how to carry out our experiments

1. Differential Fluorescence Scanning Assay (DSF Assay)

1.1 Introduction

Differential scanning fluorimetry (DSF) is a thermal shift assay technique used to measure the denaturation of proteins caused by increasing temperature, which breaks the non-covalent bonds that underlie protein folding. Meanwhile, the fluorescent dye, which gives high fluorescence in a nonpolar environment, is used in the DSF assay to probe the hydrophobic sites, which are generally embedded in the interior part of the proteins and exposed when proteins are denatured by high temperature. Thus, as the temperature increases, the ratio of denatured proteins increases, thereby elevating the intensity of the fluorescence. When the protein is fully unfolded, the intensity would peak.

1.2 Objectives

To test if the ligand-protein interaction occurs by detecting the shift of thermostability of the target protein after the addition of the small molecule ligand.

1.3 Materials

1.3.1 NaCl-Tris Buffer: 1M NaCl & 20mM Tris-HCl pH = 7.5, stored on ice

1.3.2 SYPRO red 5000x,diluted to 50x with buffer before use

1.3.3 Aca1 protein sample, stored in -80 degrees centigrade. Before use, unfreeze on ice and centrifuged at 12,000g at 4 degrees for 10mins, then test the concentration of the supernatant using Nanodrop.

1.3.4 7500 Fast Real-time PCR machine (Life technologies)

1.4 Methods

1.4.1 Configurate reactions as follows:

Protein Samples x ul 50uM (Final concentration)
50x SYPRO 4 ul 12.5x
Compounds 2 ul 1/10 concentration (10% DMSO)
Buffer 12-x ul
Total 20 ul

1.4.2 Add all ingredients by 3.2x volume into a 1.5mL centrifuge tube, and mix by gentle flicking (avoid generating too many bubbles).

1.4.3 Briefly spin down and dispense them evenly into three wells on the 96-well plate as three technical replicates.

1.4.4 Cover the plate with Optical Adhesive Film to prevent evaporation and use a 7500 Fast Real-time PCR machine (Life technologies) to read the samples.

1.4.5 The running method was designed to raise the temperature from 32°C to 81°C in 0.5°C steps, and to measure the fluorescence every 0.5°C for 30 seconds.

1.4.6 Export the results and analyze the results on the computer.

2. Protein-ligand Docking Using MOE

2.1 Protein preparation

2.1.1 Open the corresponding PDB file containing the protein crystal structure with MOE

2.1.2 MOE—RHS—QuickPrep (to calibrate the structure by protonation, adding missing H atoms, adding tethers to active center, fixing distant atoms, and determining the overall lowest potential energy configuration for the different states throughout the system)

2.1.3 Define ligand-binding pocket: MOE—Compute—Site Finder—Apply,then select a proper pocket (firstly consider the one with the largest size when no ligand presents) and create dummy atoms.

2.2 Docking a chemical ligand database

2.2.1 MOE—Compute—Dock

2.2.2 Define all parameters: Receptor: Receptor + Solvent Site: Ligand Atoms / Dummy Pharmacophore: Query Editor Ligand: the corresponding MDB file of the ligand bank Placement & Score: The more the better, compromising with the computational cost. Use the default method. Refinement: Induce fit. In this case, we chose 300 placements and 1 refined output.

3. In vivo screening using part BBa_K3423005

3.1 Introduction

Protein anti-CRISPR-associated 1 (Aca1) is the inhibitor of the promoter of gene anti-CRISPR, which exists in many phages. Without the inhibition activity of Aca1, phages will die, it is of vital importance to find an inhibitor of Aca1.

In part BBa_K3423005, Aca1 is constitutively expressed by the T7 promoter, which will subsequently inhibit the anti-CRISPR promoter, so the gene expression downstream anti-CRISPR will be inhibited until an effective Aca1 inhibitor is added artificially. Without the inhibition from Aca1, the expression of genes downstream anti-CRISPR promoters will increase significantly. Here, the gene codes green fluorescent protein is inserted downstream anti-CRISPR promoter, and thus the efficiency of the candidate compounds can be tested.

3.2 Objectives

Find an effective inhibitor of Aca1 in vivo.

3.3 Materials

3.3.1 Part BBa_K3423005 with GFP inserted after anti-CRISPR.

3.3.2 Candidate compounds dissolved in DMSO.

3.3.3 E. coli BL21 (DE3).

3.3.4 Lysogeny broth.

3.3.5 Pipettes and tips, 1.5 mL centrifuge tube, incubator, vortex mixer, orbital shaker, tabletop centrifuge, microplate reader.

3.4 Methods

3.4.1 Part BBa_K3423005 (GFP) and part BBa_K3423005 (GFP) mutation Aca1R44A is transformed into E. coli BL21 (DE3).

3.4.2 Cultured in lysogeny broth, 37 oC, 220 rpm to OD600=0.5

3.4.3 Candidate compounds are added separately, vortex completely to evenly distribute the compound precipitation in the liquid.

3.4.4 Grow at 37 oC, 220 rpm for 6 hours in 1.5 mL tube, each contains 250 ul broth

3.4.5 Measure green fluorescence intensity using a microplate reader, each well contains 200 ul. The wavelength of the excitation light is 480 nm, while the emission light is detected at 520 nm.

4. Homologous recombination

ClonExpress II One Step Cloning kit was used:

4.1 Calculate the amount of DNA for recombination by the formula.

4.2 Set up the reaction on ice

4.3 Gently pipette up and down several times to mix thoroughly. Spin briefly to bring the sample to the bottom of the tube before reaction.

4.4 Incubate at 37℃ for 30 min and immediately place the tube at 4℃ or on ice

5.1 Prepare the E. coli treated with glycerol and calcium chloride and plasmid on ice

5.2 Add the plasmids to the treated bacteria on ice and wait for 30min

5.3 Put the mix into a 40oC water bath for 90 sec

5.4 Put the mixture on ice for 2 min

5.5 Add enough LB and recovery in 30oC and 220 rpm for 40 min

6. Green fluorescence measurement

200 ul of the sample is added to a 96 wells plate, if the volume of the original sample is not enough, LB is added. Activation weave length is 480 nm, green fluorescence is detected with 520 nm using a microplate reader.