Difference between revisions of "Part:BBa K1065311"

(Usage and Biology)
(Usage and Biology)
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===Usage and Biology===
 
===Usage and Biology===
  
 
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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).
 
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 of the inverter cI. cI inhibits pLambda activity thus amilCP and EFE transcription.
 
In the dark, the autophosphorylated YF1 phosphorylates FixJ, its Response Regulator, which activates the pFixK2 promoter allowing the expression of the inverter cI. cI inhibits pLambda activity thus amilCP and EFE transcription.
 
Under constant illumination with blue light net kinase activity is strongly suppressed, consisting in a consequent inactivation of pFixK2: the outcome is AmilCP+EFE production.
 
Under constant illumination with blue light net kinase activity is strongly suppressed, consisting in a consequent inactivation of pFixK2: the outcome is AmilCP+EFE production.
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EFE enzyme was thoroughly studied by many reasearch groups. It was purified and characterized with an <i>in vitro</i> test <a href="#ref2" id="ret_ref2">[2]</a>. It was then transformed and ectopically expressed in <i>E.coli</i> <a href="#ref3" id="ret_ref3">[3]</a> and in <i>Synecocystis sp</i> <a href="#ref4" id="ret_ref4">[4]</a>.<br/>
  
 
We characterized this part in E. coli using cells NEB10b.
 
We characterized this part in E. coli using cells NEB10b.
  
 
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Revision as of 10:18, 26 September 2013

Blue-light photoinducible AmilCP + EFE producing device

This device is composed by the blue light sensor device, an inverter cassette , the reporter AmilCP and EFE (Ethylene Forming Enzyme) coding sequence. This device allows to produce AmilCP (a blue chromoprotein) and 2-Oxoglutarate Oxygenase/Decarboxylase enzyme when culture is exposed to blue light. To allow this behaviour an inverter cassette was included into the device. The cassette is composed by the cI coding sequence and the repressible promoter pLambda. Everything is under the control of promoter J23100.

This part was successfully cloned by UNITN-Trento 2013 iGEM team in order to design an ethylene producing device that is induced by blue light to speed up fruit ripening.

Parts from 2011 Uppsala-Sweden team and 2006 Berkeley team were used along with our new ethylene producing part BBa_K1065000.


SAFETY NOTES: this part is an ethylene producing system: ethylene is explosive at high concentration.

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 of the inverter cI. cI inhibits pLambda activity thus amilCP and EFE transcription. Under constant illumination with blue light net kinase activity is strongly suppressed, consisting in a consequent inactivation of pFixK2: the outcome is AmilCP+EFE production. EFE enzyme was thoroughly studied by many reasearch groups. It was purified and characterized with an in vitro test [2]. It was then transformed and ectopically expressed in E.coli [3] and in Synecocystis sp [4].
We characterized this part in E. coli using cells NEB10b.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4114
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 605
    Illegal NgoMIV site found at 677
    Illegal NgoMIV site found at 767
    Illegal NgoMIV site found at 785
    Illegal NgoMIV site found at 1297
    Illegal NgoMIV site found at 1590
    Illegal NgoMIV site found at 1684
    Illegal AgeI site found at 319
    Illegal AgeI site found at 1465
    Illegal AgeI site found at 4865
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1354
    Illegal BsaI.rc site found at 218