Difference between revisions of "Part:BBa K3815005"

 
 
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<partinfo>BBa_K3815034 short</partinfo>
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<partinfo>BBa_K3815005 short</partinfo>
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==Description of this part==
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<h3><font size="3">Targeted protein</font> </h3>
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This part is for the purification of NOP1v.This is made by adding a valine to the N-terminus of NOP1.The effect of it is the same as NOP1. We could not get NOP1 sufficiently when using ''<partinfo>BBa_K3815004</partinfo>''. Then, we thought that considering N-end-rule, the N-terminus of NOP1 might have a negative effect on the recovery of peptide. Therefore, this part is made.
  
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We used ELK16 system to purify the peptides. When this fused protein is produced, ELK16 self-assembles and precipitates. After that, taking this aggregate and adding DTT to it, the N terminal of Mxe GyrA intein is cut. Then, we can get the purpose protein. In this part, the purpose protein is NOP1v.This is a small peptide that can prevent the plants from accepting ethylene. In our experiment, we used it to inhibit the acceptance of ethylene and aging of cut flowers. We could not get NOP1 sufficiently when using BBa_K3815033, so we added a valine to the N-terminus of NOP1 according to N end rule.
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Sequence and Features<br><br>
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<h3><font size="3">Purification system</font> </h3>
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[[File:ELK16.png|300px|thumb|right|Fig2.The mechanism of ELK16]]
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In order to purify the peptide, we adopted ELK16 system that the past iGEM teams had not used. This part is composed of ELK16, Mxe GyrA intein, and PT linker. When this fused protein is produced, ELK16 self-assembles and precipitates. After that, taking this aggregate and adding DTT to it, the N terminal of Mxe GyrA intein is cut. Then, we can get the targeted protein.<br><br>
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We designed this part, however, we did not actually produce it with this part. We made this peptide with ''<partinfo>BBa_K3815010</partinfo>''.
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3815034 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3815005 SequenceAndFeatures</partinfo>
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K3815034 parameters</partinfo>
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<partinfo>BBa_K3815005 parameters</partinfo>
 
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==Reference==
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1. Hoppen, C., Müller, L., Albrecht, A.C., and Groth, G. (2019). The NOP-1 peptide derived from the central regulator of ethylene signaling EIN2 delays floral senescence in cut flowers. Sci. Rep. 9, 1287<br>
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2.Kessenbrock, M., Klein, S.M., Müller, L., Hunsche, M., Noga, G., and Groth, G. (2017). Novel Protein-Protein Inhibitor Based Approach to Control Plant Ethylene Responses: Synthetic Peptides for Ripening Control. Front. Plant Sci. 8, 1528.
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<br>
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3.Tobias, J.W., Shrader, T.E., Rocap, G., and Varshavsky, A. (1991). The N-end rule in bacteria. Science 254, 1374–1377.<br>

Latest revision as of 18:59, 21 October 2021


NOP1v-Mxe GryA intein-PT-linker-ELK16

Description of this part

Targeted protein

This part is for the purification of NOP1v.This is made by adding a valine to the N-terminus of NOP1.The effect of it is the same as NOP1. We could not get NOP1 sufficiently when using BBa_K3815004. Then, we thought that considering N-end-rule, the N-terminus of NOP1 might have a negative effect on the recovery of peptide. Therefore, this part is made.

Sequence and Features

Purification system

Fig2.The mechanism of ELK16

In order to purify the peptide, we adopted ELK16 system that the past iGEM teams had not used. This part is composed of ELK16, Mxe GyrA intein, and PT linker. When this fused protein is produced, ELK16 self-assembles and precipitates. After that, taking this aggregate and adding DTT to it, the N terminal of Mxe GyrA intein is cut. Then, we can get the targeted protein.

We designed this part, however, we did not actually produce it with this part. We made this peptide with BBa_K3815010.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 120
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 120
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 120
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 120
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 120
    Illegal NgoMIV site found at 553
  • 1000
    COMPATIBLE WITH RFC[1000]


Reference

1. Hoppen, C., Müller, L., Albrecht, A.C., and Groth, G. (2019). The NOP-1 peptide derived from the central regulator of ethylene signaling EIN2 delays floral senescence in cut flowers. Sci. Rep. 9, 1287
2.Kessenbrock, M., Klein, S.M., Müller, L., Hunsche, M., Noga, G., and Groth, G. (2017). Novel Protein-Protein Inhibitor Based Approach to Control Plant Ethylene Responses: Synthetic Peptides for Ripening Control. Front. Plant Sci. 8, 1528.
3.Tobias, J.W., Shrader, T.E., Rocap, G., and Varshavsky, A. (1991). The N-end rule in bacteria. Science 254, 1374–1377.