Team:Bordeaux/Parts/Assembly improvement

From 2014.igem.org

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#* Not compatible with RFC12 standard
#* Not compatible with RFC12 standard
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<title>Correction TD1</title>
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<a HREF="page2.html">return to project presentation</a>
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<table border="0" width="80%" cellspacing="4" bordercolor=blue>
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<TD> <img src="https://static.igem.org/mediawiki/2014/d/de/Bdx2014_Image1.png.jpg" alt="error" WIDTH=380 HEIGHT=1500></TD>
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<TD> <h1>Consensus sequences</h1>
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In the literature we read that the natural elastin got some repetition, it notice a sequence VPGXG repeated, which X is V, L, or A, it represent 9% of the whole peptide. This particular sequence were translate into DNA to make (VPGXG)x protein to know if keeping elastin properties, thus cloned into bacteria to make recombinant protein, elastin like polypeptide (ELP). We study the original peptide sequences of these proteins to bring out some consensus sequences. Then it revealed a UCST and LCST (Link to the purification page) that give ELP a transition temperature with interesting particularities (link to the transition temperature part), it’s more soluble when chilled and contrary it precipitated.
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<img src="https://static.igem.org/mediawiki/2014/5/5c/Bdx2014_Image2.png.jpg" alt="error" WIDTH=380 HEIGHT=800>
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On the same way we search others elastic proteins and find spider silks and resilin.
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<p>
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For the resilin we use the exon 1 from Drosophila melanogaster2. We identify a repetitive pattern, PSXXYGAP. So we blast it to know if the pattern were conserved into different species or specific to Drosophila.
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<img src="https://static.igem.org/mediawiki/2014/7/77/Bdx2014_image3.jpg" alt="error" WIDTH=900 HEIGHT=700>
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We take XX randomly and we bring out conserved pattern so we use it to do our resilin like polypeptide (RLP) MW-[(PSSSYGAP)(PSNSYGAP)(PSTSYGAP)(PVAYGAP)]3
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<p>
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The literature delivers some physical information3 concerning native resilin as high resilience (<90%) that permit to the protein to recover it initial shape after a high tensile stress. Thus it can be an interesting characteristic to show, so we wanted to test it on our RLP.
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<p>
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Keep following our idea we do the same on spider silks. We previously identify consensus sequence, GPGQQ, GPGGY and GPGGX. Later we find in literature4 confirmation about our consensus sequences. And we design our SLP protein, MW-[(GPGGV)2(GPGQQ)(GPGGY)]5.
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We first wanted to synthesize our SLP gene ourselves : Partie synthèse du gene
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<AREA href="page2.html" shape="rect" coords="30,80,140,140"  alt="consensus sequences">
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<AREA href="page2.html" shape="rect" coords="290,50,390,110"  alt="SLP gene synthesis">
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<AREA href="page2.html" shape="rect" coords="30,300,110,130"  alt="clonage and transformation">
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<AREA href="page2.html" shape="rect" coords="30,300,110,130"  alt="fusion protein">
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<AREA href="page2.html" shape="rect" coords="30,300,110,130"  alt="production">
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<AREA href="page2.html" shape="rect" coords="30,30,110,130"  alt="purification!">
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<AREA href="page2.html" shape="rect" coords="30,30,110,130"  alt="lyophilisation">
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<AREA href="page2.html" shape="rect" coords="30,300,110,130"  alt="characterization">
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<AREA href="page2.html" shape="rect" coords="30,30,110,130"  alt="in solution">
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{{:Team:Bordeaux/Pied}}
{{:Team:Bordeaux/Pied}}

Revision as of 21:27, 17 October 2014

We had the idea to add a new restriction site compatible with XbaI before the stop codon. This restriction site is NheI and allowed us to produce a fused protein if the 2 inserts were cloned together following this protocol:

Bdx2014 Protein fusion.png

  1. Other advantages of NheI:
    • heated-deactivation
    • compatibility with most of the others Biobricks (RFC10, 21, 23, 25, 1000)
    • obtaining a fused protein and allowing to choose the order of the genes
  2. Disadvantages of NheI:
    • Not compatible with RFC12 standard

Correction TD1 return to project presentation

error

Consensus sequences

In the literature we read that the natural elastin got some repetition, it notice a sequence VPGXG repeated, which X is V, L, or A, it represent 9% of the whole peptide. This particular sequence were translate into DNA to make (VPGXG)x protein to know if keeping elastin properties, thus cloned into bacteria to make recombinant protein, elastin like polypeptide (ELP). We study the original peptide sequences of these proteins to bring out some consensus sequences. Then it revealed a UCST and LCST (Link to the purification page) that give ELP a transition temperature with interesting particularities (link to the transition temperature part), it’s more soluble when chilled and contrary it precipitated.

error

On the same way we search others elastic proteins and find spider silks and resilin.

For the resilin we use the exon 1 from Drosophila melanogaster2. We identify a repetitive pattern, PSXXYGAP. So we blast it to know if the pattern were conserved into different species or specific to Drosophila.

error

We take XX randomly and we bring out conserved pattern so we use it to do our resilin like polypeptide (RLP) MW-[(PSSSYGAP)(PSNSYGAP)(PSTSYGAP)(PVAYGAP)]3

The literature delivers some physical information3 concerning native resilin as high resilience (<90%) that permit to the protein to recover it initial shape after a high tensile stress. Thus it can be an interesting characteristic to show, so we wanted to test it on our RLP.

Keep following our idea we do the same on spider silks. We previously identify consensus sequence, GPGQQ, GPGGY and GPGGX. Later we find in literature4 confirmation about our consensus sequences. And we design our SLP protein, MW-[(GPGGV)2(GPGQQ)(GPGGY)]5.

We first wanted to synthesize our SLP gene ourselves : Partie synthèse du gene





error consensus sequences SLP gene synthesis clonage and transformation fusion protein production purification! lyophilisation characterization in solution thread

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