Coding

Part:BBa_K1702005

Designed by: Olivia Smith, Adriana Collings   Group: iGEM15_CSU_Fort_Collins   (2015-09-17)

Trans-zeatin biosynthesis under control of the lac promoter

Biosynthesis pathway of trans-zeatin for E. coli. Trans-zeatin is a cytokinin which increases lateral bud growth, prevents leaf senescence, and increases cellular biomass of plants. Cytokinins function by signaling to activate proteins which increase transcription and cell division.

Synthesized tzs gene (from Agrobacterium tumefaciens C58) and LOG (Oryza satifa), both codon optimized for E. coli as a biobrick. The tzs gene encodes for the enzyme which converts 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate (HMBDP) into zeatin riboside 5'-phosphate. The LOG gene produces N6-dimethylallyladenine by removing the phosphoribosyl group from N6-(delta2-isopentenyl)-adenosine 5'-monophosphate. Hydroxylation of the N6-dimethylallyladenine produces biologically-active trans-zeatin. Under control of the lac promoter in DH5alpha E. coli cells, expression of the trans-zeatin biosynthesis pathway is induced by lactose or its analog IPTG.


Trans-zeatin biosynthesis pathway. The tzs gene performs the same function as the tmr gene in the process shown.

==Characterization by 2015 CSU iGEM Team==

Growth Curves:

Growth of E. coli DH5alpha cells in shake flasks with this construct over 24 hours as compared to a lac promoter control.
Growth of E. coli DH5alpha cells in shake flasks with this construct over 72 hours as compared to a lac promoter control.
Growth of E. coli DH5alpha cells in bioreactors with the construct over 72 hours as compared to a lac promoter control.

HPLC:

HPLC results after solid phase extraction procedure. A) Bioreactor lac promoter control after 72 hours B) Bioreactor trans-zeatin strain after 72 hours C) Trans-zeatin time retention standard D) Shake flask lac promoter control after 72 hours E) Shake flask trans-zeatin strain after 72 hours F) Trans-zeatin time retention standard. There is no noticeable difference in peaks between the controls and the test strains, and there are no noticeable peaks present at the time of the retention standard.


Sequence and Features


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 AgeI site found at 930
    Illegal AgeI site found at 1302
    Illegal AgeI site found at 1310
    Illegal AgeI site found at 1466
  • 1000
    COMPATIBLE WITH RFC[1000]


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