Difference between revisions of "Part:BBa K259002"

 
 
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__NOTOC__
 
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<partinfo>BBa_K259002 short</partinfo>
 
<partinfo>BBa_K259002 short</partinfo>
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This part will help users interested in protein-fusion. The usage of the part will allow in frame assembly of 2(+) proteins by:
 
This part will help users interested in protein-fusion. The usage of the part will allow in frame assembly of 2(+) proteins by:
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*Removing the stop codons from the upstream protein.
 
*Removing the stop codons from the upstream protein.
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*Acting as a translational buffer if ligation is incorrect.
 
*Acting as a translational buffer if ligation is incorrect.
  
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===Assembly Instructions====
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===Assembly Instructions===
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*Create an Assembly Standard 10 fusion on a pSB2K3 backbone plasmid as shown below ;
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  **Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part**
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[[Image:BCCS_Assembly_of_Scaffold.jpg|thumb|500px|center|The Product of Assembly between 2 proteins and the Bioscaffold Linker. ]]
  
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*Create an Assembly Standard 10 fusion giving ;
 
  
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**Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part
 
  
 
*Use the BioScaffold Specific Enzymes  
 
*Use the BioScaffold Specific Enzymes  
  
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**Restrict with BpuEI to remove upstream scar (created between Upstream Protein - Bioscaffold) and stop codons from upstream protein part.
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(i)Restrict with BpuEI to remove upstream scar (created between Upstream Protein - Bioscaffold) and stop codons from upstream protein part.<br/>
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**Ligate and you will get the intermediate product;
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(ii)Ligate and you will get the intermediate product;
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  **Upstream Protein (w/out Stop codons) - Tyr - BioScaffold Linker - Downstream Protein**
  
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Upstream Protein (w/out Stop codons) - Tyr - BioScaffold Linker - Downstream Protein
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*Restrict with BseRI to remove downstream scar.
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*Ligate to get the final product;
  
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**Restrict with BseRI to remove downstream scar.
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  **Upstream Protein - Tyr-Gly-Ser-Gly- Downstream Protein**
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**Ligate to get the final product;
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Upstream Protein - Tyr-Gly-Ser-Gly- Downstream Protein
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[[Image:BCCS_Bioscaffold_Reaction_Explanation.jpg|thumb|500px|center|The Reaction and the product. ]]
  
  

Latest revision as of 22:00, 14 October 2009

BioScaffold Linker - Removes Stop Codons & scars & replaces with a Gly-Ser linker

This part is part of a family of Bioscaffold - Linkers.

This part will help users interested in protein-fusion. The usage of the part will allow in frame assembly of 2(+) proteins by:

  • Removing the stop codons from the upstream protein.
  • Removing the scar created by the Assembly Standard 10 (RFC10) construction of 2(+) protein coding sequences.
  • Maintaining the start codon of the downstream protein.
  • Adding a flexible (Gly-Ser-Gly) linker between the two protein coding sequences.
  • Acting as a translational buffer if ligation is incorrect.

Assembly Instructions

  • Create an Assembly Standard 10 fusion on a pSB2K3 backbone plasmid as shown below ; 
 **Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part**


The Product of Assembly between 2 proteins and the Bioscaffold Linker.


  • Use the BioScaffold Specific Enzymes

(i)Restrict with BpuEI to remove upstream scar (created between Upstream Protein - Bioscaffold) and stop codons from upstream protein part.
(ii)Ligate and you will get the intermediate product; 

 **Upstream Protein (w/out Stop codons) - Tyr - BioScaffold Linker - Downstream Protein**
  • Restrict with BseRI to remove downstream scar.
  • Ligate to get the final product;
  **Upstream Protein - Tyr-Gly-Ser-Gly- Downstream Protein**
The Reaction and the product.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]