Difference between revisions of "Part:BBa K3139012:Experience"
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===Applications of BBa_K3139012=== | ===Applications of BBa_K3139012=== | ||
| + | We transferred the plasmid into BL21 strain and cultured it in LB medium to obtain supernatant after ultrasonication. Then we purified our target His-tagged protein by using Ni-NTA magnetic beads. It was predicted that TEVp cleavage would not be so complete that nine different sizes of protein bands and TEVp's own bands would appear. In the preliminary experiment, we electrophoresised the purified protein on sodium dodecyl sulfate (SDS) -12.5% (wt/vol) polyacrylamide gel, followed by Western Blot to verify the expression and cleavage effect of this plasmid in the bacteria. In the result, the band of fusion protein (75,1kDa) in sample with TEVp is deeper than the one in sample without TEVp, which strongly proves that our plasmid could express and cleave to produce proteins.(Fig.1) | ||
| + | |||
| + | Fig.1 Preliminary verification of the effect of cleavage. | ||
| + | Lane 1: protein which purified from the sample of bacteria expressing effector and TEVp simultaneously. Lane 2: protein which purified from the sample of bacteria expressing Effector only. | ||
| + | |||
| + | Comparing the results of fresh bacteria liquid and the results of bacteria liquid stored at 4 degrees for 4 days, we surprisingly find that : Both samples have the obvious band of TEVp （29.5kDa）.However, the band of large protein existed only in the fresh sample, not in the sample stored for 4 day while the smallest monomer protein (7.9kDa) produced by complete cleavage exited only in the sample stored for 4 day, which suggests that the second time cut was more complete than the first one (Fig.2). This result proves that TEVp was still able to perform the cutting function efficiently even at 4 °C. | ||
| + | |||
| + | Fig.2 Verification of the efficiency of cleavage. | ||
| + | Left: protein purified from sample of fresh engineered bacteria. Right: protein purified from sample of engineered bacteria which have been stored in 4℃ for 4 days. | ||
===User Reviews=== | ===User Reviews=== | ||
Revision as of 00:26, 21 October 2019
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Applications of BBa_K3139012
We transferred the plasmid into BL21 strain and cultured it in LB medium to obtain supernatant after ultrasonication. Then we purified our target His-tagged protein by using Ni-NTA magnetic beads. It was predicted that TEVp cleavage would not be so complete that nine different sizes of protein bands and TEVp's own bands would appear. In the preliminary experiment, we electrophoresised the purified protein on sodium dodecyl sulfate (SDS) -12.5% (wt/vol) polyacrylamide gel, followed by Western Blot to verify the expression and cleavage effect of this plasmid in the bacteria. In the result, the band of fusion protein (75,1kDa) in sample with TEVp is deeper than the one in sample without TEVp, which strongly proves that our plasmid could express and cleave to produce proteins.(Fig.1)
Fig.1 Preliminary verification of the effect of cleavage. Lane 1: protein which purified from the sample of bacteria expressing effector and TEVp simultaneously. Lane 2: protein which purified from the sample of bacteria expressing Effector only.
Comparing the results of fresh bacteria liquid and the results of bacteria liquid stored at 4 degrees for 4 days, we surprisingly find that : Both samples have the obvious band of TEVp （29.5kDa）.However, the band of large protein existed only in the fresh sample, not in the sample stored for 4 day while the smallest monomer protein (7.9kDa) produced by complete cleavage exited only in the sample stored for 4 day, which suggests that the second time cut was more complete than the first one (Fig.2). This result proves that TEVp was still able to perform the cutting function efficiently even at 4 °C.
Fig.2 Verification of the efficiency of cleavage. Left: protein purified from sample of fresh engineered bacteria. Right: protein purified from sample of engineered bacteria which have been stored in 4℃ for 4 days.
User Reviews
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