Team:Bordeaux/Parts/Assembly improvement

From 2014.igem.org

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<TD> <h1>Consensus sequences</h1>
<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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In the literature we found out that the natural elastin has some repetitions, in particular the sequence VPGXG seems to be repeated. X is V, L, or A, and it represents 9% of the whole polypeptide. This particular sequence were translated with codon optimization for E.coli to produce a (VPGXG)x protein. Elastin properties were checked to see if they are still existing after cloning into bacteria to produce the recombinant protein named elastin like polypeptide (ELP). We studied the original peptide sequences of these proteins to bring out some consensus sequences. Then it revealed that ELP has a UCST and LCST that give ELP a transition temperature with interesting particularities.
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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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We have taken XX randomly and we brought out conserved pattern so we used it to do our resilin like polypeptide with the following pattern: (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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The literature delivers some mechanical properties concerning native resilin as high resilience (<90%) that allows the protein to recover its initial folding after a high tensile stress. Thus it can be an interesting characteristic to show and use, so we wanted to test it on our RLP.
<p>
<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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Keep following our idea we do the same on spider silks. We previously identify consensus sequence, GPGQQ, GPGGY and GPGGX. Later we find in literature confirmation about our consensus sequences. And we design our SLP protein, MW-[(GPGGV)2(GPGQQ)(GPGGY)]5.
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<p>
We first wanted to synthesize our SLP gene ourselves : Partie synthèse du gene  
We first wanted to synthesize our SLP gene ourselves : Partie synthèse du gene  

Revision as of 21:32, 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 found out that the natural elastin has some repetitions, in particular the sequence VPGXG seems to be repeated. X is V, L, or A, and it represents 9% of the whole polypeptide. This particular sequence were translated with codon optimization for E.coli to produce a (VPGXG)x protein. Elastin properties were checked to see if they are still existing after cloning into bacteria to produce the recombinant protein named elastin like polypeptide (ELP). We studied the original peptide sequences of these proteins to bring out some consensus sequences. Then it revealed that ELP has a UCST and LCST that give ELP a transition temperature with interesting particularities.

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 have taken XX randomly and we brought out conserved pattern so we used it to do our resilin like polypeptide with the following pattern: (RLP) MW-[(PSSSYGAP)(PSNSYGAP)(PSTSYGAP)(PVAYGAP)]3

The literature delivers some mechanical properties concerning native resilin as high resilience (<90%) that allows the protein to recover its initial folding after a high tensile stress. Thus it can be an interesting characteristic to show and use, 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 literature 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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