DNA
BioScaffol

Part:BBa_K259002

Designed by: Petros Mina   Group: iGEM09_BCCS-Bristol   (2009-08-01)

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]


[edit]
Categories
//chassis/multihost
//direction/forward
//dna/bioscaffold
//dna/restrictionsite
//dna/spacer
Parameters
None