Coding

Part:BBa_K2665021

Designed by: Masaaki Shimazoe   Group: iGEM18_Kyoto   (2018-10-15)


SED1 secretion signal - SdrG N2 N3 domain - 6xHis tag - ybbr tag-Stalk Region - SED1 anchoring domai

Description

This part contains various parts. This part is designed for surface-display of SdrG on yeast cell wall and aggregation with yeast that has FgβF3 on its surface. Part that includes FgβF3 is BBa_K2665020

SdrG is derived from Staphylococcus aureus. SdrG combines with Fgβ very strongly. This bonding is comparable to covalent bond in strength. [1] We uses N2 and N3 domain of SdrG BBa_K2665019 for this part because it is essential and enough to combine with FgβF3.
The ybbr-tag BBa_K2665023 is also included in this part. This part was included in design of the paper, so we leaved this part just in case.

6xHis-tag BBa_K2665027 was fused for several assays of aggregation. Detail of assay is in the experience page.

We use SED1 secretion signal and SED1 anchoring domain in order to display SdrG on yeast cell wall. More detail about these parts are in the pages of BBa_K2665025 and BBa_K2665026

“Stalk Region” BBa_K2665022 is a very long linker. In order to combine yeasts, we have to provide enough room between yeasts. We referred the research that display of nanobodies on yeast cell wall. [2]

Characterization

Yeast displaying SdrG on the cell surface was captured with magnetic beads coated with anti-6xHis antibody, and after washing with PBS, the bound cells were observed with a microscope.
T--Kyoto--SdrG-anti His.jpeg
The results are shown in the figure.SdrG (His 6) expressing cells could not be recovered by recovery with His-trap magnetic beads. This may be due to the reason that the presented His6 sequence is in a situation that does not allow access of magnetic beads due to steric hindrance, or because SdrG (His 6) has a lower expression level per cell.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1080
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 664
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 3421
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1236
    Illegal SapI site found at 1556
    Illegal SapI site found at 3358

Reference

[1]L.Miles, K.Schulten, H.Gaub et al. (2018) "Molecular mechanism of extreme mechanostability in a pathogen adhesin" Science Vol.359 1527-1533
[2]C.Mcmahon, A.Baier, R.Pascolutti et al. (2018) "Yeast surface display platform for rapid discovery of conformationally selective nanobodies" Nature Structural & Molecular Biology Vol.25


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