Difference between revisions of "Part:BBa K1317001:Design"

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The strategy of our assembly is described below anyway. The gene was synthesized by ordering specific oligo with overlapping regions, and by adding a NheI site for further fusing proteins.
 
The strategy of our assembly is described below anyway. The gene was synthesized by ordering specific oligo with overlapping regions, and by adding a NheI site for further fusing proteins.
 +
 +
We used the pro-resilin exon 1 from Drosophila melanogaster. We identified a repetitive pattern, PSXXYGAP. So we blast it against the protein database of pro-resilin to check if the pattern was conserved within different species or specific to ''Drosophila''.
  
 
[[File:Bdx2014 RLPsynthesis1.png|900px]]
 
[[File:Bdx2014 RLPsynthesis1.png|900px]]
 +
 +
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.
 +
 
===Source===
 
===Source===
  
Line 19: Line 26:
  
 
===References===
 
===References===
−
Julie N. Renner et al. ''Characterization of Resilin-Based Materials for Tissue Engineering Applications'' Biomacromolecules 2012, 13, 3678−3685
+
[1] Tamburro A.M. et al. ''Molecular and supramolecular structural studies on significant repetitive sequences of resilin'' (2010) Chembiochem., 11(1), 83-93. doi: 10.1002/cbic.200900460.
 +
 
 +
[2] Linqing Li et Kristi L. Kiick. ''Resilin-Based Materials for Biomedical Applications'' (2013) ACS Macro Lett., 2(8), 635–640. doi: 10.1021/mz4002194.
 +
 
 +
[3] David H. Ardell and Svend Olav Andersen ''Tentative identification of a resilin gene in Drosophila melanogaster'' Insect Biochemistry and Molecular Biology 31 (2001) 965–970
 +
 
 +
[4] Julie N. Renner et al. ''Characterization of Resilin-Based Materials for Tissue Engineering Applications'' Biomacromolecules 2012, 13, 3678−3685
  
−
Russell E. Lyons et al. ''Comparisons of Recombinant Resilin-like Proteins: Repetitive Domains Are Sufficient to Confer Resilin-like Properties'' Biomacromolecules 2009, 10, 3009–3014
+
[5] Russell E. Lyons et al. ''Comparisons of Recombinant Resilin-like Proteins: Repetitive Domains Are Sufficient to Confer Resilin-like Properties'' Biomacromolecules 2009, 10, 3009–3014
  
−
M. Kim et al.  ''High yield expression of recombinant pro-resilin: Lactose-induced fermentation in E. coli and facile purification'' Protein Expression and Purification 52 (2007) 230–236
+
[6] M. Kim et al.  ''High yield expression of recombinant pro-resilin: Lactose-induced fermentation in E. coli and facile purification'' Protein Expression and Purification 52 (2007) 230–236
  
−
Linqing Li and Kristi L. Kiick ''Resilin-Based Materials for Biomedical Applications'' ACS Macro Lett. 2013, 2, 635−640
+
[7] Christopher M. Elvin et al. ''Synthesis and properties of crosslinked recombinant pro-resilin'' Vol 437|13 October 2005|doi:10.1038/nature04085

Latest revision as of 21:12, 16 October 2014

CDS for resilin-like polypeptide (RLP)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 300
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The gene used to assemble the biobrick has been provided by GenScript even if the assembling strategy succeeded

It allows us to have the proper sequence without mutation and with sequencing results.

The strategy of our assembly is described below anyway. The gene was synthesized by ordering specific oligo with overlapping regions, and by adding a NheI site for further fusing proteins.

We used the pro-resilin exon 1 from Drosophila melanogaster. We identified a repetitive pattern, PSXXYGAP. So we blast it against the protein database of pro-resilin to check if the pattern was conserved within different species or specific to Drosophila.

Bdx2014 RLPsynthesis1.png

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.

Source

Genomic sequence from Drosophila melanogaster

References

[1] Tamburro A.M. et al. Molecular and supramolecular structural studies on significant repetitive sequences of resilin (2010) Chembiochem., 11(1), 83-93. doi: 10.1002/cbic.200900460.

[2] Linqing Li et Kristi L. Kiick. Resilin-Based Materials for Biomedical Applications (2013) ACS Macro Lett., 2(8), 635–640. doi: 10.1021/mz4002194.

[3] David H. Ardell and Svend Olav Andersen Tentative identification of a resilin gene in Drosophila melanogaster Insect Biochemistry and Molecular Biology 31 (2001) 965–970

[4] Julie N. Renner et al. Characterization of Resilin-Based Materials for Tissue Engineering Applications Biomacromolecules 2012, 13, 3678−3685

[5] Russell E. Lyons et al. Comparisons of Recombinant Resilin-like Proteins: Repetitive Domains Are Sufficient to Confer Resilin-like Properties Biomacromolecules 2009, 10, 3009–3014

[6] M. Kim et al. High yield expression of recombinant pro-resilin: Lactose-induced fermentation in E. coli and facile purification Protein Expression and Purification 52 (2007) 230–236

[7] Christopher M. Elvin et al. Synthesis and properties of crosslinked recombinant pro-resilin Vol 437|13 October 2005|doi:10.1038/nature04085