Part:BBa_K3858014

antiGFP-High encoding

Recombinant nanobody against GFP. High affinity compared with antiGFP-Low.

Usage and Biology

In 2021, GreatBay_United tried to display this antibody onto the yeast surface.

There are two kinds of construction: Solely expression the antibody or fusion expression with GFP. To display onto surface, an existing part (Aga2) were used to lead the recombination protein to the cell membrane.

anti-GFP expression cassette

For the fusion expression version, the GFP part and anti-GFP part were linked with HCV protease cutting site. Thus, if both complex and antiGFP were successfully displayed on the surface, they will be able to sense the HCV NS3 protease in solution. The protease would cut and release the antiGFP linked to GFP, and the solely expressed anti-GFP would had chance to connect with GFP. this interaction would lead to the macroscopical agglutination phenomenon when we mix two kinds of engineered yeast together and also HCV protease.

the cassette of yeast surface display of GFP-HCV protease cut site-antiGFP complex

the two types of yeast co-culture with HCV protease in the medium

the two types of yeast co-culture without HCV protease in the medium

Because there were different affinity of antiGFPs, it could be several combination choices to design. To figure out which one can work best, we constructed two kinds GFP-antiGFP(H/L) and two kinds antiGFP(H/L), then mix them for their full combinations, thus there are 4 types.

all 4 combinations

Compared between with or without HCV protease added in the 4 co-cultures, we could make conclusion that all these types works well to sense HCV protease.

4 combinations results without HCVp added

4 combinations results with HCVp added

Reference

1. Fridy P C, Li Y, Keegan S, et al. A robust pipeline for rapid production of versatile nanobody repertoires[J]. Nature methods, 2014, 11(12): 1253-1260.
2. Toda S, McKeithan W L, Hakkinen T J, et al. Engineering synthetic morphogen systems that can program multicellular patterning[J]. Science, 2020, 370(6514): 327-331.

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]