Difference between revisions of "Part:BBa K3815008"
 
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<partinfo>BBa_K3815037 short</partinfo>
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<partinfo>BBa_K3815008 short</partinfo>
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This part is made of Hist tag for the peptide production. After producing peptide with His tag, it is recovered by Ni chromatography. Finally, adding DTT to it, we cut the N terminal of Mxe GyrA intein. Then, we can get the targeted protein.In this part, the purpose protein is LL37 derived from human. This is 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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==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 antimicrobial peptide,LL37. This is derived from <i>Homo sapiens</i>. This can inhibit the growth of both the gram-positive and gram-negative 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. This method saves the time removing the tags compared to the method using only tags without intein.
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3815037 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3815008 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℃  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 3 and 9 are the result of LL37.<br> LL37 is 4624Da, so these date shows that we could not confirm its production.
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K3815037 parameters</partinfo>
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<partinfo>BBa_K3815008 parameters</partinfo>
 
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==Reference==
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1.Ouhara, K., Komatsuzawa, H., Yamada, S., Shiba, H., Fujiwara, T., Ohara, M., Sayama, K., Hashimoto, K., Kurihara, H., and Sugai, M. (2005). Susceptibilities of periodontopathogenic and cariogenic bacteria to antibacterial peptides, {beta}-defensins and LL37, produced by human epithelial cells. J. Antimicrob. Chemother. 55, 888–896.
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<br>
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2.https://parts.igem.org/Part:BBa_K1162006

Latest revision as of 19:01, 21 October 2021


LL37-Mxe GryA intein-PT-linker-His tag

Description of this part

Targeted protein

This part is for the purification of antimicrobial peptide,LL37. This is derived from Homo sapiens. This can inhibit the growth of both the gram-positive and gram-negative 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. This method saves the time removing the tags compared to the method using only tags without intein.

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 637
    Illegal AgeI site found at 130
    Illegal AgeI site found at 331
    Illegal AgeI site found at 765
  • 1000
    COMPATIBLE WITH RFC[1000]

Purification

Fig1. SDS-PAGE of purified peptide

Expression

  • Cells were grown in 1000ml LB media at 37℃ 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 3 and 9 are the result of LL37.
LL37 is 4624Da, so these date shows that we could not confirm its production.

Reference

1.Ouhara, K., Komatsuzawa, H., Yamada, S., Shiba, H., Fujiwara, T., Ohara, M., Sayama, K., Hashimoto, K., Kurihara, H., and Sugai, M. (2005). Susceptibilities of periodontopathogenic and cariogenic bacteria to antibacterial peptides, {beta}-defensins and LL37, produced by human epithelial cells. J. Antimicrob. Chemother. 55, 888–896.
2.https://parts.igem.org/Part:BBa_K1162006