Difference between revisions of "Part:BBa K259004"

 
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<partinfo>BBa_K259004 short</partinfo>
 
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===Assembly Instructions===
 
===Assembly Instructions===
  
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*Create an Assembly Standard 10 fusion as below ;  
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*Create an Assembly Standard 10 fusion on a pSB2k3 plasmid backbone as shown below ;  
  
 
  **Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part**
 
  **Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part**
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[[Image:BCCS_Bioscaffold_Reaction_Explanation_CspCI.jpg|thumb|500px|center|The Reaction and the product. ]]
 
[[Image:BCCS_Bioscaffold_Reaction_Explanation_CspCI.jpg|thumb|500px|center|The Reaction and the product. ]]
  
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====N.B.====
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CspCI cuts with a 1 b.p discrepancy depending on the sequence. However this should not affect the restriction/ligation process as this has been taken into account and incorporated into the design. Even with the discrepancy involved the restriction/ligation process will still give the same product and in-frame fusion.
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The other restriction variants of CspCI have also been taken into account and relevant BioScaffolds are being produced to be tested. (see parts <partinfo>BBa_K259010</partinfo> <partinfo>BBa_K259009</partinfo>)
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Also see part <partinfo>BBa_K259002</partinfo>; the BpuEI - BseRI Bioscaffold.
  
  

Latest revision as of 21:58, 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 plasmid backbone 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 CspCI to remove downstream scar.
  • Ligate to get the final product;
 **Upstream Protein - Tyr-Gly-Ser-Gly- Downstream Protein**
The Reaction and the product.

N.B.

CspCI cuts with a 1 b.p discrepancy depending on the sequence. However this should not affect the restriction/ligation process as this has been taken into account and incorporated into the design. Even with the discrepancy involved the restriction/ligation process will still give the same product and in-frame fusion.

The other restriction variants of CspCI have also been taken into account and relevant BioScaffolds are being produced to be tested. (see parts BBa_K259010 BBa_K259009)

Also see part BBa_K259002; the BpuEI - BseRI Bioscaffold.


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]