Device

Part:BBa_K1065309

Designed by: Fabio Digiacomo   Group: iGEM13_UNITN-Trento   (2013-09-18)
Revision as of 08:44, 26 September 2013 by Fabius924 (Talk | contribs)

light regulated circuit producing amilGFP+EFE at dark


This part includes blue light sensor YF1, its RR fixJ,PfixK2,the reporter amilGFP and EFE+terminators: everything is under the regulation of Plac.
In the presence of blue light (470 nm) the production of the reporter amilGFP and EFE enzyme is inhibited.
In the absence of blue light the device is activated, thus producing amilGFP+EFE. 2-oxoglutarate oxygenase/decarboxylase is an Ethylene Forming Enzyme (EFE) that catalyze Ethylene biosynthesis from 2-oxoglutarate. This enzyme was firstly purified from Pseudomonas siringae pv. phaseolicola PK2, a 2-oxoglutarate-dependent ethylene producing bacterium.
This part was cloned by UNITN-Trento 2013 iGEM team for the creation of an aerobically engineered pathway for the control of fruit ripening: unlike Bba_k1065311, it doesn't have any inverter cassette, so blue light acts like an inhibitor of the circuit rather than an inducer.


Usage and Biology

YF1, the blue light sensor, is a fusion protein of the LOV blue light sensor domain of Bacillus subtilis (YtvA) and FixL histidine kinase domain (from Bradyrhizobium japonicum). In the dark, the autophosphorylated YF1 phosphorylates FixJ, its Response Regulator, which activates the pFixK2 promoter allowing the expression amilCP and EFE. Under constant illumination with blue light net kinase activity is strongly suppressed, consisting in a consequent inactivation of pFixK2.

We characterized this part in E. coli using cells NEB10b.


Safety

This part produces ethylene, a compound that can be inflammable at a high concentration (between 2.7% and 34% v,v). We suggest to manage this part carefully. (See BBa_K1065001 for more details.)


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3309
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 770
    Illegal NgoMIV site found at 842
    Illegal NgoMIV site found at 932
    Illegal NgoMIV site found at 950
    Illegal NgoMIV site found at 1462
    Illegal NgoMIV site found at 1755
    Illegal NgoMIV site found at 1849
    Illegal AgeI site found at 484
    Illegal AgeI site found at 1630
    Illegal AgeI site found at 4060
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1519
    Illegal BsaI.rc site found at 383


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