Difference between revisions of "Part:BBa K3815001"
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K3815001 short</partinfo>
 
<partinfo>BBa_K3815001 short</partinfo>
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==Usage and Biology==
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==Description of this part==
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This part is for the purfication of antimicrobial peptide, CecropinA. This is derived from <i>Hyalophora cecropia</i>. 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.<br>
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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 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.<br><br>
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<h3><font size="3">Targeted protein</font> </h3>
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This part is for the purification of the 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>
 +
 
 +
<h3><font size="3">Purification system</font> </h3>
 +
[[File:ELK16.png|300px|right|thumb|Fig1.The mechanism of ELK16]]
 +
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>
 +
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
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==Purification==
 
==Purification==
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[[File:Engineering 203 SDS-PAGE①.png|300px|thumb|right|Fig1. SDS-PAGE of purified peptide ]]
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[[File:Engineering 203 SDS-PAGE①.png|300px|thumb|right|Fig2. SDS-PAGE of purified peptide ]]
 
<h3><font size="4.5">Expression</font> </h3>
 
<h3><font size="4.5">Expression</font> </h3>
 
<ul>
 
<ul>
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<li>Cells were grown in 1000ml LB media at 37<sup>o</sup>C.
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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, IPTG was added to induce the peptide expression.
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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.
 
</ul>
 
</ul>
 
<h3><font size="4.5">Purification </font></h3>
 
<h3><font size="4.5">Purification </font></h3>
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1.<i>E.coli</i> which expressed this part were 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 are produced,it self-assembles and precipitates.<br>
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2.The aggregate was collected by centrifugation.<br>
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3.This aggregate is 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.Adding DTT to this aggregate, the targeted protein is cut out by the cleavage of intein.  
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4.SDSPAGE was performed in order to confirm the presence of it.
 
<br>
 
<br>
 
<br>
 
<br>
 
Fig1 shows the result of SDS-PAGE.
 
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 purify it sufficiently.
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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 its production.
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K3815001 parameters</partinfo>
 
<partinfo>BBa_K3815001 parameters</partinfo>
 
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==Reference==
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1.Steiner, H., Andreu, D., and Merrifield, R.B. (1988). Binding and action of cecropin and cecropin analogues: antibacterial peptides from insects. Biochim. Biophys. Acta 939, 260–266.

Latest revision as of 01:58, 22 October 2021


CecropinA-Mxe GryA intein-PT-linker-ELK16

Description of this part

Targeted protein

This part is for the purification of the 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

Fig1.The mechanism of ELK16

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.


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

Fig2. 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 its production.

Reference

1.Steiner, H., Andreu, D., and Merrifield, R.B. (1988). Binding and action of cecropin and cecropin analogues: antibacterial peptides from insects. Biochim. Biophys. Acta 939, 260–266.