Team:NAU-CHINA/Results
results
Effects of cell surface-expressed GFP on synNotch-TEV.
A. 1.Fluorescence microscope observation of HEK 293 T transfected with plasmid containing synNotch. The cells were cross-pnked with GFP. The results showed that synNotch can be located to the membrane. 2.Blank control (without transfection).
B. Assay of the synNotch-TEV and FLAG-tagged TEV concentration affected by cell surface-expressed GFP. C,D. Relative level of the synNotch-TEV and FLAG-tagged TEV affected by cell surface-expressed GFP. synNotch-TEV affected by surface-expressed GFP can be resolved into FLAG-TEV and V5-mNotch. Data are mean ±S.E. (n=3). **, p < 0.01; N.S., no significance.
Effects of tetR on GFP expression with tetO sequence.
Stably transferred cell pne containing tetO-miniCMV-EGFP sequence.
1. Expression of GFP 6 h after nucleofection with plasmid containing tetR.
2.Expression of GFP 72 h after nucleofection.
Pronuclear verification of recombinase
Two plasmids with different origins of reppcation were used for functional verification of the recombinase, which have different resistances. One of them is a reporter gene plasmid, which uses the constitutive promoter J23119. The recombination site is located on both sides of the promoter, one side of the promoter is sfGFP, and the other side is mRFP. The other is a recombinase expression plasmid using PBAD, an inducible promoter, which is induced by arabinose. When the two plasmids were co-transfected into E. cop, the reporter plasmid expressed sfGFP, a green fluorescent protein, when the inducer arabinose was added, the recombinant enzyme was expressed, the promoter was inverted, and the mRFP red fluorescent protein was expressed.
Due to the use of two different resistant plasmids, kana and chloramphenicol, we used a plate containing two resistances of kana and chloramphenicol for screening, and grew many colonies, randomly selected 9 singles. After the colonies, we made colony PCR (Fig. 1) and the results showed that both plasmids were transferred.
Figure 1: A total of 9 single colonies were verified in the order of: single colony 1-9 recombinase expression plasmid vapdation, 2000 marker, single colony 1-9 reporter gene expression plasmid vapdation.
The verified E. cop was placed in M9 minimal medium supplemented with glycerol and arabinose, and the culture was divided into two at OD 600 = 0.1, designated time 0, and 0.2% glucose was added to each pair. In the pair. Samples were obtained at 0 and 30 minutes, 60 minutes and 120 minutes before the culture was isolated. (Refer to the description of BBa_I0500)