Generator

Part:BBa_K1807007:Design

Designed by: Ivan Gyulev   Group: iGEM15_York   (2015-09-17)
Revision as of 00:42, 19 September 2015 by Isgyulev (Talk | contribs) (→‎Design Notes)

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Synthetic Phosphate-specific Transporter Operon


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 2133
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 529
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 655
    Illegal NgoMIV site found at 716
    Illegal NgoMIV site found at 1732
    Illegal NgoMIV site found at 2305
    Illegal AgeI site found at 164
    Illegal AgeI site found at 2762
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1417
    Illegal BsaI site found at 2383
    Illegal SapI site found at 1223
    Illegal SapI.rc site found at 3169


Design Notes


iGEM York 2015 generated this part by designing two overlapping G Block fragments (1900bp and 1943bp) that were used in a Gibson Assembly Reaction with BBa_K1807000.

This Phosphate-specific transporter operon has been designed and re-factored (ribosome-binding sites have been introduced between each gene). Each protein domain has been based on amino acid sequence from a metagenomic sequence. The operon coding sequences have been codon-optimized for Escherichia coli K-12 translation.


Notes on Growth

A noticeable slowing of the growth rate was observed in DH5 alpha cells even when transcription was not induced (grown on LB-Agar with appropriate antibiotic). This may be due to leaky expression (WT lacI is present in DH5 alpha rather than lacIq) increasing the translational energetic demand on the cells. It is also possible (but not confirmed) that the protein has a toxic effect on cellular metabolism.

Source

Candidatus Accumulibacter phosphatis SK-1 strain

References