Difference between revisions of "Part:BBa K3815006"
 
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<partinfo>BBa_K3815035 short</partinfo>
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<partinfo>BBa_K3815006 short</partinfo>
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This part is used His tag to purify the peptides. 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 purpose protein.In this part, the purpose 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. This sequence got from the moth. In our experiment, we used it to kill the bacteria in the vase water.
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==Description of this part==
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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><br>
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<h3><font size="3">Purification system</font> </h3>
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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
 
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===Usage and Biology===
 
===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3815035 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3815006 SequenceAndFeatures</partinfo>
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==Purification==
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[[File:Engineering 204 SDS-PAGE②.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 200μL 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 was recovered by Ni chromatography.<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.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 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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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K3815035 parameters</partinfo>
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<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.