Difference between revisions of "Part:BBa K1065311"

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
INCOMPATIBLE WITH RFC[10]
Unknown
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

@@ Line 15: / Line 15: @@
 ===Usage and Biology===
+<html>
 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 <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.
+</html>
 <!-- -->