Difference between revisions of "Part:BBa K3815010"

 
 
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<partinfo>BBa_K3815039 short</partinfo>
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<partinfo>BBa_K3815010 short</partinfo>
  
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We used His tag to purify the peptides. The peptide His-tag fused was recovered by Ni chromatography, and DTT was added to cleave the N-terminus of the intein.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_K3815038, so we added a valine to the N-terminus of NOP1 according to N end rule.
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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_K3815009</partinfo>''. Then, we thought that considering N-end-rule, the N-terminus of NOP1 might have a negative effect on the recovery of peptides. Therefore, this part is made.
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<h3><font size="3">Purification system</font> </h3>
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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>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3815039 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3815010 SequenceAndFeatures</partinfo>
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==Purification==
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[[File:Engineering 204 SDS-PAGE②.png|300px|thumb|right|Fig1. 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℃ shaking at 180 rpm.
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<li>when the OD exceeded 0.35, 1 M IPTG 200μL 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 was recovered by Ni chromatography.<br>
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2.Adding DTT to it, the targeted protein was cut out by the cleavage of intein.<br>
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3.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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Lane 5 and 11 are the result of NOP1v.<br> NOP1v is 1231Da, so this data shows that we could not confirm its production.
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K3815039 parameters</partinfo>
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<partinfo>BBa_K3815010 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.Tobias, J.W., Shrader, T.E., Rocap, G., and Varshavsky, A. (1991). The N-end rule in bacteria. Science 254, 1374–1377.

Latest revision as of 02:15, 22 October 2021


NOP1v-Mxe GryA intein-PT-linker-His tag

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_K3815009. Then, we thought that considering N-end-rule, the N-terminus of NOP1 might have a negative effect on the recovery of peptides. Therefore, this part is made.

Purification system

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 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]

Purification

Fig1. SDS-PAGE of purified peptide

Expression

  • Cells were grown in 1000ml LB media at 37℃ shaking at 180 rpm.
  • when the OD exceeded 0.35, 1 M IPTG 200μL 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 was recovered by Ni chromatography.
2.Adding DTT to it, the targeted protein was cut out by the cleavage of intein.
3.SDSPAGE was performed in order to confirm the presence of it.

Fig1 shows the result of SDS-PAGE. Lane 5 and 11 are the result of NOP1v.
NOP1v is 1231Da, so this data 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.Tobias, J.W., Shrader, T.E., Rocap, G., and Varshavsky, A. (1991). The N-end rule in bacteria. Science 254, 1374–1377.