Coding

Part:BBa_K1499003:Design

Designed by: Raman Nelakanti   Group: iGEM14_StanfordBrownSpelman   (2014-08-27)

wssI gene from wss operon


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 228
    Illegal NgoMIV site found at 628
    Illegal NgoMIV site found at 961
    Illegal AgeI site found at 275
    Illegal AgeI site found at 919
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 850

Design Notes

This gene was isolated from purified P. fluorescens genomic DNA using the following primers: 

wssI Forward: 5'-CCGGGTTTGTGTTGTCGTTC-3'
wssI Reverse: 5'-AGGCAGTCTGCTTCAAGCTC-3'

The BioBrick prefix and suffix were then added using these primers: 

Forward: 5'-GAATTCGCGGCCGCTTCTAGAGCCGGGTTTGTGTTGTCGTTC-3'
Reverse: 5'-CTGCAGCGGCCGCTACTAGTAAGGCAGTCTGCTTCAAGCTC-3'

Because there was an internal EcoRI cut-site incompatible with RFC10, we performed site-directed mutagenesis to change a single base and remove the site. We used the following primers to introduce an A->G silent mutation: 

Imut Forward:  5'-GGACGGCGGCGAGTTCTCCGGTGCGGCCAAGGCG-3'
Imut Reverse:  5'-CCGCACCGGAGAACTCGCCGCCGTCCTTGCTGAAATTGCCCACAG-3'

Source

The wss operon was extracted from P. fluorescens, an organism that produces cellulose acetate as part of a biofilm that allows it to colonize the air-liquid interface [1].

References

[1] Spiers AJ et al. (2013) Cellulose Expression in Pseudomonas fluorescens SBW25 and Other Environmental Pseudomonads in Cellulose - Medical, Pharmaceutical, and Electronic Applications. DOI: [http://cdn.intechopen.com/pdfs-wm/45637.pdf 10.5772/53736]