http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Roocfer&year=&month=2012.igem.org - User contributions [en]2024-03-28T09:20:28ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Valencia/compo_liquidTeam:Valencia/compo liquid2012-09-27T03:56:57Z<p>Roocfer: </p>
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Compo medium<br />
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<u><h4>Reagents and Materials</h4></u><br />
<ul style="list-style-type: square"><br />
<li>1’125 μl COMPO/mL distilled water</li><br />
</ul><br />
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<u><h4>Protocol</u></h4><br />
<ol><br />
<li>Pick up with a micropipette 1’125 μl COMPO in 25mL volume for each plate.</li><br />
<li>Set pH at 7’4.</li><br />
<li>Autoclave at 121ºC during 20 minutes. </li><br />
<li>Fill COMPO plus agar in the plates and leave until solidify.</li><br />
<li><u>Optative</u>: For doing solid medium used 3% agar.</li><br />
</ol><br />
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</html></div>Roocferhttp://2012.igem.org/Team:Valencia/BG-11HGlTeam:Valencia/BG-11HGl2012-09-27T03:52:42Z<p>Roocfer: </p>
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BG-11 medium<br />
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<u><h4>Reagents and Materials</h4></u><br />
<ul style="list-style-type: square"><br />
<li>Concentration BG-11:</li><br />
<ul style="list-style-type: square"><br />
<li>1’5 g/L of NaNO<sub>3</sub>.</li><br />
<li>0’039 g/l K<sub>2</sub>HPO<sub>4</sub>•3H<sub>2</sub>O.</li><br />
<li>0’075 g/l MgSO<sub>4</sub>•7H<sub>2</sub>O.</li><br />
<li>0’02g/l Na<sub>2</sub>CO<sub>3</sub>.</li><br />
<li>0’027 g/l CaCl<sub>2</sub>.</li><br />
<li>0’01g/l EDTA.</li><br />
<li>0’006 g/l ammonium Iron (III) citrate brown.</li><br />
<li>0’006 g/l citric acid anhydrous.</li><br />
</ul><br><br />
<li>Micronutrients:</li><br />
<ul style="list-style-type: square"><br />
<li>2,86 g/l H<sub>3</sub>BO<sub>3</sub>.</li><br />
<li>1,81 g/l MnCl<sub>2</sub>•4H<sub>2</sub>O.</li><br />
<li>0’222 g/l ZnSO<sub>4</sub>•7H<sub>2</sub>O.</li><br />
<li>0’391 g/l Na<sub>2</sub>MoO<sub>4•2H<sub>2</sub>O.</li><br />
<li>0’079 g/l CuSO<sub>4</sub>•5H<sub>2</sub>O.</li> <br />
<li>0’0494 g/l CoCl<sub>2</sub>•6H<sub>2</sub>O.</li><br />
</ul><br />
</ul><br />
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<br><br><br />
<u><h4>Protocol</u></h4><br />
<ol><br />
<li>Dissolve all the compounds for the BG-11 in an Erlenmeyer flask in 1 liter of deionized water and shake at room temperature until all salts are dissolved.</li><br />
<li>Dissolve all the compounds for the micronutrients in an Erlenmeyer flask in 1 liter of deionized water and shake at room temperature until all salts are dissolved. </li><br />
<li>Add 1mL of micronutrients for every liter of BG-11.</li><br />
<li>Autoclave at 121ºC during 20 minutes. </li><br />
<li>When the liquid cools down, set the pH at 7’4 for <i>Synechococcus elongatus</i> to come from Harvard. In the other hand, set the pH at 8’9 for <i>S. elongatus</i> to come from Golden. [For the S. elongatus transformed with cScB the pH must be 8,9]</li> <br />
</ol><br />
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For solid medium add 3% agar.<br />
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</html></div>Roocferhttp://2012.igem.org/Team:Valencia/BG-11HGlTeam:Valencia/BG-11HGl2012-09-27T03:51:44Z<p>Roocfer: </p>
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BG-11 medium<br />
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<div id="HomeCenterCenter"><p align="justify"><br />
<br><br />
<u><h4>Reagents and Materials</h4></u><br />
<ul style="list-style-type: square"><br />
<li>Concentration BG-11:</li><br />
<ul style="list-style-type: square"><br />
<li>1’5 g/L of NaNO<sub>3</sub>.</li><br />
<li>0’039 g/l K<sub>2</sub>HPO<sub>4</sub>•3H<sub>2</sub>O.</li><br />
<li>0’075 g/l MgSO<sub>4</sub>•7H<sub>2</sub>O.</li><br />
<li>0’02g/l Na<sub>2</sub>CO<sub>3</sub>.</li><br />
<li>0’027 g/l CaCl<sub>2</sub>.</li><br />
<li>0’01g/l EDTA.</li><br />
<li>0’006 g/l ammonium Iron (III) citrate brown.</li><br />
<li>0’006 g/l citric acid anhydrous.</li><br />
</ul><br><br />
<li>Micronutrients:</li><br />
<ul style="list-style-type: square"><br />
<li>2,86 g/l H<sub>3</sub>BO<sub>3</sub>.</li><br />
<li>1,81 g/l MnCl<sub>2</sub>•4H<sub>2</sub>O.</li><br />
<li>0’222 g/l ZnSO<sub>4</sub>•7H<sub>2</sub>O.</li><br />
<li>0’391 g/l Na<sub>2</sub>MoO<sub>4•2H<sub>2</sub>O.</li><br />
<li>0’079 g/l CuSO<sub>4</sub>•5H<sub>2</sub>O.</li> <br />
<li>0’0494 g/l CoCl<sub>2</sub>•6H<sub>2</sub>O.</li><br />
</ul><br />
</ul><br />
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<u><h4>Protocol</u></h4><br />
<ol><br />
<li>Dissolve all the compounds for the BG-11 in an Erlenmeyer flask in 1 liter of deionized water and shake at room temperature until all salts are dissolved.</li><br />
<li>Dissolve all the compounds for the micronutrients in an Erlenmeyer flask in 1 liter of deionized water and shake at room temperature until all salts are dissolved. </li><br />
<li>Add 1mL of micronutrients for every liter of BG-11.</li><br />
<li>Autoclave at 121ºC during 20 minutes. </li><br />
<li>When the liquid cools down, set the pH at 7’4 for <i>Synechococcus elongatus</i> to come from Harvard. In the other hand, set the pH at 8’9 for <i>S. elongatus</i> to come from Golden. [For the S. elongatus transformed with cScB the pH will be 8,9]</li> <br />
</ol><br />
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For solid medium add 3% agar.<br />
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</html></div>Roocferhttp://2012.igem.org/Team:Valencia/BG-11HGlTeam:Valencia/BG-11HGl2012-09-27T03:49:09Z<p>Roocfer: </p>
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BG-11 medium<br />
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<br><br />
<u><h4>Reagents and Materials</h4></u><br />
<ul style="list-style-type: square"><br />
<li>Concentration BG-11:</li><br />
<ul style="list-style-type: square"><br />
<li>1’5 g/L of NaNO<sub>3</sub>.</li><br />
<li>0’039 g/l K<sub>2</sub>HPO<sub>4</sub>•3H<sub>2</sub>O.</li><br />
<li>0’075 g/l MgSO<sub>4</sub>•7H<sub>2</sub>O.</li><br />
<li>0’02g/l Na<sub>2</sub>CO<sub>3</sub>.</li><br />
<li>0’027 g/l CaCl<sub>2</sub>.</li><br />
<li>0’01g/l EDTA.</li><br />
<li>0’006 g/l ammonium Iron (III) citrate brown.</li><br />
<li>0’006 g/l citric acid anhydrous.</li><br />
</ul><br><br />
<li>Micronutrients:</li><br />
<ul style="list-style-type: square"><br />
<li>2,86 g/l H<sub>3</sub>BO<sub>3</sub>.</li><br />
<li>1,81 g/l MnCl<sub>2</sub>•4H<sub>2</sub>O.</li><br />
<li>0’222 g/l ZnSO<sub>4</sub>•7H<sub>2</sub>O.</li><br />
<li>0’391 g/l Na<sub>2</sub>MoO<sub>4•2H<sub>2</sub>O.</li><br />
<li>0’079 g/l CuSO<sub>4</sub>•5H<sub>2</sub>O.</li> <br />
<li>0’0494 g/l CoCl<sub>2</sub>•6H<sub>2</sub>O.</li><br />
</ul><br />
</ul><br />
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<u><h4>Protocol</u></h4><br />
<ol><br />
<li>Dissolve all the compounds for the BG-11 in an Erlenmeyer flask in 1 liter of deionized water and shake at room temperature until all salts are dissolved.</li><br />
<li>Dissolve all the compounds for the micronutrients in an Erlenmeyer flask in 1 liter of deionized water and shake at room temperature until all salts are dissolved. </li><br />
<li>Add 1mL of micronutrients for every liter of BG-11.</li><br />
<li>Autoclave at 121ºC during 20 minutes. </li><br />
<li>When the liquid cools down, set the pH at 7’4 for <i>Synechococcus elongatus</i> to come from Harvard. In the other hand, set the pH at 8’9 for <i>S. elongatus</i> to come from Golden.</li><br />
</ol><br />
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For solid medium add 3% agar.<br />
</div><br />
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</html></div>Roocferhttp://2012.igem.org/Team:Valencia/BG-11HGlTeam:Valencia/BG-11HGl2012-09-27T03:45:25Z<p>Roocfer: </p>
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BG-11 medium<br />
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<u><h4>Reagents and Materials</h4></u><br />
<ul style="list-style-type: square"><br />
<li>Concentration BG-11:</li><br />
<ul style="list-style-type: square"><br />
<li>1’5 g/L of NaNO<sub>3</sub>.</li><br />
<li>0’039 g/l K<sub>2</sub>HPO<sub>4</sub>•3H<sub>2</sub>O.</li><br />
<li>0’075 g/l MgSO<sub>4</sub>•7H<sub>2</sub>O.</li><br />
<li>0’02g/l Na<sub>2</sub>CO<sub>3</sub>.</li><br />
<li>0’027 g/l CaCl<sub>2</sub>.</li><br />
<li>0’01g/l EDTA.</li><br />
<li>0’006 g/l ammonium Iron (III) citrate brown.</li><br />
<li>0’006 g/l citric acid anhydrous.</li><br />
</ul><br><br />
<li>Micronutrients:</li><br />
<ul style="list-style-type: square"><br />
<li>2,86 g/l H<sub>3</sub>BO<sub>3</sub>.</li><br />
<li>1,81 g/l MnCl<sub>2</sub>•4H<sub>2</sub>O.</li><br />
<li>0’222 g/l ZnSO<sub>4</sub>•7H<sub>2</sub>O.</li><br />
<li>0’391 g/l Na<sub>2</sub>MoO<sub>4•2H<sub>2</sub>O.</li><br />
<li>0’079 g/l CuSO<sub>4</sub>•5H<sub>2</sub>O.</li> <br />
<li>0’0494 g/l CoCl<sub>2</sub>•6H<sub>2</sub>O.</li><br />
</ul><br />
</ul><br />
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<u><h4>Protocol</u></h4><br />
<ol><br />
<li>Dissolve all the compounds for the BG-11 in an Erlenmeyer flask in 1 liter of deionized water and shake at room temperature until all salts are dissolved.</li><br />
<li>Dissolve all the compounds for the micronutrients in an Erlenmeyer flask in 1 liter of deionized water and shake at room temperature until all salts are dissolved. </li><br />
<li>Add 1mL of micronutrients for every liter of BG-11.</li><br />
<li>Autoclave at 121ºC during 20 minutes. </li><br />
<li>When the liquid cools down, set the pH at 7’4 for <i>Synechococcus elongatus</i> to come from Harvard. In the other hand, set the pH at 8’9 for <i>S. elongatus</i> to come from Golden.</li><br />
</ol><br />
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Plasmid Miniprep (<i>JETQUICK</i> kit)<br />
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<u><h4>Reagents and Materials</h4></u><br />
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<li>Solution G1 (Cell Suspension) 50mM Tris/HCl (pH 8.0); 10 mM EDTA</li><br />
<li>Solution G2 (Cell Lysis) 200 mM NaOH; 1% SDS (w/v)</li><br />
<li>Solution G3 (Neutralization/Binding) Contains acetate and guanidine hydrochloride.</li><br />
<li>Solution GX (Wash, optional) Contains guanidine hydrochloride.</li><br />
<li>Solutions G4 (Column Wash) Contains NaCl, EDTA and Tris.HCl </li><br />
</ul><br />
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<u><h4>Protocol</u></h4><br />
<ol><br />
<li><u>Harvesting Bacterial Cells</u>: E.coli cells are pelleted by centrifugation. Remove all traces of medium carefully. Then, we make sure that culture medium back-draining from the tubes wall is removed.</li><br />
<li><u>Cell Resuspending</u>: add 250 µl of solution G1 reconstituted with RNase to the pellet and resuspend the cells (by vortexing or with a pipette) until the suspension is homogeneous.</li><br />
<li><u>Cells Lysis</u>: add 250 µl of solution G2 and mix gently, but thoroughly, by inverting the tube several times. Do not vortex! Incubate at room temperature for 5 min.</li><br />
<li><u>Neutralization</u>: add 350 µl of solution G3 and mix gently but thoroughly, by inverting the tube until a homogeneous suspension is obtained. Do not vortex! Centrifuge. The mixture at room temperature and at maximum speed for 10 min.</li><br />
<li><u>Column Loading</u>: place a JETQUICK spin column into a 2 ml receiver tube (provided). Load the supernatant from step 4 into the spin column. Centrifuge at>12.000 x g for 1 min. Discard the flowthrough.</li><br />
After having emptied the receiver tube re-insert the micro-apin column into it. Add 500 µl of reconstituted buffer GX into the spin column, and centrifuge at>12.000 x g for 1 min. Discard flowthrough and place the JETQUICK column back into the same receiver tube. </li><br />
<li><u>Column Washing</u>: empty the receiver tube, and re-insert the spin column into the receiver tube. Add 500 µl of reconstituted buffer G4 and centrifuge at >12.000 x g for 1 min. Discard flowthrough and place the spin column back into the same receiver. Centrifuge again at maximum speed for 1 min. </li><br />
<li><u>Plasmid Elution</u>: higher DNA concentrations can be obtained if the elutions is carried out in only 50 µl elution buffer volume. In this case, preheat your elution buffer to 65-70ºC, add the buffer onto the center of the silica matrix of the spin column and let stand for 1 min before centrifugation. Preheated elution buffer is generally recommended when plasmids larger 5 kb are eluted. DNA eluted in water should be stored at -20ºC. Centrifuge at >12.000 x g for 2 min. </li><br />
</ol><br />
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</html></div>Roocferhttp://2012.igem.org/Team:Valencia/LigationsTeam:Valencia/Ligations2012-09-27T03:38:05Z<p>Roocfer: </p>
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Ligations<br />
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<u><h4>Reagents and Materials</h4></u><br />
<ul style="list-style-type: square"><br />
<li>25ng/µl Psb1C3.</li><br />
<li>200 ng/µl PsbAI.</li><br />
<li>0.5 µl T<sup>4</sup> ligase .</li> <br />
<li>1 µl T T<sup>4</sup> ligase buffer</li> <br />
<li>(8.5-x) µl deionized water</li><br />
</ul><br />
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<u><h4>Protocol</u></h4><br />
<ol><br />
<li>Add appropriate amount of deionized H<sub>2</sub>O to sterile 0.6 mL tube.</li><br />
<li>Add 10 μL ligation buffer to the tube.</li><br />
<li>Vortex buffer before pipetting to ensure that it is well-mixed.</li><br />
<li>Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation. It is recommended that you aliquot the Ligation Buffer into smaller quantities.</li><br />
<li>Add appropriate amount of insert to the tube.</li><br />
<li>Add appropriate amount of vector to the tube.</li><br />
<li>Add 1 μL ligase. Vortex ligase before pipetting to ensure that it is well-mixed. Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 μL, just touch your tip to the surface of the liquid when pipetting. </li><br />
<li>Incubate 5 mins on the benchtop.</li><br />
<li>Place on ice until transformation.</li><br />
<li>Generally 1 μL of ligation mix is sufficient for either chemical transformation or electroporation. The amount of salt in 1 μL ligation mix should not cause arcing. </li><br />
<li><u>Optional:</u> Heat-inactivate by incubating at 65°C for 20 mins. Then do a purification step to remove PEG.</li><br />
</ol><br />
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</html></div>Roocferhttp://2012.igem.org/Team:Valencia/genetic_engineeringTeam:Valencia/genetic engineering2012-09-27T03:35:31Z<p>Roocfer: </p>
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<br>Genetic Engineering<br><br />
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<a href="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg"><img align="right" src="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg" style="margin-left: 10px;"></a><br />
<br><br><br />
Cyanobacteria are great organisms to be used in Synthetic Biology because of their ability to capture solar energy and CO<sub>2</sub> and the fact that they can be easily genetically manipulated due to its small genome and their capacity to accept foreign DNA naturally. For our project we have chosen the cyanobacteria <i>Synechococcus elongatus</i> (figure 1) for genetic engineering because is the model organism for studying some prokaryotic processes (there is a lot of information of how to transform it) and in the last years it has become a model organism for some industrial processes, like biofuel production (Wang et al. 2012).<br><br><br />
<i>S. elongatus</i> has a circular genome of ≈2.7Mb (<a href="http://genome.kazusa.or.jp/cyanobase/SYNPCC7942" target=_blank"> fully sequenced </a>) with a GC content of 55.5%, which contains the genes for 2.612 proteins and 53 RNAs (Atsumi et al. 2009). <br />
<br><br><br><br><br />
<div align="right"><b>Figure 1: </b> <i>S. elongatus</i> PCC7942 </div><br />
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<h2><b>Transforming <i>S. elongatus</i> PCC7942 for promoter characterization:</b></h2><br />
<br><br><br />
Our main objective has been to characterize the <i>psbAI</i> promoter (<a href="https://2012.igem.org/Team:Valencia/Parts">Submitted parts</a>) of <i>Synechococcus elongatus</i> PCC7942 in order to know more about its operation and understand how this promoter would control our final construct (<a href="https://2012.igem.org/Team:Valencia/prototypes"> Designed parts</a>) to have a diel switch of AHL, the signal molecule for our <i>Aliivibrio fischeri</i> population to glow. <br><br><br />
To characterize this promoter we had two options: making <i>psbAIp::lacZ</i> fusions and monitored the β-galactosidase activity or making <i>psbAIp::luxABCDE</i> fusions and monitored the bioluminescence produced as a result of the promoter activation. We refused to use a <i>psbAIp::lacZ</i> fusion because some assays report that the <i>psbAI</i> promoter response to light cannot be properly monitored by the β-galactosidase activity (Nair et al. 2001). For this reason we choosed using vector fusions. With this aim, we cloned several fusion plasmids (pAM977 and pAM2195, table 1) in <i>Escherichia coli</i> and transformed them into <i>S. elongatus</i>, both wildtype and cscB strain. The fusion vectors were provided by Susan Golden´s lab. <br><br><br />
<center><b>Table 1: </b> Information of the two vectors used for characterize the <i>psbAI</i> promoter</center><br />
<center><a href="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png"><img src="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png" width="600" height="180"></a></center><br><br><br><br />
<br />
<b>Cloning into <i>E. coli</i></b><br />
<br><br><br />
We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this <a href="https://2012.igem.org/Team:Valencia/Transforming_ecoli">protocol</a>. For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 (figure 2) Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> <br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br><br />
<center><b>Figure 2: </b>pAM2195 vector</center><br />
<br><br><br />
<br />
<b>Transforming Coccus</b><br />
<br><br><br />
Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. <br />
As the cscB strain from Harvard was resistant to Cm, we only could transform it with the pAM977 plasmid, which gives Sp resistance. Wildtype was transformed with both plasmids (pAM977 and pAM2195).<br><br />
We used the following protocol to transform <i>Synechococcus</i>: <br><br><br />
<br />
<br><br />
<b>Protocol</b><br />
<br><br><br />
Extracted from Clerico et al. 2007:<br><br />
<ul style="list-style-type: square"><br><br />
<li>We grew 100ml of cscB and WT in BG-11 liquid medium shaking at 250rpm with constant light we used cold light fluorescent tubes. Cells had an optical density at 750nm (OD750) of 0.7, which is reach in 4 to 7 days approximately.</li><br><br />
<li>We collected 15mL of the culture and centrifuge it at 6000g for 10 min and discarded the supernatant.</li><br><br />
<li>We resuspended collected cells in 10 mL of 10mM NaCl and centrifuge them again at 6000g for 10 min.</li><br><br />
<li>After it, we resuspended the pellet in 0.3mL of BG-11M liquid medium.</li><br><br />
<li>We added 2μl of pAM2195 and pAM977 after doing a Miniprep of the clonation with a <i>JetQuick</i> kit </li><br><br />
<li>Wrap tubes in aluminum foil to keep out light and incubate at 30oC for 15 to 20h with gentle agitation.</li><br><br />
<li>And then, plate the entire 0.3mL cell suspension on BG-11 medium agar with the selective antibiotics. We used this concentration: 2μg/ml spectinomycin and 7.5μg/mL of chloramphenicol. After 7 to 10 days of incubation under standard light conditions transformed colonies should appear.</li><br><br />
<li>After colonies appearance pick single transformants and grow them on fresh liquid BG-11M agar plate with selective antibiotic (this is done to ensure that all colonies have incorporated the trans gene as S. elongatus has multiple copies of its chromosome).</li><br><br />
<li>After 5 to 7 days of growth, cyanobacteria can be used to inoculate a BG-11 liquid culture with the selective antibiotic.</li></ul><br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG"><img src="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG" width="458" height="343"></a></center><br />
<center><b>Figure 3: </b>A view of our transformed cells</center><br />
<br><br />
<b>Results</b><br />
<br><br />
We weren´t able to obtain transformants with the wild type strain of <i>S. elongatus</i>. <br />
But we obtained resistant colonies to Sp of the cscB strain transformed with the pAM977 vector (figure 4). This was achieved the last week of lab work, so due to the slowly growth rate of <i>S. elongatus</i>, we haven´t have the time to grow the transformants in liquid medium and start with the bioluminescence measures to characterize the promoter. However, we will try to have some results for the Jamboree concerning this part.<br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"><img src="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"></a></center><br />
<center><b>Figure 4: </b> pAM977 transformants</center><br />
<br><br><br />
<br />
<h2><b>Transformation of <i>E. coli</i> for AHL production to test bioluminescent response in <i>A. fischeri</i>:</b></h2><br><br />
Another part of our project included transforming <i>E. coli</i> with the registry part <a href="http://partsregistry.org/Part:BBa_K084014"> BBa_K084014 </a> in order to make <i>E. coli</i> produce AHL under IPTG regulation to test the bioluminescent response in A. fischeri. To achieve this goal we cloned the part in DH5α <i>E. coli</i> strains using this <a href="https://www.facebook.com/l.php?u=http%3A%2F%2Fpartsregistry.org%2FHelp%3ADistribution_Kits&h=oAQFOZXUl">protocol</a>(figure 5). The part (869 bp) is included inside the pSB1A2 (2079bp) BioBrick.<br><br><br />
<center><a href="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"><img src="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"></a></center><br />
<center><b>Figure 5:</b>The whole BBa_K084014 part has a size of 2948bp. </center><br />
<br><br />
We designed an experimental procedure to test different experimental conditions for the production of AHL.<br><br><br />
<center><b>Table 2: </b> Experimental design for AHL production </center><br />
<center><a href="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png"><img src="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png" width="600" height="140"></a></center><br><br />
<br />
<br>When we wanted to test if <i>A. fischeri</i> was able to glow with the AHL produced by <i>E. coli</i> we discovered that the strain we had been growing in the lab was not <i> V. fischeri</i>, but <i>V.mediterrani</i> (GREAT!).<br><br />
We will try to obtain <i>V. fischeri</i> as soon as possible to test this.<br><br />
</p><br />
<br><br />
<h2><b>Building our BioBricks</b></h2><br />
<br><br />
<b><i>psbAI</i> BioBrick</b><br />
<br><br><br />
Our promoter with the aproppiate <a href="http://partsregistry.org/Help:Assembly_standard_25"> suffix and preffix </a> (289bp) was synthesized by Genscript and cloned into a puc57-Kan (2579bp) vector. We successfully digested the part and ligated it into the psb1C3 BioBrick from the parts registry (figure 6, 7 and 8). For this aim we followed the openwetware protocols, which you can find here. <br><br><br />
<br />
<b>Complete Construct</b><br />
<br><br><br />
Our final objective is to have a S. elongatus capable to produce AHL during the night. For this we designed this new BioBrick controled by the psbAI promoter which is active in normal light conditions.<a href="https://2012.igem.org/Team:Valencia/prototypes"> Here </a> you will find all the information concerning the different parts of the BioBrick.<br><br />
We successfully cloned all the parts in DH5α E. coli strains using this <a href="https://2012.igem.org/Team:Valencia/Transforming_ecoli">protocol</a>. We used the <a href="http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf">BioBrick Assembly Manual</a> protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3.<br><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/f/fc/DSC02872_VLC.jpg"> &nbsp; &nbsp;<br />
<img src="https://static.igem.org/mediawiki/2012/6/6b/Electro_VLCXXX.jpg" width="350" height="270"><br />
</center><br />
<center><b>Figures 6 & 7: </b> Succesfully we build our BioBrick. In the electrophoresis the band that corresponds to the part (3868bp) is the one surrounded with a red triangle </center><br />
<br><br><br />
<center><img src="https://static.igem.org/mediawiki/2012/c/ce/VLC_PsB1C3%2BpsbAI.png"></center><br />
<center><b>Figure 8: </b> <i>psbAI</i> promoter inside the pSB1C3</center><br />
<br />
<strong>References</strong><br />
<hr><hr><br />
<br><br />
Atsumi, S., Higashide, W., and Liao, J. C. (2009) Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. <i>Nat Biotechnol</i>. 27:1177-1180<br><br><br />
Clerico, E. M., Ditty, J. L. & Golden, S.S. (2007) Specialized Techniques for Site-Directed Mutagenesis in Cyanobacteria. <i>Methods in Molecular Biology.</i> 362:153–172.<br><br><br />
Nair, U., Thomas, C. & Golden, S. S. (2001) Functional Elements of the Strong <i>psbAI</i> Promoter of Synechococcus elongatus PCC 7942. <i>J. Bacteriology</i>, 183:1740–1747.<br><br><br />
Wang, B., Wang J., Zhang, W. & Meldrum, D. R. (2012) Application of Synthetic Biology in cyanobacteria and algae. Frontiers in Microbiology, doi: 10.3389/fmicb.2012.00344<br><br><br />
<br />
</p><br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/genetic_engineeringTeam:Valencia/genetic engineering2012-09-27T03:31:05Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Genetic Engineering<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
<a href="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg"><img align="right" src="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg" style="margin-left: 10px;"></a><br />
Cyanobacteria are great organisms to be used in Synthetic Biology because of their ability to capture solar energy and CO<sub>2</sub> and the fact that they can be easily genetically manipulated due to its small genome and their capacity to accept foreign DNA naturally. For our project we have chosen the cyanobacteria <i>Synechococcus elongatus</i> (figure 1) for genetic engineering because is the model organism for studying some prokaryotic processes (there is a lot of information of how to transform it) and in the last years it has become a model organism for some industrial processes, like biofuel production (Wang et al. 2012).<br><br><br />
<i>S. elongatus</i> has a circular genome of ≈2.7Mb (<a href="http://genome.kazusa.or.jp/cyanobase/SYNPCC7942" target=_blank"> fully sequenced </a>) with a GC content of 55.5%, which contains the genes for 2.612 proteins and 53 RNAs (Atsumi et al. 2009). <br />
<br><br><br><br><br><br><br />
<div align="right"><b>Figure 1: </b> <i>S. elongatus</i> PCC7942 </div><br />
<br><br />
<br />
<h2><b>Transforming <i>S. elongatus</i> PCC7942 for promoter characterization:</b></h2><br />
<br><br><br />
Our main objective has been to characterize the <i>psbAI</i> promoter (<a href="https://2012.igem.org/Team:Valencia/Parts">Submitted parts</a>) of <i>Synechococcus elongatus</i> PCC7942 in order to know more about its operation and understand how this promoter would control our final construct (<a href="https://2012.igem.org/Team:Valencia/prototypes"> Designed parts</a>) to have a diel switch of AHL, the signal molecule for our <i>Aliivibrio fischeri</i> population to glow. <br><br><br />
To characterize this promoter we had two options: making <i>psbAIp::lacZ</i> fusions and monitored the β-galactosidase activity or making <i>psbAIp::luxABCDE</i> fusions and monitored the bioluminescence produced as a result of the promoter activation. We refused to use a <i>psbAIp::lacZ</i> fusion because some assays report that the <i>psbAI</i> promoter response to light cannot be properly monitored by the β-galactosidase activity (Nair et al. 2001). For this reason we choosed using vector fusions. With this aim, we cloned several fusion plasmids (pAM977 and pAM2195, table 1) in <i>Escherichia coli</i> and transformed them into <i>S. elongatus</i>, both wildtype and cscB strain. The fusion vectors were provided by Susan Golden´s lab. <br><br><br />
<center><b>Table 1: </b> Information of the two vectors used for characterize the <i>psbAI</i> promoter</center><br />
<center><a href="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png"><img src="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png" width="600" height="180"></a></center><br><br><br><br />
<br />
<b>Cloning into <i>E. coli</i></b><br />
<br><br><br />
We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this <a href="https://2012.igem.org/Team:Valencia/Transforming_ecoli">protocol</a>. For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 (figure 2) Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> <br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br><br />
<center><b>Figure 2: </b>pAM2195 vector</center><br />
<br><br><br />
<br />
<b>Transforming Coccus</b><br />
<br><br><br />
Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. <br />
As the cscB strain from Harvard was resistant to Cm, we only could transform it with the pAM977 plasmid, which gives Sp resistance. Wildtype was transformed with both plasmids (pAM977 and pAM2195).<br><br />
We used the following protocol to transform <i>Synechococcus</i>: <br><br><br />
<br />
<br><br />
<b>Protocol</b><br />
<br><br><br />
Extracted from Clerico et al. 2007:<br><br />
<ul style="list-style-type: square"><br><br />
<li>We grew 100ml of cscB and WT in BG-11 liquid medium shaking at 250rpm with constant light we used cold light fluorescent tubes. Cells had an optical density at 750nm (OD750) of 0.7, which is reach in 4 to 7 days approximately.</li><br><br />
<li>We collected 15mL of the culture and centrifuge it at 6000g for 10 min and discarded the supernatant.</li><br><br />
<li>We resuspended collected cells in 10 mL of 10mM NaCl and centrifuge them again at 6000g for 10 min.</li><br><br />
<li>After it, we resuspended the pellet in 0.3mL of BG-11M liquid medium.</li><br><br />
<li>We added 2μl of pAM2195 and pAM977 after doing a Miniprep of the clonation with a <i>JetQuick</i> kit </li><br><br />
<li>Wrap tubes in aluminum foil to keep out light and incubate at 30oC for 15 to 20h with gentle agitation.</li><br><br />
<li>And then, plate the entire 0.3mL cell suspension on BG-11 medium agar with the selective antibiotics. We used this concentration: 2μg/ml spectinomycin and 7.5μg/mL of chloramphenicol. After 7 to 10 days of incubation under standard light conditions transformed colonies should appear.</li><br><br />
<li>After colonies appearance pick single transformants and grow them on fresh liquid BG-11M agar plate with selective antibiotic (this is done to ensure that all colonies have incorporated the trans gene as S. elongatus has multiple copies of its chromosome).</li><br><br />
<li>After 5 to 7 days of growth, cyanobacteria can be used to inoculate a BG-11 liquid culture with the selective antibiotic.</li></ul><br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG"><img src="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG" width="458" height="343"></a></center><br />
<center><b>Figure 3: </b>A view of our transformed cells</center><br />
<br><br />
<b>Results</b><br />
<br><br />
We weren´t able to obtain transformants with the wild type strain of <i>S. elongatus</i>. <br />
But we obtained resistant colonies to Sp of the cscB strain transformed with the pAM977 vector (figure 4). This was achieved the last week of lab work, so due to the slowly growth rate of <i>S. elongatus</i>, we haven´t have the time to grow the transformants in liquid medium and start with the bioluminescence measures to characterize the promoter. However, we will try to have some results for the Jamboree concerning this part.<br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"><img src="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"></a></center><br />
<center><b>Figure 4: </b> pAM977 transformants</center><br />
<br><br><br />
<br />
<h2><b>Transformation of <i>E. coli</i> for AHL production to test bioluminescent response in <i>A. fischeri</i>:</b></h2><br><br />
Another part of our project included transforming <i>E. coli</i> with the registry part <a href="http://partsregistry.org/Part:BBa_K084014"> BBa_K084014 </a> in order to make <i>E. coli</i> produce AHL under IPTG regulation to test the bioluminescent response in A. fischeri. To achieve this goal we cloned the part in DH5α <i>E. coli</i> strains using this <a href="https://www.facebook.com/l.php?u=http%3A%2F%2Fpartsregistry.org%2FHelp%3ADistribution_Kits&h=oAQFOZXUl">protocol</a>(figure 5). The part (869 bp) is included inside the pSB1A2 (2079bp) BioBrick.<br><br><br />
<center><a href="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"><img src="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"></a></center><br />
<center><b>Figure 5:</b>The whole BBa_K084014 part has a size of 2948bp. </center><br />
<br><br />
We designed an experimental procedure to test different experimental conditions for the production of AHL.<br><br><br />
<center><b>Table 2: </b> Experimental design for AHL production </center><br />
<center><a href="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png"><img src="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png" width="600" height="140"></a></center><br><br />
<br />
<br>When we wanted to test if <i>A. fischeri</i> was able to glow with the AHL produced by <i>E. coli</i> we discovered that the strain we had been growing in the lab was not <i> V. fischeri</i>, but <i>V.mediterrani</i> (GREAT!).<br><br />
We will try to obtain <i>V. fischeri</i> as soon as possible to test this.<br><br />
</p><br />
<br><br />
<h2><b>Building our BioBricks</b></h2><br />
<br><br />
<b><i>psbAI</i> BioBrick</b><br />
<br><br><br />
Our promoter with the aproppiate <a href="http://partsregistry.org/Help:Assembly_standard_25"> suffix and preffix </a> (289bp) was synthesized by Genscript and cloned into a puc57-Kan (2579bp) vector. We successfully digested the part and ligated it into the psb1C3 BioBrick from the parts registry (figure 6, 7 and 8). For this aim we followed the openwetware protocols, which you can find here. <br><br><br />
<br />
<b>Complete Construct</b><br />
<br><br><br />
Our final objective is to have a S. elongatus capable to produce AHL during the night. For this we designed this new BioBrick controled by the psbAI promoter which is active in normal light conditions.<a href="https://2012.igem.org/Team:Valencia/prototypes"> Here </a> you will find all the information concerning the different parts of the BioBrick.<br><br />
We successfully cloned all the parts in DH5α E. coli strains using this <a href="https://2012.igem.org/Team:Valencia/Transforming_ecoli">protocol</a>. We used the <a href="http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf">BioBrick Assembly Manual</a> protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3.<br><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/f/fc/DSC02872_VLC.jpg"> &nbsp; &nbsp;<br />
<img src="https://static.igem.org/mediawiki/2012/6/6b/Electro_VLCXXX.jpg" width="350" height="270"><br />
</center><br />
<center><b>Figures 6 & 7: </b> Succesfully we build our BioBrick. In the electrophoresis the band that corresponds to the part (3868bp) is the one surrounded with a red triangle </center><br />
<br><br><br />
<center><img src="https://static.igem.org/mediawiki/2012/c/ce/VLC_PsB1C3%2BpsbAI.png"></center><br />
<center><b>Figure 8: </b> <i>psbAI</i> promoter inside the pSB1C3</center><br />
<br />
<strong>References</strong><br />
<hr><hr><br />
<br><br />
Atsumi, S., Higashide, W., and Liao, J. C. (2009) Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. <i>Nat Biotechnol</i>. 27:1177-1180<br><br><br />
Clerico, E. M., Ditty, J. L. & Golden, S.S. (2007) Specialized Techniques for Site-Directed Mutagenesis in Cyanobacteria. <i>Methods in Molecular Biology.</i> 362:153–172.<br><br><br />
Nair, U., Thomas, C. & Golden, S. S. (2001) Functional Elements of the Strong <i>psbAI</i> Promoter of Synechococcus elongatus PCC 7942. <i>J. Bacteriology</i>, 183:1740–1747.<br><br><br />
Wang, B., Wang J., Zhang, W. & Meldrum, D. R. (2012) Application of Synthetic Biology in cyanobacteria and algae. Frontiers in Microbiology, doi: 10.3389/fmicb.2012.00344<br><br><br />
<br />
</p><br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/genetic_engineeringTeam:Valencia/genetic engineering2012-09-27T03:28:58Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Genetic Engineering<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
<a href="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg"><img align="right" src="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg" style="margin-left: 10px;"></a><br />
Cyanobacteria are great organisms to be used in Synthetic Biology because of their ability to capture solar energy and CO<sub>2</sub> and the fact that they can be easily genetically manipulated due to its small genome and their capacity to accept foreign DNA naturally. For our project we have chosen the cyanobacteria <i>Synechococcus elongatus</i> (figure 1) for genetic engineering because is the model organism for studying some prokaryotic processes (there is a lot of information of how to transform it) and in the last years it has become a model organism for some industrial processes, like biofuel production (Wang et al. 2012).<br><br><br />
<i>S. elongatus</i> has a circular genome of ≈2.7Mb (<a href="http://genome.kazusa.or.jp/cyanobase/SYNPCC7942" target=_blank"> fully sequenced </a>) with a GC content of 55.5%, which contains the genes for 2.612 proteins and 53 RNAs (Atsumi et al. 2009). <br />
<br><br><br><br><br><br><br />
<div align="right"><b>Figure 1: </b> <i>S. elongatus</i> PCC7942 </div><br />
<br><br />
<br />
<h2><b>Transforming <i>S. elongatus</i> PCC7942 for promoter characterization:</b></h2><br />
<br><br><br />
Our main objective has been to characterize the <i>psbAI</i> promoter (<a href="https://2012.igem.org/Team:Valencia/Parts">Submitted parts</a>) of <i>Synechococcus elongatus</i> PCC7942 in order to know more about its operation and understand how this promoter would control our final construct (<a href="https://2012.igem.org/Team:Valencia/prototypes"> Designed parts</a>) to have a diel switch of AHL, the signal molecule for our <i>Aliivibrio fischeri</i> population to glow. <br><br><br />
To characterize this promoter we had two options: making <i>psbAIp::lacZ</i> fusions and monitored the β-galactosidase activity or making <i>psbAIp::luxABCDE</i> fusions and monitored the bioluminescence produced as a result of the promoter activation. We refused to use a <i>psbAIp::lacZ</i> fusion because some assays report that the <i>psbAI</i> promoter response to light cannot be properly monitored by the β-galactosidase activity (Nair et al. 2001). For this reason we choosed using vector fusions. With this aim, we cloned several fusion plasmids (pAM977 and pAM2195, table 1) in <i>Escherichia coli</i> and transformed them into <i>S. elongatus</i>, both wildtype and cscB strain. The fusion vectors were provided by Susan Golden´s lab. <br><br><br />
<center><b>Table 1: </b> Information of the two vectors used for characterize the <i>psbAI</i> promoter</center><br />
<center><a href="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png"><img src="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png" width="600" height="180"></a></center><br><br><br><br />
<br />
<b>Cloning into <i>E. coli</i></b><br><br />
We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this <a href="https://2012.igem.org/Team:Valencia/Transforming_ecoli">protocol</a>. For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 (figure 2) Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> <br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br><br />
<center><b>Figure 2: </b>pAM2195 vector</center><br><br><br />
<b>Transforming Coccus</b><br><br />
Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. <br />
As the cscB strain from Harvard was resistant to Cm, we only could transform it with the pAM977 plasmid, which gives Sp resistance. Wildtype was transformed with both plasmids (pAM977 and pAM2195).<br><br />
We used the following protocol to transform <i>Synechococcus</i>: <br><br><br />
<br />
<br />
<br><br />
<b>Protocol</b><br />
<br><br />
Extracted from Clerico et al. 2007:<br><br />
<ul style="list-style-type: square"><br><br />
<li>We grew 100ml of cscB and WT in BG-11 liquid medium shaking at 250rpm with constant light we used cold light fluorescent tubes. Cells had an optical density at 750nm (OD750) of 0.7, which is reach in 4 to 7 days approximately.</li><br><br />
<li>We collected 15mL of the culture and centrifuge it at 6000g for 10 min and discarded the supernatant.</li><br><br />
<li>We resuspended collected cells in 10 mL of 10mM NaCl and centrifuge them again at 6000g for 10 min.</li><br><br />
<li>After it, we resuspended the pellet in 0.3mL of BG-11M liquid medium.</li><br><br />
<li>We added 2μl of pAM2195 and pAM977 after doing a Miniprep of the clonation with a <i>JetQuick</i> kit </li><br><br />
<li>Wrap tubes in aluminum foil to keep out light and incubate at 30oC for 15 to 20h with gentle agitation.</li><br><br />
<li>And then, plate the entire 0.3mL cell suspension on BG-11 medium agar with the selective antibiotics. We used this concentration: 2μg/ml spectinomycin and 7.5μg/mL of chloramphenicol. After 7 to 10 days of incubation under standard light conditions transformed colonies should appear.</li><br><br />
<li>After colonies appearance pick single transformants and grow them on fresh liquid BG-11M agar plate with selective antibiotic (this is done to ensure that all colonies have incorporated the trans gene as S. elongatus has multiple copies of its chromosome).</li><br><br />
<li>After 5 to 7 days of growth, cyanobacteria can be used to inoculate a BG-11 liquid culture with the selective antibiotic.</li></ul><br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG"><img src="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG" width="458" height="343"></a></center><br />
<center><b>Figure 3: </b>A view of our transformed cells</center><br />
<br><br />
<b>Results</b><br />
<br><br />
We weren´t able to obtain transformants with the wild type strain of <i>S. elongatus</i>. <br />
But we obtained resistant colonies to Sp of the cscB strain transformed with the pAM977 vector (figure 4). This was achieved the last week of lab work, so due to the slowly growth rate of <i>S. elongatus</i>, we haven´t have the time to grow the transformants in liquid medium and start with the bioluminescence measures to characterize the promoter. However, we will try to have some results for the Jamboree concerning this part.<br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"><img src="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"></a></center><br />
<center><b>Figure 4: </b> pAM977 transformants</center><br />
<br><br><br />
<br />
<h2><b>Transformation of <i>E. coli</i> for AHL production to test bioluminescent response in <i>A. fischeri</i>:</b></h2><br><br />
Another part of our project included transforming <i>E. coli</i> with the registry part <a href="http://partsregistry.org/Part:BBa_K084014"> BBa_K084014 </a> in order to make <i>E. coli</i> produce AHL under IPTG regulation to test the bioluminescent response in A. fischeri. To achieve this goal we cloned the part in DH5α <i>E. coli</i> strains using this <a href="https://www.facebook.com/l.php?u=http%3A%2F%2Fpartsregistry.org%2FHelp%3ADistribution_Kits&h=oAQFOZXUl">protocol</a>(figure 5). The part (869 bp) is included inside the pSB1A2 (2079bp) BioBrick.<br><br><br />
<center><a href="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"><img src="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"></a></center><br />
<center><b>Figure 5:</b>The whole BBa_K084014 part has a size of 2948bp. </center><br />
<br><br />
We designed an experimental procedure to test different experimental conditions for the production of AHL.<br><br><br />
<center><b>Table 2: </b> Experimental design for AHL production </center><br />
<center><a href="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png"><img src="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png" width="600" height="140"></a></center><br><br />
<br />
<br>When we wanted to test if <i>A. fischeri</i> was able to glow with the AHL produced by <i>E. coli</i> we discovered that the strain we had been growing in the lab was not <i> V. fischeri</i>, but <i>V.mediterrani</i> (GREAT!).<br><br />
We will try to obtain <i>V. fischeri</i> as soon as possible to test this.<br><br />
</p><br />
<br><br />
<h2><b>Building our BioBricks</b></h2><br />
<br><br />
<b><i>psbAI</i> BioBrick</b><br />
<br><br><br />
Our promoter with the aproppiate <a href="http://partsregistry.org/Help:Assembly_standard_25"> suffix and preffix </a> (289bp) was synthesized by Genscript and cloned into a puc57-Kan (2579bp) vector. We successfully digested the part and ligated it into the psb1C3 BioBrick from the parts registry (figure 6, 7 and 8). For this aim we followed the openwetware protocols, which you can find here. <br><br><br />
<br />
<b>Complete Construct</b><br />
<br><br><br />
Our final objective is to have a S. elongatus capable to produce AHL during the night. For this we designed this new BioBrick controled by the psbAI promoter which is active in normal light conditions.<a href="https://2012.igem.org/Team:Valencia/prototypes"> Here </a> you will find all the information concerning the different parts of the BioBrick.<br><br />
We successfully cloned all the parts in DH5α E. coli strains using this <a href="https://2012.igem.org/Team:Valencia/Transforming_ecoli">protocol</a>. We used the <a href="http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf">BioBrick Assembly Manual</a> protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3.<br><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/f/fc/DSC02872_VLC.jpg"> &nbsp; &nbsp;<br />
<img src="https://static.igem.org/mediawiki/2012/6/6b/Electro_VLCXXX.jpg" width="350" height="270"><br />
</center><br />
<center><b>Figures 6 & 7: </b> Succesfully we build our BioBrick. In the electrophoresis the band that corresponds to the part (3868bp) is the one surrounded with a red triangle </center><br />
<br><br><br />
<center><img src="https://static.igem.org/mediawiki/2012/c/ce/VLC_PsB1C3%2BpsbAI.png"></center><br />
<center><b>Figure 8: </b> <i>psbAI</i> promoter inside the pSB1C3</center><br />
<br />
<strong>References</strong><br />
<hr><hr><br />
<br><br />
Atsumi, S., Higashide, W., and Liao, J. C. (2009) Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. <i>Nat Biotechnol</i>. 27:1177-1180<br><br><br />
Clerico, E. M., Ditty, J. L. & Golden, S.S. (2007) Specialized Techniques for Site-Directed Mutagenesis in Cyanobacteria. <i>Methods in Molecular Biology.</i> 362:153–172.<br><br><br />
Nair, U., Thomas, C. & Golden, S. S. (2001) Functional Elements of the Strong <i>psbAI</i> Promoter of Synechococcus elongatus PCC 7942. <i>J. Bacteriology</i>, 183:1740–1747.<br><br><br />
Wang, B., Wang J., Zhang, W. & Meldrum, D. R. (2012) Application of Synthetic Biology in cyanobacteria and algae. Frontiers in Microbiology, doi: 10.3389/fmicb.2012.00344<br><br><br />
<br />
</p><br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/genetic_engineeringTeam:Valencia/genetic engineering2012-09-27T03:26:52Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Genetic Engineering<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
<a href="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg"><img align="right" src="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg" style="margin-left: 10px;"></a><br />
Cyanobacteria are great organisms to be used in Synthetic Biology because of their ability to capture solar energy and CO<sub>2</sub> and the fact that they can be easily genetically manipulated due to its small genome and their capacity to accept foreign DNA naturally. For our project we have chosen the cyanobacteria <i>Synechococcus elongatus</i> (figure 1) for genetic engineering because is the model organism for studying some prokaryotic processes (there is a lot of information of how to transform it) and in the last years it has become a model organism for some industrial processes, like biofuel production (Wang et al. 2012).<br><br><br />
<i>S. elongatus</i> has a circular genome of ≈2.7Mb (<a href="http://genome.kazusa.or.jp/cyanobase/SYNPCC7942" target=_blank"> fully sequenced </a>) with a GC content of 55.5%, which contains the genes for 2.612 proteins and 53 RNAs (Atsumi et al. 2009). <br />
<br><br><br><br><br><br><br />
<div align="right"><b>Figure 1: </b> <i>S. elongatus</i> PCC7942 </div><br />
<br><br />
<br />
<h2><b>Transforming <i>S. elongatus</i> PCC7942 for promoter characterization:</b></h2><br />
<br><br><br />
Our main objective has been to characterize the <i>psbAI</i> promoter (<a href="https://2012.igem.org/Team:Valencia/Parts">Submitted parts</a>) of <i>Synechococcus elongatus</i> PCC7942 in order to know more about its operation and understand how this promoter would control our final construct (<a href="https://2012.igem.org/Team:Valencia/prototypes"> Designed parts</a>) to have a diel switch of AHL, the signal molecule for our <i>Aliivibrio fischeri</i> population to glow. <br><br><br />
To characterize this promoter we had two options: making <i>psbAIp::lacZ</i> fusions and monitored the β-galactosidase activity or making <i>psbAIp::luxABCDE</i> fusions and monitored the bioluminescence produced as a result of the promoter activation. We refused to use a <i>psbAIp::lacZ</i> fusion because some assays report that the <i>psbAI</i> promoter response to light cannot be properly monitored by the β-galactosidase activity (Nair et al. 2001). For this reason we choosed using vector fusions. With this aim, we cloned several fusion plasmids (pAM977 and pAM2195, table 1) in <i>Escherichia coli</i> and transformed them into <i>S. elongatus</i>, both wildtype and cscB strain. The fusion vectors were provided by Susan Golden´s lab. <br><br><br />
<center><b>Table 1: </b> Information of the two vectors used for characterize the <i>psbAI</i> promoter</center><br />
<center><a href="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png"><img src="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png" width="600" height="180"></a></center><br><br><br><br />
<br />
<b>Cloning into <i>E. coli</i></b><br />
We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this <a href="https://2012.igem.org/Team:Valencia/Transforming_ecoli">protocol</a>. For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 (figure 2) Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> <br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br><br />
<center><b>Figure 2: </b>pAM2195 vector</center><br><br><br />
<b>Transforming Coccus</b><br />
Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. <br />
As the cscB strain from Harvard was resistant to Cm, we only could transform it with the pAM977 plasmid, which gives Sp resistance. Wildtype was transformed with both plasmids (pAM977 and pAM2195).<br><br />
We used the following protocol to transform <i>Synechococcus</i>: <br><br><br />
<br />
<br />
<br><br />
<b>Protocol</b><br />
<br><br />
Extracted from Clerico et al. 2007:<br><br />
<ul style="list-style-type: square"><br><br />
<li>We grew 100ml of cscB and WT in BG-11 liquid medium shaking at 250rpm with constant light we used cold light fluorescent tubes. Cells had an optical density at 750nm (OD750) of 0.7, which is reach in 4 to 7 days approximately.</li><br><br />
<li>We collected 15mL of the culture and centrifuge it at 6000g for 10 min and discarded the supernatant.</li><br><br />
<li>We resuspended collected cells in 10 mL of 10mM NaCl and centrifuge them again at 6000g for 10 min.</li><br><br />
<li>After it, we resuspended the pellet in 0.3mL of BG-11M liquid medium.</li><br><br />
<li>We added 2μl of pAM2195 and pAM977 after doing a Miniprep of the clonation with a <i>JetQuick</i> kit </li><br><br />
<li>Wrap tubes in aluminum foil to keep out light and incubate at 30oC for 15 to 20h with gentle agitation.</li><br><br />
<li>And then, plate the entire 0.3mL cell suspension on BG-11 medium agar with the selective antibiotics. We used this concentration: 2μg/ml spectinomycin and 7.5μg/mL of chloramphenicol. After 7 to 10 days of incubation under standard light conditions transformed colonies should appear.</li><br><br />
<li>After colonies appearance pick single transformants and grow them on fresh liquid BG-11M agar plate with selective antibiotic (this is done to ensure that all colonies have incorporated the trans gene as S. elongatus has multiple copies of its chromosome).</li><br><br />
<li>After 5 to 7 days of growth, cyanobacteria can be used to inoculate a BG-11 liquid culture with the selective antibiotic.</li></ul><br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG"><img src="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG" width="458" height="343"></a></center><br />
<center><b>Figure 3: </b>A view of our transformed cells</center><br />
<br><br />
<h2><b>Results</b></h2><br />
<br><br />
We weren´t able to obtain transformants with the wild type strain of <i>S. elongatus</i>. <br />
But we obtained resistant colonies to Sp of the cscB strain transformed with the pAM977 vector (figure 4). This was achieved the last week of lab work, so due to the slowly growth rate of <i>S. elongatus</i>, we haven´t have the time to grow the transformants in liquid medium and start with the bioluminescence measures to characterize the promoter. However, we will try to have some results for the Jamboree concerning this part.<br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"><img src="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"></a></center><br />
<center><b>Figure 4: </b> pAM977 transformants</center><br />
<br><br />
<br />
<h2><b>Transformation of <i>E. coli</i> for AHL production to test bioluminescent response in <i>A. fischeri</i>:</b></h2><br><br />
Another part of our project included transforming <i>E. coli</i> with the registry part <a href="http://partsregistry.org/Part:BBa_K084014"> BBa_K084014 </a> in order to make <i>E. coli</i> produce AHL under IPTG regulation to test the bioluminescent response in A. fischeri. To achieve this goal we cloned the part in DH5α <i>E. coli</i> strains using this <a href="https://www.facebook.com/l.php?u=http%3A%2F%2Fpartsregistry.org%2FHelp%3ADistribution_Kits&h=oAQFOZXUl">protocol</a>(figure 5). The part (869 bp) is included inside the pSB1A2 (2079bp) BioBrick.<br><br><br />
<center><a href="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"><img src="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"></a></center><br />
<center><b>Figure 5:</b>The whole BBa_K084014 part has a size of 2948bp. </center><br />
<br><br />
We designed an experimental procedure to test different experimental conditions for the production of AHL.<br><br><br />
<center><b>Table 2: </b> Experimental design for AHL production </center><br />
<center><a href="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png"><img src="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png" width="600" height="140"></a></center><br><br />
<br />
<br>When we wanted to test if <i>A. fischeri</i> was able to glow with the AHL produced by <i>E. coli</i> we discovered that the strain we had been growing in the lab was not <i> V. fischeri</i>, but <i>V.mediterrani</i> (GREAT!).<br><br />
We will try to obtain <i>V. fischeri</i> as soon as possible to test this.<br><br />
</p><br />
<br><br />
<h2><b>Building our BioBricks</b></h2><br />
<br><br />
<b><i>psbAI</i> BioBrick</b><br />
<br><br><br />
Our promoter with the aproppiate <a href="http://partsregistry.org/Help:Assembly_standard_25"> suffix and preffix </a> (289bp) was synthesized by Genscript and cloned into a puc57-Kan (2579bp) vector. We successfully digested the part and ligated it into the psb1C3 BioBrick from the parts registry (figure 6, 7 and 8). For this aim we followed the openwetware protocols, which you can find here. <br><br><br />
<br />
<b>Complete Construct</b><br />
<br><br><br />
Our final objective is to have a S. elongatus capable to produce AHL during the night. For this we designed this new BioBrick controled by the psbAI promoter which is active in normal light conditions.<a href="https://2012.igem.org/Team:Valencia/prototypes"> Here </a> you will find all the information concerning the different parts of the BioBrick.<br><br />
We successfully cloned all the parts in DH5α E. coli strains using this <a href="https://2012.igem.org/Team:Valencia/Transforming_ecoli">protocol</a>. We used the <a href="http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf">BioBrick Assembly Manual</a> protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3.<br><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/f/fc/DSC02872_VLC.jpg"> &nbsp; &nbsp;<br />
<img src="https://static.igem.org/mediawiki/2012/6/6b/Electro_VLCXXX.jpg" width="350" height="270"><br />
</center><br />
<center><b>Figures 6 & 7: </b> Succesfully we build our BioBrick. In the electrophoresis the band that corresponds to the part (3868bp) is the one surrounded with a red triangle </center><br />
<br><br><br />
<center><img src="https://static.igem.org/mediawiki/2012/c/ce/VLC_PsB1C3%2BpsbAI.png"></center><br />
<center><b>Figure 8: </b> <i>psbAI</i> promoter inside the pSB1C3</center><br />
<br />
<strong>References</strong><br />
<hr><hr><br />
<br><br />
Atsumi, S., Higashide, W., and Liao, J. C. (2009) Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. <i>Nat Biotechnol</i>. 27:1177-1180<br><br><br />
Clerico, E. M., Ditty, J. L. & Golden, S.S. (2007) Specialized Techniques for Site-Directed Mutagenesis in Cyanobacteria. <i>Methods in Molecular Biology.</i> 362:153–172.<br><br><br />
Nair, U., Thomas, C. & Golden, S. S. (2001) Functional Elements of the Strong <i>psbAI</i> Promoter of Synechococcus elongatus PCC 7942. <i>J. Bacteriology</i>, 183:1740–1747.<br><br><br />
Wang, B., Wang J., Zhang, W. & Meldrum, D. R. (2012) Application of Synthetic Biology in cyanobacteria and algae. Frontiers in Microbiology, doi: 10.3389/fmicb.2012.00344<br><br><br />
<br />
</p><br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/genetic_engineeringTeam:Valencia/genetic engineering2012-09-27T03:25:32Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Genetic Engineering<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
<a href="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg"><img align="right" src="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg" style="margin-left: 10px;"></a><br />
Cyanobacteria are great organisms to be used in Synthetic Biology because of their ability to capture solar energy and CO<sub>2</sub> and the fact that they can be easily genetically manipulated due to its small genome and their capacity to accept foreign DNA naturally. For our project we have chosen the cyanobacteria <i>Synechococcus elongatus</i> (figure 1) for genetic engineering because is the model organism for studying some prokaryotic processes (there is a lot of information of how to transform it) and in the last years it has become a model organism for some industrial processes, like biofuel production (Wang et al. 2012).<br><br><br />
<i>S. elongatus</i> has a circular genome of ≈2.7Mb (<a href="http://genome.kazusa.or.jp/cyanobase/SYNPCC7942" target=_blank"> fully sequenced </a>) with a GC content of 55.5%, which contains the genes for 2.612 proteins and 53 RNAs (Atsumi et al. 2009). <br />
<br><br><br><br><br><br><br />
<div align="right"><b>Figure 1: </b> <i>S. elongatus</i> PCC7942 </div><br />
<br><br />
<br />
<h2><b>Transforming <i>S. elongatus</i> PCC7942 for promoter characterization:</b></h2><br />
<br><br><br />
Our main objective has been to characterize the <i>psbAI</i> promoter (<a href="https://2012.igem.org/Team:Valencia/Parts">Submitted parts</a>) of <i>Synechococcus elongatus</i> PCC7942 in order to know more about its operation and understand how this promoter would control our final construct (<a href="https://2012.igem.org/Team:Valencia/prototypes"> Designed parts</a>) to have a diel switch of AHL, the signal molecule for our <i>Aliivibrio fischeri</i> population to glow. <br><br><br />
To characterize this promoter we had two options: making <i>psbAIp::lacZ</i> fusions and monitored the β-galactosidase activity or making <i>psbAIp::luxABCDE</i> fusions and monitored the bioluminescence produced as a result of the promoter activation. We refused to use a <i>psbAIp::lacZ</i> fusion because some assays report that the <i>psbAI</i> promoter response to light cannot be properly monitored by the β-galactosidase activity (Nair et al. 2001). For this reason we choosed using vector fusions. With this aim, we cloned several fusion plasmids (pAM977 and pAM2195, table 1) in <i>Escherichia coli</i> and transformed them into <i>S. elongatus</i>, both wildtype and cscB strain. The fusion vectors were provided by Susan Golden´s lab. <br><br><br />
<center><b>Table 1: </b> Information of the two vectors used for characterize the <i>psbAI</i> promoter</center><br />
<center><a href="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png"><img src="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png" width="600" height="180"></a></center><br><br><br><br />
<br />
<h2><b>Cloning into <i>E. coli</i></b></h2><br />
We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this <a href="https://2012.igem.org/Team:Valencia/Transforming_ecoli">protocol</a>. For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 (figure 2) Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> <br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br><br />
<center><b>Figure 2: </b>pAM2195 vector</center><br><br><br />
<h2><b>Transforming Coccus</b></h2><br />
Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. <br />
As the cscB strain from Harvard was resistant to Cm, we only could transform it with the pAM977 plasmid, which gives Sp resistance. Wildtype was transformed with both plasmids (pAM977 and pAM2195).<br><br />
We used the following protocol to transform <i>Synechococcus</i>: <br><br><br />
<br />
<br />
<br><br />
<h2><b>Protocol</b></h2><br />
<br><br />
Extracted from Clerico et al. 2007:<br><br />
<ul style="list-style-type: square"><br><br />
<li>We grew 100ml of cscB and WT in BG-11 liquid medium shaking at 250rpm with constant light we used cold light fluorescent tubes. Cells had an optical density at 750nm (OD750) of 0.7, which is reach in 4 to 7 days approximately.</li><br><br />
<li>We collected 15mL of the culture and centrifuge it at 6000g for 10 min and discarded the supernatant.</li><br><br />
<li>We resuspended collected cells in 10 mL of 10mM NaCl and centrifuge them again at 6000g for 10 min.</li><br><br />
<li>After it, we resuspended the pellet in 0.3mL of BG-11M liquid medium.</li><br><br />
<li>We added 2μl of pAM2195 and pAM977 after doing a Miniprep of the clonation with a <i>JetQuick</i> kit </li><br><br />
<li>Wrap tubes in aluminum foil to keep out light and incubate at 30oC for 15 to 20h with gentle agitation.</li><br><br />
<li>And then, plate the entire 0.3mL cell suspension on BG-11 medium agar with the selective antibiotics. We used this concentration: 2μg/ml spectinomycin and 7.5μg/mL of chloramphenicol. After 7 to 10 days of incubation under standard light conditions transformed colonies should appear.</li><br><br />
<li>After colonies appearance pick single transformants and grow them on fresh liquid BG-11M agar plate with selective antibiotic (this is done to ensure that all colonies have incorporated the trans gene as S. elongatus has multiple copies of its chromosome).</li><br><br />
<li>After 5 to 7 days of growth, cyanobacteria can be used to inoculate a BG-11 liquid culture with the selective antibiotic.</li></ul><br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG"><img src="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG" width="458" height="343"></a></center><br />
<center><b>Figure 3: </b>A view of our transformed cells</center><br />
<br><br />
<h2><b>Results</b></h2><br />
<br><br />
We weren´t able to obtain transformants with the wild type strain of <i>S. elongatus</i>. <br />
But we obtained resistant colonies to Sp of the cscB strain transformed with the pAM977 vector (figure 4). This was achieved the last week of lab work, so due to the slowly growth rate of <i>S. elongatus</i>, we haven´t have the time to grow the transformants in liquid medium and start with the bioluminescence measures to characterize the promoter. However, we will try to have some results for the Jamboree concerning this part.<br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"><img src="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"></a></center><br />
<center><b>Figure 4: </b> pAM977 transformants</center><br />
<br><br />
<br />
<h2><b>Transformation of <i>E. coli</i> for AHL production to test bioluminescent response in <i>A. fischeri</i>:</b></h2><br><br />
Another part of our project included transforming <i>E. coli</i> with the registry part <a href="http://partsregistry.org/Part:BBa_K084014"> BBa_K084014 </a> in order to make <i>E. coli</i> produce AHL under IPTG regulation to test the bioluminescent response in A. fischeri. To achieve this goal we cloned the part in DH5α <i>E. coli</i> strains using this <a href="https://www.facebook.com/l.php?u=http%3A%2F%2Fpartsregistry.org%2FHelp%3ADistribution_Kits&h=oAQFOZXUl">protocol</a>(figure 5). The part (869 bp) is included inside the pSB1A2 (2079bp) BioBrick.<br><br><br />
<center><a href="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"><img src="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"></a></center><br />
<center><b>Figure 5:</b>The whole BBa_K084014 part has a size of 2948bp. </center><br />
<br><br />
We designed an experimental procedure to test different experimental conditions for the production of AHL.<br><br><br />
<center><b>Table 2: </b> Experimental design for AHL production </center><br />
<center><a href="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png"><img src="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png" width="600" height="140"></a></center><br><br />
<br />
<br>When we wanted to test if <i>A. fischeri</i> was able to glow with the AHL produced by <i>E. coli</i> we discovered that the strain we had been growing in the lab was not <i> V. fischeri</i>, but <i>V.mediterrani</i> (GREAT!).<br><br />
We will try to obtain <i>V. fischeri</i> as soon as possible to test this.<br><br />
</p><br />
<br><br />
<h2><b>Building our BioBricks</b></h2><br />
<br><br />
<b><i>psbAI</i> BioBrick</b><br />
<br><br><br />
Our promoter with the aproppiate <a href="http://partsregistry.org/Help:Assembly_standard_25"> suffix and preffix </a> (289bp) was synthesized by Genscript and cloned into a puc57-Kan (2579bp) vector. We successfully digested the part and ligated it into the psb1C3 BioBrick from the parts registry (figure 6, 7 and 8). For this aim we followed the openwetware protocols, which you can find here. <br><br><br />
<br />
<b>Complete Construct</b><br />
<br><br><br />
Our final objective is to have a S. elongatus capable to produce AHL during the night. For this we designed this new BioBrick controled by the psbAI promoter which is active in normal light conditions.<a href="https://2012.igem.org/Team:Valencia/prototypes"> Here </a> you will find all the information concerning the different parts of the BioBrick.<br><br />
We successfully cloned all the parts in DH5α E. coli strains using this <a href="https://2012.igem.org/Team:Valencia/Transforming_ecoli">protocol</a>. We used the <a href="http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf">BioBrick Assembly Manual</a> protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3.<br><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/f/fc/DSC02872_VLC.jpg"> &nbsp; &nbsp;<br />
<img src="https://static.igem.org/mediawiki/2012/6/6b/Electro_VLCXXX.jpg" width="350" height="270"><br />
</center><br />
<center><b>Figures 6 & 7: </b> Succesfully we build our BioBrick. In the electrophoresis the band that corresponds to the part (3868bp) is the one surrounded with a red triangle </center><br />
<br><br><br />
<center><img src="https://static.igem.org/mediawiki/2012/c/ce/VLC_PsB1C3%2BpsbAI.png"></center><br />
<center><b>Figure 8: </b> <i>psbAI</i> promoter inside the pSB1C3</center><br />
<br />
<strong>References</strong><br />
<hr><hr><br />
<br><br />
Atsumi, S., Higashide, W., and Liao, J. C. (2009) Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. <i>Nat Biotechnol</i>. 27:1177-1180<br><br><br />
Clerico, E. M., Ditty, J. L. & Golden, S.S. (2007) Specialized Techniques for Site-Directed Mutagenesis in Cyanobacteria. <i>Methods in Molecular Biology.</i> 362:153–172.<br><br><br />
Nair, U., Thomas, C. & Golden, S. S. (2001) Functional Elements of the Strong <i>psbAI</i> Promoter of Synechococcus elongatus PCC 7942. <i>J. Bacteriology</i>, 183:1740–1747.<br><br><br />
Wang, B., Wang J., Zhang, W. & Meldrum, D. R. (2012) Application of Synthetic Biology in cyanobacteria and algae. Frontiers in Microbiology, doi: 10.3389/fmicb.2012.00344<br><br><br />
<br />
</p><br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/genetic_engineeringTeam:Valencia/genetic engineering2012-09-27T03:16:58Z<p>Roocfer: </p>
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<a href="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg"><img align="right" src="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg" style="margin-left: 10px;"></a><br />
Cyanobacteria are great organisms to be used in Synthetic Biology because of their ability to capture solar energy and CO<sub>2</sub> and the fact that they can be easily genetically manipulated due to its small genome and their capacity to accept foreign DNA naturally. For our project we have chosen the cyanobacteria <i>Synechococcus elongatus</i> (figure 1) for genetic engineering because is the model organism for studying some prokaryotic processes (there is a lot of information of how to transform it) and in the last years it has become a model organism for some industrial processes, like biofuel production (Wang et al. 2012).<br><br><br />
<i>S. elongatus</i> has a circular genome of ≈2.7Mb (<a href="http://genome.kazusa.or.jp/cyanobase/SYNPCC7942" target=_blank"> fully sequenced </a>) with a GC content of 55.5%, which contains the genes for 2.612 proteins and 53 RNAs (Atsumi et al. 2009). <br />
<br><br><br><br><br><br><br />
<div align="right"><b>Figure 1: </b> <i>S. elongatus</i> PCC7942 </div><br />
<br><br />
<br />
<h2><b>Transforming <i>S. elongatus</i> PCC7942 for promoter characterization:</b></h2><br />
<br><br><br />
Our main objective has been to characterize the <i>psbAI</i> promoter (<a href="https://2012.igem.org/Team:Valencia/Parts">Submitted parts</a>) of <i>Synechococcus elongatus</i> PCC7942 in order to know more about its operation and understand how this promoter would control our final construct (<a href="https://2012.igem.org/Team:Valencia/prototypes"> Designed parts</a>) to have a diel switch of AHL, the signal molecule for our <i>Aliivibrio fischeri</i> population to glow. <br><br><br />
To characterize this promoter we had two options: making <i>psbAIp::lacZ</i> fusions and monitored the β-galactosidase activity or making <i>psbAIp::luxABCDE</i> fusions and monitored the bioluminescence produced as a result of the promoter activation. We refused to use a <i>psbAIp::lacZ</i> fusion because some assays report that the <i>psbAI</i> promoter response to light cannot be properly monitored by the β-galactosidase activity (Nair et al. 2001). For this reason we choosed using vector fusions. With this aim, we cloned several fusion plasmids (pAM977 and pAM2195, table 1) in <i>Escherichia coli</i> and transformed them into <i>S. elongatus</i>, both wildtype and cscB strain. The fusion vectors were provided by Susan Golden´s lab. <br><br><br />
<center><b>Table 1: </b> Information of the two vectors used for characterize the <i>psbAI</i> promoter</center><br />
<center><a href="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png"><img src="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png" width="600" height="180"></a></center><br><br><br><br />
<br />
<h2><b>Cloning into <i>E. coli</i></b></h2><br />
We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this <a href="https://2012.igem.org/Team:Valencia/LB_Agar">protocol</a>. For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 (figure 2) Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> <br />
<br />
<h2><b>Transforming Coccus</b></h2><br />
Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. <br />
As the cscB strain from Harvard was resistant to Cm, we only could transform it with the pAM977 plasmid, which gives Sp resistance. Wildtype was transformed with both plasmids (pAM977 and pAM2195).<br><br />
We used the following protocol to transform <i>Synechococcus</i>: <br><br><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br><br />
<center><b>Figure 2: </b>pAM2195 vector</center><br />
<br><br />
<h2><b>Protocol</b></h2><br />
<br><br />
Extracted from Clerico et al. 2007:<br><br />
<ul style="list-style-type: square"><br><br />
<li>We grew 100ml of cscB and WT in BG-11 liquid medium shaking at 250rpm with constant light we used cold light fluorescent tubes. Cells had an optical density at 750nm (OD750) of 0.7, which is reach in 4 to 7 days approximately.</li><br><br />
<li>We collected 15mL of the culture and centrifuge it at 6000g for 10 min and discarded the supernatant.</li><br><br />
<li>We resuspended collected cells in 10 mL of 10mM NaCl and centrifuge them again at 6000g for 10 min.</li><br><br />
<li>After it, we resuspended the pellet in 0.3mL of BG-11M liquid medium.</li><br><br />
<li>We added 2μl of pAM2195 and pAM977 after doing a Miniprep of the clonation with a <i>JetQuick</i> kit </li><br><br />
<li>Wrap tubes in aluminum foil to keep out light and incubate at 30oC for 15 to 20h with gentle agitation.</li><br><br />
<li>And then, plate the entire 0.3mL cell suspension on BG-11 medium agar with the selective antibiotics. We used this concentration: 2μg/ml spectinomycin and 7.5μg/mL of chloramphenicol. After 7 to 10 days of incubation under standard light conditions transformed colonies should appear.</li><br><br />
<li>After colonies appearance pick single transformants and grow them on fresh liquid BG-11M agar plate with selective antibiotic (this is done to ensure that all colonies have incorporated the trans gene as S. elongatus has multiple copies of its chromosome).</li><br><br />
<li>After 5 to 7 days of growth, cyanobacteria can be used to inoculate a BG-11 liquid culture with the selective antibiotic.</li></ul><br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG"><img src="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG" width="458" height="343"></a></center><br />
<center><b>Figure 3: </b>A view of our transformed cells</center><br />
<br><br />
<h2><b>Results</b></h2><br />
<br><br />
We weren´t able to obtain transformants with the wild type strain of <i>S. elongatus</i>. <br />
But we obtained resistant colonies to Sp of the cscB strain transformed with the pAM977 vector (figure 4). This was achieved the last week of lab work, so due to the slowly growth rate of <i>S. elongatus</i>, we haven´t have the time to grow the transformants in liquid medium and start with the bioluminescence measures to characterize the promoter. However, we will try to have some results for the Jamboree concerning this part.<br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"><img src="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"></a></center><br />
<center><b>Figure 4: </b> pAM977 transformants</center><br />
<br><br />
<br />
<h2><b>Transformation of <i>E. coli</i> for AHL production to test bioluminescent response in <i>A. fischeri</i>:</b></h2><br><br />
Another part of our project included transforming <i>E. coli</i> with the registry part <a href="http://partsregistry.org/Part:BBa_K084014"> BBa_K084014 </a> in order to make <i>E. coli</i> produce AHL under IPTG regulation to test the bioluminescent response in A. fischeri. To achieve this goal we cloned the part in DH5α <i>E. coli</i> strains using this <a href="https://www.facebook.com/l.php?u=http%3A%2F%2Fpartsregistry.org%2FHelp%3ADistribution_Kits&h=oAQFOZXUl">protocol</a>(figure 5). The part (869 bp) is included inside the pSB1A2 (2079bp) BioBrick.<br><br><br />
<center><a href="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"><img src="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"></a></center><br />
<center><b>Figure 5:</b>The whole BBa_K084014 part has a size of 2948bp. </center><br />
<br><br />
We designed an experimental procedure to test different experimental conditions for the production of AHL.<br><br><br />
<center><b>Table 2: </b> Experimental design for AHL production </center><br />
<center><a href="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png"><img src="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png" width="600" height="140"></a></center><br><br />
<br />
<br>When we wanted to test if <i>A. fischeri</i> was able to glow with the AHL produced by <i>E. coli</i> we discovered that the strain we had been growing in the lab was not <i> V. fischeri</i>, but <i>V.mediterrani</i> (GREAT!).<br><br />
We will try to obtain <i>V. fischeri</i> as soon as possible to test this.<br><br />
</p><br />
<br><br />
<h2><b>Building our BioBricks</b></h2><br />
<br><br />
<b><i>psbAI</i> BioBrick</b><br />
<br><br><br />
Our promoter with the aproppiate <a href="http://partsregistry.org/Help:Assembly_standard_25"> suffix and preffix </a> (289bp) was synthesized by Genscript and cloned into a puc57-Kan (2579bp) vector. We successfully digested the part and ligated it into the psb1C3 BioBrick from the parts registry (figure 6, 7 and 8). For this aim we followed the openwetware protocols, which you can find here. <br><br><br />
<br />
<b>Complete Construct</b><br />
<br><br><br />
Our final objective is to have a S. elongatus capable to produce AHL during the night. For this we designed this new BioBrick controled by the psbAI promoter which is active in normal light conditions.<a href="https://2012.igem.org/Team:Valencia/prototypes"> Here </a> you will find all the information concerning the different parts of the BioBrick.<br><br />
We successfully cloned all the parts in DH5α E. coli strains using this <a href="https://2012.igem.org/Team:Valencia/Transforming_ecoli">protocol</a>. We used the <a href="http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf">BioBrick Assembly Manual</a> protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3.<br><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/f/fc/DSC02872_VLC.jpg"> &nbsp; &nbsp;<br />
<img src="https://static.igem.org/mediawiki/2012/6/6b/Electro_VLCXXX.jpg" width="350" height="270"><br />
</center><br />
<center><b>Figures 6 & 7: </b> Succesfully we build our BioBrick. In the electrophoresis the band that corresponds to the part (3868bp) is the one surrounded with a red triangle </center><br />
<br><br><br />
<center><img src="https://static.igem.org/mediawiki/2012/c/ce/VLC_PsB1C3%2BpsbAI.png"></center><br />
<center><b>Figure 8: </b> <i>psbAI</i> promoter inside the pSB1C3</center><br />
<br />
<strong>References</strong><br />
<hr><hr><br />
<br><br />
Atsumi, S., Higashide, W., and Liao, J. C. (2009) Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. <i>Nat Biotechnol</i>. 27:1177-1180<br><br><br />
Clerico, E. M., Ditty, J. L. & Golden, S.S. (2007) Specialized Techniques for Site-Directed Mutagenesis in Cyanobacteria. <i>Methods in Molecular Biology.</i> 362:153–172.<br><br><br />
Wang, B., Wang J., Zhang, W. & Meldrum, D. R. (2012) Application of Synthetic Biology in cyanobacteria and algae. Frontiers in Microbiology. doi: 10.3389/fmicb.2012.00344<br><br><br />
<br />
</p><br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/menuTeam:Valencia/menu2012-09-27T02:49:09Z<p>Roocfer: </p>
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<li class="top"><a href="https://2012.igem.org/Team:Valencia/Team" target="_self" class="top_link"><span>Team</span></a><br />
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<li><a href="https://2012.igem.org/Team:Valencia/Team" target="_self">Team description</a></li><br />
<li><a href="https://2012.igem.org/Team:Valencia/profiles" target="_self">Members</a></li><br />
<li><a href="https://2012.igem.org/Team:Valencia/Acknowledgments" target="_self">Acknowledgments</a></li><br />
<li><a href="https://2012.igem.org/Team:Valencia/gallery;" target="_self">Gallery</a></li><br />
</ul><br />
</li><br />
<br />
<br />
<li class="top"><a href="https://2012.igem.org/Team:Valencia/labwork" target="_self" class="top_link"><span>Lab Work</span></a><br />
<ul class="sub"><br />
<li class="tap"><a href="https://2012.igem.org/Team:Valencia/labwork" target="_self">What we did</a></li><br />
<li><a href="https://2012.igem.org/Team:Valencia/Parts" target="_self">Submitted parts</a></li><br />
<li class="tap"><a href="https://2012.igem.org/Team:Valencia/prototypes" target="_self">Designed Biobricks</a></li><br />
<li><a href="https://2012.igem.org/Team:Valencia/notebook" target="_self">Notebook</a></li><br />
<li><a href="https://2012.igem.org/Team:Valencia/protocols" target="_self">Protocols</a></li><br />
</ul><br />
</li><br />
<br />
<br />
<li class="top"><a href="https://2012.igem.org/Team:Valencia/achivements" target="_self" class="top_link"><span>Achievements</span></a><br />
<ul class="sub"><br />
<li><a href="https://2012.igem.org/Team:Valencia/achivements" target="_self">Achievements</a></li><br />
<li><a href="https://2012.igem.org/Team:Valencia/judging" target="_self">Judging criteria</a></li><br />
<li><a href="https://2012.igem.org/Team:Valencia/collaboration" target="_self">Collaborations</a></li><br />
</ul><br />
</li><br />
<br />
</div></div>Roocferhttp://2012.igem.org/Team:Valencia/labworkTeam:Valencia/labwork2012-09-27T02:41:58Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<br />
<br />
<html><br />
<br><br><br />
<div id="Titulos"><br />
Lab Work<br />
</div><br />
<br><br />
<div id="HomeRight"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ad/Experimental_design1.png" width="230" height="240"><br />
</div><br />
<br><br />
<div id="HomeCenter2"><br />
<p align="justify"><p>In this section we want to tell everybody our lab-experience.</p><br />
<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><p>We want to show you the psbAI promoter genetic characterization, and its sequence, composition, function, regulation or behaviour. <a href="https://2012.igem.org/Team:Valencia/Parts"> Here </a>you can know more about the constructor and how it works. </p></li></ul><br />
<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><p>About Biobricks, they have been designed for produce AHL during the night been controled by psbAI promoter; and constructor parts and structure are explained <a href="https://2012.igem.org/Team:Valencia/prototypes"> here </a>.</p></li></ul><br />
<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><p>If you want know more about our lab-routine, don't forget glance through <a href="https://2012.igem.org/Team:Valencia/notebook">Notebook</a> section. Have been three months full of anecdotes, and there can find the development of project, our decisions, the victories and failures or gladness and troubles.</p></li></ul><br />
<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><p> <a href="https://2012.igem.org/Team:Valencia/protocols">Protocols</a> is a part dedicated to show everybody our 'work planing'. <br />
</p></li><br />
</ul><br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/labworkTeam:Valencia/labwork2012-09-27T02:39:15Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<br />
<br />
<html><br />
<br><br><br />
<div id="Titulos"><br />
Lab Work<br />
</div><br />
<br><br />
<div id="HomeRight"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ad/Experimental_design1.png" width="230" height="240"><br />
</div><br />
<br><br />
<div id="HomeCenter2"><br />
<p align="justify"><p>In this section we want to tell everybody our lab-experience.</p><br />
<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><p>We want to show you the psbAI promoter genetic characterization, and its sequence, composition, function, regulation or behaviour. <a href="https://2012.igem.org/Team:Valencia/Parts"> Here </a>you can know more about the constructor and how it works. </p></li></ul><br />
<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><p>About Biobricks, they have been designed for produce AHL during the night been controled by psbAI promoter; and constructor parts and structure are explained <a href="https://2012.igem.org/Team:Valencia/prototypes"> here </a>.</p></li></ul><br />
<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><p>If you want know more about our lab-routine, don't forget glance through <a href="https://2012.igem.org/Team:Valencia/prototypes">Notebook</a> section. Have been three months full of anecdotes, and there can find the development of project, our decisions, the victories and failures or gladness and troubles.</p></li></ul><br />
<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><p> <a href="https://2012.igem.org/Team:Valencia/prototypes">Protocols</a> is a part dedicated to show everybody our 'work planing'. <br />
</p></li><br />
</ul><br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/labworkTeam:Valencia/labwork2012-09-27T02:37:20Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<br />
<br />
<html><br />
<br><br><br />
<div id="Titulos"><br />
Lab Work<br />
</div><br />
<br><br />
<div id="HomeRight"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ad/Experimental_design1.png" width="230" height="240"><br />
</div><br />
<br><br />
<div id="HomeCenter2"><br />
<p align="justify"><p>In this section we want to tell everybody our lab-experience.</p><br />
<br><br />
<p>We want to show you the psbAI promoter genetic characterization, and its sequence, composition, function, regulation or behaviour. Here you can know more about the constructor and how it works. </p><br />
<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><p>About Biobricks, they have been designed for produce AHL during the night been controled by psbAI promoter; and constructor parts and structure are explained <a href="https://2012.igem.org/Team:Valencia/prototypes"> here </a>.</p></li></ul><br />
<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><p>If you want know more about our lab-routine, don't forget glance through <a href="https://2012.igem.org/Team:Valencia/prototypes">Notebook</a> section. Have been three months full of anecdotes, and there can find the development of project, our decisions, the victories and failures or gladness and troubles.</p></li></ul><br />
<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><p> <a href="https://2012.igem.org/Team:Valencia/prototypes">Protocols</a> is a part dedicated to show everybody our 'work planing'. <br />
</p></li><br />
</ul><br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/labworkTeam:Valencia/labwork2012-09-27T02:36:48Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<br />
<br />
<html><br />
<br><br><br />
<div id="Titulos"><br />
Lab Work<br />
</div><br />
<br><br />
<div id="HomeRight"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ad/Experimental_design1.png" width="230" height="240"><br />
</div><br />
<br><br />
<div id="HomeCenter2"><br />
<p align="justify"><p>In this section we want to tell everybody our lab-experience.</p><br />
<br><br />
<p>We want to show you the psbAI promoter genetic characterization, and its sequence, composition, function, regulation or behaviour. Here you can know more about the constructor and how it works. </p><br />
<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><p>About Biobricks, they have been designed for produce AHL during the night been controled by psbAI promoter; and constructor parts and structure are explained <a href="https://2012.igem.org/Team:Valencia/prototypes"> here </a>.</p></li></ul><br />
<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><p>If you want know more about our lab-routine, don't forget glance through <a href="https://2012.igem.org/Team:Valencia/prototypes">Notebook</a> section. Have been three months full of anecdotes, and there can find the development of project, our decisions, the victories and failures or gladness and troubles.</p></li></ul><br />
<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><p> <a href="https://2012.igem.org/Team:Valencia/prototypes>Protocols</a> is a part dedicated to show everybody our 'work planing'. <br />
</p></li><br />
</ul><br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/prototypesTeam:Valencia/prototypes2012-09-27T02:29:59Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Designed Biobricks<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
Our final objective is to have a <i>S. elongatus</i> capable to produce AHL during the night. For this we designed this new BioBrick controled by the <i>psbAI</i> promoter which is active in normal light conditions. To have an inverse response to light we have put the cI repressor after the <i>psbAI</i> promoter which binds to the lambda promoter inhibiting the luxI transcription, when <i>psbAI</i> is active. During the night, when the promoter is less active, the repressor will not be active, allowing the expression of luxI, and therefore having AHL.<br><br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2012/archive/f/fd/20120926234950%21VLC_Construct.png"></center><br />
<center><b>Figure 1.</b> Our idea of the construct</center><br />
<br />
<b>Parts Information</b>:<br><br><br />
<a href="http://partsregistry.org/Part:BBa_K754000">BBa_K754000 </a> : <i>S. elongatus</i> PCC7942 <i>psbAI</i> promoter. This is our submitted part for the registry. This promoter has a light dependent regulation being active in normal light conditions. Read more here.<br><br />
<a href="http://partsregistry.org/Part:BBa_Q04510">BBa_Q04510 </a>: Consists in the next parts:<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>: RBS<br></li><br />
</ul> <br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_C0051">C0051</a>: cI repressor from E. coli phage lambda <br />
Coding region for the cI repressor based on cI repressor from bacteriophage lambda modified with an LVA tail for rapid degradation of the protein. cI repressor binds to the cI regulator.<br></li><br />
</ul><br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_B0015">B0015</a>: double terminator<br><br />
This is the most commonly used terminator. It seems to be reliable<br></li><br />
</ul><br />
<br />
<ul style="list-style-type: square"> <br />
<li><a href="http://partsregistry.org/Part:BBa_R0051">R0051</a>: promoter (lambda cI regulated)<br><br />
The cI regulated promoter is based on the pR promoter from bacteriophage lambda. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. cI binding results in repression of transcription.<br></li><br />
</ul><br />
<br />
<br />
<a href="http://partsregistry.org/Part:BBa_C0061">BBa_C0061 </a>: Autoinducer synthetase for AHL<br><br />
The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes. This expression is up-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound HSL. This complex binds to a palindromic site on the promoter, increasing the rate of transcription.<br><br />
<br />
<a href="http://partsregistry.org/Part:BBa_B1002">BBa_B1002</a>: Terminator<br><br />
<br />
<br><br><br />
<strong>Constructing the BioBrick</strong><br />
<br><br><br />
<br />
We've ligated the part <a href="http://partsregistry.org/Part:BBa_K754000">BBa_K754000 </a> (psbAI promoter) with the psb1C3 vector. <br />
<center><img src="https://static.igem.org/mediawiki/2012/c/ce/VLC_PsB1C3%2BpsbAI.png"></center><br />
<center><b>Figure 2.</b> psbAI promoter into the psb1C3 vector</center><br />
</p><br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/prototypesTeam:Valencia/prototypes2012-09-27T02:26:06Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Designed Biobricks<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
Our final objective is to have a <i>S. elongatus</i> capable to produce AHL during the night. For this we designed this new BioBrick controled by the <i>psbAI</i> promoter which is active in normal light conditions. To have an inverse response to light we have put the cI repressor after the <i>psbAI</i> promoter which binds to the lambda promoter inhibiting the luxI transcription, when <i>psbAI</i> is active. During the night, when the promoter is less active, the repressor will not be active, allowing the expression of luxI, and therefore having AHL.<br><br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2012/archive/f/fd/20120926234950%21VLC_Construct.png"></center><br />
<center>Figure 1. Our idea of the construct</center><br />
<br />
<b>Parts Information</b>:<br><br><br />
<a href="http://partsregistry.org/Part:BBa_K754000">BBa_K754000 </a> : <i>S. elongatus</i> PCC7942 <i>psbAI</i> promoter. This is our submitted part for the registry. This promoter has a light dependent regulation being active in normal light conditions. Read more here.<br><br />
<a href="http://partsregistry.org/Part:BBa_Q04510">BBa_Q04510 </a>: Consists in the next parts:<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>: RBS<br></li><br />
</ul> <br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_C0051">C0051</a>: cI repressor from E. coli phage lambda <br />
Coding region for the cI repressor based on cI repressor from bacteriophage lambda modified with an LVA tail for rapid degradation of the protein. cI repressor binds to the cI regulator.<br></li><br />
</ul><br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_B0015">B0015</a>: double terminator<br><br />
This is the most commonly used terminator. It seems to be reliable<br></li><br />
</ul><br />
<br />
<ul style="list-style-type: square"> <br />
<li><a href="http://partsregistry.org/Part:BBa_R0051">R0051</a>: promoter (lambda cI regulated)<br><br />
The cI regulated promoter is based on the pR promoter from bacteriophage lambda. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. cI binding results in repression of transcription.<br></li><br />
</ul><br />
<br />
<br />
<a href="http://partsregistry.org/Part:BBa_C0061">BBa_C0061 </a>: Autoinducer synthetase for AHL<br><br />
The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes. This expression is up-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound HSL. This complex binds to a palindromic site on the promoter, increasing the rate of transcription.<br><br />
<br />
<a href="http://partsregistry.org/Part:BBa_B1002">BBa_B1002</a>: Terminator<br><br />
<br />
<br><br><br />
<strong>Constructing the BioBrick</strong><br />
<br><br><br />
<br />
We've ligated the part <a href="http://partsregistry.org/Part:BBa_K754000">BBa_K754000 </a> (psbAI promoter) with the psb1C3 vector. <br />
<center><img src="https://static.igem.org/mediawiki/2012/c/ce/VLC_PsB1C3%2BpsbAI.png"></center><br />
<center>Figure 2.psbAI promoter into the psb1C3 vector</center><br />
</p><br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/prototypesTeam:Valencia/prototypes2012-09-27T02:23:43Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Designed Biobricks<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
Our final objective is to have a <i>S. elongatus</i> capable to produce AHL during the night. For this we designed this new BioBrick controled by the <i>psbAI</i> promoter which is active in normal light conditions. To have an inverse response to light we have put the cI repressor after the <i>psbAI</i> promoter which binds to the lambda promoter inhibiting the luxI transcription, when <i>psbAI</i> is active. During the night, when the promoter is less active, the repressor will not be active, allowing the expression of luxI, and therefore having AHL.<br><br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2012/archive/f/fd/20120926234950%21VLC_Construct.png"></center><br />
<br />
<b>Parts Information</b>:<br><br><br />
<a href="http://partsregistry.org/Part:BBa_K754000">BBa_K754000 </a> : <i>S. elongatus</i> PCC7942 <i>psbAI</i> promoter. This is our submitted part for the registry. This promoter has a light dependent regulation being active in normal light conditions. Read more here.<br><br />
<a href="http://partsregistry.org/Part:BBa_Q04510">BBa_Q04510 </a>: Consists in the next parts:<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>: RBS<br></li><br />
</ul> <br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_C0051">C0051</a>: cI repressor from E. coli phage lambda <br />
Coding region for the cI repressor based on cI repressor from bacteriophage lambda modified with an LVA tail for rapid degradation of the protein. cI repressor binds to the cI regulator.<br></li><br />
</ul><br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_B0015">B0015</a>: double terminator<br><br />
This is the most commonly used terminator. It seems to be reliable<br></li><br />
</ul><br />
<br />
<ul style="list-style-type: square"> <br />
<li><a href="http://partsregistry.org/Part:BBa_R0051">R0051</a>: promoter (lambda cI regulated)<br><br />
The cI regulated promoter is based on the pR promoter from bacteriophage lambda. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. cI binding results in repression of transcription.<br></li><br />
</ul><br />
<br />
<br />
<a href="http://partsregistry.org/Part:BBa_C0061">BBa_C0061 </a>: Autoinducer synthetase for AHL<br><br />
The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes. This expression is up-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound HSL. This complex binds to a palindromic site on the promoter, increasing the rate of transcription.<br><br />
<br />
<a href="http://partsregistry.org/Part:BBa_B1002">BBa_B1002</a>: Terminator<br><br />
<br />
<br><br><br />
<strong>Constructing the BioBrick</strong><br />
<br><br><br />
<br />
We've ligated the part <a href="http://partsregistry.org/Part:BBa_K754000">BBa_K754000 </a> (psbAI promoter) with the psb1C3 vector. <br />
<center><img src="https://static.igem.org/mediawiki/2012/c/ce/VLC_PsB1C3%2BpsbAI.png"></center><br />
</p><br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/prototypesTeam:Valencia/prototypes2012-09-27T02:21:27Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Designed Biobricks<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
Our final objective is to have a <i>S. elongatus</i> capable to produce AHL during the night. For this we designed this new BioBrick controled by the <i>psbAI</i> promoter which is active in normal light conditions. To have an inverse response to light we have put the cI repressor after the <i>psbAI</i> promoter which binds to the lambda promoter inhibiting the luxI transcription, when <i>psbAI</i> is active. During the night, when the promoter is less active, the repressor will not be active, allowing the expression of luxI, and therefore having AHL.<br><br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2012/archive/f/fd/20120926234950%21VLC_Construct.png"></center><br />
<br />
<b>Parts Information</b>:<br><br><br />
<a href="http://partsregistry.org/Part:BBa_K754000">BBa_K754000 </a> : <i>S. elongatus</i> PCC7942 <i>psbAI</i> promoter. This is our submitted part for the registry. This promoter has a light dependent regulation being active in normal light conditions. Read more here.<br><br />
<a href="http://partsregistry.org/Part:BBa_Q04510">BBa_Q04510 </a>: Consists in the next parts:<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>: RBS<br></li><br />
</ul> <br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_C0051">C0051</a>: cI repressor from E. coli phage lambda <br />
Coding region for the cI repressor based on cI repressor from bacteriophage lambda modified with an LVA tail for rapid degradation of the protein. cI repressor binds to the cI regulator.<br></li><br />
</ul><br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_B0015">B0015</a>: double terminator<br><br />
This is the most commonly used terminator. It seems to be reliable<br></li><br />
</ul><br />
<br />
<ul style="list-style-type: square"> <br />
<li><a href="http://partsregistry.org/Part:BBa_R0051">R0051</a>: promoter (lambda cI regulated)<br><br />
The cI regulated promoter is based on the pR promoter from bacteriophage lambda. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. cI binding results in repression of transcription.<br></li><br />
</ul><br />
<br />
<br />
<a href="http://partsregistry.org/Part:BBa_C0061">BBa_C0061 </a>: Autoinducer synthetase for AHL<br><br />
The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes. This expression is up-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound HSL. This complex binds to a palindromic site on the promoter, increasing the rate of transcription.<br><br />
<br />
<a href="http://partsregistry.org/Part:BBa_B1002">BBa_B1002</a>: Terminator<br><br />
<br />
<br><br><br />
<strong>Constructing the BioBrick</strong><br />
<br><br><br />
<br />
We ligate the part <a href="http://partsregistry.org/Part:BBa_K754000">BBa_K754000 </a> (psbAI promoter) with the psb1C3 vector. <br />
<center><img src="https://static.igem.org/mediawiki/2012/c/ce/VLC_PsB1C3%2BpsbAI.png"></center><br />
</p><br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/genetic_engineeringTeam:Valencia/genetic engineering2012-09-27T02:16:09Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Genetic Engineering<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
<a href="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg"><img align="right" src="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg" style="margin-left: 10px;"></a><br />
Cyanobacteria are great organisms to be used in Synthetic Biology because of their ability to capture solar energy and CO<sub>2</sub> and the fact that they can be easily genetically manipulated due to its small genome and their capacity to accept foreign DNA naturally. For our project we have chosen the cyanobacteria <i>Synechococcus elongatus</i> for genetic engineering because is the model organism for studying some prokaryotic processes (there is a lot of information of how to transform it) and in the last years it has become a model organism for some industrial processes, like biofuel production (Wang et al. 2012).<br><br><br />
<i>S. elongatus</i> has a circular genome of ≈2.7Mb (<a href="http://genome.kazusa.or.jp/cyanobase/SYNPCC7942" target=_blank"> fully sequenced </a>) with a GC content of 55.5%, which contains the genes for 2.612 proteins and 53 RNAs (Atsumi et al. 2009). <br />
<br><br><br><br><br />
<br />
<h2><b>Transforming <i>S. elongatus</i> PCC7942 for promoter characterization:</b></h2><br />
<br><br><br />
Our main objective has been to characterize the <i>psbAI</i> promoter (<a href="https://2012.igem.org/Team:Valencia/Parts">Submitted parts</a>) of <i>Synechococcus elongatus</i> PCC7942 in order to know more about its operation and understand how this promoter would control our final construct (<a href="https://2012.igem.org/Team:Valencia/prototypes"> Designed parts</a>) to have a diel switch of AHL, the signal molecule for our <i>Aliivibrio fischeri</i> population to glow. <br><br><br />
To characterize this promoter we had two options: making <i>psbAIp::lacZ</i> fusions and monitored the β-galactosidase activity or making <i>psbAIp::luxABCDE</i> fusions and monitored the bioluminescence produced as a result of the promoter activation. We refused to use a <i>psbAIp::lacZ</i> fusion because some assays report that the <i>psbAI</i> promoter response to light cannot be properly monitored by the β-galactosidase activity (Nair et al. 2001). For this reason we choosed using vector fusions. With this aim, we cloned several fusion plasmids (pAM977 and pAM2195) in <i>Escherichia coli</i> and transformed them into <i>S. elongatus</i>, both wildtype and cscB strain. The fusion vectors were provided by Susan Golden´s lab. <br><br><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png"><img src="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png" width="600" height="180"></a></center><br><br><br><br />
<br />
<h2><b>Cloning into <i>E. coli</i></b></h2><br />
We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this protocol (link). For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> <br />
<br />
<h2><b>Transforming Coccus</b></h2><br />
Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. <br />
As the cscB strain from Harvard was resistant to Cm, we only could transform it with the pAM977 plasmid, which gives Sp resistance. Wildtype was transformed with both plasmids (pAM977 and pAM2195).<br><br />
We used the following protocol to transform <i>Synechococcus</i>: <br><br><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br />
<br><br />
<h2><b>Protocol</b></h2><br />
<br><br />
Extracted from Clerico et al. 2007:<br><br />
<ul style="list-style-type: square"><br><br />
<li>We grew 100ml of cscB and WT in BG-11 liquid medium (see how we grown them and our recipe here) shaking at 250rpm with constant light we used cold light fluorescent tubes. Cells had an optical density at 750nm (OD750) of 0.7, which is reach in 4 to 7 days approximately.</li><br><br />
<li>We collected 15mL of the culture and centrifuge it at 6000g for 10 min and discarded the supernatant.</li><br><br />
<li>We resuspended collected cells in 10 mL of 10mM NaCl and centrifuge them again at 6000g for 10 min.</li><br><br />
<li>After it, we resuspended the pellet in 0.3mL of BG-11M liquid medium.</li><br><br />
<li>We added 2μl of pAM2195 and pAM977 after doing a Miniprep of the clonation with a JetQuick kit (link al kit?)</li><br><br />
<li>Wrap tubes in aluminum foil to keep out light and incubate at 30oC for 15 to 20h with gentle agitation.</li><br><br />
<li>And then, plate the entire 0.3mL cell suspension on BG-11 medium agar (see here the recipe) with the selective antibiotics. We used this concentration: 2μg/ml spectinomycin and 7.5μg/mL of chloramphenicol. After 7 to 10 days of incubation under standard light conditions transformed colonies should appear.</li><br><br />
<li>After colonies appearance pick single transformants and grow them on fresh liquid BG-11M agar plate with selective antibiotic (this is done to ensure that all colonies have incorporated the trans gene as S. elongatus has multiple copies of its chromosome).</li><br><br />
<li>After 5 to 7 days of growth, cyanobacteria can be used to inoculate a BG-11 liquid culture with the selective antibiotic.</li></ul><br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG"><img src="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG" width="458" height="343"></a></center><br />
<br><br />
<h2><b>Results</b></h2><br />
<br><br />
We weren´t able to obtain transformants with the wild type strain of <i>S. elongatus</i>. <br />
But we obtained resistant colonies to Sp of the cscB strain transformed with the pAM977 vector (figure x). This was achieved the last week of lab work, so due to the slowly growth rate of <i>S. elongatus</i>, we haven´t have the time to grow the transformants in liquid medium and start with the bioluminescence measures to characterize the promoter. However, we will try to have some results for the Jamboree concerning this part.<br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"><img src="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"></a></center><br />
<br><br />
<br />
<h2><b>Transformation of <i>E. coli</i> for AHL production to test bioluminescent response in <i>A. fischeri</i>:</b></h2><br><br />
Another part of our project included transforming <i>E. coli</i> with the registry part <a href="http://partsregistry.org/Part:BBa_K084014"> BBa_K084014 </a> in order to make <i>E. coli</i> produce AHL under IPTG regulation to test the bioluminescent response in A. fischeri. To achieve this goal we cloned the part in DH5α <i>E. coli</i> strains using this <a href="https://www.facebook.com/l.php?u=http%3A%2F%2Fpartsregistry.org%2FHelp%3ADistribution_Kits&h=oAQFOZXUl">protocol</a>(figure 1). The part (869 bp) is included inside the pSB1A2 (2079bp) BioBrick.<br><br><br />
<center><a href="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"><img src="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"></a></center><br><br />
We designed an experimental procedure to test different experimental conditions for the production of AHL.<br><br><br />
<center><a href="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png"><img src="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png" width="600" height="140"></a></center><br><br />
<br />
<br>When we wanted to test if <i>A. fischeri</i> was able to glow with the AHL produced by <i>E. coli</i> we discovered that the strain we had been growing in the lab WAS NOT VIBRIO FISCHERI, but VIBRIO MEDITERRANEI, a non bioluminescent Vibrio strain (GREAT!).<br><br />
We couldn´t go further with this experiment.<br><br />
</p><br />
<br><br />
<h2><b>Building our BioBricks</b></h2><br />
<br><br />
<b><i>psbAI</i> BioBrick</b><br />
<br><br><br />
Our promoter with the aproppiate <a href="http://partsregistry.org/Help:Assembly_standard_25"> suffix and preffix </a> (289bp) was synthesized by Genscript and cloned into a puc57-Kan (2579bp) vector. We successfully digested the part and ligated it into the psb1C3 BioBrick from the parts registry (figure 1 and 2). For this aim we followed the openwetware protocols, which you can find here. <br><br />
<br />
<b>Complete Construct</b><br />
<br><br><br />
Our final objective is to have a S. elongatus capable to produce AHL during the night. For this we designed this new BioBrick controled by the psbAI promoter which is active in normal light conditions.<a href="https://2012.igem.org/Team:Valencia/prototypes"> Here </a> you will find all the information concerning the different parts of the BioBrick.<br><br />
We successfully cloned all the parts in DH5α E. coli strains using this protocol (link). We used the BioBrick Assembly Manual (link) protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3.<br><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/f/fc/DSC02872_VLC.jpg"> &nbsp; &nbsp;<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/6/6b/Electro_VLCXXX.jpg" width="350" height="270"><br />
<br />
<br><br><br />
<center><img src="https://static.igem.org/mediawiki/2012/c/ce/VLC_PsB1C3%2BpsbAI.png"></center><br />
</p><br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/genetic_engineeringTeam:Valencia/genetic engineering2012-09-27T02:10:21Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Genetic Engineering<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
<a href="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg"><img align="right" src="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg" style="margin-left: 10px;"></a><br />
Cyanobacteria are great organisms to be used in Synthetic Biology because of their ability to capture solar energy and CO<sub>2</sub> and the fact that they can be easily genetically manipulated due to its small genome and their capacity to accept foreign DNA naturally. For our project we have chosen the cyanobacteria <i>Synechococcus elongatus</i> for genetic engineering because is the model organism for studying some prokaryotic processes (there is a lot of information of how to transform it) and in the last years it has become a model organism for some industrial processes, like biofuel production (Wang et al. 2012).<br><br><br />
<i>S. elongatus</i> has a circular genome of ≈2.7Mb (<a href="http://genome.kazusa.or.jp/cyanobase/SYNPCC7942" target=_blank"> fully sequenced </a>) with a GC content of 55.5%, which contains the genes for 2.612 proteins and 53 RNAs (Atsumi et al. 2009). <br />
<br><br><br><br><br />
<br />
<h2><b>Transforming <i>S. elongatus</i> PCC7942 for promoter characterization:</b></h2><br />
<br><br><br />
Our main objective has been to characterize the <i>psbAI</i> promoter (<a href="https://2012.igem.org/Team:Valencia/Parts">Submitted parts</a>) of <i>Synechococcus elongatus</i> PCC7942 in order to know more about its operation and understand how this promoter would control our final construct (<a href="https://2012.igem.org/Team:Valencia/prototypes"> Designed parts</a>) to have a diel switch of AHL, the signal molecule for our <i>Aliivibrio fischeri</i> population to glow. <br><br><br />
To characterize this promoter we had two options: making <i>psbAIp::lacZ</i> fusions and monitored the β-galactosidase activity or making <i>psbAIp::luxABCDE</i> fusions and monitored the bioluminescence produced as a result of the promoter activation. We refused to use a <i>psbAIp::lacZ</i> fusion because some assays report that the <i>psbAI</i> promoter response to light cannot be properly monitored by the β-galactosidase activity (Nair et al. 2001). For this reason we choosed using vector fusions. With this aim, we cloned several fusion plasmids (pAM977 and pAM2195) in <i>Escherichia coli</i> and transformed them into <i>S. elongatus</i>, both wildtype and cscB strain. The fusion vectors were provided by Susan Golden´s lab. <br><br><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png"><img src="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png" width="600" height="180"></a></center><br><br><br><br />
<br />
<h2><b>Cloning into <i>E. coli</i></b></h2><br />
We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this protocol (link). For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> <br />
<br />
<h2><b>Transforming Coccus</b></h2><br />
Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. <br />
As the cscB strain from Harvard was resistant to Cm, we only could transform it with the pAM977 plasmid, which gives Sp resistance. Wildtype was transformed with both plasmids (pAM977 and pAM2195).<br><br />
We used the following protocol to transform <i>Synechococcus</i>: <br><br><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br />
<br><br />
<h2><b>Protocol</b></h2><br />
<br><br />
Extracted from Clerico et al. 2007:<br><br />
<ul style="list-style-type: square"><br><br />
<li>We grew 100ml of cscB and WT in BG-11 liquid medium (see how we grown them and our recipe here) shaking at 250rpm with constant light we used cold light fluorescent tubes. Cells had an optical density at 750nm (OD750) of 0.7, which is reach in 4 to 7 days approximately.</li><br><br />
<li>We collected 15mL of the culture and centrifuge it at 6000g for 10 min and discarded the supernatant.</li><br><br />
<li>We resuspended collected cells in 10 mL of 10mM NaCl and centrifuge them again at 6000g for 10 min.</li><br><br />
<li>After it, we resuspended the pellet in 0.3mL of BG-11M liquid medium.</li><br><br />
<li>We added 2μl of pAM2195 and pAM977 after doing a Miniprep of the clonation with a JetQuick kit (link al kit?)</li><br><br />
<li>Wrap tubes in aluminum foil to keep out light and incubate at 30oC for 15 to 20h with gentle agitation.</li><br><br />
<li>And then, plate the entire 0.3mL cell suspension on BG-11 medium agar (see here the recipe) with the selective antibiotics. We used this concentration: 2μg/ml spectinomycin and 7.5μg/mL of chloramphenicol. After 7 to 10 days of incubation under standard light conditions transformed colonies should appear.</li><br><br />
<li>After colonies appearance pick single transformants and grow them on fresh liquid BG-11M agar plate with selective antibiotic (this is done to ensure that all colonies have incorporated the trans gene as S. elongatus has multiple copies of its chromosome).</li><br><br />
<li>After 5 to 7 days of growth, cyanobacteria can be used to inoculate a BG-11 liquid culture with the selective antibiotic.</li></ul><br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG"><img src="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG" width="458" height="343"></a></center><br />
<br><br />
<h2><b>Results</b></h2><br />
<br><br />
We weren´t able to obtain transformants with the wild type strain of <i>S. elongatus</i>. <br />
But we obtained resistant colonies to Sp of the cscB strain transformed with the pAM977 vector (figure x). This was achieved the last week of lab work, so due to the slowly growth rate of <i>S. elongatus</i>, we haven´t have the time to grow the transformants in liquid medium and start with the bioluminescence measures to characterize the promoter. However, we will try to have some results for the Jamboree concerning this part.<br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"><img src="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"></a></center><br />
<br><br />
<br />
<h2><b>Transformation of <i>E. coli</i> for AHL production to test bioluminescent response in <i>A. fischeri</i>:</b></h2><br><br />
Another part of our project included transforming <i>E. coli</i> with the registry part <a href="http://partsregistry.org/Part:BBa_K084014"> BBa_K084014 </a> in order to make <i>E. coli</i> produce AHL under IPTG regulation to test the bioluminescent response in A. fischeri. To achieve this goal we cloned the part in DH5α <i>E. coli</i> strains using this <a href="https://www.facebook.com/l.php?u=http%3A%2F%2Fpartsregistry.org%2FHelp%3ADistribution_Kits&h=oAQFOZXUl">protocol</a>(figure 1). The part (869 bp) is included inside the pSB1A2 (2079bp) BioBrick.<br><br><br />
<center><a href="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"><img src="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"></a></center><br><br />
We designed an experimental procedure to test different experimental conditions for the production of AHL.<br><br><br />
<center><a href="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png"><img src="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png" width="600" height="140"></a></center><br><br />
<br />
<br>When we wanted to test if <i>A. fischeri</i> was able to glow with the AHL produced by <i>E. coli</i> we discovered that the strain we had been growing in the lab WAS NOT VIBRIO FISCHERI, but VIBRIO MEDITERRANEI, a non bioluminescent Vibrio strain (GREAT!).<br><br />
We couldn´t go further with this experiment.<br><br />
</p><br />
<br><br />
<h2><b>Building our BioBricks</b></h2><br />
<br><br />
<i>psbAI</i> BioBrick<br><br />
Our promoter with the aproppiate <a href="http://partsregistry.org/Help:Assembly_standard_25"> suffix and preffix </a> (289bp) was synthesized by Genscript and cloned into a puc57-Kan (2579bp) vector. We successfully digested the part and ligated it into the psb1C3 BioBrick from the parts registry (figure 1 and 2). For this aim we followed the openwetware protocols, which you can find here. <br><br />
<br />
Complete Construct<br><br />
Our final objective is to have a S. elongatus capable to produce AHL during the night. For this we designed this new BioBrick controled by the psbAI promoter which is active in normal light conditions.<a href="https://2012.igem.org/Team:Valencia/prototypes"> Here </a> you will find all the information concerning the different parts of the BioBrick.<br><br />
We successfully cloned all the parts in DH5α E. coli strains using this protocol (link). We used the BioBrick Assembly Manual (link) protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3.<br><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/f/fc/DSC02872_VLC.jpg"> &nbsp; &nbsp;<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/6/6b/Electro_VLCXXX.jpg" width="350" height="270"><br />
<br />
<br><br><br />
<br />
<img src="https://2012.igem.org/File:VLC_PsB1C3%2BpsbAI.png"><br />
<br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/genetic_engineeringTeam:Valencia/genetic engineering2012-09-27T01:42:38Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Genetic Engineering<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
<a href="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg"><img align="right" src="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg" style="margin-left: 10px;"></a><br />
Cyanobacteria are great organisms to be used in Synthetic Biology because of their ability to capture solar energy and CO<sub>2</sub> and the fact that they can be easily genetically manipulated due to its small genome and their capacity to accept foreign DNA naturally. For our project we have chosen the cyanobacteria <i>Synechococcus elongatus</i> for genetic engineering because is the model organism for studying some prokaryotic processes (there is a lot of information of how to transform it) and in the last years it has become a model organism for some industrial processes, like biofuel production (Wang et al. 2012).<br><br><br />
<i>S. elongatus</i> has a circular genome of ≈2.7Mb (<a href="http://genome.kazusa.or.jp/cyanobase/SYNPCC7942" target=_blank"> fully sequenced </a>) with a GC content of 55.5%, which contains the genes for 2.612 proteins and 53 RNAs (Atsumi et al. 2009). <br />
<br><br><br><br><br />
<br />
<h2><b>Transforming <i>S. elongatus</i> PCC7942 for promoter characterization:</b></h2><br />
<br><br><br />
Our main objective has been to characterize the <i>psbAI</i> promoter (<a href="https://2012.igem.org/Team:Valencia/Parts">Submitted parts</a>) of <i>Synechococcus elongatus</i> PCC7942 in order to know more about its operation and understand how this promoter would control our final construct (<a href="https://2012.igem.org/Team:Valencia/prototypes"> Designed parts</a>) to have a diel switch of AHL, the signal molecule for our <i>Aliivibrio fischeri</i> population to glow. <br><br><br />
To characterize this promoter we had two options: making <i>psbAIp::lacZ</i> fusions and monitored the β-galactosidase activity or making <i>psbAIp::luxABCDE</i> fusions and monitored the bioluminescence produced as a result of the promoter activation. We refused to use a <i>psbAIp::lacZ</i> fusion because some assays report that the <i>psbAI</i> promoter response to light cannot be properly monitored by the β-galactosidase activity (Nair et al. 2001). For this reason we choosed using vector fusions. With this aim, we cloned several fusion plasmids (pAM977 and pAM2195) in <i>Escherichia coli</i> and transformed them into <i>S. elongatus</i>, both wildtype and cscB strain. The fusion vectors were provided by Susan Golden´s lab. <br><br><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png"><img src="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png" width="600" height="180"></a></center><br><br><br><br />
<br />
<h2><b>Cloning into <i>E. coli</i></b></h2><br />
We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this protocol (link). For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> <br />
<br />
<h2><b>Transforming Coccus</b></h2><br />
Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. <br />
As the cscB strain from Harvard was resistant to Cm, we only could transform it with the pAM977 plasmid, which gives Sp resistance. Wildtype was transformed with both plasmids (pAM977 and pAM2195).<br><br />
We used the following protocol to transform <i>Synechococcus</i>: <br><br><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br />
<br><br />
<h2><b>Protocol</b></h2><br />
<br><br />
Extracted from Clerico et al. 2007:<br><br />
<ul style="list-style-type: square"><br><br />
<li>We grew 100ml of cscB and WT in BG-11 liquid medium (see how we grown them and our recipe here) shaking at 250rpm with constant light we used cold light fluorescent tubes. Cells had an optical density at 750nm (OD750) of 0.7, which is reach in 4 to 7 days approximately.</li><br><br />
<li>We collected 15mL of the culture and centrifuge it at 6000g for 10 min and discarded the supernatant.</li><br><br />
<li>We resuspended collected cells in 10 mL of 10mM NaCl and centrifuge them again at 6000g for 10 min.</li><br><br />
<li>After it, we resuspended the pellet in 0.3mL of BG-11M liquid medium.</li><br><br />
<li>We added 2μl of pAM2195 and pAM977 after doing a Miniprep of the clonation with a JetQuick kit (link al kit?)</li><br><br />
<li>Wrap tubes in aluminum foil to keep out light and incubate at 30oC for 15 to 20h with gentle agitation.</li><br><br />
<li>And then, plate the entire 0.3mL cell suspension on BG-11 medium agar (see here the recipe) with the selective antibiotics. We used this concentration: 2μg/ml spectinomycin and 7.5μg/mL of chloramphenicol. After 7 to 10 days of incubation under standard light conditions transformed colonies should appear.</li><br><br />
<li>After colonies appearance pick single transformants and grow them on fresh liquid BG-11M agar plate with selective antibiotic (this is done to ensure that all colonies have incorporated the trans gene as S. elongatus has multiple copies of its chromosome).</li><br><br />
<li>After 5 to 7 days of growth, cyanobacteria can be used to inoculate a BG-11 liquid culture with the selective antibiotic.</li></ul><br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG"><img src="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG" width="458" height="343"></a></center><br />
<br><br />
<h2><b>Results</b></h2><br />
<br><br />
We weren´t able to obtain transformants with the wild type strain of <i>S. elongatus</i>. <br />
But we obtained resistant colonies to Sp of the cscB strain transformed with the pAM977 vector (figure x). This was achieved the last week of lab work, so due to the slowly growth rate of <i>S. elongatus</i>, we haven´t have the time to grow the transformants in liquid medium and start with the bioluminescence measures to characterize the promoter. However, we will try to have some results for the Jamboree concerning this part.<br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"><img src="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"></a></center><br />
<br><br />
<br />
<h2><b>Transformation of <i>E. coli</i> for AHL production to test bioluminescent response in <i>A. fischeri</i>:</b></h2><br><br />
Another part of our project included transforming <i>E. coli</i> with the registry part <a href="http://partsregistry.org/Part:BBa_K084014"> BBa_K084014 </a> in order to make <i>E. coli</i> produce AHL under IPTG regulation to test the bioluminescent response in A. fischeri. To achieve this goal we cloned the part in DH5α <i>E. coli</i> strains using this <a href="https://www.facebook.com/l.php?u=http%3A%2F%2Fpartsregistry.org%2FHelp%3ADistribution_Kits&h=oAQFOZXUl">protocol</a>(figure 1). The part (869 bp) is included inside the pSB1A2 (2079bp) BioBrick.<br><br><br />
<center><a href="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"><img src="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"></a></center><br><br />
We designed an experimental procedure to test different experimental conditions for the production of AHL.<br><br><br />
<center><a href="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png"><img src="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png" width="600" height="140"></a></center><br><br />
<br />
<br>When we wanted to test if <i>A. fischeri</i> was able to glow with the AHL produced by <i>E. coli</i> we discovered that the strain we had been growing in the lab WAS NOT VIBRIO FISCHERI, but VIBRIO MEDITERRANEI, a non bioluminescent Vibrio strain (GREAT!).<br><br />
We couldn´t go further with this experiment.<br><br />
</p><br />
<br><br />
<h2><b>Building our BioBricks</b></h2><br />
<br><br />
<i>psbAI</i> BioBrick<br><br />
Our promoter with the aproppiate suffix and preffix (289bp) (<a href="http://partsregistry.org/Help:Assembly_standard_25>") was synthesized by Genscript and cloned into a puc57-Kan (2579bp) vector. We successfully digested the part and ligated it into the psb1C3 BioBrick from the parts registry (figure 1 and 2). For this aim we followed the openwetware protocols, which you can find here. <br><br />
<br />
Complete Construct<br><br />
Our final objective is to have a S. elongatus capable to produce AHL during the night. For this we designed this new BioBrick controled by the psbAI promoter which is active in normal light conditions.<a href="https://2012.igem.org/Team:Valencia/prototypes"> Here </a> you will find all the information concerning the different parts of the BioBrick.<br><br />
We successfully cloned all the parts in DH5α E. coli strains using this protocol (link al protocolo). We used the BioBrick Assembly Manual (link) protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3.<br><br />
<br />
<br />
<div id=HomeLeft><img src="https://static.igem.org/mediawiki/2012/f/fc/DSC02872_VLC.jpg"><br><br />
</div><br />
<br />
<br />
<br />
<br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/genetic_engineeringTeam:Valencia/genetic engineering2012-09-27T01:39:46Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Genetic Engineering<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
<a href="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg"><img align="right" src="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg" style="margin-left: 10px;"></a><br />
Cyanobacteria are great organisms to be used in Synthetic Biology because of their ability to capture solar energy and CO<sub>2</sub> and the fact that they can be easily genetically manipulated due to its small genome and their capacity to accept foreign DNA naturally. For our project we have chosen the cyanobacteria <i>Synechococcus elongatus</i> for genetic engineering because is the model organism for studying some prokaryotic processes (there is a lot of information of how to transform it) and in the last years it has become a model organism for some industrial processes, like biofuel production (Wang et al. 2012).<br><br><br />
<i>S. elongatus</i> has a circular genome of ≈2.7Mb (<a href="http://genome.kazusa.or.jp/cyanobase/SYNPCC7942" target=_blank"> fully sequenced </a>) with a GC content of 55.5%, which contains the genes for 2.612 proteins and 53 RNAs (Atsumi et al. 2009). <br />
<br><br><br><br><br />
<br />
<h2><b>Transforming <i>S. elongatus</i> PCC7942 for promoter characterization:</b></h2><br />
<br><br><br />
Our main objective has been to characterize the <i>psbAI</i> promoter (<a href="https://2012.igem.org/Team:Valencia/Parts">Submitted parts</a>) of <i>Synechococcus elongatus</i> PCC7942 in order to know more about its operation and understand how this promoter would control our final construct (<a href="https://2012.igem.org/Team:Valencia/prototypes"> Designed parts</a>) to have a diel switch of AHL, the signal molecule for our <i>Aliivibrio fischeri</i> population to glow. <br><br><br />
To characterize this promoter we had two options: making <i>psbAIp::lacZ</i> fusions and monitored the β-galactosidase activity or making <i>psbAIp::luxABCDE</i> fusions and monitored the bioluminescence produced as a result of the promoter activation. We refused to use a <i>psbAIp::lacZ</i> fusion because some assays report that the <i>psbAI</i> promoter response to light cannot be properly monitored by the β-galactosidase activity (Nair et al. 2001). For this reason we choosed using vector fusions. With this aim, we cloned several fusion plasmids (pAM977 and pAM2195) in <i>Escherichia coli</i> and transformed them into <i>S. elongatus</i>, both wildtype and cscB strain. The fusion vectors were provided by Susan Golden´s lab. <br><br><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png"><img src="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png" width="600" height="180"></a></center><br><br><br><br />
<br />
<h2><b>Cloning into <i>E. coli</i></b></h2><br />
We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this protocol (link). For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> <br />
<br />
<h2><b>Transforming Coccus</b></h2><br />
Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. <br />
As the cscB strain from Harvard was resistant to Cm, we only could transform it with the pAM977 plasmid, which gives Sp resistance. Wildtype was transformed with both plasmids (pAM977 and pAM2195).<br><br />
We used the following protocol to transform <i>Synechococcus</i>: <br><br><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br />
<br><br />
<h2><b>Protocol</b></h2><br />
<br><br />
Extracted from Clerico et al. 2007:<br><br />
<ul style="list-style-type: square"><br><br />
<li>We grew 100ml of cscB and WT in BG-11 liquid medium (see how we grown them and our recipe here) shaking at 250rpm with constant light we used cold light fluorescent tubes. Cells had an optical density at 750nm (OD750) of 0.7, which is reach in 4 to 7 days approximately.</li><br><br />
<li>We collected 15mL of the culture and centrifuge it at 6000g for 10 min and discarded the supernatant.</li><br><br />
<li>We resuspended collected cells in 10 mL of 10mM NaCl and centrifuge them again at 6000g for 10 min.</li><br><br />
<li>After it, we resuspended the pellet in 0.3mL of BG-11M liquid medium.</li><br><br />
<li>We added 2μl of pAM2195 and pAM977 after doing a Miniprep of the clonation with a JetQuick kit (link al kit?)</li><br><br />
<li>Wrap tubes in aluminum foil to keep out light and incubate at 30oC for 15 to 20h with gentle agitation.</li><br><br />
<li>And then, plate the entire 0.3mL cell suspension on BG-11 medium agar (see here the recipe) with the selective antibiotics. We used this concentration: 2μg/ml spectinomycin and 7.5μg/mL of chloramphenicol. After 7 to 10 days of incubation under standard light conditions transformed colonies should appear.</li><br><br />
<li>After colonies appearance pick single transformants and grow them on fresh liquid BG-11M agar plate with selective antibiotic (this is done to ensure that all colonies have incorporated the trans gene as S. elongatus has multiple copies of its chromosome).</li><br><br />
<li>After 5 to 7 days of growth, cyanobacteria can be used to inoculate a BG-11 liquid culture with the selective antibiotic.</li></ul><br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG"><img src="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG" width="458" height="343"></a></center><br />
<br><br />
<h2><b>Results</b></h2><br />
<br><br />
We weren´t able to obtain transformants with the wild type strain of <i>S. elongatus</i>. <br />
But we obtained resistant colonies to Sp of the cscB strain transformed with the pAM977 vector (figure x). This was achieved the last week of lab work, so due to the slowly growth rate of <i>S. elongatus</i>, we haven´t have the time to grow the transformants in liquid medium and start with the bioluminescence measures to characterize the promoter. However, we will try to have some results for the Jamboree concerning this part.<br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"><img src="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"></a></center><br />
<br><br />
<br />
<h2><b>Transformation of <i>E. coli</i> for AHL production to test bioluminescent response in <i>A. fischeri</i>:</b></h2><br><br />
Another part of our project included transforming <i>E. coli</i> with the registry part <a href="http://partsregistry.org/Part:BBa_K084014"> BBa_K084014 </a> in order to make <i>E. coli</i> produce AHL under IPTG regulation to test the bioluminescent response in A. fischeri. To achieve this goal we cloned the part in DH5α <i>E. coli</i> strains using this <a href="https://www.facebook.com/l.php?u=http%3A%2F%2Fpartsregistry.org%2FHelp%3ADistribution_Kits&h=oAQFOZXUl">protocol</a>(figure 1). The part (869 bp) is included inside the pSB1A2 (2079bp) BioBrick.<br><br><br />
<center><a href="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"><img src="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"></a></center><br><br />
We designed an experimental procedure to test different experimental conditions for the production of AHL.<br><br><br />
<center><a href="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png"><img src="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png" width="600" height="140"></a></center><br><br />
<br />
<br>When we wanted to test if <i>A. fischeri</i> was able to glow with the AHL produced by <i>E. coli</i> we discovered that the strain we had been growing in the lab WAS NOT VIBRIO FISCHERI, but VIBRIO MEDITERRANEI, a non bioluminescent Vibrio strain (GREAT!).<br><br />
We couldn´t go further with this experiment.<br><br />
</p><br />
<br><br />
Building our BioBricks<br><br />
<i>psbAI</i> BioBrick<br><br />
Our promoter with the aproppiate suffix and preffix (289bp) (<a href="http://partsregistry.org/Help:Assembly_standard_25>") was synthesized by Genscript and cloned into a puc57-Kan (2579bp) vector. We successfully digested the part and ligated it into the psb1C3 BioBrick from the parts registry (figure 1 and 2). For this aim we followed the openwetware protocols, which you can find here. <br><br />
<br />
Complete Construct<br><br />
Our final objective is to have a S. elongatus capable to produce AHL during the night. For this we designed this new BioBrick controled by the psbAI promoter which is active in normal light conditions.<a href="https://2012.igem.org/Team:Valencia/prototypes"> Here</a> (a designed part) you will find all the information concerning the different parts of the BioBrick.<br><br />
We successfully cloned all the parts in DH5α E. coli strains using this protocol (link al protocolo). We used the BioBrick Assembly Manual (link) protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3.<br><br />
<br />
<br />
<div id=HomeLeft><img src="https://static.igem.org/mediawiki/2012/f/fc/DSC02872_VLC.jpg"><br><br />
</div><br />
<br />
<br />
<br />
<br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/genetic_engineeringTeam:Valencia/genetic engineering2012-09-27T01:22:12Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Genetic Engineering<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
<a href="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg"><img align="right" src="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg" style="margin-left: 10px;"></a><br />
Cyanobacteria are great organisms to be used in Synthetic Biology because of their ability to capture solar energy and CO<sub>2</sub> and the fact that they can be easily genetically manipulated due to its small genome and their capacity to accept foreign DNA naturally. For our project we have chosen the cyanobacteria <i>Synechococcus elongatus</i> for genetic engineering because is the model organism for studying some prokaryotic processes (there is a lot of information of how to transform it) and in the last years it has become a model organism for some industrial processes, like biofuel production (Wang et al. 2012).<br><br><br />
<i>S. elongatus</i> has a circular genome of ≈2.7Mb (<a href="http://genome.kazusa.or.jp/cyanobase/SYNPCC7942" target=_blank"> fully sequenced </a>) with a GC content of 55.5%, which contains the genes for 2.612 proteins and 53 RNAs (Atsumi et al. 2009). <br />
<br><br><br><br><br />
<br />
<h2><b>Transforming <i>S. elongatus</i> PCC7942 for promoter characterization:</b></h2><br />
<br><br><br />
Our main objective has been to characterize the <i>psbAI</i> promoter (<a href="https://2012.igem.org/Team:Valencia/Parts">Submitted parts</a>) of <i>Synechococcus elongatus</i> PCC7942 in order to know more about its operation and understand how this promoter would control our final construct (<a href="https://2012.igem.org/Team:Valencia/prototypes"> Designed parts</a>) to have a diel switch of AHL, the signal molecule for our <i>Aliivibrio fischeri</i> population to glow. <br><br><br />
To characterize this promoter we had two options: making <i>psbAIp::lacZ</i> fusions and monitored the β-galactosidase activity or making <i>psbAIp::luxABCDE</i> fusions and monitored the bioluminescence produced as a result of the promoter activation. We refused to use a <i>psbAIp::lacZ</i> fusion because some assays report that the <i>psbAI</i> promoter response to light cannot be properly monitored by the β-galactosidase activity (Nair et al. 2001). For this reason we choosed using vector fusions. With this aim, we cloned several fusion plasmids (pAM977 and pAM2195) in <i>Escherichia coli</i> and transformed them into <i>S. elongatus</i>, both wildtype and cscB strain. The fusion vectors were provided by Susan Golden´s lab. <br><br><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png"><img src="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png" width="600" height="180"></a></center><br><br><br><br />
<br />
<h2><b>Cloning into <i>E. coli</i></b></h2><br />
We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this protocol (link). For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> <br />
<br />
<h2><b>Transforming Coccus</b></h2><br />
Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. <br />
As the cscB strain from Harvard was resistant to Cm, we only could transform it with the pAM977 plasmid, which gives Sp resistance. Wildtype was transformed with both plasmids (pAM977 and pAM2195).<br><br />
We used the following protocol to transform <i>Synechococcus</i>: <br><br><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br />
<br><br />
<h2><b>Protocol</b></h2><br />
<br><br />
Extracted from Clerico et al. 2007:<br><br />
<ul style="list-style-type: square"><br><br />
<li>We grew 100ml of cscB and WT in BG-11 liquid medium (see how we grown them and our recipe here) shaking at 250rpm with constant light we used cold light fluorescent tubes (like this one). Cells had an optical density at 750nm (OD750) of 0.7, which is reach in 4 to 7 days approximately.</li><br><br />
<li>We collected 15mL of the culture and centrifuge it at 6000g for 10 min and discarded the supernatant.</li><br><br />
<li>We resuspended collected cells in 10 mL of 10mM NaCl and centrifuge them again at 6000g for 10 min.</li><br><br />
<li>After it, we resuspended the pellet in 0.3mL of BG-11M liquid medium.</li><br><br />
<li>We added 2μl of pAM2195 and pAM977 after doing a Miniprep of the clonation with a JetQuick kit (link al kit?)</li><br><br />
<li>Wrap tubes in aluminum foil to keep out light and incubate at 30oC for 15 to 20h with gentle agitation.</li><br><br />
<li>And then, plate the entire 0.3mL cell suspension on BG-11 medium agar (see here the recipe) with the selective antibiotics. We used this concentration: 2μg/ml spectinomycin and 7.5μg/mL of chloramphenicol. After 7 to 10 days of incubation under standard light conditions transformed colonies should appear.</li><br><br />
<li>After colonies appearance pick single transformants and grow them on fresh liquid BG-11M agar plate with selective antibiotic (this is done to ensure that all colonies have incorporated the trans gene as S. elongatus has multiple copies of its chromosome).</li><br><br />
<li>After 5 to 7 days of growth, cyanobacteria can be used to inoculate a BG-11 liquid culture with the selective antibiotic.</li></ul><br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG"><img src="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG" width="458" height="343"></a></center><br />
<br><br />
<h2><b>Results</b></h2><br />
<br><br />
We weren´t able to obtain transformants with the wild type strain of <i>S. elongatus</i>. <br />
But we obtained resistant colonies to Sp of the cscB strain transformed with the pAM977 vector (figure x). This was achieved the last week of lab work, so due to the slowly growth rate of <i>S. elongatus</i>, we haven´t have the time to grow the transformants in liquid medium and start with the bioluminescence measures to characterize the promoter. However, we will try to have some results for the Jamboree concerning this part.<br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"><img src="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"></a></center><br />
<br><br />
<br />
<h2><b>Transformation of <i>E. coli</i> for AHL production to test bioluminescent response in <i>A. fischeri</i>:</b></h2><br><br />
Another part of our project included transforming <i>E. coli</i> with the registry part <a href="http://partsregistry.org/Part:BBa_K084014"> BBa_K084014 </a> in order to make <i>E. coli</i> produce AHL under IPTG regulation to test the bioluminescent response in A. fischeri. To achieve this goal we cloned the part in DH5α <i>E. coli</i> strains using this protocol (link al registro que te dice como usar las partes) (figure 1). The part (869 bp) is included inside the pSB1A2 (2079bp) BioBrick.<br><br><br />
<center><a href="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"><img src="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"></a></center><br><br />
We designed an experimental procedure to test different experimental conditions for the production of AHL.<br><br><br />
<center><a href="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png"><img src="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png" width="600" height="140"></a></center><br><br />
<br />
<br>When we wanted to test if <i>A. fischeri</i> was able to glow with the AHL produced by <i>E. coli</i> we discovered that the strain we had been growing in the lab WAS NOT VIBRIO FISCHERI, but VIBRIO MEDITERRANEI, a non bioluminescent Vibrio strain (GREAT!).<br><br />
We couldn´t go further with this experiment.<br><br />
</p><br />
<br><br />
Building our BioBricks<br><br />
<i>psbAI</i> BioBrick<br><br />
Our promoter with the aproppiate suffix and preffix (289bp) (links a la wiki donde los explica) was synthesized by Genscript and cloned into a puc57-Kan (2579bp) vector. We successfully digested the part and ligated it into the psb1C3 BioBrick from the parts registry (figure 1 and 2). For this aim we followed the openwetware protocols, which you can find here. <br><br />
<br />
Complete Construct<br><br />
Our final objective is to have a S. elongatus capable to produce AHL during the night. For this we designed this new BioBrick controled by the psbAI promoter which is active in normal light conditions. Here (link a designed part) you will find all the information concerning the different parts of the BioBrick.<br><br />
We successfully cloned all the parts in DH5α E. coli strains using this protocol (link al protocolo). We used the BioBrick Assembly Manual (link) protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3.<br><br />
<br />
<br />
<div id=HomeLeft><img src="https://static.igem.org/mediawiki/2012/f/fc/DSC02872_VLC.jpg"><br><br />
</div><br />
<br />
<br />
<br />
<br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/genetic_engineeringTeam:Valencia/genetic engineering2012-09-27T01:20:37Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Genetic Engineering<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
<a href="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg"><img align="right" src="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg" style="margin-left: 10px;"></a><br />
Cyanobacteria are great organisms to be used in Synthetic Biology because of their ability to capture solar energy and CO<sub>2</sub> and the fact that they can be easily genetically manipulated due to its small genome and their capacity to accept foreign DNA naturally. For our project we have chosen the cyanobacteria <i>Synechococcus elongatus</i> for genetic engineering because is the model organism for studying some prokaryotic processes (there is a lot of information of how to transform it) and in the last years it has become a model organism for some industrial processes, like biofuel production (Wang et al. 2012).<br><br><br />
<i>S. elongatus</i> has a circular genome of ≈2.7Mb (<a href="http://genome.kazusa.or.jp/cyanobase/SYNPCC7942" target=_blank"> fully sequenced </a>) with a GC content of 55.5%, which contains the genes for 2.612 proteins and 53 RNAs (Atsumi et al. 2009). <br />
<br><br><br><br><br />
<br />
<h2><b>Transforming <i>S. elongatus</i> PCC7942 for promoter characterization:</b></h2><br />
<br><br><br />
Our main objective has been to characterize the <i>psbAI</i> promoter (<a href="https://2012.igem.org/Team:Valencia/Parts">Submitted parts</a>) of <i>Synechococcus elongatus</i> PCC7942 in order to know more about its operation and understand how this promoter would control our final construct (<a href="https://2012.igem.org/Team:Valencia/prototypes" Designed parts</a>) to have a diel switch of AHL, the signal molecule for our <i>Aliivibrio fischeri</i> population to glow. <br><br><br />
To characterize this promoter we had two options: making <i>psbAIp::lacZ</i> fusions and monitored the β-galactosidase activity or making <i>psbAIp::luxABCDE</i> fusions and monitored the bioluminescence produced as a result of the promoter activation. We refused to use a <i>psbAIp::lacZ</i> fusion because some assays report that the <i>psbAI</i> promoter response to light cannot be properly monitored by the β-galactosidase activity (Nair et al. 2001). For this reason we choosed using vector fusions. With this aim, we cloned several fusion plasmids (pAM977 and pAM2195) in <i>Escherichia coli</i> and transformed them into <i>S. elongatus</i>, both wildtype and cscB strain. The fusion vectors were provided by Susan Golden´s lab. <br><br><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png"><img src="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png" width="600" height="180"></a></center><br><br><br><br />
<br />
<h2><b>Cloning into <i>E. coli</i></b></h2><br />
We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this protocol (link). For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> <br />
<br />
<h2><b>Transforming Coccus</b></h2><br />
Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. <br />
As the cscB strain from Harvard was resistant to Cm, we only could transform it with the pAM977 plasmid, which gives Sp resistance. Wildtype was transformed with both plasmids (pAM977 and pAM2195).<br><br />
We used the following protocol to transform <i>Synechococcus</i>: <br><br><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br />
<br><br />
<h2><b>Protocol</b></h2><br />
<br><br />
Extracted from Clerico et al. 2007:<br><br />
<ul style="list-style-type: square"><br><br />
<li>We grew 100ml of cscB and WT in BG-11 liquid medium (see how we grown them and our recipe here) shaking at 250rpm with constant light we used cold light fluorescent tubes (like this one). Cells had an optical density at 750nm (OD750) of 0.7, which is reach in 4 to 7 days approximately.</li><br><br />
<li>We collected 15mL of the culture and centrifuge it at 6000g for 10 min and discarded the supernatant.</li><br><br />
<li>We resuspended collected cells in 10 mL of 10mM NaCl and centrifuge them again at 6000g for 10 min.</li><br><br />
<li>After it, we resuspended the pellet in 0.3mL of BG-11M liquid medium.</li><br><br />
<li>We added 2μl of pAM2195 and pAM977 after doing a Miniprep of the clonation with a JetQuick kit (link al kit?)</li><br><br />
<li>Wrap tubes in aluminum foil to keep out light and incubate at 30oC for 15 to 20h with gentle agitation.</li><br><br />
<li>And then, plate the entire 0.3mL cell suspension on BG-11 medium agar (see here the recipe) with the selective antibiotics. We used this concentration: 2μg/ml spectinomycin and 7.5μg/mL of chloramphenicol. After 7 to 10 days of incubation under standard light conditions transformed colonies should appear.</li><br><br />
<li>After colonies appearance pick single transformants and grow them on fresh liquid BG-11M agar plate with selective antibiotic (this is done to ensure that all colonies have incorporated the trans gene as S. elongatus has multiple copies of its chromosome).</li><br><br />
<li>After 5 to 7 days of growth, cyanobacteria can be used to inoculate a BG-11 liquid culture with the selective antibiotic.</li></ul><br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG"><img src="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG" width="458" height="343"></a></center><br />
<br><br />
<h2><b>Results</b></h2><br />
<br><br />
We weren´t able to obtain transformants with the wild type strain of <i>S. elongatus</i>. <br />
But we obtained resistant colonies to Sp of the cscB strain transformed with the pAM977 vector (figure x). This was achieved the last week of lab work, so due to the slowly growth rate of <i>S. elongatus</i>, we haven´t have the time to grow the transformants in liquid medium and start with the bioluminescence measures to characterize the promoter. However, we will try to have some results for the Jamboree concerning this part.<br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"><img src="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"></a></center><br />
<br><br />
<br />
<h2><b>Transformation of <i>E. coli</i> for AHL production to test bioluminescent response in <i>A. fischeri</i>:</b></h2><br><br />
Another part of our project included transforming <i>E. coli</i> with the registry part <a href="http://partsregistry.org/Part:BBa_K084014"> BBa_K084014 </a> in order to make <i>E. coli</i> produce AHL under IPTG regulation to test the bioluminescent response in A. fischeri. To achieve this goal we cloned the part in DH5α <i>E. coli</i> strains using this protocol (link al registro que te dice como usar las partes) (figure 1). The part (869 bp) is included inside the pSB1A2 (2079bp) BioBrick.<br><br><br />
<center><a href="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"><img src="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"></a></center><br><br />
We designed an experimental procedure to test different experimental conditions for the production of AHL.<br><br><br />
<center><a href="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png"><img src="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png" width="600" height="140"></a></center><br><br />
<br />
<br>When we wanted to test if <i>A. fischeri</i> was able to glow with the AHL produced by <i>E. coli</i> we discovered that the strain we had been growing in the lab WAS NOT VIBRIO FISCHERI, but VIBRIO MEDITERRANEI, a non bioluminescent Vibrio strain (GREAT!).<br><br />
We couldn´t go further with this experiment.<br><br />
</p><br />
<br><br />
Building our BioBricks<br><br />
<i>psbAI</i> BioBrick<br><br />
Our promoter with the aproppiate suffix and preffix (289bp) (links a la wiki donde los explica) was synthesized by Genscript and cloned into a puc57-Kan (2579bp) vector. We successfully digested the part and ligated it into the psb1C3 BioBrick from the parts registry (figure 1 and 2). For this aim we followed the openwetware protocols, which you can find here. <br><br />
<br />
Complete Construct<br><br />
Our final objective is to have a S. elongatus capable to produce AHL during the night. For this we designed this new BioBrick controled by the psbAI promoter which is active in normal light conditions. Here (link a designed part) you will find all the information concerning the different parts of the BioBrick.<br><br />
We successfully cloned all the parts in DH5α E. coli strains using this protocol (link al protocolo). We used the BioBrick Assembly Manual (link) protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3.<br><br />
<br />
<br />
<div id=HomeLeft><img src="https://static.igem.org/mediawiki/2012/f/fc/DSC02872_VLC.jpg"><br><br />
</div><br />
<br />
<br />
<br />
<br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/genetic_engineeringTeam:Valencia/genetic engineering2012-09-27T01:02:15Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Genetic Engineering<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
<a href="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg"><img align="right" src="https://static.igem.org/mediawiki/2012/f/fe/VLC_Synel.jpg" style="margin-left: 10px;"></a><br />
Cyanobacteria are great organisms to be used in Synthetic Biology because of their ability to capture solar energy and CO<sub>2</sub> and the fact that they can be easily genetically manipulated due to its small genome and their capacity to accept foreign DNA naturally. For our project we have chosen the cyanobacteria <i>Synechococcus elongatus</i> for genetic engineering because is the model organism for studying some prokaryotic processes (there is a lot of information of how to transform it) and in the last years it has become a model organism for some industrial processes, like biofuel production (Wang et al. 2012).<br><br><br />
<i>S. elongatus</i> has a circular genome of ≈2.7Mb (fully sequenced link a la base de datos) with a GC content of 55.5%, which contains the genes for 2.612 proteins and 53 RNAs (Atsumi et al. 2009). <br />
<br><br><br><br><br />
<br />
<h2><b>Transforming <i>S. elongatus</i> PCC7942 for promoter characterization:</b></h2><br />
<br><br><br />
Our main objective has been to characterize the <i>psbAI</i> promoter (link a submitted parts) of <i>Synechococcus elongatus</i> PCC7942 in order to know more about its operation and understand how this promoter would control our final construct (link a prototipo) to have a diel switch of AHL, the signal molecule for our <i>Aliivibrio fischeri</i> population to glow. <br><br><br />
To characterize this promoter we had two options: making <i>psbAIp::lacZ</i> fusions and monitored the β-galactosidase activity or making <i>psbAIp::luxABCDE</i> fusions and monitored the bioluminescence produced as a result of the promoter activation. We refused to use a <i>psbAIp::lacZ</i> fusion because some assays report that the <i>psbAI</i> promoter response to light cannot be properly monitored by the β-galactosidase activity (Nair et al. 2001). For this reason we choosed using vector fusions. With this aim, we cloned several fusion plasmids (pAM977 and pAM2195) in <i>Escherichia coli</i> and transformed them into <i>S. elongatus</i>, both wildtype and cscB strain. The fusion vectors were provided by Susan Golden´s lab. <br><br><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png"><img src="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png" width="600" height="180"></a></center><br><br><br><br />
<br />
<h2><b>Cloning into <i>E. coli</i></b></h2><br />
We successfully cloned all the vectors in DH5α <i>E. coli</i> strains. For<i> E. coli</i> transformation we used this protocol (link). For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> <br />
<br />
<h2><b>Transforming Coccus</b></h2><br />
Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. <br />
As the cscB strain from Harvard was resistant to Cm, we only could transform it with the pAM977 plasmid, which gives Sp resistance. Wildtype was transformed with both plasmids (pAM977 and pAM2195).<br><br />
We used the following protocol to transform <i>Synechococcus</i>: <br><br><br />
<br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"><img src="https://static.igem.org/mediawiki/2012/a/a2/VLC12_AM2195.png"></a></center><br />
<br><br />
<h2><b>Protocol</b></h2><br />
<br><br />
Extracted from Clerico et al. 2007:<br><br />
<ul style="list-style-type: square"><br><br />
<li>We grew 100ml of cscB and WT in BG-11 liquid medium (see how we grown them and our recipe here) shaking at 250rpm with constant light we used cold light fluorescent tubes (like this one). Cells had an optical density at 750nm (OD750) of 0.7, which is reach in 4 to 7 days approximately.</li><br><br />
<li>We collected 15mL of the culture and centrifuge it at 6000g for 10 min and discarded the supernatant.</li><br><br />
<li>We resuspended collected cells in 10 mL of 10mM NaCl and centrifuge them again at 6000g for 10 min.</li><br><br />
<li>After it, we resuspended the pellet in 0.3mL of BG-11M liquid medium.</li><br><br />
<li>We added 2μl of pAM2195 and pAM977 after doing a Miniprep of the clonation with a JetQuick kit (link al kit?)</li><br><br />
<li>Wrap tubes in aluminum foil to keep out light and incubate at 30oC for 15 to 20h with gentle agitation.</li><br><br />
<li>And then, plate the entire 0.3mL cell suspension on BG-11 medium agar (see here the recipe) with the selective antibiotics. We used this concentration: 2μg/ml spectinomycin and 7.5μg/mL of chloramphenicol. After 7 to 10 days of incubation under standard light conditions transformed colonies should appear.</li><br><br />
<li>After colonies appearance pick single transformants and grow them on fresh liquid BG-11M agar plate with selective antibiotic (this is done to ensure that all colonies have incorporated the trans gene as S. elongatus has multiple copies of its chromosome).</li><br><br />
<li>After 5 to 7 days of growth, cyanobacteria can be used to inoculate a BG-11 liquid culture with the selective antibiotic.</li></ul><br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG"><img src="https://static.igem.org/mediawiki/2012/d/dd/VLC_transformingcoccus.JPG" width="458" height="343"></a></center><br />
<br><br />
<h2><b>Results</b></h2><br />
<br><br />
We weren´t able to obtain transformants with the wild type strain of <i>S. elongatus</i>. <br />
But we obtained resistant colonies to Sp of the cscB strain transformed with the pAM977 vector (figure x). This was achieved the last week of lab work, so due to the slowly growth rate of <i>S. elongatus</i>, we haven´t have the time to grow the transformants in liquid medium and start with the bioluminescence measures to characterize the promoter. However, we will try to have some results for the Jamboree concerning this part.<br />
<br><br />
<center><a href="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"><img src="https://static.igem.org/mediawiki/2012/a/a9/VLC_Transformant2.png"></a></center><br />
<br><br />
<br />
<h2><b>Transformation of <i>E. coli</i> for AHL production to test bioluminescent response in <i>A. fischeri</i>:</b></h2><br><br />
Another part of our project included transforming <i>E. coli</i> with the registry part <a href="http://partsregistry.org/Part:BBa_K084014"> BBa_K084014 </a> in order to make <i>E. coli</i> produce AHL under IPTG regulation to test the bioluminescent response in A. fischeri. To achieve this goal we cloned the part in DH5α <i>E. coli</i> strains using this protocol (link al registro que te dice como usar las partes) (figure 1). The part (869 bp) is included inside the pSB1A2 (2079bp) BioBrick.<br><br><br />
<center><a href="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"><img src="https://static.igem.org/mediawiki/2012/5/5a/VLC_Electroforesis23-08.png"></a></center><br><br />
We designed an experimental procedure to test different experimental conditions for the production of AHL.<br><br><br />
<center><a href="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png"><img src="https://static.igem.org/mediawiki/2012/b/b3/VLC_TableIPTG.png" width="600" height="140"></a></center><br><br />
<br />
<br>When we wanted to test if <i>A. fischeri</i> was able to glow with the AHL produced by <i>E. coli</i> we discovered that the strain we had been growing in the lab WAS NOT VIBRIO FISCHERI, but VIBRIO MEDITERRANEI, a non bioluminescent Vibrio strain (GREAT!).<br><br />
We couldn´t go further with this experiment.<br><br />
</p><br />
<br />
<br />
<br />
<div id=HomeLeft><img src="https://static.igem.org/mediawiki/2012/f/fc/DSC02872_VLC.jpg"><br><br />
</div><br />
<br />
<br />
<br />
<br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/prototypesTeam:Valencia/prototypes2012-09-26T23:55:41Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Designed Biobricks<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
Our final objective is to have a <i>S. elongatus</i> capable to produce AHL during the night. For this we designed this new BioBrick controled by the <i>psbAI</i> promoter which is active in normal light conditions. To have an inverse response to light we have put the cI repressor after the <i>psbAI</i> promoter which binds to the lambda promoter inhibiting the luxI transcription, when <i>psbAI</i> is active. During the night, when the promoter is less active, the repressor will not be active, allowing the expression of luxI, and therefore having AHL.<br><br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2012/archive/f/fd/20120926234950%21VLC_Construct.png"></center><br />
<br />
<b>Parts Information</b>:<br><br><br />
<a href="http://partsregistry.org/Part:BBa_K754000">BBa_K754000 </a> : <i>S. elongatus</i> PCC7942 <i>psbAI</i> promoter. This is our submitted part for the registry. This promoter has a light dependent regulation being active in normal light conditions. Read more here.<br><br />
<a href="http://partsregistry.org/Part:BBa_Q04510">BBa_Q04510 </a>: Consists in the next parts:<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>: RBS<br></li><br />
</ul> <br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_C0051">C0051</a>: cI repressor from E. coli phage lambda <br />
Coding region for the cI repressor based on cI repressor from bacteriophage lambda modified with an LVA tail for rapid degradation of the protein. cI repressor binds to the cI regulator.<br></li><br />
</ul><br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_B0015">B0015</a>: double terminator<br><br />
This is the most commonly used terminator. It seems to be reliable<br></li><br />
</ul><br />
<br />
<ul style="list-style-type: square"> <br />
<li><a href="http://partsregistry.org/Part:BBa_R0051">R0051</a>: promoter (lambda cI regulated)<br><br />
The cI regulated promoter is based on the pR promoter from bacteriophage lambda. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. cI binding results in repression of transcription.<br></li><br />
</ul><br />
<br />
<br />
<a href="http://partsregistry.org/Part:BBa_C0061">BBa_C0061 </a>: Autoinducer synthetase for AHL<br><br />
The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes. This expression is up-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound HSL. This complex binds to a palindromic site on the promoter, increasing the rate of transcription.<br><br />
<br />
<a href="http://partsregistry.org/Part:BBa_B1002">BBa_B1002</a>: Terminator<br><br />
<br />
<br><br><br />
<strong>Constructing the BioBrick</strong><br />
<br><br><br />
We successfully cloned all the parts in DH5α E. coli strains using this protocol (link al protocolo). <br />
We used the BioBrick Assembly Manual (link) protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3. Read the protocol we followed here.<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2012/c/ce/VLC_PsB1C3%2BpsbAI.png"></center><br />
</p><br />
</html></div>Roocferhttp://2012.igem.org/File:VLC_PsB1C3%2BpsbAI.pngFile:VLC PsB1C3+psbAI.png2012-09-26T23:53:04Z<p>Roocfer: uploaded a new version of &quot;File:VLC PsB1C3+psbAI.png&quot;</p>
<hr />
<div></div>Roocferhttp://2012.igem.org/File:VLC_PsB1C3%2BpsbAI.pngFile:VLC PsB1C3+psbAI.png2012-09-26T23:51:53Z<p>Roocfer: </p>
<hr />
<div></div>Roocferhttp://2012.igem.org/Team:Valencia/prototypesTeam:Valencia/prototypes2012-09-26T23:50:40Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Designed Biobricks<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
Our final objective is to have a <i>S. elongatus</i> capable to produce AHL during the night. For this we designed this new BioBrick controled by the <i>psbAI</i> promoter which is active in normal light conditions. To have an inverse response to light we have put the cI repressor after the <i>psbAI</i> promoter which binds to the lambda promoter inhibiting the luxI transcription, when <i>psbAI</i> is active. During the night, when the promoter is less active, the repressor will not be active, allowing the expression of luxI, and therefore having AHL.<br><br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2012/archive/f/fd/20120926234950%21VLC_Construct.png"></center><br />
<br />
<b>Parts Information</b>:<br><br><br />
<a href="http://partsregistry.org/Part:BBa_K754000">BBa_K754000 </a> : <i>S. elongatus</i> PCC7942 <i>psbAI</i> promoter. This is our submitted part for the registry. This promoter has a light dependent regulation being active in normal light conditions. Read more here.<br><br />
<a href="http://partsregistry.org/Part:BBa_Q04510">BBa_Q04510 </a>: Consists in the next parts:<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>: RBS<br></li><br />
</ul> <br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_C0051">C0051</a>: cI repressor from E. coli phage lambda <br />
Coding region for the cI repressor based on cI repressor from bacteriophage lambda modified with an LVA tail for rapid degradation of the protein. cI repressor binds to the cI regulator.<br></li><br />
</ul><br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_B0015">B0015</a>: double terminator<br><br />
This is the most commonly used terminator. It seems to be reliable<br></li><br />
</ul><br />
<br />
<ul style="list-style-type: square"> <br />
<li><a href="http://partsregistry.org/Part:BBa_R0051">R0051</a>: promoter (lambda cI regulated)<br><br />
The cI regulated promoter is based on the pR promoter from bacteriophage lambda. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. cI binding results in repression of transcription.<br></li><br />
</ul><br />
<br />
<br />
<a href="http://partsregistry.org/Part:BBa_C0061">BBa_C0061 </a>: Autoinducer synthetase for AHL<br><br />
The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes. This expression is up-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound HSL. This complex binds to a palindromic site on the promoter, increasing the rate of transcription.<br><br />
<br />
<a href="http://partsregistry.org/Part:BBa_B1002">BBa_B1002</a>: Terminator<br><br />
<br />
<br><br><br />
<strong>Constructing the BioBrick</strong><br />
<br><br><br />
We successfully cloned all the parts in DH5α E. coli strains using this protocol (link al protocolo). <br />
We used the BioBrick Assembly Manual (link) protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3. Read the protocol we followed here.<br />
<br />
</p><br />
</html></div>Roocferhttp://2012.igem.org/File:VLC_Construct.pngFile:VLC Construct.png2012-09-26T23:49:50Z<p>Roocfer: uploaded a new version of &quot;File:VLC Construct.png&quot;</p>
<hr />
<div></div>Roocferhttp://2012.igem.org/File:VLC_Construct.pngFile:VLC Construct.png2012-09-26T23:45:36Z<p>Roocfer: uploaded a new version of &quot;File:VLC Construct.png&quot;</p>
<hr />
<div></div>Roocferhttp://2012.igem.org/Team:Valencia/prototypesTeam:Valencia/prototypes2012-09-26T23:34:04Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Designed Biobricks<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
Our final objective is to have a <i>S. elongatus</i> capable to produce AHL during the night. For this we designed this new BioBrick controled by the <i>psbAI</i> promoter which is active in normal light conditions. To have an inverse response to light we have put the cI repressor after the <i>psbAI</i> promoter which binds to the lambda promoter inhibiting the luxI transcription, when <i>psbAI</i> is active. During the night, when the promoter is less active, the repressor will not be active, allowing the expression of luxI, and therefore having AHL.<br><br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2012/f/fd/VLC_Construct.png"></center><br />
<br />
<b>Parts Information</b>:<br><br><br />
<a href="http://partsregistry.org/Part:BBa_K754000">BBa_K754000 </a> : <i>S. elongatus</i> PCC7942 <i>psbAI</i> promoter. This is our submitted part for the registry. This promoter has a light dependent regulation being active in normal light conditions. Read more here.<br><br />
<a href="http://partsregistry.org/Part:BBa_Q04510">BBa_Q04510 </a>: Consists in the next parts:<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>: RBS<br></li><br />
</ul> <br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_C0051">C0051</a>: cI repressor from E. coli phage lambda <br />
Coding region for the cI repressor based on cI repressor from bacteriophage lambda modified with an LVA tail for rapid degradation of the protein. cI repressor binds to the cI regulator.<br></li><br />
</ul><br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_B0015">B0015</a>: double terminator<br><br />
This is the most commonly used terminator. It seems to be reliable<br></li><br />
</ul><br />
<br />
<ul style="list-style-type: square"> <br />
<li><a href="http://partsregistry.org/Part:BBa_R0051">R0051</a>: promoter (lambda cI regulated)<br><br />
The cI regulated promoter is based on the pR promoter from bacteriophage lambda. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. cI binding results in repression of transcription.<br></li><br />
</ul><br />
<br />
<br />
<a href="http://partsregistry.org/Part:BBa_C0061">BBa_C0061 </a>: Autoinducer synthetase for AHL<br><br />
The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes. This expression is up-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound HSL. This complex binds to a palindromic site on the promoter, increasing the rate of transcription.<br><br />
<br />
<a href="http://partsregistry.org/Part:BBa_B1002">BBa_B1002</a>: Terminator<br><br />
<br />
<br><br><br />
<strong>Constructing the BioBrick</strong><br />
<br><br><br />
We successfully cloned all the parts in DH5α E. coli strains using this protocol (link al protocolo). <br />
We used the BioBrick Assembly Manual (link) protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3. Read the protocol we followed here.<br />
<br />
</p><br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/prototypesTeam:Valencia/prototypes2012-09-26T23:17:36Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Designed Biobricks<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
Our final objective is to have a <i>S. elongatus</i> capable to produce AHL during the night. For this we designed this new BioBrick controled by the <i>psbAI</i> promoter which is active in normal light conditions. To have an inverse response to light we have put the cI repressor after the <i>psbAI</i> promoter which binds to the lambda promoter inhibiting the luxI transcription, when <i>psbAI</i> is active. During the night, when the promoter is less active, the repressor will not be active, allowing the expression of luxI, and therefore having AHL.<br><br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2012/f/fd/VLC_Construct.png"></center><br />
<br />
Parts Information:<br><br><br />
<a href="http://partsregistry.org/Part:BBa_K754000">BBa_K754000 </a> : <i>S. elongatus</i> PCC7942 <i>psbAI</i> promoter. This is our submitted part for the registry. This promoter has a light dependent regulation being active in normal light conditions. Read more here.<br><br />
<a href="http://partsregistry.org/Part:BBa_Q04510">BBa_Q04510 </a>: Consists in the next parts:<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_B0034">B0034</a>: RBS<br></li><br />
</ul> <br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_C0051">C0051</a>: cI repressor from E. coli phage lambda <br />
Coding region for the cI repressor based on cI repressor from bacteriophage lambda modified with an LVA tail for rapid degradation of the protein. cI repressor binds to the cI regulator.<br></li><br />
</ul><br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_B0015">B0015</a>: double terminator<br><br />
This is the most commonly used terminator. It seems to be reliable<br></li><br />
</ul><br />
<br />
<ul style="list-style-type: square"> <br />
<li><a href="http://partsregistry.org/Part:BBa_R0051">R0051</a>: promoter (lambda cI regulated)<br><br />
The cI regulated promoter is based on the pR promoter from bacteriophage lambda. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. cI binding results in repression of transcription.<br></li><br />
</ul><br />
<br />
<br />
<a href="http://partsregistry.org/Part:BBa_C0061">BBa_C0061 </a>: Autoinducer synthetase for AHL<br><br />
The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes. This expression is up-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound HSL. This complex binds to a palindromic site on the promoter, increasing the rate of transcription.<br><br />
<br />
<a href="http://partsregistry.org/Part:BBa_B1002">BBa_B1002</a>: Terminator<br><br />
<br />
<br><br><br />
<strong>Constructing the BioBrick</strong><br />
<br><br><br />
We successfully cloned all the parts in DH5α E. coli strains using this protocol (link al protocolo). <br />
We used the BioBrick Assembly Manual (link) protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3. Read the protocol we followed here.<br />
<br />
</p><br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/prototypesTeam:Valencia/prototypes2012-09-26T23:10:11Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Designed Biobricks<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
Our final objective is to have a <i>S. elongatus</i> capable to produce AHL during the night. For this we designed this new BioBrick controled by the <i>psbAI</i> promoter which is active in normal light conditions. To have an inverse response to light we have put the cI repressor after the <i>psbAI</i> promoter which binds to the lambda promoter inhibiting the luxI transcription, when <i>psbAI</i> is active. During the night, when the promoter is less active, the repressor will not be active, allowing the expression of luxI, and therefore having AHL.<br><br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2012/f/fd/VLC_Construct.png"></center><br />
<br />
Parts Information:<br><br><br />
<a href="http://partsregistry.org/Part:BBa_K754000">BBa_K754000 </a> : <i>S. elongatus</i> PCC7942 <i>psbAI</i> promoter. This is our submitted part for the registry. This promoter has a light dependent regulation being active in normal light conditions. Read more here.<br><br />
<a href="http://partsregistry.org/Part:BBa_Q04510">BBa_Q04510 </a>: Consists in the next parts:<br><br />
<br />
<ul style="list-style-type: square"><br />
<li><a href="http://partsregistry.org/Part:BBa_B0015">B0015</a>: double terminator<br><br />
This is the most commonly used terminator. It seems to be reliable<br></li><br />
</ul><br />
<br />
<ul style="list-style-type: square"> <br />
<li><a href="http://partsregistry.org/Part:BBa_R0051">R0051</a>: promoter (lambda cI regulated)<br><br />
The cI regulated promoter is based on the pR promoter from bacteriophage lambda. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. cI binding results in repression of transcription.<br></li><br />
</ul><br />
<br />
<br />
<a href="http://partsregistry.org/Part:BBa_C0061">BBa_C0061 </a>: Autoinducer synthetase for AHL<br><br />
The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes. This expression is up-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound HSL. This complex binds to a palindromic site on the promoter, increasing the rate of transcription.<br><br />
<br />
<a href="http://partsregistry.org/Part:BBa_B1002">BBa_B1002</a>: Terminator<br><br />
- Artifical terminator with %T≈85<br><br />
- 6bp stem, 4nt loop<br><br />
<br><br><br />
<strong>Constructing the BioBrick</strong><br />
<br><br><br />
We successfully cloned all the parts in DH5α E. coli strains using this protocol (link al protocolo). <br />
We used the BioBrick Assembly Manual (link) protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3. Read the protocol we followed here.<br />
<br />
</p><br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/prototypesTeam:Valencia/prototypes2012-09-26T22:32:47Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Designed Biobricks<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
Our final objective is to have a <i>S. elongatus</i> capable to produce AHL during the night. For this we designed this new BioBrick controled by the <i>psbAI</i> promoter which is active in normal light conditions. To have an inverse response to light we have put the cI repressor after the <i>psbAI</i> promoter which binds to the lambda promoter inhibiting the luxI transcription, when <i>psbAI</i> is active. During the night, when the promoter is less active, the repressor will not be active, allowing the expression of luxI, and therefore having AHL.<br><br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2012/f/fd/VLC_Construct.png"></center><br />
<br />
Parts Information:<br><br><br />
BBa_K754000 (link): <i>S. elongatus</i> PCC7942 <i>psbAI</i> promoter. This is our submitted part for the registry. This promoter has a light dependent regulation being active in normal light conditions. Read more here.<br><br />
BBa_Q04510 (link): Consists in the next parts:<br><br />
- B0034: RBS <br><br />
- C0051: cI repressor from E. coli phage lambda <br><br />
Coding region for the cI repressor based on cI repressor from bacteriophage lambda modified with an LVA tail for rapid degradation of the protein. cI repressor binds to the cI regulator.<br><br />
- B0015: double terminator<br><br />
This is the most commonly used terminator. It seems to be reliable<br><br />
-R0051: promoter (lambda cI regulated)<br><br />
The cI regulated promoter is based on the pR promoter from bacteriophage lambda. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. cI binding results in repression of transcription.<br><br />
BBa_C0061 (link): Autoinducer synthetase for AHL<br><br />
The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes. This expression is up-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound HSL. This complex binds to a palindromic site on the promoter, increasing the rate of transcription.<br><br />
BBa_B1002 (link): Terminator<br><br />
- Artifical terminator with %T≈85<br><br />
- 6bp stem, 4nt loop<br><br />
<br />
<strong>Constructing the BioBrick</strong><br />
We successfully cloned all the parts in DH5α E. coli strains using this protocol (link al protocolo). <br />
We used the BioBrick Assembly Manual (link) protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3. Read the protocol we followed here.<br />
<br />
</p><br />
</html></div>Roocferhttp://2012.igem.org/Team:Valencia/prototypesTeam:Valencia/prototypes2012-09-26T22:27:57Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br />
<br>Designed Biobricks<br><br />
<br><br><br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
Our final objective is to have a <i>S. elongatus</i> capable to produce AHL during the night. For this we designed this new BioBrick controled by the <i>psbAI</i> promoter which is active in normal light conditions. To have an inverse response to light we have put the cI repressor after the <i>psbAI</i> promoter which binds to the lambda promoter inhibiting the luxI transcription, when <i>psbAI</i> is active. During the night, when the promoter is less active, the repressor will not be active, allowing the expression of luxI, and therefore having AHL.<br><br><br />
<br />
<center><img src="https://2012.igem.org/File:VLC_transformingcoccus.JPG"></center><br />
<br />
Parts Information:<br><br><br />
BBa_K754000 (link): <i>S. elongatus</i> PCC7942 <i>psbAI</i> promoter. This is our submitted part for the registry. This promoter has a light dependent regulation being active in normal light conditions. Read more here.<br><br />
BBa_Q04510 (link): Consists in the next parts:<br><br />
- B0034: RBS <br><br />
- C0051: cI repressor from E. coli phage lambda <br><br />
Coding region for the cI repressor based on cI repressor from bacteriophage lambda modified with an LVA tail for rapid degradation of the protein. cI repressor binds to the cI regulator.<br><br />
- B0015: double terminator<br><br />
This is the most commonly used terminator. It seems to be reliable<br><br />
-R0051: promoter (lambda cI regulated)<br><br />
The cI regulated promoter is based on the pR promoter from bacteriophage lambda. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. cI binding results in repression of transcription.<br><br />
BBa_C0061 (link): Autoinducer synthetase for AHL<br><br />
The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes. This expression is up-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound HSL. This complex binds to a palindromic site on the promoter, increasing the rate of transcription.<br><br />
BBa_B1002 (link): Terminator<br><br />
- Artifical terminator with %T≈85<br><br />
- 6bp stem, 4nt loop<br><br />
<br />
<strong>Constructing the BioBrick</strong><br />
We successfully cloned all the parts in DH5α E. coli strains using this protocol (link al protocolo). <br />
We used the BioBrick Assembly Manual (link) protocol to ligate our parts, but only were able to ligate the psbAI promoter to psb1C3. Read the protocol we followed here.<br />
<br />
</p><br />
</html></div>Roocferhttp://2012.igem.org/File:VLC_transformingcoccus.JPGFile:VLC transformingcoccus.JPG2012-09-26T22:13:46Z<p>Roocfer: </p>
<hr />
<div></div>Roocferhttp://2012.igem.org/Team:Valencia/EngineeringTeam:Valencia/Engineering2012-09-26T22:04:48Z<p>Roocfer: </p>
<hr />
<div>{{:Team:Valencia/menu}}<br />
{{:Team:Valencia/fondoweb}}<br />
<html><br />
<div id="Titulos"><br />
<br><br><br />
Bioreactor<br />
</div><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
<br />
<b>CO2 Pump</b><br />
<br><br><br />
<br />
To enhance a faster growth rate in our Synechococcus broth culture and taking advantage of the bacterium being photosynthetic, we decided to make a device with a CO2 pump in order to make the transformations quickly. <br />
<br><br><br />
<br />
<u>Materials:</u><br />
<br><br><br />
<br />
<ul style="list-style-type: square"><br />
<li>A bottle of water (1.5 litres) + a cap so the bottle remains hermetically sealed.</li><br />
<li>PVC tubes.</li><br />
<li>Rapid fixation glue.</li><br />
<li>30g of sugar.</li><br />
<li>One spoon of yeast (Saccharomyces cerevisae).</li><br />
<li>Water.</li><br />
<li>Methylene blue.</li><br />
</ul><br />
<br />
<br><br><br />
(FOTO)<br />
<br><br><br />
<br />
<u>Construction:</u><br />
<br><br><br />
<br />
First of all, we bored holes to the bottle and sealed the edges. Then we cut the tubes so they fit in the holes properly. Fill in one third of the botle approximately with water at 100ºC. Then add up the sugar. After that, shake the bottle so the sugar dissolves in the water. Add the spoon of yeast and shake again.<br />
<br><br><br />
Fresh yeast is a living organism, a micro fungus able to perform fermentation: in absence of oxygen converts the sugar into alcohol and liberates CO<sub>2</sub>.<br />
<br><br><br />
At last, pour 1 litre of Methylene blue into another bottle, used as a contamination control device, in order to purify the air that got into our culture. And finally we connect the air pumps to keep shaking the coccus.<br />
<br />
<br><br><br />
(FOTO)<br />
<br><br><br />
<br />
The results of our experiment were not the expected, having proved that our cyanobacteria grew up faster if we provided an extra input of CO2,bu the culture didn’t develop well due to contamination problems. We do not know if the yeast caused the problem or the bottle just wasn’t properly sealed.<br />
</div><br />
<br />
<br><br><br><br><br />
<div id="Titulos"><br />
<br><br><br />
Advanced continuous coculture system<br />
</div><br />
<br><br><br />
<br />
<div id="HomeCenterCenter"><br />
<p align="justify"><br />
Our furthest goal is to build a continuous culture system with spatial and temporal decoupling from the photosynthetic and the bioluminescent module, totally autonomous. <br />
<br><br><br />
<h3><u>Design:</u></h3><br />
<br><br />
Set 2 separate culture modules, a flat wide one for <i>S. elongatus cscB</i> as a solar module and a smaller compact one for <i>A. fischeri</i> as a biobulb. Prepare an open system so that there can be gas exchange from the cultures with the atmosphere (to let the system at as a CO<sub>2</sub> sink), but with a Pasteurian design opening to avoid contamination from deposition.<br />
<br><br><br />
Set tubing connecting both cultures, protecting each end with a syringe filter 0.45 microns of pore size. The fluid medium dynamics is borne by a pair of peristaltic reversible-flow pumps, which switch flow direction every 30-60 seconds to avoid collapsing any side of the membranes with jammed cells. The membranes allow the exchange of gases, ions, water, sucrose and AHL, but not cells, so that populations do not mix. This is fundamental to guarantee the light-efficiency of each compartment, without having cyanobacteria shading light emission from <i>A. fischeri</i>, and permitting a spatial decoupling of the photosynthetic module and the biolamp.<br />
<br><br><br />
Cell density would be regulated by output tubes from each culture which would recycle broth after passing it through a filter/skimmer. This process would be regulated by a turbidimeter (in theory) or by sampling-and-testing, at least in the first experimental prototype.<br />
<br><br><br />
Water loss due to evaporation would be solved by a water-level-wise lever opening a valve which would let in some distilled water from a small deposit, when the volume of the liquid falls below certain threshold.<br />
<br><br><br />
A microcontroller would be used as hardware to coordinate pump flow switch, input signals from turbidimeters and control of the cell density control system.<br />
<br><br><br />
All electric devices would be powered by a small solar panel, to preserve to the last detail the energetic autonomy of the system.<br />
<br><br><br />
Routine analyses of the system would be carried out on sucrose, oxygen and AHL levels in the liquid medium. Luminescence would be measured at night to acquire an experimental Light/Time curve.<br />
<br><br><br />
[Future design development: Achieve a knock-out/directed mutagenesis of the luxI autoinducer genes in <i>A. fischeri</i> to unable isolated autoinduction. This would render the system totally dependent on the AHL production of the transformed <i>S. elongatus</i>. In such situation, turning valves of different membrane pore size at the cell-stopping sections would result extremely useful, to manually switch on/off the diffusion of AHL from the <i>Synechococcus</i> culture to the biolamp. This enables an element of human control to turn on/off the lights over the normal day/night fashion].<br />
<br><br><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2012/5/58/Advanced_coculture_vlc_BBFJFJBFJBFJ.JPG"><br />
</center><br />
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<b>Figure a:</b> Advanced continuous cocultive device, showing in both culture compartments<br> with optical cell density monitors (green diodes), reversible-flow pumps at th sides,<br> bacteriological (Fb) and activated carbon (Fc) filtering and medium recycle system <br>at the middle. Evaporation loss automatic replenisher at the top (H<sub>2</sub>O).<br />
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<h3><u>Results:</u></h3><br><br />
Construction of inter-flask pumping system with 0.45micron pore membranes.<br><br />
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</html></div>Roocferhttp://2012.igem.org/Team:Valencia/genetic_engineeringTeam:Valencia/genetic engineering2012-09-26T18:29:52Z<p>Roocfer: </p>
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<br>Genetic Engineering<br><br />
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¿¿Nuestro objetivo y constructo final??<br><br><br />
Cyanobacteria are great organisms to be used in Synthetic Biology because of their ability to capture solar energy and CO2 and the fact that they can be easily genetically manipulated due to its small genome and their capacity to accept foreign DNA naturally. For our project we have chosen the cyanobacteria <i>Synechococcus elongatus</i> for genetic engineering because is the model organism for studying some prokaryotic processes (there is a lot of information of how to transform it) and in the last years it has become a model organism for some industrial processes, like biofuel production (Wang et al. 2012).<br><br><br />
S. elongatus has a circular genome of ≈2.7Mb (fully sequenced link a la base de datos) with a GC content of 55.5%, which contains the genes for 2.612 proteins and 53 RNAs (Atsumi et al. 2009). <br />
¿¿Nuestro objetivo y constructo final??<br><br><br><br><br />
<br />
<strong>Transforming Synechococcus elongatus PCC7942 for promoter characterization:</strong><br />
<br><br><br />
Our main objective has been to characterize the psbAI promoter (link a submitted parts) of Synechococcus elongatus PCC7942 in order to know more about its operation and understand how this promoter would control our final construct (link a prototipo) to have a diel switch of AHL, the signal molecule for our Aliivibrio fischeri population to glow. <br />
To characterize this promoter we had two options: making psbAIp: lacZ fusions and monitored the β-galactosidase activity or making psbAIp: luxABCDE fusions and monitored the bioluminescence produced as a result of the promoter activation. We refused to use a psbAIp::lacZ fusion because some assays report that the psbAI promoter response to light cannot be properly monitored by the β-galactosidase activity (Nair et al. 2001). For this reason we choosed using psbAIp:luxABCDE vector fusions. With this aim, we cloned several fusion plasmids (pAM977 and pAM2195) in E. coli and transformed them into S. elongatus, both wildtype and cscB strain. The fusion vectors were provided by Susan Golden´s lab. <br><br><br />
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<center><img src="https://static.igem.org/mediawiki/2012/f/fa/VLC_Tableplasmids.png"></center><br><br><br><br />
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<strong>Cloning into E. coli</strong><br />
We successfully cloned all the vectors in DH5α E. coli strains. For E. coli transformation we used this protocol (link). For the selective selection for pAM977 we used ampicillin (1μl/ml) and for pAM2195 Chloramphenicol (0.7μl/ml). The lack of spectrophotometer or other apparatus to measure optical density in our lab impede us to determine the concentration of our vectors.<br><br> <br />
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<strong>Transforming Coccus</strong><br />
Once cloned we transformed a wild type strain provided from the Physiology, Genetic and Microbiology departments of University of Alicante and the cscB strain provided from the Wyss Institute in Harvard. <br />
As the cscB strain from Harvard was resistant to Cm, we only could transform it with the pAM977 plasmid, which gives Sp resistance. Wildtype was transformed with both plasmids (pAM977 and pAM2195)<br />
We used the following protocol to transform Synechococcus. <br><br><br />
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Protocol<br><br />
¿Link o esto?<br><br />
Extracted from Clerico et al. 2007:<br><br />
- We grew 100ml of cscB and WT in BG-11 liquid medium (see how we grown them and our recipe here) shaking at 250rpm with constant light we used cold light fluorescent tubes (like this one). Cells had an optical density at 750nm (OD750) of 0.7, which is reach in 4 to 7 days approximately.<br><br />
- We collected 15mL of the culture and centrifuge it at 6000g for 10 min and discarded the supernatant.<br><br />
- We resuspended collected cells in 10 mL of 10mM NaCl and centrifuge them again at 6000g for 10 min.<br><br />
- After it, we resuspended the pellet in 0.3mL of BG-11M liquid medium.<br><br />
- We added 2μl of pAM2195 and pAM977 after doing a Miniprep of the clonation with a JetQuick kit (link al kit?)<br><br />
- Wrap tubes in aluminum foil to keep out light and incubate at 30oC for 15 to 20h with gentle agitation.<br><br />
- And then, plate the entire 0.3mL cell suspension on BG-11 medium agar (see here the recipe) with the selective antibiotics. We used this concentration: 2μg/ml spectinomycin and 7.5μg/mL of chloramphenicol. After 7 to 10 days of incubation under standard light conditions transformed colonies should appear.<br><br />
- After colonies appearance pick single transformants and grow them on fresh liquid BG-11M agar plate with selective antibiotic (this is done to ensure that all colonies have incorporated the trans gene as S. elongatus has multiple copies of its chromosome).<br><br />
- After 5 to 7 days of growth, cyanobacteria can be used to inoculate a BG-11 liquid culture with the selective antibiotic.<br><br><br />
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<strong>Results</strong><br />
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¿Fotos placas?<br />
We weren´t able to obtain transformants with the wild type strain of S. elongatus (razones?). <br />
But we obtained resistant colonies to Sp of the cscB strain transformed with the pAM977 vector (figure x). This was achieved the last week of lab work, so due to the slowly growth rate of S. elongatus, we haven´t have the time to grow the transformants in liquid medium and start with the bioluminescence measures to characterize the promoter. However, we will try to have some results for the Jamboree concerning this part.<br />
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Following steps?: Poner como habríamos medido la luminiscencia en caso de haber tenido crecimiento????<br />
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<strong>Transformation of E. coli for AHL production to test bioluminescent response in A. fischeri:</strong><br><br />
Another part of our project included transforming E. coli with the registry part BBa_K084014<br />
(link) in order to make E. coli produce AHL under IPTG regulation to test the bioluminescent response in A. fischeri. To achieve this goal we cloned the part in DH5α E. coli strains using this protocol (link al registro que te dice como usar las partes) (figure 1). The part (869 bp) is included inside the pSB1A2 (2079bp) BioBrick.<br> <br />
We designed an experimental procedure to test different experimental conditions for the production of AHL.<br><br />
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<br>When we wanted to test if A. fischeri was able to glow with the AHL produced by E. coli we discovered that the strain we had been growing in the lab WAS NOT VIBRIO FISCHERI, but VIBRIO MEDITERRANI, a non bioluminescent Vibrio strain (GREAT!).<br><br />
We couldn´t go further with this experiment.<br><br />
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