Difference between revisions of "Part:BBa K1065311"

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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.
 
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.
  
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<html><center><img style="width:700px;"src="https://static.igem.org/mediawiki/2013/5/59/BluelightEFE.jpg"></center></html>
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<html><center><img style="width:700px;"src="https://static.igem.org/mediawiki/2013/a/ac/Tn-2013Bluelig311.png"></center></html>
  
 
Parts from 2011 Uppsala-Sweden team and 2006 Berkeley team were used along with our new ethylene producing part <partinfo>BBa_K1065000</partinfo>.
 
Parts from 2011 Uppsala-Sweden team and 2006 Berkeley team were used along with our new ethylene producing part <partinfo>BBa_K1065000</partinfo>.
  
  
−
SAFETY NOTES: this part is an ethylene producing system: ethylene is explosive at high concentration, we suggest to handle this parte carefully. Cell cultures should be opened under a chemical hood.
+
===Safety notes===
 +
This part is an ethylene producing system: ethylene is explosive at high concentration, we suggest to handle this parte carefully. Cell cultures should be opened under a chemical hood.
 +
 
 
===Usage and Biology===
 
===Usage and Biology===
  
 
<html>
 
<html>
−
YF1, the blue light sensor, is a fusion protein of the LOV blue light sensor domain of <i>Bacillus subtilis</i> (YtvA) and FixL histidine kinase domain (from <i>Bradyrhizobium japonicum</i>) (1, 2).<BR>
+
YF1, the blue light sensor, is a fusion protein of the LOV blue light sensor domain of <i>Bacillus subtilis</i> (YtvA) and FixL histidine kinase domain (from <i>Bradyrhizobium japonicum</i>). <a href="#ref1">[1]</a> <a href="#ref2">[2]</a><BR />
 
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.<BR>
 
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.<BR>
 
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.<BR>
 
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.<BR>
−
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">[3]</a>.It was then transformed and ectopically expressed in <i>E.coli</i> <a href="#ref3" id="ret_ref3">[4]</a> and in <i>Synecocystis sp</i> <a href="#ref4" id="ret_ref4">[5]</a>.<br/>
+
EFE enzyme was thoroughly studied by many reasearch groups. It was purified and characterized with an <i>in vitro</i> test <a href="#ref3">[3]</a>.It was then transformed and ectopically expressed in <i>E.coli</i> <a href="#ref4">[4]</a> and in <i>Synecocystis sp</i> <a href="#ref5">[5]</a>.<br/>
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<BR>
+
<BR> </html>
−
We characterized this part in <i>E. coli</i> using cells NEB10beta and have some preliminary results. We are in the process of acquiring gas-chromatocgraohic measurements in order to test light dependent EFE production.
+
===Characterization before the jamboree===
−
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 produced.
+
 
 +
Before the European jamboree we characterized this part in <i>E. coli</i> using cells NEB10beta and had some preliminary results: 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 produced.
 +
 
  
−
</html>
 
 
<html><center><img style="width:500px;"src="https://static.igem.org/mediawiki/2013/7/7e/Tn-2013Pelletts.png"></center>
 
<html><center><img style="width:500px;"src="https://static.igem.org/mediawiki/2013/7/7e/Tn-2013Pelletts.png"></center>
 
<center><p style="width:600px; margin-bottom:60px; text-align:justify">
 
<center><p style="width:600px; margin-bottom:60px; text-align:justify">
 
<b>Cells transformed with BBa_K1065311: pellets after induction time.</b>  
 
<b>Cells transformed with BBa_K1065311: pellets after induction time.</b>  
−
When cultures reached an OD=0.7 we started the induction test with a blue LED (2) for 10 hours. On the other hand, the control (1) was covered with aluminum foil and kept in complete darkness for about 10 hours. A substantial difference between pelletts is clearly visible. amilCP production probably reflects ethylene synthesis (not measured yet). We are in the process of characterizing this part with gas-chromatographic measurement.
+
When cultures reached an OD=0.7 we started the induction test with a blue LED (2) for 10 hours. On the other hand, the control (1) was covered with aluminum foil and kept in complete darkness for about 10 hours. A substantial difference between pelletts is clearly visible. amilCP production probably reflects ethylene synthesis (not measured yet). We are in the process of characterizing this part with gas-chromatographic measurements.
 
</p></center>
 
</p></center>
  
 
</html>
 
</html>
  
−
===References===
+
===Characterization after the jamboree: ethylene production controlled by light presence===
−
<html><ol>
+
After the European jamboree we tested the circuit and acquired gas-chromatographic measurements in order to test light dependent EFE production.
−
<li>Moglich A, Ayers RA and Moffat K. (2009) Design and Signaling Mechanism of Light-Regulated Histidine Kinases. J. Mol. Bio. 385, 5, 1433-1444.</li>
+
 
−
<li>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</li>
+
 
−
<li>Nagahama K, Ogawa T, Fujii T, Tazaki M, Tanase S, et al. (1991) Purification and properties of an ethylene-forming enzyme from <I>Pseudomonas syringae </I>pv.<I> phaseolicola</I> PK2. Journal of General Microbiology 137: 2281–2286.</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>
+
<html><center><img style="width:500px;"src="https://static.igem.org/mediawiki/2013/0/0c/Tn2013_ethylene_311.png"></center>
−
<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>
+
<center><p style="width:600px; margin-bottom:60px; text-align:justify">
 +
<b>Ethylene formation upon photoinduction.</b>  
 +
E. coli NEB10β cells transformed with BBa_K1065311 were grown in the dark until O.D.= 0.7 was reached. The culture was then split in two samples, one in the dark and the other exposed to a blue LED. After 16 hours from the induction we measured the amount of ethylene produced with the micro GC. Ethylene is produced upon blue light exposure (92 &plusmn; 15 ppm), while it is not produced in the dark.
 +
</p></center>
 +
 
 +
</html>
 +
 
 +
We repeated the experiment with several colonies in order to demonstrate its repeatability since we previously noticed that the circuit (<partinfo>BBa_k1065310</partinfo>) behavior was not always consistent. We observed some unfunctional colonies, some others producing ethylene in the control and some with a not complete and defined shutdown of the system in the dark.
 +
For these reasons we also characterized the same circuit without the inverter (<partinfo>BBa_k1065309</partinfo>) to see if the switch would be sharper and obtain better defined results.
 +
 
 +
 
  
−
<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>
 
  
  
−
</ol></html>
 
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
Line 56: Line 66:
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1065311 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1065311 SequenceAndFeatures</partinfo>
 +
 +
===References===
 +
<html><ol>
 +
<li><a id="ref1"></a>Moglich A, Ayers RA and Moffat K. (2009) Design and Signaling Mechanism of Light-Regulated Histidine Kinases. J. Mol. Bio. 385, 5, 1433-1444.</li>
 +
<li><a id="ref2"></a>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</li>
 +
<li><a id="ref3"></a>Nagahama K, Ogawa T, Fujii T, Tazaki M, Tanase S, et al. (1991) Purification and properties of an ethylene-forming enzyme from <I>Pseudomonas syringae </I>pv.<I> phaseolicola</I> PK2. Journal of General Microbiology 137: 2281–2286.</li>
 +
<li><a id="ref4"></a>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><a id="ref5"></a>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><a id="ref7"></a>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>
 +
</ol></html>
  
  

Latest revision as of 22:50, 28 October 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 behavior 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, we suggest to handle this parte carefully. Cell cultures should be opened under a chemical hood.

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). [1] [2]
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].

Characterization before the jamboree

Before the European jamboree we characterized this part in E. coli using cells NEB10beta and had some preliminary results: 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 produced.


Cells transformed with BBa_K1065311: pellets after induction time. When cultures reached an OD=0.7 we started the induction test with a blue LED (2) for 10 hours. On the other hand, the control (1) was covered with aluminum foil and kept in complete darkness for about 10 hours. A substantial difference between pelletts is clearly visible. amilCP production probably reflects ethylene synthesis (not measured yet). We are in the process of characterizing this part with gas-chromatographic measurements.

Characterization after the jamboree: ethylene production controlled by light presence

After the European jamboree we tested the circuit and acquired gas-chromatographic measurements in order to test light dependent EFE production.


Ethylene formation upon photoinduction. E. coli NEB10β cells transformed with BBa_K1065311 were grown in the dark until O.D.= 0.7 was reached. The culture was then split in two samples, one in the dark and the other exposed to a blue LED. After 16 hours from the induction we measured the amount of ethylene produced with the micro GC. Ethylene is produced upon blue light exposure (92 ± 15 ppm), while it is not produced in the dark.

We repeated the experiment with several colonies in order to demonstrate its repeatability since we previously noticed that the circuit (BBa_K1065310) behavior was not always consistent. We observed some unfunctional colonies, some others producing ethylene in the control and some with a not complete and defined shutdown of the system in the dark. For these reasons we also characterized the same circuit without the inverter (BBa_K1065309) to see if the switch would be sharper and obtain better defined results.





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

References

  1. Moglich A, Ayers RA and Moffat K. (2009) Design and Signaling Mechanism of Light-Regulated Histidine Kinases. J. Mol. Bio. 385, 5, 1433-1444.
  2. 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
  3. 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.
  4. 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.
  5. 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.
  6. 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