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 streptavidine-tag (wich can bind biotin). Between those tag we add a TEV clivation site in order to give the possibility of decrease the lengh 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.
  
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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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Which can be constructed as : GSG-WSHPQPEL-GSG-ASQFYLNE-GSG-HHHHHH
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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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Glycin and serine residues are added to the sequence in order to give flexibility and space between tags and clivation site.
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We retro-translate this sequence and optimized it for ''E. coli''.
  
 
===Source===
 
===Source===

Revision as of 16:49, 26 September 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 streptavidine-tag (wich can bind biotin). Between those tag we add a TEV clivation site in order to give the possibility of decrease the lengh of this multi-tag.

Our tag is composed of : StrepTag-TEV-6His.

Which can be constructed as : GSG-WSHPQPEL-GSG-ASQFYLNE-GSG-HHHHHH

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

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

Source

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

References