Difference between revisions of "Part:BBa K3815004"
 
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This part is for the purfication 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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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.
 
[[File:Ethylene_pathway2.png|200px|thumb|right|Fig2. Ethylene pathway]]<br><br><br><br><br><br><br><br><br>
 
[[File:Ethylene_pathway2.png|200px|thumb|right|Fig2. Ethylene pathway]]<br><br><br><br><br><br><br><br><br>
 
<h3><font size="3">Purification system</font> </h3>
 
<h3><font size="3">Purification system</font> </h3>
 
[[File:ELK16.png|300px|right|thumb|Fig3.The mechanism of ELK16]]
 
[[File:ELK16.png|300px|right|thumb|Fig3.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>
+
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>
  
  

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.