Difference between revisions of "Part:BBa K3815001"
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<h3><font size="4.5">Purification </font></h3>
 
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1.<i>E.coli</i> which expressed this part was lysed with sonification.<br>
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1.When this fused protein were produced, it self-assembled and precipitated<br>
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2.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.This aggregate was collected by centrifugation.<br>
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3.Adding DTT to this aggregate, the targeted protein was cut out by the cleavage of intein.  
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4.Adding DTT to this aggregate, the targeted protein was cut out by the cleavage of intein.  
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Revision as of 16:45, 19 October 2021


CecropinA-Mxe GryA intein-PT-linker-ELK16

Usage and Biology

This part is for the purfication of antimicrobial peptide, CecropinA. This is derived from Hyalophora cecropia. The antimicrobial peptide has the 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.
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, we cut the N terminal of Mxe GyrA intein. Then, we can get the targeted protein.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 814
  • 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, IPTG 2ml was added to induce the peptide expression.

Purification

1.When this fused protein were produced, it self-assembled and precipitated
2.The aggregate was collected by centrifugation.
3.Adding DTT to this aggregate, the targeted protein was cut out by the cleavage of intein.

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 purify it sufficiently.