Difference between revisions of "Part:BBa K3139013"
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<partinfo>BBa_K3139013 short</partinfo> | <partinfo>BBa_K3139013 short</partinfo> | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
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| + | The optimized anti-<i>plasmodium</i> fusion protein contains all the anti-<i>plasmodium</i> peptides uploaded ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K3139000 BBa_K3139000], [https://parts.igem.org/wiki/index.php?title=Part:BBa_K3139002 BBa_K3139002],[https://parts.igem.org/wiki/index.php?title=Part:BBa_K3139003 BBa_K3139003], [https://parts.igem.org/wiki/index.php?title=Part:BBa_K3139004 BBa_K3139004],[https://parts.igem.org/wiki/index.php?title=Part:BBa_K3139005 BBa_K3139005],[https://parts.igem.org/wiki/index.php?title=Part:BBa_K3139006 BBa_K3139006], [https://parts.igem.org/wiki/index.php?title=Part:BBa_K3139007 BBa_K3139007],[https://parts.igem.org/wiki/index.php?title=Part:BBa_K3139008 BBa_K3139008], [https://parts.igem.org/wiki/index.php?title=Part:BBa_K3139009 BBa_K3139009]), TEV protease recognition and cleavage sites ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K3139011 BBa_K3139011]) between each 2 peptide coding domains, and a His-tag is attached on the N-terminal for purification. They were found in some reviews in this field and chosen by our team members as their efficient effect reported. With reports of each sigle peptide, we primarily acquire the anti-<i>plasmodium</i> effect of each peptide. And we finally confirmed its sequence with help from mathmatical model. The copies and types of the peptides were selected elaborately. | ||
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| + | ===Characterize=== | ||
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| + | 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 anti-<i>plasmodium</i> effector fusion protein (75,1kDa) in sample without TEVp is darker than the one in sample with TEVp, which strongly proves that our plasmid could express and be cleaved to produce single peptide.(Fig.1) | ||
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| + | <html> | ||
| + | <img src="https://2019.igem.org/wiki/images/2/2d/T--NAU-CHINA--Basic_Part_1.png"width="300"/> | ||
| + | </html> | ||
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| + | Fig.1 Preliminary verification of the effect of cleavage.<BR> | ||
| + | Lane 1: protein which purified from the sample of bacteria expressing effector and TEVp simultaneously. <BR> | ||
| + | Lane 2: protein which purified from the sample of bacteria expressing Effector only. | ||
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| + | [[File:T--NAU-CHINA--MESS TEV.jpg|500px|Figure.2]] | ||
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| + | Figure.2 Mass spectrometry analysis of cleavaged fusion protein | ||
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| + | The mass spectrometry analysis (Figure.2) shows that His-A, the smallest component of the fusion protein, was detected. | ||
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Latest revision as of 00:12, 22 October 2019
Optimized anti-plasmodium fusion protein
Usage and Biology
The optimized anti-plasmodium fusion protein contains all the anti-plasmodium peptides uploaded (BBa_K3139000, BBa_K3139002,BBa_K3139003, BBa_K3139004,BBa_K3139005,BBa_K3139006, BBa_K3139007,BBa_K3139008, BBa_K3139009), TEV protease recognition and cleavage sites (BBa_K3139011) between each 2 peptide coding domains, and a His-tag is attached on the N-terminal for purification. They were found in some reviews in this field and chosen by our team members as their efficient effect reported. With reports of each sigle peptide, we primarily acquire the anti-plasmodium effect of each peptide. And we finally confirmed its sequence with help from mathmatical model. The copies and types of the peptides were selected elaborately.
Characterize
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 anti-plasmodium effector fusion protein (75,1kDa) in sample without TEVp is darker than the one in sample with TEVp, which strongly proves that our plasmid could express and be cleaved to produce single peptide.(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.
Figure.2 Mass spectrometry analysis of cleavaged fusion protein
The mass spectrometry analysis (Figure.2) shows that His-A, the smallest component of the fusion protein, was detected.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 409
- 1000COMPATIBLE WITH RFC[1000]