Part:BBa_K3123000

CBD Nanobody Luciferase with FKPA

This part codes for two nanobody binders attached to a subunit of a split-luciferase system to detect the presence of cannabidiol (CBD). The first nanobody, the anchor binder, is linked to the large subunit of the split-luciferase protein; the anchor binder binds with CBD with high specificity. The second nanobody, the dimer binder, is linked to the small subunit of the split-luciferase protein. Both binder sequences are preceded by a PelB leader sequence, which transports the amino acid chain to the periplasm of the cell to be folded. Each binder sequence also contains an Avi tag and a His tag. The Avi tag allows the user to biotinylate the protein, which allows it to be used in many ways with streptavidin. The His tag allows for protein purification of the nanobodies using a nickel trap column. The dimer binder will only bind to the anchor binder/CBD complex, after the anchor binder and CBD have already bound together. By joining these binders together, the split-luciferase subunits have a higher likelihood of interacting and forming a functional luciferase protein. This luciferase protein will catalyze a reaction with a luciferin molecule and result in a readable luminescent signal, effectively acting as a measurement proxy for CBD. These two binder+subunit sequences are flanked by a protein coding region for FKPA, a chaperone protein that stabilizes protein folding in the periplasm of bacteria. This part is regulated by a lac operon and uses biobrick BBa_B0010, a T1 terminator, in order to produce a single mRNA strand.

Applications of BBa_K3123001

A major theoretical application of this device is in measuring CBD concentrations during production. This general nanobody/luciferase biosensor can also be generalized to utilize binders for other small molecule analytes once binders have been identified.

Washington iGEM 2019: Characterization

Unfortunately, this part remains untested by the Washington iGEM 2019 team as we had trouble with assembling this construct through both gBlock synthesis and digestion/ligation, using the cut sites mentioned on the part design page [1], and through homology based assembly¹.

For the problems with assembly through gBlock synthesis and digestion/ligation, we believe that the problems may have arisen from using BglII and BamHI cut sites to stitch together this part. The DNA cut sites for these restriction enyzmes are very similar, and using BamHI on the dimer binder fragment may have cut both restriction sites, causing the final ligation to ligate in the wrong order and thus result in the wrong construction. It is possible that removing one of these added cut sites and replacing it with another compatible site may allow this part to be constructed properly.

References

1. Kostylev M, Otwell AE, Richardson RE, Suzuki Y (2015) Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies. PLoS ONE 10(9): e0137466. https://doi.org/10.1371/journal.pone.0137466

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1977
  Illegal BamHI site found at 1307
  Illegal XhoI site found at 1929
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 290
  Illegal NgoMIV site found at 1404
  Illegal AgeI site found at 711
  Illegal AgeI site found at 1825
  
• 1000
  COMPATIBLE WITH RFC[1000]