Part:BBa_K2653001
GFP-nano
The nanobody of GFP.
Usage and Biology
The antibody GFP-nano is designed to bind with both Trim21 and the antigen GFP to prove that the P.R.PREDATOR really works. It belongs to the single-chain variable fragment(scFv) family, which has the ability to bind with GFP, but is much smaller than the common antibody of GFP(only 364 bps). In the project GFP-nano successfully bound with target GFP. The GFP-nano works as a core in our system to degrade the proof of concept target GFP as the tool to bind with it. In the design we link GFP-nano with hIgG1-Fc, so that it can work as the bridge between target GFP and Trim21, and finally help form a ternary complex of GFP, GFP-nano and Trim21. Then the E3 ubiquitin ligase Trim21 would tag the complex with the ubiquitin mark, and then employ the proteasome-ubiquitin system to degrade the target GFP protein.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 35
Source
-The sequence of GFP-nano comes from a Korean patent:patent NO.10-2012-0049206.
Experimental Validation
Sequencing
This part is sequenced as correct after construction.
PCR
Methods
The PCR is performed with Premix EX Taq by Takara.
F-Prime: 5’- aatccttagctttcgctaaggatgatttctg-3’
R-Prime: 5’- ccttgcccttttttgccgga-3’
The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1％ Agarose glu. The result of the agarose electrophoresis was shown on the picture below.
Figure 2. Electrophoresis showing the production of four miRNA lockers by asymmetric PCR.
Enzyme digestion test
Methods
After the assembly ,the plasmid was transferred into the Competent E. coli DH5α). After culturing overnight in LB,we minipreped the plasmid for cutting. The preparation of the plasmid was performed with OMEGA e.Z.N.A Plasmid Mini Kit I(200). The cutting procedure was performed with EcoRI and SpeI restriction endonuclease bought from TAKARA.
The plasmid was cutted in a 10μL system at 37 ℃ for 4 hours. The Electrophoresis was performed on a 1％ Agarose glu.
The result of the agarose electrophoresis was shown on the picture above.
Functional Test
Figure1. HEK293T cells were transfected with GFP PREDATOR plasmid and GFP-expressing plasmid, fluorescence images indicated a significantly decrease in GFP fluorescence.
Results showed the complexes precipitated with ProteinA/G by Fc fragment which directly linked to GFP nanobody contained GFP protein (detected by Western Blotting), this verified the binding between GFP and GFP nanobody.
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