Difference between revisions of "Part:BBa K1915001:Experience"

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===Applications of BBa_K1915001===
 
===Applications of BBa_K1915001===
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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.
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After induction, the culture was centrifuged and then separated into pellet and supernatant fractions. The pellet was then lysed with a lysis buffer and then sonicated. Then the sonicate was centrifuged at 20,000 RPM for 20.0 minutes. The pellet was then resuspended in 1x PBS. Finally, purification of the resuspended pellet was carried out using a Ni-NTA resin column.
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[[File:T--Georgia_State--LT10_in_pSB1C3_restriction_digest_EcoR1_and_Pst1.jpg]]
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'''Figure 1: Gel of inducible lac promoter+LTNF10 in pSB1C3 digested with EcoRI and PstI'''
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''Lanes 1,3,5,7, and 9 contained the plasmid with a single digest using EcoRI''
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''Lanes 2,4,6,8, and 10 contained  the plasmid with a double digest using EcoRI and PstI and the inducible lac promoter + LTNF10 can be seen as the second band''
  
  
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[[File:T--Georgia_State--LT10_Coomassie3.png|none|500px]]
 
[[File:T--Georgia_State--LT10_Coomassie3.png|none|500px]]
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'''Figure 1: Coomassie Gel Stain of LTNF10 in BL21 E. coli after Protein Induction.'''
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'''Figure 2: 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.''
 
''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.''
 
''Fraction P3 consisted of sonicated lysate. Fractions F1, Wash 1, Wash 2, Elution 1, 2, 3, and 4 from Ni-NTA column.''
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[[File:T--Georgia State--LTNF10 Western.png|none|500px]]
 
[[File:T--Georgia State--LTNF10 Western.png|none|500px]]
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'''Figure 2: Western blot of LT10 in BL21 E. coli after Protein Induction.'''
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'''Figure 3: Western blot of LT10 in BL21 E. coli after Protein Induction.'''
 
''Western blot of SDS-PAGE with His antibody.''
 
''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.''
 
''Fraction P3 consisted of sonicated lysate. Fractions F1, Wash 1, Wash 2, Elution 1, 2, 3, and 4 from Ni-NTA column.''

Latest revision as of 17:28, 29 October 2016


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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.

After induction, the culture was centrifuged and then separated into pellet and supernatant fractions. The pellet was then lysed with a lysis buffer and then sonicated. Then the sonicate was centrifuged at 20,000 RPM for 20.0 minutes. The pellet was then resuspended in 1x PBS. Finally, purification of the resuspended pellet was carried out using a Ni-NTA resin column.


T--Georgia State--LT10 in pSB1C3 restriction digest EcoR1 and Pst1.jpg

Figure 1: Gel of inducible lac promoter+LTNF10 in pSB1C3 digested with EcoRI and PstI Lanes 1,3,5,7, and 9 contained the plasmid with a single digest using EcoRI Lanes 2,4,6,8, and 10 contained the plasmid with a double digest using EcoRI and PstI and the inducible lac promoter + LTNF10 can be seen as the second band



T--Georgia State--LT10 Coomassie3.png

Figure 2: 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.



T--Georgia State--LTNF10 Western.png

Figure 3: 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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