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Team:Bordeaux/Parts/Assembly improvement
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Revision as of 21:47, 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:
- 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
- Disadvantages of NheI:
- Not compatible with RFC12 standard
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Consensus sequencesIn 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.
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 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 |
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