Part:BBa_K3123001
CBD Nanobody Luciferase
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. 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: Construction and Use
Our team set out to construct this biobrick using two methods: homology based cloning1, similar to a Gibson Assembly, and synthesized DNA cloning.
Homology Based Cloning
For this method, we used two DNA sequences: one was the CBD Anchor Binder and the other was the CBD Dimer Binder. Both of these binders were linked to their respective split-luciferase subunits and were inserted into the pADL2-23c vector.
Primers were designed to add homology to the CBD Dimer Binder sequence as an insert into the CBD Anchor Binder plasmid and were elongated using Q5 Phusion polymerase from NEB. After homology was added, the products were treated with DpnI to remove the original plasmids; these products were then directly transformed into chemically competent DH5α E. coli at an optimized molar ratio, similar to the standard ratio used for a Gibson Assembly. These transformations were then plated, with several successful plasmid constructions appearing.
gBlock Assembly
Using DNA synthesis, we have yet to construct a correct plasmid. 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.
Washington iGEM 2019: Characterization
Unfortunately, this part could not be verified by the Washington iGEM 2019 team as our characterization experiments yielded primarily negative results.
As the description of this part shows, it is intended to act as a biosensor for CBD by dimerizing the anchor and binder nanobodies, which are in turn attached to split-luciferase subunits that can also bind to form a functional luciferase. This luciferase catalyzes a reaction with furimazine, which produces furimamide and a luminescent signal.
Methods
We constructed test plasmids as described above and tested the periplasm extract of the positive constructs. The periplasm extract was released from an IPTG induced culture using TES buffer. We incubated the periplasm extracts to CBD for an hour and then added furimazine from the Nano-Glo Live Cell Assay System. These samples were then plated (or prepared on the plate before adding furimazine) to a black 96 well plate and scanned through a luminescent plate reader.
Results
Unfortunately, as we moved to testing this construct with CBD and furimazine, a luciferin, our measurements of luminescence on average showed no signal. Because of these results, we sequenced this initial construct and found that the backbone vector we had used was incorrect. The plasmid we had used as the backbone was an empty pADL-23c and was missing the coding sequence for the anchor binder. We quickly moved to reconstruct the plasmid with the correct components and sequence verified it. Despite this, assay results from this newly constructed plasmid also returned mostly zeroes. There are many different possible explanations, but we believe a very plausible one to be that the lack of FKPA, a chaperone protein, contributed to incorrect folding of the nanobodies and subsequently a loss of function. More experiments need to be run in order to determine the cause. A compilation of our data generated from several luciferase assays can be found here.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1977
Illegal BamHI site found at 1307
Illegal XhoI site found at 1929 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000COMPATIBLE WITH RFC[1000]
//function/sensor
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