Difference between revisions of "Part:BBa K1065309"
 
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<partinfo>BBa_K1065309 short</partinfo>
 
<partinfo>BBa_K1065309 short</partinfo>
  
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<BR>This part includes blue light sensor YF1, its RR FixJ, pFixK2, the reporter amilGFP and EFE: 2-oxoglutarate oxygenase/decarboxylase is an Ethylene Forming Enzyme that catalyzes ethylene biosynthesis from 2-oxoglutarate. This enzyme was initially purified from <i>Pseudomonas siringae pv. phaseolicola PK2</I>, a 2-oxoglutarate-dependent ethylene producing bacterium.<br><br>
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<BR>This part includes blue light sensor YF1, its RR FixJ, pFixK2,<html><a href="#ref1" id="ret_ref1">[1]</a></html><html><a href="#ref2" id="ret_ref2">[2]</a></html> the reporter amilGFP and EFE: 2-oxoglutarate oxygenase/decarboxylase is an Ethylene Forming Enzyme that catalyzes ethylene biosynthesis from 2-oxoglutarate <html><a href="#ref3" id="ret_ref3">[3]</a></html> <html><a href="#ref4" id="ret_ref4">[4]</a></html>. This enzyme was initially purified from <i>Pseudomonas siringae pv. phaseolicola PK2</I>, a 2-oxoglutarate-dependent ethylene producing bacterium.<html><a href="#ref5" id="ret_ref5">[5]</a></html> YF1 and FixJ production is under the regulation of pLac.<BR><BR>
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YF1 and FixJ production are under the regulation of pLac.<BR><BR>
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<html><center><img style="width:800px;"src="https://static.igem.org/mediawiki/2013/2/26/Tn-2013Blueligh309.png"></center></html>
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<BR>
 
In the presence of blue light (470 nm) the production of the reporter amilGFP and EFE enzyme is inhibited.<BR>
 
In the presence of blue light (470 nm) the production of the reporter amilGFP and EFE enzyme is inhibited.<BR>
 
In the absence of blue light the device is activated, thus producing amilGFP+EFE.<BR>
 
In the absence of blue light the device is activated, thus producing amilGFP+EFE.<BR>
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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>).<BR>
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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>)<html><a href="#ref1" id="ret_ref1">[1]</a></html> <html><a href="#ref2" id="ret_ref2">[2]</a></html>.<BR>
 
In the dark, the autophosphorylated YF1 phosphorylates FixJ, its Response Regulator, which activates the pFixK2 promoter allowing the expression amilGFP and EFE.
 
In the dark, the autophosphorylated YF1 phosphorylates FixJ, its Response Regulator, which activates the pFixK2 promoter allowing the expression amilGFP and EFE.
 
Under constant illumination with blue light net kinase activity is strongly suppressed, consisting in a consequent inactivation of pFixK2.
 
Under constant illumination with blue light net kinase activity is strongly suppressed, consisting in a consequent inactivation of pFixK2.
  
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At the moment we characterized only part <partinfo>BBa_K1065311</partinfo>, the blue light dependent circuit producing amilGFP+EFE that contains the inverter cassette.
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At the moment we characterized only part <partinfo>BBa_K1065311</partinfo>, the blue light dependent circuit producing amilCP+EFE that contains the inverter cassette.
  
 
===Safety===
 
===Safety===
 
<html>
 
<html>
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Ethylene is a compound that can be explosive at high concentrations (between 2.7% and 34% v,v). We suggest to manage this part carefully. (See <a href="https://parts.igem.org/Part:BBa_K1065002">BBa_K1065002</a> for more details.) <BR><BR><BR></html>
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Ethylene is a compound that can be explosive at high concentrations (between 2.7% and 34% v,v). We suggest to manage this part carefully. (See <a href="https://parts.igem.org/Part:BBa_K1065002">BBa_K1065002</a> for more details.) <BR></html>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1065309 SequenceAndFeatures</partinfo>
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===Control of ethylene production with the photoinducible circuit===
  
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<hr></html>
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We produced ethylene with two different blue light circuits: one with an inverter <partinfo>BBa_K1065311</partinfo> and one without the inverter cassette <partinfo>BBa_K1065309</partinfo>. Part <partinfo>BBa_K1065311</partinfo> produced ethylene with some inconsistent results (i.e. not all colonies were functional, check the corresponding part page for more info). To obtain a more functional system, with a tight switch control, we also built and characterized a circuit without inverter that produces ethylene in the dark.
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<BR>
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<BR>
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<html><center><img style="width:500px;"src="https://static.igem.org/mediawiki/2013/9/95/Tn-2013_ethylene_309.png"></center>
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<center><p style="width:600px; margin-bottom:60px; text-align:justify">
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<b>Ethylene formation in the absence of light.</b>
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</b> <i>E. coli</i> NEB10&beta; transformed with <a href="https://parts.igem.org/Part:BBa_K1065309">BBa_1065309</a> was grown until O.D. 0.7 was reached. The culture was then split and kept under the two different conditions. In the dark we could appreciate ethylene production (37 &plusmn; 15 ppm) instead in the presence of blue light there was no ethylene produced.
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</p></center></html>
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<html><center><img style="width:400px;"src="https://static.igem.org/mediawiki/2013/d/dc/Tn-2013_bluelight_kinetic.JPG"></center></html><br>
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Also in this case not every colony behaved correctly and sometimes we saw ethylene in the control or just no ethylene at all. However the on/off switch was better defined with this part. Further experiments need to be done to obtain the perfect and complete switch, for instance we could remove the reporter gene before the EFE sequence: this could be the right move to get a more efficient behavior.
  
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<!-- Uncomment this to enable Functional Parameter display
 
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===Functional Parameters===
 
 
<partinfo>BBa_K1065309 parameters</partinfo>
 
<partinfo>BBa_K1065309 parameters</partinfo>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1065309 SequenceAndFeatures</partinfo>
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===References===
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<html>
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<a id="ref1"></a>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.<br>
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<a id="ref2"></a>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<br>
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<a id="ref3"></a>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.<br>
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<a id="ref4"></a>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.<br>
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<a id="ref5"></a>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.
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</html>

Latest revision as of 22:45, 28 October 2013

light regulated circuit producing amilGFP+EFE at dark


This part includes blue light sensor YF1, its RR FixJ, pFixK2,[1][2] the reporter amilGFP and EFE: 2-oxoglutarate oxygenase/decarboxylase is an Ethylene Forming Enzyme that catalyzes ethylene biosynthesis from 2-oxoglutarate [3] [4]. This enzyme was initially purified from Pseudomonas siringae pv. phaseolicola PK2, a 2-oxoglutarate-dependent ethylene producing bacterium.[5] YF1 and FixJ production 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.

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 an 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)[1] [2].
In the dark, the autophosphorylated YF1 phosphorylates FixJ, its Response Regulator, which activates the pFixK2 promoter allowing the expression amilGFP and EFE. Under constant illumination with blue light net kinase activity is strongly suppressed, consisting in a consequent inactivation of pFixK2.

At the moment we characterized only part BBa_K1065311, the blue light dependent circuit producing amilCP+EFE that contains the inverter cassette.

Safety

Ethylene is a compound that can be explosive at high concentrations (between 2.7% and 34% v,v). We suggest to manage this part carefully. (See BBa_K1065002 for more details.)

Control of ethylene production with the photoinducible circuit

We produced ethylene with two different blue light circuits: one with an inverter BBa_K1065311 and one without the inverter cassette BBa_K1065309. Part BBa_K1065311 produced ethylene with some inconsistent results (i.e. not all colonies were functional, check the corresponding part page for more info). To obtain a more functional system, with a tight switch control, we also built and characterized a circuit without inverter that produces ethylene in the dark.

Ethylene formation in the absence of light. E. coli NEB10β transformed with BBa_1065309 was grown until O.D. 0.7 was reached. The culture was then split and kept under the two different conditions. In the dark we could appreciate ethylene production (37 ± 15 ppm) instead in the presence of blue light there was no ethylene produced.


Also in this case not every colony behaved correctly and sometimes we saw ethylene in the control or just no ethylene at all. However the on/off switch was better defined with this part. Further experiments need to be done to obtain the perfect and complete switch, for instance we could remove the reporter gene before the EFE sequence: this could be the right move to get a more efficient behavior.

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

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.