Regulatory

Part:BBa_K2255004:Design

Designed by: Camille Garcia   Group: iGEM17_Aix-Marseille   (2017-08-28)


Xylella fastidiosa constitutive promoter + strong RBS


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

This part was design to be a constitutive promoter with a strong RBS for the bacteria Xylella fastidiosa. The best way to have a strong constitutive promoter is to take the upstream part of a strong constitutive gene.

But as X. fastidiosa hasn't genes well study we choose to look at Escherichia coli constitutive genes and found their X. fastidiosa orthologues. Firstly, we found Best Bidirectional Hits (BBH) between Escherichia coli str. K-12 substr. MG1655 genes and X. fastidiosa 9a5c ones.

Secondly, with the tool [http://rsat-tagc.univ-mrs.fr/rsat/RSAT_home.cgi rsat][1], for each selected gene, we took its upstream sequence to the ATG. With the tool [http://www.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb BPROM] [2], we then chose the sequence with the highest score predicted promoter. We chose XF_RS01885, which is the BBH of purA, which is a strong constitutive gene of E. coli and code for an adenylosuccinate synthetase.

T--Aix-Marseille--M13pIII-SoftBerry.jpeg

Finally, we tried to find the Ribosome Binding Site (RBS) consensus in Xylella fastidiosa. To do so we searched for the anti Shine-Dalgarno sequence within the Xylella fastidiosa 16S ribosomal RNA gene (accession number: NR_041779). The consensus we found is AGGAGG. Since the RBS is supposed to be 6 to 12 nucleotide upstream the ATG, we modified the sequence and added the Rfc10 prefix and suffix region.

Source

It came from the genomic sequence of Xylella fastidiosa 9a5c.

References

  1. ↑ van Helden, J. (2003). Regulatory sequence analysis tools. Nucleic Acids Res. 2003 Jul 1;31(13):3593-6. [Pubmed 12824373]
  2. ↑ V. Solovyev, A Salamov (2011) Automatic Annotation of Microbial Genomes and Metagenomic Sequences. In Metagenomics and its Applications in Agriculture, Biomedicine and Environmental Studies (Ed. R.W. Li), Nova Science Publishers, p. 61-78