Part:BBa_K4011010
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 576
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 424
Illegal SapI.rc site found at 2320
None |