Team:ZJU-China/Protocol
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We obtained csgA sequence from the genome of E. Coli MG1655 by PCR and inserted it into Pet26(+). The recombinant plasmid was transfromed into BL21(DE3) and shaken overnight in LB with antibiotics. After reaching an optical density of 0.6~0.8 at 600 nm, the protein expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 25℃. We centrifuged the solution at 12000 rmp for 15mins, and cell pellets were resuspended gently with lysis buffer followed by sonication at the power of 300w for 30mins. The supernatant after centrifugation was kept for future purification.
2. Purification and Analysis• Agarose Gel Electrophoresis
Dissolve the agarose powder in TAE. Heat it to near-boiling point, cool sufficiently and pour the solution into a cast with a comb. After solidification, set gel tray into cuvette, filled with 1x TAE buffer. Load samples into the walls created by the comb. Run gel at 90-120V for 20-40min. Check image under UV light.
• Ni-NTA Affinity Purification
Centrifuge the E. coli Lysates at 12000rmp at 4℃ for 20 minutes. Wash the column with 5ml sterile water, 8 mol/L urea, and sterile water. Add 0.1 mol/L Nickel Sulfate until the column changes from white to blue (Ni). Remove the excess Nickel with sterile water. Balance the column with wash solution. Add supernatant into the Ni-NTA column. Control the flow rate to about 0.5-1ml/min. Collect sample with EP tube.
• SDS-PAGE
Gel production:Put two sealed glass plates together and clamp in a stand to make a mold. Mix H2O, 30%(Acr-Bise), gel buffer, SDS, TEMED and 10%APS to make a gel solution. Pour the solution into the mold without creating bubbles, and add water. After the polymerization of the separating gel, the water is discard and add the stacking gel solution which is made similarly.
Sample preparation: Add the sample buffer, and thus SDS in excess to the proteins, and the sample is then heated to 95℃ for five minutes.
Load 15-20 ul supernatant per lane on a protein gel. Load 10 ul prestained protein ladder. Run protein gel at 200V until dye front reaches the bottom of the gel.
Stain with coomassie blue for 1h and destain by destaining solution overnight .
• Cango Red Assay
As an amyloid protein, we dyed curli by Cango Red to indicate the expression and characteristic of curli. Centrifuge curli pellet at 12,000g for 5 mins. Resuspend gently by LB and add Cango Red to 30μg/ml CR and incubate at RT for 30 mins. Centrifuge at 14,000rpm for 5mins. Set LB with equal CR as the blank. Measure the absorbance of supernatant at 480nm for CR and 600nm for cell concentration. Compare the expression in CsgA knockout Defect bacteria with that of our recombinant recombinant strain.
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