP-HRP)
 
</gallery>
 
</gallery>
  
−
==== Expression in pOCC97, SDS-PAGE [https://parts.igem.org/Part:BBa_K3037000 (BBa_3037000)] ====
+
==== 2) Expression in pOCC97, SDS-PAGE [https://parts.igem.org/Part:BBa_K3037000 (BBa_3037000)] ====
 +
 
 +
Expression of MBP-HRP seen in the graph (right)
  
 
<gallery mode="packed", caption="Assay to determine the protein expression", widths=400px, heights=200px>
 
<gallery mode="packed", caption="Assay to determine the protein expression", widths=400px, heights=200px>
−
File:T--TU_Dresden--Comparison_expression_HRP_full_construct_BBa_K3037000.png|<span style="color:#0000ff"> ''SDS-PAGE''  </span> (Comparison of expression of different proteins in the vector before and after optimization)
+
File:T--TU_Dresden--Comparison_expression_HRP_full_construct_BBa_K3037000.png|<span style="color:#0000ff"> ''SDS-PAGE''  </span> (Comparison of expression of full construct (BBa_K3037003) and MBP-HRP (BBa_K3037008) in the pOCC97 (BBa_K3037000) before and after optimization)
 
</gallery>
 
</gallery>
  
 
== Sequence ==
 
== Sequence ==
  
−
NOTE: For some reason the specified scar when aploaded the sequence was the one of the RFC25 standard but the registry shows the one of the RFC23
+
NOTE: Please be aware, that by combining all the basic parts used for this composite part, the registry automatically inserted a RFC23 scar. However, the design and our cloning strategy is based on RFC25.
  
 
<partinfo>BBa_K3037008 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3037008 SequenceAndFeatures</partinfo>
  
 +
== Reference ==
 +
 +
[1] https://www.genscript.com/bacterial-soluble-protein-expression-MBP-tag.html
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 00:37, 22 October 2019

MBP+HRP

MBP+HRP
Function Expression
Use in Escherichia coli
RFC standard Freiburg RFC25 standard
Backbone pSB1C3
Experimental Backbone pOCC97
Submitted by Team: TU_Dresden 2019



Overview

The TU Dresden 2019 team designed this fusion protein in order to increase the expression of HRP. For creating this fusion protein, the RFC 25 standard was used (more information).

HRP was inserted into the pSB1C3 vector for transformation and expressed with pOCC97 (BBa_K3037000) in Escherichia coli.

The HRP was made from BBa_K1800002and adapted to the Freiburg RFC25 standard.

Biology

The metalloenzyme horseradishperoxidase (HRP) is widely used in many biochemical and immunological applications. This enzyme on its own does not give any visual readout, but however, upon addition of a suitable substrate, HRP oxidizes this substrate and yields a colour change and this can be spectrophotometrically analysed. One such common example for chromogenic substrate is TMB (3,3',5,5'-Tetramethylbenzidine) which is oxidized by HRP. Additionally, an advantage of using HRP includes its stability and small size, which results in the reduced interference during the conjugation reactions such as for secondary antibody-HRP conjugation. Moreover, it is economical in comparison with other alternative enzymes like the alkaline phosphatase.

MBP is a well-established, reliable protein-tag that is meant to increase the solubility of the proteins it is fused to. Therefore it limits the risk of accumulation of over expressed recombinant protein in inclusion bodies and increases the total protein yield. It can also improve expression of difficult enzymes like Cas9. It can further be used to purify proteins via affinity chromatography in amylose resin.[1]

You can find more information in the pages of the individual parts of this composite: BBa_K3037007 BBa_K3037001

Characterization

Outline

1) Expression in pOCC97, growth curve (BBa_3037000)

2) Expression in pOCC97, SDS-PAGE (BBa_3037000)

Experiments in Detail

1) Expression in pOCC97, growth curve (BBa_3037000)

  • Growth curve (Normal bacterial Growth curve is seen during the expression of MBP-HRP)

2) Expression in pOCC97, SDS-PAGE (BBa_3037000)

Expression of MBP-HRP seen in the graph (right)

  • SDS-PAGE (Comparison of expression of full construct (BBa_K3037003) and MBP-HRP (BBa_K3037008) in the pOCC97 (BBa_K3037000) before and after optimization)

Sequence

NOTE: Please be aware, that by combining all the basic parts used for this composite part, the registry automatically inserted a RFC23 scar. However, the design and our cloning strategy is based on RFC25.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1357
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 381
    Illegal BglII site found at 1599
    Illegal XhoI site found at 1683
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 79

Reference

[1] https://www.genscript.com/bacterial-soluble-protein-expression-MBP-tag.html