Difference between revisions of "Part:BBa K3815004"
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3815004 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3815004 SequenceAndFeatures</partinfo>
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==Purification==
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[[File:Engineering 203 SDS-PAGE①.png|300px|thumb|right|Fig1. SDS-PAGE of purified peptide ]]
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<h3><font size="4.5">Expression</font> </h3>
 +
<ul>
 +
<li>Cells were grown in 1000ml LB media at 37<sup>o</sup>C shaking at 180 rpm.
 +
<li>when the OD exceeded 0.35, 1 M IPTG 2ml was added to induce the peptide expression.
 +
<li>Incubate at 30℃ at 180rpm for 6 hours after adding IPTG.
 +
</ul>
 +
<h3><font size="4.5">Purification </font></h3>
 +
1.When this fused protein were produced, it self-assembled and precipitated<br>
 +
2.The aggregate was collected by centrifugation.<br>
 +
3.Adding 40mM DTT to this aggregate, the targeted protein was cut out by the cleavage of intein.<br>
 +
4.SDSPAGE was performed in order to confirm the presence of it.
 +
<br>
 +
<br>
 +
Fig1 shows the result of SDS-PAGE.
 +
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  

Revision as of 10:05, 20 October 2021


NOP1-Mxe GryA intein-PT-linker-ELK16

Descriotion of this part

Targeted protein

This part is for the purfication of the small peptide, NOP1. It can prevent the plants from accepting ethylene. In our experiment, we used it to inhibit the acceptance of ethylene that promotes the wilting of a plant and aging of cut flowers.

Purification system

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 Gyr 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.


Usage and Biology

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

Fig1. 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.