Difference between revisions of "Part:BBa K3139016"

 
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===Usage and Biology===
 
===Usage and Biology===
  
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In the anti-plasmodium system of NAU-2019, the amount of both TEV protease [https://parts.igem.org/wiki/index.php?title=Part:BBa_K3139012 BBa_K3139012]and anti-plasmodium fusion protein [https://parts.igem.org/wiki/index.php?title=Part:BBa_K3139013 BBa_K3139013]should be kept at a certain proportion to avoid a waste of cell resources.
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If the TEV protease is excessive, it shall be wasted though the fusion proteins are cleaved completely. If the fusion protein is excessive, it cannot be cleaved completely with insufficient TEV protease.
 +
 +
By testing different promoter combinations of TEVp and fusion protein, we tried to find out the most economical combination which has the least waste and most complete cleavage with the help of mathematic model.
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To measure, we planned to replace the promoter of TEVp, and in this part we chose promoter J23111, and acquired a middle effect among all 3 promoters.
 
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===Characterization of BBa_K3139016===
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====Background====
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In our design, we described an anti-Plasmodium system with TEV protease cleavage system (Fig.1), anti-Plasmodium fusion protein system and the HasA secretion system. In the anti-Plasmodium system of NAU-2019, the amount of both TEV protease and anti-Plasmodium fusion protein should be kept at a certain proportion to avoid a waste of cell resources.
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[[File:T--NAU-CHINA--promoter.jpg|500px|Figure.1]]
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'''Figure.1 Recombinant plasmid used'''
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If the TEV protease is excessive, it shall be wasted though the fusion proteins are cleaved completely. If the fusion protein is excessive, it cannot be cleaved completely with insufficient TEV protease.
 +
 +
By testing different promoter combinations of TEVp and fusion protein, we tried to find out the most economical combination which has the least waste and most complete cleavage with the help of mathematic model.
 +
 +
====Characterization====
 +
 +
[[File:T--NAU-CHINA--system.jpg|500px|Figure.2]]
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'''Figure.2 Mechanism of anti-Plasmodium system'''
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'''1.Anti-Plasmodium fusion protein.'''
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'''2.TEV protease with hasA signal peptide, unfolded and nonfunctioning.'''
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'''3.1 and 2are secreted by has secretion complex.'''
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'''4.Anti-Plasmodium fusion protein is cleaved by TEV protease and turns functioning.'''
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'''5.HasA signal peptide is cutted off, the TEV protease is completely functioning. The protease is mostly folded and functioning when it is secreted out of the cell.'''
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We constructed 3 recombinant plasmids (Fig.2). To obtain the best cleavage efficiency, we planned to replace the promoter of TEVp, observing the change of cleavage efficiency, and finally chose the most suitable promoter. We selected three promoters: J23111，J23110 and J23106, from the family of constitutive promoter parts, to construct 3 plasmids. We transformed these three plasmids into S. marcescens JCM11315, and cultured it in LB medium for 60h to obtain supernatant after ultrasonication. Then we purified our target His tagged protein by using Ni-NTA magnetic beads.
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====Results====
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The WB result shows several bands(Fig.3). We defined the smallest monomer protein band (7.2kd) completely produced by cutting as the representative of cutting effect, and the band of TEVp (29.5kDa) represents the promoter strength of TEVp. From the grayscale of the TEVp and monomer protein bands, we find out the cutting effect of the strongest promoter J23111 was weak than the second strong promoter J23106. It’s also supporting the results of our model that changing the strength of promoters can change the cutting effect and the concentration of single effectors in extracellular, providing theoretical support for the practical application of our project.
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[[File:T--NAU-CHINA--Collection 1.jpg|500px|Figure.3]]
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'''Figure.3 Cleavage effect under three promoters of TEVp.'''
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'''Lane 1: protein purified from supernatant of bacteria liquid expressing TEVp with promoter J23110. '''
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'''Lane 2: protein purified from supernatant of bacteria liquid expressing TEVp with promoter J23111.'''
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'''Lane 3: protein purified from supernatant of bacteria liquid expressing TEVp with promoter J23106.'''
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3139016 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3139016 SequenceAndFeatures</partinfo>

Revision as of 19:12, 21 October 2019


J23100-fusion protein-B0015-J23111-TEVp-TEVp site-HasA-B0010



Characterization of BBa_K3139016

Background

In our design, we described an anti-Plasmodium system with TEV protease cleavage system (Fig.1), anti-Plasmodium fusion protein system and the HasA secretion system. In the anti-Plasmodium system of NAU-2019, the amount of both TEV protease and anti-Plasmodium fusion protein should be kept at a certain proportion to avoid a waste of cell resources.

Figure.1 Figure.1 Recombinant plasmid used

If the TEV protease is excessive, it shall be wasted though the fusion proteins are cleaved completely. If the fusion protein is excessive, it cannot be cleaved completely with insufficient TEV protease.

By testing different promoter combinations of TEVp and fusion protein, we tried to find out the most economical combination which has the least waste and most complete cleavage with the help of mathematic model.

Characterization

Figure.2 Figure.2 Mechanism of anti-Plasmodium system

1.Anti-Plasmodium fusion protein.

2.TEV protease with hasA signal peptide, unfolded and nonfunctioning.

3.1 and 2are secreted by has secretion complex.

4.Anti-Plasmodium fusion protein is cleaved by TEV protease and turns functioning.

5.HasA signal peptide is cutted off, the TEV protease is completely functioning. The protease is mostly folded and functioning when it is secreted out of the cell.

We constructed 3 recombinant plasmids (Fig.2). To obtain the best cleavage efficiency, we planned to replace the promoter of TEVp, observing the change of cleavage efficiency, and finally chose the most suitable promoter. We selected three promoters: J23111，J23110 and J23106, from the family of constitutive promoter parts, to construct 3 plasmids. We transformed these three plasmids into S. marcescens JCM11315, and cultured it in LB medium for 60h to obtain supernatant after ultrasonication. Then we purified our target His tagged protein by using Ni-NTA magnetic beads.

Results

The WB result shows several bands(Fig.3). We defined the smallest monomer protein band (7.2kd) completely produced by cutting as the representative of cutting effect, and the band of TEVp (29.5kDa) represents the promoter strength of TEVp. From the grayscale of the TEVp and monomer protein bands, we find out the cutting effect of the strongest promoter J23111 was weak than the second strong promoter J23106. It’s also supporting the results of our model that changing the strength of promoters can change the cutting effect and the concentration of single effectors in extracellular, providing theoretical support for the practical application of our project.

Figure.3 Figure.3 Cleavage effect under three promoters of TEVp.

Lane 1: protein purified from supernatant of bacteria liquid expressing TEVp with promoter J23110.

Lane 2: protein purified from supernatant of bacteria liquid expressing TEVp with promoter J23111.

Lane 3: protein purified from supernatant of bacteria liquid expressing TEVp with promoter J23106.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 2232
    Illegal NheI site found at 2255
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 444
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 2582
    Illegal SapI.rc site found at 2930