6.20-6.23: Screening over the first chemical library (800 candidates)

6.24-5.30: Screening over the Second chemical library (2,000 candidates)

7.10: Optimize the Condition for DSF assay (three factors including protein concentration, dye concentration, and buffer type)

7.14-7.20: Use different Concentrations of protein (5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 uM) with 2.5x SYPRO red dye (final concentration) and found out that 50 uM concentration was suitable for detection.

8.1-8.10: Use different concentrations of fluorescence dye (SYPRO red) to optimize the detection of protein stability shift using 100 uM DNA as a positive control, and found out that 12.5x final concentration is the best.

8.16-8.30: Purchase the top 30 chemical candidates and test them with the system described below and search for possible candidates.

8.13: Amplify the GFP fragment with PCR (EcoRI and HindIII sites were added by the primers).

8.16: Enzyme EcoRI and HindIII were used to cut the amplified fragment and plasmid pUCP20 from Yang Lab, the processed two parts were then ligated by ligase.

8.17: Plasmid pUCP20-GFP was amplified by DH5α, and primer M13 fwd was used for the sequencing of the GFP region.

8.18: The promoter region of acr was synthesized by IDT, the origin lac promoter of pUCP20-GFP was replaced by the acr promoter using homologous recombination.

8.19: Plasmid pUCP20-GFP-acr promoter was transformed into BL21-CodonPlus (DE3)-RIL to test the efficiency of the acr promoter. BL21-CodonPlus (DE3)-RIL turned green.

8.20-8.25: Gene aca1 was added to pRSFDuet-1 using homologous recombination. (aca1 was synthesized by IDT).

8.26-8.30: Plasmids pRSFDuet-1-aca1 and pUCP20-GFP-acr promoter were co-transformed into BL21-CodonPlus (DE3)-RIL. With the presence of IPTG, which can induce the expression of Aca1, the E.Coli was not as green as that without IPTG.

9.1-9.5: BL21-CodonPlus (DE3)-RIL containing plasmids pRSFDuet-1-aca1(pRSFDuet-1-aca1R44A for positive control) and the pUCP20-GFP-acr promoter was cultured in LB to OD₆₀₀ =0.5 with 500uM IPTG, then the candidate compounds were added separately to 105ul bacteria liquid (concentration of compounds are 39uM). After overnight growth at 37^oC 220 rpm, green fluorescence was observed with a microplate reader. Select those who have the closest green fluorescence to the aca1R44A one.

9.6-9.10: Increase the concentration of the selected compounds and repeat the experiments.

10.10-10.15: DSF was used to detect the binding of the compound and Aca1.

10.16-10.22: Designed 60 chemically modified versions of our chemical candidate, and used Molecular Operating Environment to estimated the strength of protein-ligand interaction.