Part:BBa_K1033902:Experience
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Applications of BBa_K1033902
1.Construction, transformation, and validation of tsPurple protein
The designed plasmid of pET29a(+)-tsPurple and pET29a(+)-meffBlue were synthesized by SnapGene. We transformed the synthetic plasmids into BL21 E. coli.
The plasmid of pET29a(+)-tsPurple and pET29a(+)-meffBlue were extracted using AxyPerp Fast Plasmid Miniprep Kit. The coding gene of the tsPurple protein and the amplified by using the recombinant plasmid pET29a(+)-tsPurple as a template and tsPurple-F and tsPurple-R as primers. The coding gene of the meffBlue protein and the amplified by using the recombinant plasmid pET29a(+)-meffBlue as a template and meffBlue-F and meffBlue-R as primers. The plasmid PCR results were verified by agarose gel electrophoresis.
Conclusion:
BL21-pET29a(+)-tsPurple recombinant plasmid was guided into BL21 E.coli successfully(682bp).
BL21-pET29a(+)-meffBlue recombinant plasmid was guided into BL21 E.coli successfully(660bp).
2. Expression, collection and purification of our target proteins
Fermentation broth for inducing protein expression by adding IPTG for tsPurple and meffBlue. After 24 hours of inducing protein expression, we centrifuged the fermentation broth and collected the bacteria.
Conclusion:
The cell pellet was collected by harvesting 50mL culture after 24h of induction followed by
centrifugation at 4 degrees and 6000 rpm for 10min. The bacterial cells of the tsPurple protein and the meffBlue
protein showed obvious color, indicating that the protein expression level was high.
After that, we performed ultrasonic disruption, collected the supernatant after centrifugation, used two 10KDa membranes to perform ultrafiltration to purify the protein, then performed nickel affinity chromatography to further purify the protein, and finally performed ultrafiltration to concentrate the protein.
We perform SDS-PAGE gel electrophoresis on the tsPurple protein and the meffBlue protein to detect the protein purification effect and observe the protein expression. Next, we did the Time of Flight Mass Spectrometer for the tsPurple protein samples and the meffBlue protein samples. We used the BCA protein assay to determine the concentration of the tsPurple protein and the meffBlue protein. Finally, we used Swiss-Model to model the three-dimensional structure of the tsPurple protein and the meffBlue protein.
1· tsPurple- The culture without IPTG induction.
2· tsPurple- Supernatant without IPTG induction after sonication.
3· tsPurple- The sedimentation without IPTG induction and after sonication.
4· tsPurple- Supernatant sample without IPTG induction after sonication.
5· tsPurple- The sedimentation after IPTG induction and ultrasound.
6· tsPurple- The culture after IPTG induction.
7· tsPurple- Protein sample after the ultrafiltration (diluted 5 times).
8. tsPurple- Protein after GST affinity chromatography.
9· tsPurple- Purified protein sample.
1· meffBlue- The culture without IPTG induction.
2·meffBlue - Supernatant without IPTG induction after sonication.
3· meffBlue - The sedimentation without IPTG induction and after sonication.
4· meffBlue - Supernatant sample without IPTG induction after sonication.
5· meffBlue - The sedimentation after IPTG induction and ultrasound.
6· meffBlue - The culture after IPTG induction.
7· meffBlue - Protein sample after the ultrafiltration (diluted 5 times).
8. meffBlue - Protein after GST affinity chromatography.
9·meffBlue - Purified protein sample.
Conclusion:
The protein gel preliminarily proved that the molecular mass of the tsPurple protein and the meffBlue protein
were correct, which one is consistent with the expected molecular mass of tsPurple protein (the molecular mass
of tsPurple protein is about 26.9 kDa).,and the other one is consistent with the expected molecular mass of
meffBlue protein (the molecular mass of meffBlue protein is about 26.3 kDa).Compared with lane 5, 6, and 7,
lanes 1, 2, 3 and 4 indicate that more tsPurple protein and more meffBlue protein can be obtained with IPTG
induction. As is shown in lane 8, the concentration of protein was increased after ultrafiltration
concentration. Lane 9 shows that the purification effect of protein after nickel affinity chromatography was
better, and the impurity protein was less than before affinity chromatography. In conclusion, it can be seen
that our expression and purification strategy is effective.
Conclusion:
We performed a Time of Flight Mass Spectrometer on the purified HIS-tagged tsPurple protein. The predicted
molecular mass of this protein is about 26.9KDa. The result of TOF-Mass Spectrometry showed that the specific
molecular mass of tsPurple protein is 26.27Da (the value of the sharpest peak is shown as the molecular mass of
tsPurple protein).
Conclusion:
We performed a Time of Flight Mass Spectrometer on the purified HIS-tagged meffBlue protein. The predicted molecular mass of this protein is about 26.3KDa. The result of TOF-Mass Spectrometry showed that the specific molecular mass of meffBlue protein is 26.27Da (the value of the sharpest peak is shown as the molecular mass of meffBlue protein).
We used the ultramicro spectrophotometer to measure the concentration of tsPurple protein. The concentration of tsPurple samples was 0.7533 mg/ml.
We used the ultramicro spectrophotometer to measure the concentration of meffBlue protein. The concentration of meffBlue samples was 1.42mg/ml.
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Conclusion:
We used Swiss-Model to simulate the three-dimensional structure of meffBlue protein. The above figures showed the modeling result of Swiss-Model.
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Conclusion:
We used Swiss-Model to simulate the three-dimensional structure of tsPurple protein. The above figures showed the modeling result of Swiss-Model.
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