Difference between revisions of "Part:BBa K4202009:Experience"

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Applications of BBa_K4202009

1.Protrein expression verification

To confirm that the optimized sequence could be produced by E. coli, we transformed the plasmid pET-28a (+)-ACCBP into BL21 (DE3) strain and cultured them in kanamycin resistant LB plates at 37 ^o C overnight. The next day, the overnight-cultured solution was transferred into 5 ml kanamycin resistant LB medium at a ratio of 1:20. The solution was cultured at 37°C, 220 rpm until OD₆₀₀ reached 0.6-0.8. Then 0.1 mM IPTG, 10% glycerol and 1mM CaCl₂ was added to induce the protein expression at 8^oC

At the end of induction, the bacterial solution was centrifuged at 12000 rpm at 4°C and the bacteria were collected. After washing the precipitation once with PBS, the precipitation were resuspended in a 1.5ml EP tube with 1mL PBS (appropriate amount of PMSF can be added).The bacteria were disrupted by ultrasonic disruption until the suspension was clear. The products are used for SDS-PAGE.

Result: Acorrding to the result of SDS-PAGE, we can found that the more protein were both in the supernatant and precipitation. And the more protein are in the precipitation, indicating that under this condition majority of the protein are insoluble.

<Fig 1-6 SDS-PAGE and Coomassie brilliantblue staining results of supernatant and sediment of BL21(DE3) strain with PET-28a(+)-ACCBP. The lanes have been indicated .

2.Protein expression and purification

The overnight-cultured bacterial fluid was inoculated into 500 ml Kanamycin resistant LB medium at the ratio of 1:100. The solution was cultured at 37 ^° C , 220rpm until OD₆₀₀ reached 0.6-0.8 and protein was induced at 8 ^oC with 1mM IPTG, 1% glycerol and 1mM CaCl2 for 48h. Then we collect the bacterium and resuspend in a special protein extract buffer (containing 10% glycerol, 500 mM PH 7.5 Tris-HCl buffer, and 0.01mM PMSF ). The next steps are same as 1 above .
Notes: In order to get a better protein expression effect, we also use palsmids Pet28a(+)-ACCBP, Pet28a(+)-Δ16 ACCBP, strains BL21(DE3) and shuffle T7 for this experiment .

Result : The purified ACCBP and Δ16 ACCBP protein were obtained by multiple combination of plasmids and strains. The protein will be used in further experiments.

Fig 1-7 SDS-PAGE and Coomassie brilliantblue staining results of protein purification. A: The purification result of ACCBP; B: The purification result of Δ16 ACCBP. The results show that both proteins have a considerable abundance in eluent 2 and 3 although there are some non-objective bands shown due to proteolysis.

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