Part:BBa_K1915001:Experience

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Applications of BBa_K1915001

After successful sequencing results in DH5α LTNF10 was transformed into BL21 E. coli for protein expression. Once transformed, a 5.0 mL overnight culture was created. The next morning it was subcultured into 300.0 mL of Terrific Broth and 300.0 µL of chloramphenicol. The culture was allowed to grow in a shaker/incubator at 37.0˚C until an inducible OD600 was reached. Then the culture was induced with 1.0 mM IPTG and placed back into the shaker/incubator for 3 hours. Finally, the culture was placed into a 4.0˚C refrigerator overnight.

Figure 1: Coomassie Gel Stain of LTNF10 in BL21 E. coli after Protein Induction. Coomassie gel stain of SDS-PAGE run at 100 volts for 45 minutes. Fraction P3 consisted of sonicated lysate. Fractions F1, Wash 1, Wash 2, Elution 1, 2, 3, and 4 from Ni-NTA column.

Figure 2: Western blot of LT10 in BL21 E. coli after Protein Induction. Western blot of SDS-PAGE with His antibody. Fraction P3 consisted of sonicated lysate. Fractions F1, Wash 1, Wash 2, Elution 1, 2, 3, and 4 from Ni-NTA column.

Elution 2 exhibited the strongest signal after visualization of western blot.

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