From 2014.igem.org

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Project presentation:

Earlier this year we spoke about plastics and we agreed that plastics are useful for many reasons, they’re durable, flexible and can be molded. But plastics also are problems because of the destruction of ecosystems, pollution of some natural resources, and the reduction of dump area.

So we try to make E.Coli produce elastic protein, and this is ELASTICOLI.

We select 3 elastic proteins, the elastin from mammals, the resilin from insect and the dragline silks from spiders. The elastin is responsible of the elasticity of the skin and muscles. The resilin enables insects to jump or pivot their wings efficiently to fly. Finally the dragline silk allow spiders to dangle because of the high tensile strength and extensibility.

Consensus sequence:

In the literature1 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.

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.

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

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

References: 1- Doreen M. Floss1, Kai Schallau2, Stefan Rose-John. ELASTIN-like polypeptides revolutionize recombinant protein expression and their biomedical application. Trends in Biotechnology Vol.28 No.1 (http://www.ncbi.nlm.nih.gov/pubmed/19897265)

2- David H. Ardell , Svend Olav Andersen. TENTATIVE identification of a resilin gene in Drosophila melanogaster. Insect Biochemistry and Molecular Biology 31 (2001) 965–970 (http://www.ncbi.nlm.nih.gov/pubmed/11483432)

3- Christopher M. Elvin, Andrew G. Carr, Mickey G. Huson. SYNTHESIS and properties of crosslinked recombinantpro-resilin. Vol 437|13 October 2005|doi:10.1038/nature04085 (http://www.ncbi.nlm.nih.gov/pubmed/16222249)

4- Michael B. Hinman, Justin A. Jones and Randolph V. Lewis. SYNTHETIC spider silk: a modular fiber. TIBTECH SEPTEMBER 2000 (Vol. 18) (http://www.ncbi.nlm.nih.gov/pubmed/10942961)