Part:BBa_K5314007

AM.Sed + EGFP

We used the cell wall anchoring sequence AM.Sed (from Aureobasidium Melanogenum BZ-11) to ligate to EGFP to add a cell wall anchoring sequence to EGFP, giving it the ability to be secreted outside the cell. We tested the expression strength by observing fluorescence intensity and fluorescence position to test the expression intensity. However, the anchoring efficiency is not perfect, most of the fluorescence is inside the cell. We speculate that it may be the result of inefficient anchoring of C-terminal sequences. In the future we will optimise the C-terminal structure and advise the future iGEM teams.

Fig 1. AM.Sed GFP Plasmid map

Fig 2. AM.Sed-EGFP was introduced into Aureobasidium Melanogenum BZ-11 and cultured for 48h and observed under 488nm excitation fluorescence (right) respectively.

Source

Organism: AM.Sed (Aureobasidium Melanogenum BZ-11)

Sequence and Features

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
  COMPATIBLE WITH RFC[1000]

References:

[1] Jaafar, L., & Zueco, J. (2004). Characterization of a glycosylphosphatidylinositol-bound cell-wall protein (GPI-CWP) in Yarrowia lipolytica. Microbiology (Reading, England), 150(Pt 1), 53–60. https://doi.org/10.1099/mic.0.26430-0

[2] Fujii, T., Hitoshi Shimoi, & Yuzuru Iimura. (1999b). Structure of the glucan-binding sugar chain of Tip1p, a cell wall protein of Saccharomyces cerevisiae.Biochimica et Biophysica Acta (BBA) - General Subjects, 1427(2), 133–144. https://doi.org/10.1016/s0304-4165(99)00012-4