Difference between revisions of "Part:BBa K1065311"
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<li>Fukuda H, Ogawa T, Ishihara K, Fujii T, Nagahama K, et al. (1992) Molecular cloning in Escherichia coli, expression, and nucleotide sequence of the gene for the ethylene-forming enzyme of <I>Pseudomonas syringae </I>pv.<I> phaseolicola</I> PK2. Biochem Biophys Res Commun 188: 826–832.</li> | <li>Fukuda H, Ogawa T, Ishihara K, Fujii T, Nagahama K, et al. (1992) Molecular cloning in Escherichia coli, expression, and nucleotide sequence of the gene for the ethylene-forming enzyme of <I>Pseudomonas syringae </I>pv.<I> phaseolicola</I> PK2. Biochem Biophys Res Commun 188: 826–832.</li> | ||
<li>Guerrero F, Carbonell. V., Cossu M, Correddu D, Jones PR (2012) Ethylene Synthesis and Regulated Expression of Recombinant Protein in <I>Synechocystis sp.</I> PCC 6803. PLoS ONE 7(11): e50470.</li> | <li>Guerrero F, Carbonell. V., Cossu M, Correddu D, Jones PR (2012) Ethylene Synthesis and Regulated Expression of Recombinant Protein in <I>Synechocystis sp.</I> PCC 6803. PLoS ONE 7(11): e50470.</li> | ||
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+ | <li>A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000</li> | ||
+ | <li>Octamerization of CI repressor is needed for effective repression of PRM and efficient switching from lysogeny. Ian B. Dodd,1 Alison J. Perkins, Daniel Tsemitsidis, and J. Barry Egan , Genes and Development (Vol 15, No. 22) 3013-3022: 2001</li> | ||
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Revision as of 20:58, 28 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 [3].It was then transformed and ectopically expressed in E.coli [4] and in Synecocystis sp [5].
We characterized this part in E. coli using cells NEB10b and have preliminary results.
We are in the process of acquiring gas-chromatocgraohic measurements in order to test light dependent EFE production.
Up to now, we were only able to observe amilCP production upon blue light illumination: since the blue reporter correctly appeared only in the induced control, we think that ethylene could be properly detected.
Cells transformed with BBa_K1065311: pellets after induction time. We induced cultures snice O.D. reached 0.7 for about 10 hours with a blue LED (2) instead the control (1) was covered with aluminum foil and taken in complete darkness. we can notice a substantial difference between pelletts' colors. AmilCP production probably reflects ethylene synthesis (not measured yet). We are in the process of characterizing this part more properly and getting some gas-chromatographic.
References
- Moglich A, Ayers RA and Moffat K. (2009) Design and Signaling Mechanism of Light-Regulated Histidine Kinases. J. Mol. Bio. 385, 5, 1433-1444.
- Ohlendorf, R., Vidavski, R.R., Eldar, A., Moffat, K. & Möglich, A.(2012). From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. J. Mol. Biol., 416: 534: 542
- Nagahama K, Ogawa T, Fujii T, Tazaki M, Tanase S, et al. (1991) Purification and properties of an ethylene-forming enzyme from Pseudomonas syringae pv. phaseolicola PK2. Journal of General Microbiology 137: 2281–2286.
- Fukuda H, Ogawa T, Ishihara K, Fujii T, Nagahama K, et al. (1992) Molecular cloning in Escherichia coli, expression, and nucleotide sequence of the gene for the ethylene-forming enzyme of Pseudomonas syringae pv. phaseolicola PK2. Biochem Biophys Res Commun 188: 826–832.
- Guerrero F, Carbonell. V., Cossu M, Correddu D, Jones PR (2012) Ethylene Synthesis and Regulated Expression of Recombinant Protein in Synechocystis sp. PCC 6803. PLoS ONE 7(11): e50470.
- A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000
- Octamerization of CI repressor is needed for effective repression of PRM and efficient switching from lysogeny. Ian B. Dodd,1 Alison J. Perkins, Daniel Tsemitsidis, and J. Barry Egan , Genes and Development (Vol 15, No. 22) 3013-3022: 2001
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 4114
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1354
Illegal BsaI.rc site found at 218