Coding

Part:BBa_K1839002:Design

Designed by: Patrik D'haeseleer   Group: iGEM15_SF_Bay_Area_DIYBio   (2015-09-18)


Ava_3856 ATP-grasp ligase


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 uses the native cyanobacterial DNA sequence from Anabaena variabilis, with two small but important changes:

1. We removed an EcoRI restriction site by by making a synonymous single-nucleotide change: GAATTC -> GAATTT.

2. We accidentally forgot to include the stop codon in our design. Luckily the BioBrick suffix does include an in-frame TAG stop codon, but if you express the gene directly from this biobrick, you can expect an extra tyrosine at the C terminal of the protein.



Source

Table S1 in [Balskus and Walsh, 2010] contains the gene locus tags and protein accession numbers for the genes in the shinorine biosynthesis gene cluster. Table S4 contains the PCR primers used for cloning the gene cluster.

Ava_3857 corresponds to protein accession number ABA23461.1. The exact DNA sequence can be found in the genome sequence here: http://www.ncbi.nlm.nih.gov/nuccore/75699950?from=4801723&to=4803099&sat=4&sat_key=105749970&report=gbwithparts

Note that the DNA sequence reported by that link is the reverse complement, since the gene is on the opposite strand in the genome sequence!

Essential references:

  • Balskus EP, Walsh CT. The genetic and molecular basis for sunscreen biosynthesis in cyanobacteria. Science. 2010 Sep 24;329(5999):1653-6. http://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20813918/
  • Gao Q, Garcia-Pichel F. An ATP-grasp ligase involved in the last biosynthetic step of the iminomycosporine shinorine in Nostoc punctiforme ATCC 29133. J Bacteriol. 2011 Nov;193(21):5923-8. http://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21890703/

References