Difference between revisions of "Part:BBa K2255003:Design"

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===Design Notes===
 
===Design Notes===
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We wanted to create a multi-tag with a his-tag (attachment to Co or Ni column) and a Strep-tag <ref name= Strep>Schmidt, Thomas GM; Skerra, Arne (2007). "The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins". Nature Protocols. 2 (6): 1528–35. PMID 17571060. doi:10.1038/nprot.2007.209</ref> (wich has an affinity towards Strep-Tactin®). Between those tag, we added a TEV <ref name=TEV>Parks TD, Leuther KK, Howard ED, Johnston SA, Dougherty WG (February 1994). "Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase". Anal. Biochem. 216 (2): 413–7. PMID 8179197. doi:10.1006/abio.1994.1060</ref> cutting site to give the possibility to decrease the length of this multi-tag.
  
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[[File:T--Aix-Marseille--M13pIII-SoftBerry.jpeg|400px|right]]
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Our tag is composed of StrepTag-TEV-6His, which can be constructed as GSG-WSHPQPEL-GSG-ASQFYLNE-GSG-HHHHHH
  
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Firstly, we found best bidirectional hit (BBH) between Escherichia coli str. K-12 substr. MG1655 genes and ''Xylella fastidiosa'' 9a5c ones. In order to have a strong constitutive promoter we look at highly expressed genes from ''E.coli''.<ref>S, K., J, M., A, C. & D, K. Characterizations of highly expressed genes of four fast-growing bacteria., Characterizations of Highly Expressed Genes of Four Fast-Growing Bacteria. J Bacteriol 183, 183, 5025, 5025–5040 (2001).</ref>
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Glycine and serine residues are added to the sequence in order to give flexibility and space between the tags and the cutting site.
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Secontly, with the tool rsat, for each gene selected we take the upstream sequence from the previous gene to the ATG And with the tool BPROM we choose the sequence with predicted box with the best score. We choose XF_RS01885 which is the BBH of purA, which code for an adenylosuccinate synthetase.
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Finaly, we tried to find the ribosome binding site (RBS) consensus in ''Xylella fastidiosa''. To do so we search for the anti-Shine dalgarno sequence with ''Xylella fastidiosa'' 16S ribosomal RNA gene (accession number : NR_041779). The consus found is : AGGAGG. The RBS is supposed to be 6 to 12 nucleotide upstream the ATG. So we modified the sequence. And we added Rfc10 prefix and suffix region.
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We retro-translated this sequence and optimized it for ''E. coli''.
  
 
===Source===
 
===Source===
  
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This part is made with the consensus sequence of streptavidine tag , histidine tag and TEV protease clivation site.
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This part is made with the consensus sequence of the streptavidine tag, the histidine tag and the TEV protease cutting site.
  
 
===References===
 
===References===
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<references/>

Latest revision as of 11:31, 6 October 2017


Multi-Tag (Rfc 25)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

We wanted to create a multi-tag with a his-tag (attachment to Co or Ni column) and a Strep-tag [1] (wich has an affinity towards Strep-Tactin®). Between those tag, we added a TEV [2] cutting site to give the possibility to decrease the length of this multi-tag.

Our tag is composed of StrepTag-TEV-6His, which can be constructed as GSG-WSHPQPEL-GSG-ASQFYLNE-GSG-HHHHHH

Glycine and serine residues are added to the sequence in order to give flexibility and space between the tags and the cutting site.

We retro-translated this sequence and optimized it for E. coli.

Source

This part is made with the consensus sequence of the streptavidine tag, the histidine tag and the TEV protease cutting site.

References

  1. ↑ Schmidt, Thomas GM; Skerra, Arne (2007). "The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins". Nature Protocols. 2 (6): 1528–35. PMID 17571060. doi:10.1038/nprot.2007.209
  2. ↑ Parks TD, Leuther KK, Howard ED, Johnston SA, Dougherty WG (February 1994). "Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase". Anal. Biochem. 216 (2): 413–7. PMID 8179197. doi:10.1006/abio.1994.1060