Difference between revisions of "Part:BBa K342002:Design"
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===Design Notes===
 
===Design Notes===
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This part has been designed thanks to the results of the publication <A HREF="http://2010.igem.org/Team:INSA-Lyon/Project/References#et1>Banki et al.</A>
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The sequencing of this part is difficult because of the repetition of phasin's sequence.
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<font face="serif" size="3">
  
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<p>This part is a self-cleaving fusion protein. It has been designed thanks to the results of the publication
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<a href="http://2010.igem.org/Team:INSA-Lyon/Project/References#et1"><font color= black><i><u>Banki et al.</i></u></font></a>
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<p>The phasin protein binds to the granule membrane. The second phasin makes the binding stronger and then increases the rate of purified protein during the step of granules extraction. The intein is a self-cleaving protein which breaks under pH=6 and allows the release of the protein of interest fused at the end of the sequence</p>
  
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<p>The prefix biobrick standard used contains a <a href="http://2009.igem.org/Team:Paris/Parts_RBS#bottom"><font color= black><i><u>perfect RBS</u></i></a>. <br/>
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The suffix is a silver standard fusion suffix: <i>5' ACTAGT A GCGGCCG CTGCAG 3'</i>.<br/>
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The phasin sequence is the same as the biobrick <a href="https://parts.igem.org/Part:BBa_K208001"><font color= "black"><i><u>BBa_K208001</u></i></font></a> from the Utah 2009 team.<br/>
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The intein is from the self-cleaving protein sequence delta.I-CM mini intein from <i>mycobacterium tuberculosis</i> described in <a href="http://www.faqs.org/patents/app/20090098611"><font color= black><i><u>this patent</i></u></font></a>. We contacted David W. Wood to confirm the exact sequence they used.<br/>
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The intergenic regions are the same as the one described in <a href="http://2010.igem.org/Team:INSA-Lyon/Project/References#et1"><font color= black><i><u>Banki et al.</i></u></font></a>
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<p>This part should have been synthesized by Mr gene in once but due to technical issues we had to redesign two separated fusion biobricks and ligate them.</p>
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<p>The sequencing of this part confirmed the expected sequence.</p>
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===Source===
 
===Source===
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Phasin protein from <i>Ralstonia eutropha H16</i><br>
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Optimized intein from <i>Mycobacterium tuberculosis (Mtu)</i> recA<br>
 
Ligation of two synthetized parts : Phasin and Phasin-Intein
 
Ligation of two synthetized parts : Phasin and Phasin-Intein
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</html>
  
 
===References===
 
===References===
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"Novel and economical purification of recombinant proteins: Intein-mediated protein purification using in vivo polyhydroxybutyrate (PHB) matrix association", Mahmoud Reza Banki, Tillman U.Gerngross and David W. Wood, 2005, Protein Science, 14:1387–1395

Latest revision as of 17:52, 7 November 2010

Phasin-Phasin Intein (for silver standard fusion)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1693
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1279
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This part is a self-cleaving fusion protein. It has been designed thanks to the results of the publication Banki et al.

The phasin protein binds to the granule membrane. The second phasin makes the binding stronger and then increases the rate of purified protein during the step of granules extraction. The intein is a self-cleaving protein which breaks under pH=6 and allows the release of the protein of interest fused at the end of the sequence

The prefix biobrick standard used contains a perfect RBS.
The suffix is a silver standard fusion suffix: 5' ACTAGT A GCGGCCG CTGCAG 3'.
The phasin sequence is the same as the biobrick BBa_K208001 from the Utah 2009 team.
The intein is from the self-cleaving protein sequence delta.I-CM mini intein from mycobacterium tuberculosis described in this patent. We contacted David W. Wood to confirm the exact sequence they used.
The intergenic regions are the same as the one described in Banki et al.

This part should have been synthesized by Mr gene in once but due to technical issues we had to redesign two separated fusion biobricks and ligate them.

The sequencing of this part confirmed the expected sequence.

Source

Phasin protein from Ralstonia eutropha H16
Optimized intein from Mycobacterium tuberculosis (Mtu) recA
Ligation of two synthetized parts : Phasin and Phasin-Intein

References

"Novel and economical purification of recombinant proteins: Intein-mediated protein purification using in vivo polyhydroxybutyrate (PHB) matrix association", Mahmoud Reza Banki, Tillman U.Gerngross and David W. Wood, 2005, Protein Science, 14:1387–1395