Difference between revisions of "Part:BBa K2653001"
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<partinfo>BBa_K2653001 short</partinfo>
 
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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.
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The nanobody of GFP.
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
===Usage and Biology===

Revision as of 12:55, 17 October 2018


GFP-nano

The nanobody of GFP. 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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 35



Usage and Biology

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.

Source

The sequence of GFP-nano we use came from a patent.

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.

T--NUDT_CHINA--Parts-GFP-nano.jpg

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