Difference between revisions of "Part:BBa K1088052:Design"

 
(→‎Design Notes)
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1) Design primers for amplification of the N-terminal protein/domain that includes BamHI-site
 
1) Design primers for amplification of the N-terminal protein/domain that includes BamHI-site
 +
 
a) Forward primer: 5'-cgctTCTAGAgNNN...NNN-3' - includes XbaI-site and sequence complementary to DNA sequence of N-terminal protein/domain
 
a) Forward primer: 5'-cgctTCTAGAgNNN...NNN-3' - includes XbaI-site and sequence complementary to DNA sequence of N-terminal protein/domain
 +
 
b) Reverse primer: 5'-atatGGATCCNNN...NNN-3' - includes BamHI-site and sequence complementary to DNA sequence of N-terminal protein/domain (OBS! do NOT include stop-codons of coding sequences)
 
b) Reverse primer: 5'-atatGGATCCNNN...NNN-3' - includes BamHI-site and sequence complementary to DNA sequence of N-terminal protein/domain (OBS! do NOT include stop-codons of coding sequences)
 +
 
2) PCR amplify BioBrick with designed primer
 
2) PCR amplify BioBrick with designed primer
 +
 
3) digest pSB1C3-Linker:GFP and PCR product with XbaI and BamHI
 
3) digest pSB1C3-Linker:GFP and PCR product with XbaI and BamHI
−
4) ligate the digested pSB1C3-Linker:GFP and PCR product to create a brick with your protein/domain fused with GFP
 
−
 
  
 +
4) ligate the digested pSB1C3-Linker:GFP and PCR product to create a brick with your protein/domain fused with GFP
  
 
===Source===
 
===Source===

Revision as of 22:10, 16 March 2015


GFP reporter with flexible linker at N-terminus for creation of GFP fusions


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 674


Design Notes

The linker was added according to step 1-4 as specified on the "Design" page of the flexible linker

Using standard RFC[10] assembly of two parts creates scarsites (tactag). Transcription of the scarsite encodes: "uac uag" of which uag is a stop codon that would render the linker useless. Thus, standard assembly can not be employed for fusing proteins/domains.

1) Design primers for amplification of the N-terminal protein/domain that includes BamHI-site

a) Forward primer: 5'-cgctTCTAGAgNNN...NNN-3' - includes XbaI-site and sequence complementary to DNA sequence of N-terminal protein/domain

b) Reverse primer: 5'-atatGGATCCNNN...NNN-3' - includes BamHI-site and sequence complementary to DNA sequence of N-terminal protein/domain (OBS! do NOT include stop-codons of coding sequences)

2) PCR amplify BioBrick with designed primer

3) digest pSB1C3-Linker:GFP and PCR product with XbaI and BamHI

4) ligate the digested pSB1C3-Linker:GFP and PCR product to create a brick with your protein/domain fused with GFP

Source

BBa_I13401 and BBa_K1088051

References