Difference between revisions of "Part:BBa K3815003"
 
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<partinfo>BBa_K3815032 short</partinfo>
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<partinfo>BBa_K3815003 short</partinfo>
  
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This part is used for ELK16 system to purify the peptides. When this fused protein is produced, the part of 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 purpose 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 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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[[File:ELK16.png|300px|right|thumb|Fig1.The mechanism of ELK16]]
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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 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>
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3815032 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3815003 SequenceAndFeatures</partinfo>
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[[File:Engineering 203 SDS-PAGE①.png|300px|thumb|right|Fig2. 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 2ml 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 self-assembled and precipitated<br>
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2.The aggregate was 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.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, 7,and 11 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_K3815032 parameters</partinfo>
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<partinfo>BBa_K3815003 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 18:41, 21 October 2021


LL37-Mxe GryA intein-PT-linker-ELK16

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 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
    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
  • 1000
    COMPATIBLE WITH RFC[1000]
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 3, 7,and 11 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