Team:NYMU-Taipei/Results
Results
ACE2-Spike Binding Affinity
Plasmid construction
We successfully constructed a plasmid that has ACE2 RBD coding sequence with a bacterial expression backbone and 6x histag.
plasmid overview on snapgene(3953bp) gel electrophoresis digested by BamHI(1061) and EcoRI(1791)
Protease Cleaving Activity
Plasmid construction
We successfully constructed a plasmid that has pepP coding sequence with a bacterial expression backbone and 6x histag.
plasmid overview on snapgene(3056bp) gel electrophoresis digested by XbaI(70) and HindIII(1590)
we expressed the construct through E. coli Bl21, purify with Ni-NTA column and tested the correctness through nanodrop, SDS-page and Western blotting.
Nanodrop result and concentration(protein presented in the A280 peak*
*Note that the concentration is different from the experiment concentration since the elution buffer is full of imidazo. We use an exchance membrane to remove them to present interference of experimental condition.
SDS-page result
Western Blot result
We constructed four fragments from SARS-CoV-2 spike protein(4.2kDa each) with a pepP cleavage site on each of them to test the cleavage activity (1st fragment comes up with a unfavored result).
plasmid overview on snapgene(1799bp) gel electrophoresis digested by XbaI(70) and HindIII(333)
We also expressed the construct through E. coli Bl21, purify with Ni-NTA column and tested the correctness through nanodrop and SDS-page. Special tricine SDS page was used here since the protein is only 4kDa.
Protease-ACE2 Cleaving Activity
Plasmid construction
We successfully constructed a plasmid that has pepP + ACE2 RBD coding sequence with a bacterial expression backbone and 6x histag
plasmid overview on snapgene(5312bp) gel electrophoresis digested by XbaI(5270) and NheI(1329)
We are using the same spike fragments as pepP cleaving activity.
Gel conjugation
polyHEMA making
Because polyHEMA is a mesh aggregation of thermal polymerization, we experieced different heat condition to find out the best product method.
The result showed that althought microwave oven is much faster than dry oven, it is hard for us to control the stability of temperature of our gel. Instead, if we use dry oven in 75-85 degree Celsius, the gel will easily reach the perfact situation, which is jelly-like status and without bubble or withering.
*the heat mentioned above is the step after heating in microwave oven.
75 degree Celsius in dry oven for 2 hours
Microwave for 25 seconds
Microwave for 20 seconds
Microwave for 15 seconds
Microwave for 10 seconds
We use Potassium permanganate to oxidase the hydroxyl group on polyHEMA. After oxidation, we used FTIR to detect whether the functional group on polyHEMA had changed from R-OH to R-COOH. The result indicated that the site of classical carboxyl group adsorbing had a obvious increase.
