Team:UC Chile/Protocols

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[[File: Chile_pipeting.jpg| 300px| right]]
[[File: Chile_pipeting.jpg| 300px| right]]
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<p>Here you will find various protocols we have used during the development of our project. We have separated them into 4 main sections: <br /><br />  [[#General Protocols]] <br /> [[#Cyanobacteria Protocols]] <br /> [[#Bactomithril Protocols]] <br /> [[#Characterization Protocols]] </p>
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<p>Here you will find various protocols we have used during the development of our project. We have separated them into 3 main sections: <br /><br />  [[#General Protocols]] <br /> [[#Cyanobacteria Protocols]] <br /> [[#Characterization Protocols]] </p>
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<div id="General Protocols">
<div id="General Protocols">
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Concentrate the DNA to be transformed into Synechocystis to 1µg/µL in 10µL for each transformation (A total of 10 ug of the transformation plasmid).  
Concentrate the DNA to be transformed into Synechocystis to 1µg/µL in 10µL for each transformation (A total of 10 ug of the transformation plasmid).  
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Inoculate Synechocystis PCC 6803 into a sterile 250mL Erlenmeyer flask with 150mL* of liquid BG-11 and grow culture until you reach an OD730nm of 0.8 – 1.0 (That should take between 6 to 10 days dependending on the amount of initial inoculum).  
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Inoculate Synechocystis PCC 6803 into a sterile 250mL Erlenmeyer flask with 150mL* of liquid BG-11 and grow culture until you reach an OD730nm of 0.4 – 0.6, never above 0.8 (That should take between 6 to 10 days dependending on the amount of initial inoculum).  
* For each 50 mL of Synechocystis PCC 6803 you will have 5 individual transformations.  
* For each 50 mL of Synechocystis PCC 6803 you will have 5 individual transformations.  
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<div id="Reinoculation">
<div id="Reinoculation">
<h2>Reinoculation</h2>
<h2>Reinoculation</h2>
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Protocol right here!
 
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</div>
 
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<div id="Cyanobacterial DNA extraction">
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<br />
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<h2>Cyanobacterial DNA extraction</h2>
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This is a standard protocol for propagating cultures. The amount of initial innoculum will depend on when you will need a specific OD. Refer to [https://2012.igem.org/Team:UC_Chile/Results/Growthcurve growth curves] to calculate your needed inoculum through a standard C*V = constant relation.
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</div>
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<p>Protocol goes here!</p>
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<div id="Bactomithril Protocol">
 
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<h1>Bactomithril Protocols</h1>
 
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</div>
 
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<p>You can find a complete protocol to produce spider-silk fibers in the following paper:
 
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Teulé, F., Cooper, A. R., Furin, W. a, Bittencourt, D., Rech, E. L., Brooks, A., & Lewis, R. V. (2009). A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning. Nature protocols, 4(3), 341-55. doi:10.1038/nprot.2008.250
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All work must be done under sterile conditions.
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<br><br>Other easier protocols are in the following adresses:<br>
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http://www.pnas.org/content/107/32/14059.long<br>
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<li>Autoclave a flask with enough volume to harbor at least twice the volume you require</li>
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http://onlinelibrary.wiley.com/doi/10.1002/jbm.a.34353/full</p>
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<li>Measure and add BG-11 media to flask</li>
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<li>Add axenic Synechocystis to flask. Inoculum must be in range 5-20% v/v of final volume</li>
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</ul>
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</div>
<div id="Characterization Protocols">
<div id="Characterization Protocols">
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<div id="SyneGrowth">
<div id="SyneGrowth">
<h2>Methods for the characterization of Synechocystis PCC 6803 growth curve</h2>
<h2>Methods for the characterization of Synechocystis PCC 6803 growth curve</h2>
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This are the methods we used for setting up the growth curve experiment of our Synechocystis PCC 6803 which is further described [[Team:UC_Chile2/Characterization | here ]].
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These are the methods we used for setting up the growth curve experiment of our Synechocystis PCC 6803 which is further described [[Team:UC_Chile2/Characterization | here ]].
<h3>Materials</h3>
<h3>Materials</h3>
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<div id="Luciferase Assay">
<div id="Luciferase Assay">
<h2>Promoters Luciferase Assay</h2>
<h2>Promoters Luciferase Assay</h2>
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We measured:
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(1) Luminiscence of LuxAB genes (from ''Xenorhabdus luminescens'' and ''Vibrio fischeri'') placed under [http://partsregistry.org/Part:BBa_K743003 Pta promoter] in Synechocystis upon induction with decanal and dodecanal. Both testing results at [http://partsregistry.org/Part:BBa_K743014:Experience] and [http://partsregistry.org/Part:BBa_K743015:Experience]
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(2) Luminiscence of [http://partsregistry.org/Part:BBa_K325905 Bacterial Lux reporter
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CDEG pλ AB] upon induction with decanal and dodecanal. Testing results [http://partsregistry.org/Part:BBa_K325905:Experience here]
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Controls used:
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Positive control: E. coli with arabinose-induced [http://partsregistry.org/Part:BBa_K325909 Luxbrick]
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</div>
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Negative control 1: E. coli wt cells
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</div>
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Negative control 2: Synechocystis PCC6803 wt cells
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Method
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E. coli and Synechocystis PCC 6803 transformed with the target constructs and negative controls were grown in liquid media at 37 °C (E. coli) and 30 °C (Synechocystis PCC 6803).
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Two hours after the beginning of the experiment, OD at 600 nm was measured for K325905 and Top10 in order to reinoculate at 0.24 OD (600 nm) in a total volume of 1.5 mL.
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Aftewards, LuxBrick was induced with Arabinose to a concentration of 3 mM and left in shaker at 30°C along with K325905 and Top10.
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After two hours, 100 µl of transformed bacteria and negative controls were inoculated in a 96 plate well. Every sample was inoculated in octuplicate. Luminiscence of the wells was measured by illuminometer.
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Several plate measurements were done to the plate with decanal and dodecanal at different concentrations (including 0 concentration).
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Note: This experience was done twice. Measurements were done at 14 and 16 hour of circadian rhythm in order to evaluate production of LuxAB under transaldolase promoter.
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Results at hour 14
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<h1>References</h1>
<h1>References</h1>
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[2]Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO. (2009). "Enzymatic assembly of DNA molecules up to several hundred kilobases". Nature Methods 6 (5): 343–345. doi:10.1038/nmeth.1318
[2]Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO. (2009). "Enzymatic assembly of DNA molecules up to several hundred kilobases". Nature Methods 6 (5): 343–345. doi:10.1038/nmeth.1318
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Latest revision as of 08:36, 26 October 2012

Project: Luxilla - Pontificia Universidad Católica de Chile, iGEM 2012