Coding

Part:BBa_K4488027:Design

Designed by: Jessica, Sylvie, Kawana   Group: iGEM22_Sydney_Australia   (2022-10-11)


Neae-intimin Construct Displaying anti-sfGFP Nanobody H


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]


Design Notes

Firstly, we created a plasmid that contained Neae-intimin, a bacterial protein which can display other proteins (i.e. our nanobodies) on the surface of E. coli. Our Neae part contained a lacz-alpha gene flanked by Golden Gate cloning sites so that we could clone in our nanobodies after the fact. This means that this first stage of cloning was just taking the Neae-intimin gene and inserting it into pUS250.

Then, the second round of cloning involved inserting the nanobody into pUS250 with Neae-intimin. Esp3I Gold Gate sites that are present on either side of the lacZalpha region in the Neae part were used to this which means that when we cloned in our nanobodies, they replaced the lacz-alpha.


Source

The anti-sfGFP nanobody was adapted from PDB: 3OGO-H (see BBa_K4488005 for further detail). The Neae-intimin sequence was modified from Salema & Fernandez (2017) (BBa_K4488000).

References

Salema, V., & Fernández, L. Á. (2017). Escherichia coli surface display for the selection of nanobodies. Microbial Biotechnology, 10(6), 1468-1484. https://doi.org/https://doi.org/10.1111/1751-7915.12819