Difference between revisions of "Part:BBa K4202009:Experience"
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===Applications of BBa_K4202009=== | ===Applications of BBa_K4202009=== | ||
| − | <h3>Protrein expression verification</h3> | + | <h3>1.Protrein expression verification</h3> |
<p>To confirm that the optimized sequence could be produced by <i>E. coli</i>, we transformed the plasmid pET-28a (+)-ACCBP into BL21 (DE3) strain and cultured them in kanamycin resistant LB plates at 37 <sup>o</sup> 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 OD<sub>600</sub> reached 0.6-0.8. Then 0.1 mM IPTG, 10% glycerol and 1mM CaCl<sub>2</sub> was added to induce the protein expression at 8<sup>o</sup>C</p> | <p>To confirm that the optimized sequence could be produced by <i>E. coli</i>, we transformed the plasmid pET-28a (+)-ACCBP into BL21 (DE3) strain and cultured them in kanamycin resistant LB plates at 37 <sup>o</sup> 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 OD<sub>600</sub> reached 0.6-0.8. Then 0.1 mM IPTG, 10% glycerol and 1mM CaCl<sub>2</sub> was added to induce the protein expression at 8<sup>o</sup>C</p> | ||
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https://static.igem.wiki/teams/4202/wiki/m1-accbp.png | https://static.igem.wiki/teams/4202/wiki/m1-accbp.png | ||
| − | <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><<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> |
===User Reviews=== | ===User Reviews=== | ||
Revision as of 06:39, 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 .
User Reviews
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