Difference between revisions of "Part:BBa K4202009:Experience"
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<p><b>Result</b>: 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.</p> | <p><b>Result</b>: 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.</p> | ||
| − | + | <div align="center">[[File:m1-accbp.png|600px]]</div> | |
<p><<b>Fig 1-6</b> SDS-PAGE and Coomassie brilliantblue staining results of supernatant and sediment of BL21(DE3) strain with PET-28a(+)-ACCBP. The lanes have been indicated .</p> | <p><<b>Fig 1-6</b> SDS-PAGE and Coomassie brilliantblue staining results of supernatant and sediment of BL21(DE3) strain with PET-28a(+)-ACCBP. The lanes have been indicated .</p> | ||
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| + | <h3>2.Protein expression and purification</h3> | ||
| + | <p> | ||
| + | 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 <sup>°</sup> C , 220rpm until OD<sub>600</sub> reached 0.6-0.8 and protein was induced at 8 <sup>o</sup>C 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 . | ||
===User Reviews=== | ===User Reviews=== | ||
Revision as of 06:49, 12 October 2022
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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 8oC
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 .
User Reviews
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