Difference between revisions of "Part:BBa K1489000:Design"

 
 
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<partinfo>BBa_K1489000 short</partinfo>
 
<partinfo>BBa_K1489000 short</partinfo>
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===Design Notes===
 
===Design Notes===
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EcoRI site in middle of Bovine Galectin gene was removed, codons were optimized for expression in E. coli.
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UMaryland 2014 is interested in anchoring a galectin in the outer membrane of E. coli in an effort to bind to carbohydrate chains on an oyster pathogen. While an oyster galectin exists (BBa_K1489005), it is much larger than mammalian galectins of similar function. Bovine galectin 1 (BtGal1) was chosen to serve as a model galectin to anchor in the outer membrane due to its smaller size and its simpler quaternary structure; Bovine galectin binds as a dimer while the oyster galectin, CvGal1, is believed to bind as a tetramer.
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The pBAD promoter was chosen for protein expression due to previous experience with arabinose induction and a belief in tighter expression than the pLac promoter. An additional terminator was added at the end of the DNA sequence to increase the efficiency of transcription termination.
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While designing the biobrick sequence we discovered an internal EcoRI site in the middle of the coding sequence. This RE site was removed during codon optimization for expression in E. coli.
  
  
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===References===
 
===References===
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Bourne Y, Bolgiano B, Liao DI, et al. Crosslinking of mammalian lectin (galectin-1) by complex biantennary saccharides. Nat Struct Biol. 1994;1(12):863-70.

Latest revision as of 00:14, 17 October 2014

pBAD-RBS-Bovine Galectin-1-B0012 Terminator


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
    Illegal XhoI site found at 499
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

UMaryland 2014 is interested in anchoring a galectin in the outer membrane of E. coli in an effort to bind to carbohydrate chains on an oyster pathogen. While an oyster galectin exists (BBa_K1489005), it is much larger than mammalian galectins of similar function. Bovine galectin 1 (BtGal1) was chosen to serve as a model galectin to anchor in the outer membrane due to its smaller size and its simpler quaternary structure; Bovine galectin binds as a dimer while the oyster galectin, CvGal1, is believed to bind as a tetramer.

The pBAD promoter was chosen for protein expression due to previous experience with arabinose induction and a belief in tighter expression than the pLac promoter. An additional terminator was added at the end of the DNA sequence to increase the efficiency of transcription termination.

While designing the biobrick sequence we discovered an internal EcoRI site in the middle of the coding sequence. This RE site was removed during codon optimization for expression in E. coli.


Source

Sequence generated from Bos taurus genome, reoptimized for E. coli expression.

References

Bourne Y, Bolgiano B, Liao DI, et al. Crosslinking of mammalian lectin (galectin-1) by complex biantennary saccharides. Nat Struct Biol. 1994;1(12):863-70.