Difference between revisions of "Part:BBa K3815006"
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<partinfo>BBa_K3815006 short</partinfo>
 
<partinfo>BBa_K3815006 short</partinfo>
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This part is made of His tag for peptide production. After producing peptide with His tag, it is recovered by Ni chromatography. Finally, adding DTT, we cut the N terminal of Mxe GyrA intein. Then, we can get the targeted protein.In this part, the targeted protein is CecropinA derived from a moth. This is the antimicrobial peptide that can inhibit the growth of both gram-negative bacteria and gram-positive bacteria. In our experiment, we used it to kill the bacteria in the vase water.
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<h3><font size="4.5">Descriotion of this part</font> </h3>
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<h3><font size="3">Targeted protein</font> </h3>
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This part is for the purfication of antimicrobial peptide, CecropinA. This is derived from <i>Hyalophora cecropia</i>. This can inhibit the growth of both gram-negative bacteria and gram-positive bacteria. In our experiment, we used it to kill the bacteria in the vase water.<br><br>
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<h3><font size="3">Purification system</font> </h3>
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3815006 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3815006 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>
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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 1, 5,and 9 are the result of CecropinA.<br>  CecropinA is 4051Da, so these date shows that we could not confirm CecA production.
  
 
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Revision as of 10:28, 20 October 2021


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

Descriotion of this part

Targeted protein

This part is for the purfication of antimicrobial peptide, CecropinA. This is derived from Hyalophora cecropia. This can inhibit the growth of both gram-negative bacteria and gram-positive bacteria. In our experiment, we used it to kill the bacteria in the vase water.

Purification system

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 67
    Illegal AgeI site found at 346
  • 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 1, 5,and 9 are the result of CecropinA.
CecropinA is 4051Da, so these date shows that we could not confirm CecA production.