Coding

Part:BBa_K4011010

Designed by: Meilun Wu   Group: iGEM21_LINKS_China   (2021-10-21)
Revision as of 11:22, 21 October 2021 by Azahraonng (Talk | contribs)


Ma-sfGFP-CBM3


Ma-Mut is a mutated protein tag derived from the protein secretion tag (Ma) of mating type alpha Saccharomyces cerevisiae. In yeast cells, proteins with the Ma tag will be secreted out of the cell. This year, we will use Ma-Mut to enable secretion of proteins fused with cellulose binding domain 3 (CBM3) from yeast. CBM3 BBa_K4011000 is an artificial protein derived from Ruminiclostridium thermocellum (Protein Data Bank (PDB) accession: 1NBC) and will bind to cellulose fibers. This will allow for modification of our bacterial cellulose membrane produced by a co-culture of Komagataeibacter and yeast. We will use Ma_Mut to construct composite parts Ma-sfGFP-CBM3 BBa_K4011010 and Ma-CBM3-NT2RepCT-CBM3 BBa_K4011011.

Other teams can utilize our Ma sequence for S. cerevisiae protein secretion.

Usage and Biology

The alpha-factor preproleader sequence is responsible for protein secretion in mating type alpha Saccharomyces cerevisiae. Mα_Mut is derived from the preproleader sequence and first characterized by Aza et al in 2021, who mutated the sequence to increase protein secretion efficiency.

Natural Mα contains 89 amino acids with three functional regions: a pre-region (19 amino acids), pro-region (64 amino acids), and a spacer (6 amino acids). During S. cerevisiae modification, the preproleader will translocate the protein across the Endoplasmic Reticulum (ER), and the pre-region will be cleaved. Then, the pro-region will direct the protein to the Golgi Apparutus for final modifications before release into media.

CBM3s allow for fused proteins to be bound to cellulose, and sfGFP have fluorescent properties suitable for easy detection.

Source

Ma comes from S. cerevisiae, sfGFP is a highly characterized sequence used in synbio, CBM3 comes from Ruminiclostridium thermocellum.

Characterization

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 576
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 948
    Illegal AgeI site found at 3079
    Illegal AgeI site found at 3123
    Illegal AgeI site found at 3295
    Illegal AgeI site found at 3361
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 424
    Illegal SapI.rc site found at 2320


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Categories
Parameters
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