Metodologia - BrainStorming

[nathaliaraquelx]

Combinação SynBio e o artigo que estamos usando como base:

• [ ] Expressão de ACE2 em bactéria (qual abordagem utilizar?)

• [ ] Quais plasmídeos utilizar?

• [ ] Qual cepa fazer a transfecção?

• [ ] ACE2 fusionada com Fc;

• [ ] Purificação e SDS-PAGE da ACE2;

• [ ] Contrução do teste: pCL2 as N-terminal fusions to Aga2p followed by eGFP;

• [ ] Miniprep;

• [ ] Screening:

cells were washed twice with PBSA and stained with 50 nM biotinylated RBD monomer (10 ml) for 1 hour at room temperature

• [ ] Citometria de Fluxo;

To fit yeast binding data to KD values, Alexa Fluor 647 mean fluorescence intensities were extracted from the GFP-positive population, background subtracted using secondary only controls, normalized to the highest fluorescent population, and fit to the Hill equation without cooperativity in Python.

• [ ] In vitro binding and activity characterization of inactivation mutations in ACE2-Fc;

• [ ] Pseudotyped SARS-CoV-2 neutralization IC50 curves for WT and engineered ACE2(614)-Fc and ACE2(740)-Fc molecules.

• [ ] Live virus qPCR assays of three viral genes (N, E, RdRp) were run 16 hours post-infection using host genes BGUS or ACTB as normalization controls.

[nathaliaraquelx]

• [ ] Estudar o uso dos marcadores: Alex-fluor-488 and Alex-fluor-546