Part:BBa_K2625000

Alpha-Agglutin 2 (Aga2) fused to GFP

Usage and Biology

This part was constructed by our team in order to test the function of the Saccharomyces cerevisiae cell wall anchoring protein Aga2 by fusing it to GFP. The expression of this protein sequence allows for locating GFP at the yeast cell surface.

Alpha-Agglutin 2 (Aga2) binds to the S.cerevisiae cell wall protein Alpha agglutin 1 (Aga1) with a double disulfide bond. In order to check the protein function, GFP was fused to the 3’-end of Aga2. A Kozak sequence was added at the 5’-end of the Aga2 to increase chances of expression, and to allow for better protein function of the individual proteins, a flexible linker (GGGS)x3 was added in between the proteins. Figure 1 shows how the plasmid has been constructed:

Figure 1: Plasmid design with Aga2 and GFP, TEF1 promoter, CYC1 terminator, the GGGS linker and the Kozac sequence. The plasmid of origin is from pSB1C3.

Characterization

Figure 2: GFP expression after culturing S. cerevisiae overnight in liquid Delft + His media. On the left: pictures of yeasts cells, on the right: pictures of the same cells under fluorescence light exposure

Figure 3: The gel electrophoresis result for the miniprepped plasmid digestion with NcoI. There are 2 bands: one of 1851 bp and the other 1266 bp. The ladder used is GeneRuler 1kb.

For testing the efficiency of GFP expression, fluorescence has been checked with a microscope (results in Figure 2). S. cerevisiae has been transformed with a p416Tef plasmid containing Aga2 and GFP, then it has been grown in Delft + His media overnight at 20°. After harvesting, it was resuspended in water and put on a microscope slide for testing. It has been observed that Aga2-GFP is secreted and it concentrates around what it seems to be the nucleus and, in minor quantity, in the surface. For specific information about the experiments and the results please check out [http://2018.igem.org/Team:Chalmers-Gothenburg/Labwork#Lab_journal].
Gene sequence has been verified by Gel Electrophoresis (extracted plasmid pSB1C3-Aga2GFP digested with NcoI:Figure 3) and by gene sequencing. To read more about the results check out [http://2018.igem.org/Team:Chalmers-Gothenburg/Labwork]

References

Tanaka, T., & Kondo, A. (2015). Cell-surface display of enzymes by the yeast Saccharomyces cerevisiae for synthetic biology. FEMS yeast research, 15(1), 1-9.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 974