Part:BBa_K4016023

HA-Trim21-LD3

This composite part is made up of Trim21 and LD3 sequence. Trim21 is the fuction module in our design which lead to the ubiquitination and degradation of the target module.The introduction of LD3 makes it possible to combine with BcL-XL, which link to the GFP nanobody, thus lead to GFP degradation.As designed,the part can be used as a small molecules mediated system with GFP reporter.

Usage and Biology

Bcl-XL and LD3 are used in our project as an “OFF system”, based on their disassociation ability in response to A1155463. We replaced the PRYSPRY structure of truncated trim21 with the Bcl-xL and LD3 protein pair, and attached Trim21 to the end of LD3 to achieve specific degradation of the GFP protein.

Our composite part Part:BBa_K4016023 is a testing part reacting with BBa_K4016024. To test the result of the trim21-BcL-XL-LD3 interaction, if this can work, we can make an “OFF switch” in a system mediated by small molecules.

Figure1. Schematic figure of BBa_K4016023 and BBa_K4016024

Characterization

This part BBa_K4016023 was cloned in pXQ107 plasmid and transfected into HEK293T cell lines using Invitrogen LipofectamineTM 3000.It was validated through four ways: PCR, Sequence, and functional testing.

PCR

The PCR is performed with Green Taq Mix by Vazyme.

F-Prime：5’CTAGCGTTTAAACTTAAGCTTGCCACCATGGAGTACCCCTACGACGTGCCCGACTAC 3’

R-Prime：5’GTGGTGGTGGTGGTGCtcgaGCCGTTCAGCAGCGCCAGCCTG 3’

The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1% Agarose gel.

Enzyme Digestion

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 cutting procedure was performed with Hind III EcoR I restriction endonuclease bought. The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose glu.

Sequecing

The plasmid was sequenced correct.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 204
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 161
  
• 1000
  COMPATIBLE WITH RFC[1000]

Functional test

This part (BBa_K4016023) was tested together with Part:BBa_K4016024

Method

• 1.Cell transfection

(1)Seed HEK293T cells into 6-well cell culture plates.

(2)Culture for 16 h before transfection

(3)Total plasmid mixes of 800ng per well are mixed thoroughly in DMEM before a polyethylenimine (PEI) solution (1 mg/ml) is added into the plasmid mixture in a ratio of 1:5 (plasmid weight/PEI weight)

(4)The plasmid–PEI mixture is vortexed and incubated at room temperature for 15 min. The mixture is then added into the cells and incubated for at least 6 h.

(5)Cells are then changed into fresh medium and culture for 18 h before subculture.

Reference

[1] Giordano-Attianese, G. et al. A computationally designed chimeric antigen receptor provides a small-molecule safety switch for T-cell therapy. Nat Biotechnol 38, 426–432 (2020).