Difference between revisions of "Part:BBa K1065311"
 
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__NOTOC__
 
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<partinfo>BBa_K1065000 short</partinfo>
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<partinfo>BBa_K1065311 short</partinfo>
  
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<html>2-oxoglutarate oxygenase/decarboxylase is an Ethylene Forming Enzyme (EFE) that catalyze Ethylene biosynthesis from 2-oxoglutarate. This enzyme was firstly purified  from <I>Pseudomonas Siringae</I> pv. <I>phaseolicola</I> PK2, a 2-oxoglutarate-dependent ethylene producing bacterium <a href="#ref1" id="ret_ref1">[1]</a>. <br/> This part was cloned by the iGEM Trento 2013 team for the creation of an aerobically engineered pathway for the control of fruit ripening. The part has been successfully operated while controlled by AraC-pBAD in pSB1C3 (BBa_K1065001) using <i>E.coli</i> as chassis. Further information about this part and its characterization can be found in the <a href="http://2013.igem.org/Team:UNITN-Trento">iGEM Trento 2013 wiki</a>.
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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.
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<br/><h3 style="margin-bottom:10px;">Please note that this part has a modified Prefix and Suffix compatible to RFC25 (Freiburg Assembly). Check the design section for more information.</h3></html>
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Everything is under the control of promoter J23100.  
  
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<!-- Add more about the biology of this part here-->
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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.
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===Usage and Biology===
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<html>
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The 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/>
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</html>
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===Safety===
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<html>
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This part produces ethylene, a compound that can be inflammable at a concentration between 2.7 to 36%. We characterized this part under the control of an AraC-pBAD promoter. With a air volume/culture volume ratio = 4, we detected about 200 ppm of Ethylene. This concentration is not dangerous and not inflammable. However we suggest to manage this part carefully. (See <a href="https://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a> for more details.)
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<hr></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>
  
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===Characterization===
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Parts from 2011 Uppsala-Sweden team and 2006 Berkeley team were used along with our new ethylene producing part <partinfo>BBa_K1065000</partinfo>.
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<html>
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This part was characterized with with two different inducible systems in Neb10beta cells:<br/>
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<ul>
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<li>under the control of an AraC-pBAD promoter inducible by arabinose (<a href="https://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a>;</li>
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<li>under the control of a photoinducible circuit (<a href="https://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>).</li>
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</ul>
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</html>
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===EFE characterization with AraC-pBAD promoter===
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<html>
 
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<center><img style="width:800px;"src="https://static.igem.org/mediawiki/2013/6/6f/Tn-20130627-Efe_Toxicity_test-PLOT.png"></center>
 
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<center><p style="width:600px; margin-bottom:60px; text-align:justify"><b>Figure 1 Effect of EFE on cell growth</b>. Cell density was measured at different time points to determine the effect of EFE expression. Cells were grown at 37 °C in LB until it was reached an OD of 0.6. The cells were then splitted in four samples of equal volume. Two samples were then induced with 5 mM Arabinose. Induced samples show a slowed growth rate,
 
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as espected (5mM arabinose is a strong induction that causes stress on cells). However, cell growth is not completely inhibited so EFE is not highly toxic</p></center>
 
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<h3>Ethylene detection through Micro Gas-Chromatography</h3><br/>
 
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<center><img style="width:800px;"src="https://static.igem.org/mediawiki/2013/c/c4/Tn-2013_ETH_detection.jpg"></center>
 
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<center><p style="width:600px; margin-bottom:60px; text-align:justify"><b>Figure 2</b> Ethylene detection with an Agilent 3000 Micro GC set up with a plot U column. Cells were grown until O.D.600 reached 0.5. The cells were then splitted in two samples of equal volume (3 ml) and putted into an hermetically closed vial with a septum with a rubber cap. One of the two sample was induced with 5 mM Arabinose. The vials were left in the thermoshaker for 4 hours. After that, the vials were connected to a micro GC and a measure was taken. Panel A: a 1.5 ml sample induced (green curve) and 3 ml sample induced (red curve) showed a characteristic peak corresponding to Ethylene. On the other hand, the 3 ml not induced sample (blue curve) didn't show the peak. Ethylene was estimated to be 61 ± 15 ppm for the 1.5 ml culture and  101 ± 15 ppm for the 3 ml culture. Panel B: picture of the vial connected to the micro GC.</p></center>
 
  
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<h3>Kinetic assay for Ethylene production</h3>
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===Safety notes===
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<center><img style="width:800px" src="https://static.igem.org/mediawiki/2013/0/00/Tn-2013_kinetic_EFE_plot-2.png"></center>
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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.
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<center><p style="width:600px; margin-bottom:60px; text-align:justify"><b>Figure 3</b> Kinetic assay for Ethylene production. Cells were grown until an O.D. of 0.5 - 0.8 and then connected to the micro GC, while in agitation on a thermoblock at 37 &deg;C for the entire duration of the experiment. Every 45/60 mins a meausure was taken for a total of about 8 hours. Samples were induced at two differents O.D.600 and this had big effect on the amount of ethylene produced. However, it seems that the Ethylene concentration in the air space reached saturation after only two hours. The red dashed line indicates the amount of ethylene detected with a culture left in the thermoshaker for the all duration of the experiment and subjected to only one measurement. As expected an higher value of ethylene was measured due to the minimal gas loss with this approach.</p></center>
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<h3>Acceleration of fruit ripening</h3>
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===Usage and Biology===
  
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<h4><center>This device was used for accelerating fruit ripening. Many type of fruit were tested and ripened successfully. For more information and details please visit the <a href="http://2013.igem.org/Team:UNITN-Trento/Project/Fruit_ripening">UNITN wiki page </a>.</center></h4>
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<html>
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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>). <a href="#ref1">[1]</a> <a href="#ref2">[2]</a><BR />
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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>
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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>
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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="#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> </html>
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===Characterization before the jamboree===
  
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<center><img style="width:600px" src="https://static.igem.org/mediawiki/2013/6/64/Tn-2013_Application_on_fruit_for_part_wiki.jpg"></center>
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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.
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<center><p style="width:600px; margin-bottom:60px; text-align:justify"><b>Figure 4</b> Acceleration of fruit ripening. Panel A: our system was exploited for the acceleration of fruit ripening. We designed an hermetically closed jar with a rubber hose connector. These jars contained our test-fruit and each one was connected to a flask. The flasks contained 300 ml of induced (or not) culture when O.D.600 reached 0.8. The flasks contained a cultures maintained at 37 °C using a laboratory heating plate while stirring. For four to six days, every morning the culture in the flasks was substituted with a new induced (or not) culture. Furthermore, non-modified jars (i.e.: with no connector) were adopted to contain the negative control fruit samples (no-cells). Panel B: ripening of plums. Plum exposed to ethylene, show a more advanced stage of ripening after 4 days of treatment when compared to two negative controls (no-cells and BBa_K1065001 not induced).</p></center>
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<html><center><img style="width:500px;"src="https://static.igem.org/mediawiki/2013/7/7e/Tn-2013Pelletts.png"></center>
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<center><p style="width:600px; margin-bottom:60px; text-align:justify">
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<b>Cells transformed with BBa_K1065311: pellets after induction time.</b>  
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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.
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</p></center>
  
 
</html>
 
</html>
  
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===EFE characterization with a photoinducble circuit===
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===Characterization after the jamboree: ethylene production controlled by light presence===
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After the European jamboree we tested the circuit and acquired gas-chromatographic measurements in order to test light dependent EFE production.
  
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We characterized this part (<a href="https://parts.igem.org/Part:BBa_K1065311">BBa_K1065311</a>) in E. coli using cells NEB10b and have preliminary results.
 
  
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<html><center><img style="width:500px;"src="https://static.igem.org/mediawiki/2013/7/7e/Tn-2013Pelletts.png"></center>
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<html><center><img style="width:500px;"src="https://static.igem.org/mediawiki/2013/0/0c/Tn2013_ethylene_311.png"></center>
 
<center><p style="width:600px; margin-bottom:60px; text-align:justify">
 
<center><p style="width:600px; margin-bottom:60px; text-align:justify">
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<b>Frigure 5 Cells transformed with BBa_K1065311: pellets after induction time.</b>  
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<b>Ethylene formation upon photoinduction.</b>  
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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.
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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>
 
</p></center>
  
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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.
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</html>
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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.
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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.
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K1065000 parameters</partinfo>
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1065000 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K1065311 SequenceAndFeatures</partinfo>
  
 
===References===
 
===References===
 
<html><ol>
 
<html><ol>
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<li>Goto M, Shiday I, Akitaway T, Hyodoh, (1985). Ethylene production by the Kudzu strains of <I>Pseudomonas syringae</I> pv. <I>phaseolicola</I> causing halo blight in Pueraria lobata (Willd) Ohwi. Plant and Cell Physiology 26, 141-150.</li>
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<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>
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<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>
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<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>
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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>
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<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>
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<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 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>
 
</ol></html>
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K1065000 parameters</partinfo>
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<partinfo>BBa_K1065311 parameters</partinfo>
 
<!-- -->
 
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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