Difference between revisions of "Part:BBa K4390065"
 
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'''This part is not compatible with BioBrick RFC10 assembly but is compatible with the iGEM Type IIS Part standard [[Help:Standards/Assembly/Type_IIS|which is also accepted by iGEM.]]'''
 
'''This part is not compatible with BioBrick RFC10 assembly but is compatible with the iGEM Type IIS Part standard [[Help:Standards/Assembly/Type_IIS|which is also accepted by iGEM.]]'''
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'''This is a level 1 part formed by assembly of the following level 0 parts:'''
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{| class="wikitable" style="margin:auto"
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|-
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| Promoter || [[part:BBa_J23100|J23100]]
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|-
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| RBS || [[part:BBa_B0034|B0034]]
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|-
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| N part || [[part:BBa_K4390016|K4390016]]
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|-
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| O-C part || [[part:BBa_K4390064|K4390064]]
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|-
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| Terminator || [[part:BBa_K4390001|K4390001]]
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|}
  
 
===Usage and Biology===
 
===Usage and Biology===
 
SpyTag is a 13 amino acid-long protein that spontaneously reacts with the SpyCatcher protein (12.3 kDa). Together they form an intermolecular isopeptide bond. This provides an alternative to His-tag purification techniques. Insertion of either the SpyTag or the SpyCatcher sequence into a recombinant protein will allow its purification.
 
SpyTag is a 13 amino acid-long protein that spontaneously reacts with the SpyCatcher protein (12.3 kDa). Together they form an intermolecular isopeptide bond. This provides an alternative to His-tag purification techniques. Insertion of either the SpyTag or the SpyCatcher sequence into a recombinant protein will allow its purification.
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The green fluorescent protein (GFP) is a protein that can produce bright green colour. Due to these properties, it has always been used as reporter of protein expression. Protein fused with sfGFP will turn the colony green if expressed, which can be used to identify the ability of certain genes to be expressed in other organism like E. coli.
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By adding a SpyTag to sfGFP, this can be used to see how efficient the SpyCatcher is at binding the SpyTag bound to the sfGFP (as a control, to see the binding efficiency of binding to the tagged proteins in general).
  
 
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Latest revision as of 23:54, 13 October 2022


His-tagged SpyTagged sfGFP expression construct

This part is not compatible with BioBrick RFC10 assembly but is compatible with the iGEM Type IIS Part standard which is also accepted by iGEM.

This is a level 1 part formed by assembly of the following level 0 parts:

Promoter J23100
RBS B0034
N part K4390016
O-C part K4390064
Terminator K4390001

Usage and Biology

SpyTag is a 13 amino acid-long protein that spontaneously reacts with the SpyCatcher protein (12.3 kDa). Together they form an intermolecular isopeptide bond. This provides an alternative to His-tag purification techniques. Insertion of either the SpyTag or the SpyCatcher sequence into a recombinant protein will allow its purification.

The green fluorescent protein (GFP) is a protein that can produce bright green colour. Due to these properties, it has always been used as reporter of protein expression. Protein fused with sfGFP will turn the colony green if expressed, which can be used to identify the ability of certain genes to be expressed in other organism like E. coli.

By adding a SpyTag to sfGFP, this can be used to see how efficient the SpyCatcher is at binding the SpyTag bound to the sfGFP (as a control, to see the binding efficiency of binding to the tagged proteins in general).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 11
    Illegal NheI site found at 34
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]