Difference between revisions of "Part:BBa K3815006"
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K3815006 short</partinfo>
 
<partinfo>BBa_K3815006 short</partinfo>
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<h3><font size="4.5">Descriotion of this part</font> </h3>
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==Description of this part==
 
<h3><font size="3">Targeted protein</font> </h3>
 
<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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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><br>
 
<h3><font size="3">Purification system</font> </h3>
 
<h3><font size="3">Purification system</font> </h3>
 +
This part is composed of His tag, Mxe GyrA intein, PT linker, and targeted protein. This is for the peptide intein tag purification. Producing peptide with His tag, it is recovered by Ni chromatography. After that, adding DTT to it, the N terminal of Mxe GyrA intein is cut. Then, we get the targeted protein.
  
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Sequence and Features
 
<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
===Usage and Biology===
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<ul>
 
<ul>
 
<li>Cells were grown in 1000ml LB media at 37<sup>o</sup>C shaking at 180 rpm.
 
<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>when the OD exceeded 0.35, 1 M IPTG 200μL was added to induce the peptide expression.
 
<li>Incubate at 30℃ at 180rpm for 6 hours after adding IPTG.
 
<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.When this fused protein were produced, it self-assembled and precipitated<br>
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1.When this fused protein were produced, it was recovered by Ni chromatography.<br>
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2.The aggregate was collected by centrifugation.<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.Adding 40mM DTT to this aggregate, 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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4.SDSPAGE was performed in order to confirm the presence of it.
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<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 confirm CecA production.
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The lane　1 and 6 are the result of CecropinA.<br>  CecropinA is 4051Da, so these date shows that we could not confirm CecA production.
  
 
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<!-- Uncomment this to enable Functional Parameter display  
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<partinfo>BBa_K3815006 parameters</partinfo>
 
<partinfo>BBa_K3815006 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 02:12, 22 October 2021


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

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

This part is composed of His tag, Mxe GyrA intein, PT linker, and targeted protein. This is for the peptide intein tag purification. Producing peptide with His tag, it is recovered by Ni chromatography. After that, adding DTT to it, the N terminal of Mxe GyrA intein is cut. Then, we get the targeted protein.

Sequence and Features 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 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. The lane　1 and 6 are the result of CecropinA.
CecropinA is 4051Da, so these date shows that we could not confirm CecA 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.