Part:BBa_K4016024

GFPnano-BCl-XL

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 a GFPnano antibody to the end of Bcl-XL to achieve specific degradation of the GFP protein.

The antibody GFP-nano is designed to bind with both Trim21 and the antigen GFP to prove that the system works well. In the design we link GFP-nano with BcL-XL, so that it can work as the bridge between target GFP and Trim21.

Figure1. Schematic figure of BBa_K4016024 and BBa_K4016023

Characterization

This part BBa_K4016024 was cloned in pXQ109 plasmid and transfected into HEK293T cell lines using Invitrogen LipofectamineTM 3000.This part is validated through four ways: PCR, Sequence, and functional testing.

PCR

The PCR is performed with Green Taq Mix by Vazyme.

F-Prime：5’CTAGCGTTTAAACTTAAGCTTGCCACCATGgagtctgggggag 3’

R-Prime：5’gctgtagtccaggatTCCGTACAGTTCCACGAAGGT 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
  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
  

Functional test

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

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).