Difference between revisions of "Part:BBa K4016024"
Line 11: Line 11:
  
  
−
https://2021.igem.org/wiki/images/b/b5/T--NUDT_CHINA--Part_SchematicFigure_23-24.png
+
<html>
 +
 
 +
<figure class="figure">
 +
<img src="https://static.igem.org/mediawiki/parts/3/39/T--NUDT_CHINA--Part_SchematicFigure_23-24_.png
 +
" class="figure-img img-fluid rounded"  height="350px">
 +
 
 +
</figure>
 +
 
 +
</html>
 
Figure1. Schematic figure of BBa_K4016024 and BBa_K4016023
 
Figure1. Schematic figure of BBa_K4016024 and BBa_K4016023
  

Revision as of 17:11, 20 October 2021


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.

  • 2.Dual luciferase assay

(1)Wash HEK293T cells in 6-well plate with PBS and trypsinize prior to resuspension in fresh complete medium in a 15 ml microcentrifuge tube.

(2)Dispense 100ul of cell suspension (approximately 30000 cells per well) into 96 well plates.

(3)Capture the fluorescent image before apply blue light.(24h after transfection)

(4)Apply the experiment group with blue light stimulus (480nm, stimulate 2 seconds with a 58 second-interval) for 24/48/72 h before sampling and analysis assay. Capture the fluorescent image at 48/72 h respectively

(5)At 96h , add Reporter cell lysates in 96 well plates. (100uL per well)

(6)After extensive lysis, centrifugation at 10000-15000g for 3-5 min. Take the supernatant for assay.

(7)Thaw firefly luciferase assay reagent and Renilla luciferase assay buffer, and bring to room temperature. Renilla luciferase assay substrate (100x) was placed on an ice bath or on an ice box for later use.

(8)Prepare Renilla luciferase assay working solution by adding Renilla luciferase assay substrate (100x) at 1:100 in an amount of 100 µ l per sample.

(9)Switch on the microplate reader, set the assay interval to 2 s and the assay time to 10 s.

(10)Take 20 to 100 ul of each sample for assay

(11)Add 100 ul of firefly luciferase assay reagent, measure the RLU (relative light unit) after mixing. Reporter cell lysate was used as a blank control.

(12)Add 100 ul of Renilla luciferase assay working solution

(13)The RLU value obtained from the Fluc assay was divided by the RLU value obtained from the Rluc assay. The degree of reporter gene activation of interest was compared between different samples according to the ratio obtained.


Result

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).