Difference between revisions of "Part:BBa K3815004"
 
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<partinfo>BBa_K3815033 short</partinfo>
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<partinfo>BBa_K3815004 short</partinfo>
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This part is used for ELK16 system to purify the peptides. When this fused protein is produced, the part of ELK16 self-assembles and precipitates. After that, taking this aggregate and adding DTT to it, we cut the N terminal of Mxe GyrA intein. Then, we can get the purpose protein. In this part, the purpose protein is NOP1.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.
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==Description of this part==
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<!-- Add more about the biology of this part here
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<h3><font size="3">Targeted protein</font> </h3>
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===Usage and Biology===
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[[File:Ethylene_pathway.png|200px|thumb|right|Fig1. Ethylene pathway]]
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<br><br>
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This part is for the purification of NOP1. This is a peptide known to prolong the life of flowering plants and can inhibit ethylene-dependent senescence. The signaling pathway triggered by the binding of ethylene to ETR1 inactivates CTR1 kinase and inhibits the phosphorylation of the ethylene regulatory factor EIN2, thereby activating the expression of ethylene response genes. Therefore, NOP-1, a peptide derived from the nuclear localization signal of EIN2, can regulate senescence signaling by binding to the GAF domain of ETR1 and arresting intra- and intermolecular downstream signaling of the receptor. In fact, it was confirmed that flower senescence of flowering plants treated with NOP-1 was suppressed.
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[[File:Ethylene_pathway2.png|200px|thumb|right|Fig2. Ethylene pathway]]<br><br><br><br><br><br><br><br><br>
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<h3><font size="3">Purification system</font> </h3>
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[[File:ELK16.png|300px|right|thumb|Fig3.The mechanism of ELK16]]
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In order to purify the peptide, we adopted the 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>
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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'>Sequence and Features</span><br><br><br><br><br><br>
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<partinfo>BBa_K3815033 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3815004 SequenceAndFeatures</partinfo>
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==Purification==
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[[File:Engineering 203 SDS-PAGE①.png|300px|thumb|right|Fig4. SDS-PAGE of purified peptide ]]
  
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<h3><font size="4.5">Expression</font> </h3>
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<ul>
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<li>Cells were grown in 1000ml LB media at 37<sup>o</sup>C shaking at 180 rpm.
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<li>when the OD exceeded 0.35, 1 M IPTG 2ml was added to induce the peptide expression.
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<li>Incubate at 30℃ at 180rpm for 6 hours after adding IPTG.
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</ul>
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<h3><font size="4.5">Purification </font></h3>
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1.When this fused protein were produced, it self-assembled and precipitated<br>
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2.The aggregate was collected by centrifugation.<br>
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3.Adding 40mM DTT to this aggregate, the targeted protein was cut out by the cleavage of intein.<br>
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4.SDSPAGE was performed in order to confirm the presence of it.
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<br>
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<br>
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Fig1 shows the result of SDS-PAGE.
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The lane 4, 8,and 12 are the result of NOP1.<br>  NOP1 is 1132Da, so these date shows that we could not confirm its production.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K3815033 parameters</partinfo>
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<partinfo>BBa_K3815004 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.
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<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.

Latest revision as of 02:10, 22 October 2021


NOP1-Mxe GryA intein-PT-linker-ELK16

Description of this part

Targeted protein

Fig1. Ethylene pathway



This part is for the purification of NOP1. This is a peptide known to prolong the life of flowering plants and can inhibit ethylene-dependent senescence. The signaling pathway triggered by the binding of ethylene to ETR1 inactivates CTR1 kinase and inhibits the phosphorylation of the ethylene regulatory factor EIN2, thereby activating the expression of ethylene response genes. Therefore, NOP-1, a peptide derived from the nuclear localization signal of EIN2, can regulate senescence signaling by binding to the GAF domain of ETR1 and arresting intra- and intermolecular downstream signaling of the receptor. In fact, it was confirmed that flower senescence of flowering plants treated with NOP-1 was suppressed.

Fig2. Ethylene pathway









Purification system

Fig3.The mechanism of ELK16

In order to purify the peptide, we adopted the 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.


Sequence and Features






Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 117
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 117
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 117
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 117
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 117
    Illegal NgoMIV site found at 550
  • 1000
    COMPATIBLE WITH RFC[1000]

Purification

Fig4. SDS-PAGE of purified peptide

Expression

  • Cells were grown in 1000ml LB media at 37oC shaking at 180 rpm.
  • when the OD exceeded 0.35, 1 M IPTG 2ml was added to induce the peptide expression.
  • Incubate at 30℃ at 180rpm for 6 hours after adding IPTG.

Purification

1.When this fused protein were produced, it self-assembled and precipitated
2.The aggregate was collected by centrifugation.
3.Adding 40mM DTT to this aggregate, the targeted protein was cut out by the cleavage of intein.
4.SDSPAGE was performed in order to confirm the presence of it.

Fig1 shows the result of SDS-PAGE. The lane 4, 8,and 12 are the result of NOP1.
NOP1 is 1132Da, so these date shows that we could not confirm its production.

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.