Difference between revisions of "Team:CCA San Diego/Measurement"

 
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<h2 style="font-size:79px">Measurement</h2>
 
<h2 style="font-size:79px">Measurement</h2>
<h4>See <a href="https://2017.igem.org/Team:CCA_San_Diego/InterLab">Interlab page</a> for more details.</h4><br><br>
 
  
<center><h6>First we standardized our measurements in both LUDOX-HS40 and water by taking a set of measurements in a spectrophotometer and calculating a correction factor to convert our data into the standardized units that iGEM requested (OD<subscript>600</subscript>).  
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<h6>CCA_San_Diego created a method for measuring the effective nature of PAH degradation by substituting the media’s standard carbon source for PAH compounds, therefore rendering the bacteria without an energy supply unless the pathway of PAH degradation succeeds and efficiently leads to the creation of two substrates: salicylate and phthalate (which supply carbon for the Krebs Cycle in E. coli). By doing this, growth curves can be effectively used to monitor the degradation efficiency and progression over time as bacterial colonies grow.</h6>
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<center><img src="https://static.igem.org/mediawiki/2017/a/a1/InterlabFigure1.png" width="100%" height="auto"></center>
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<img src="https://static.igem.org/mediawiki/2017/c/c2/Measurements_graph.png" width="100%" height="auto">
<br><h6>Figure 1: Absorbance measurement of LUDOX 100% and H2O and correction factor table.</h6>
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<br><h6>Figure 1. Time course biotransformation experiments using crude oil samples from Pennsylvania (PA), Saudi Arabia, and Ecuador, measuring absorbance at 600nm of E.coli BL21 recombinant cultures containing the fluorene and phenanthrene catabolic pathways or control vectors. MM=M9 minimal medium.</h6>
 
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<br><h6>Next we used a serial dilution of fluorescein to generate a standard curve of the concentration, which we used to correct our cell-based GFP readings later in the experiment to ensure the accuracy of our measurements.
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<h6>Results show growth in oil or isolated PAH is equivalent to easy to access carbon source (Glucose).</h6>
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<h6>Carbon is the building block of organic molecules and makes up a large portion of the structures in life forms. Thus to grow and reproduce, a method of breaking down and accessing carbon is necessary. In this context, using a singular carbon source in a growth setup for bacteria allows us to isolate if the bacteria degrade and process complex hydrocarbons to form molecules capable of facilitating life and growth. This measurement technique is very useful in testing bioremediation projects that involve degradation</h6>
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<h6>In our tests, Fluorene or Phenanthrene was used a singular carbon source. Since bacteria need some source of carbon to grow, they must be using the only carbon source present when growing. The growth curves indicate that the bacteria with the pathways inserted are growing at rates comparable with a common carbon source, Glucose. This means the bacteria are degrading the PAHs and consuming the substrate products.</h6>
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<br><h6>Figure 2: FITC standard curve plotted from the data of 12 dilutions of concentration of 4 replicates</h6>
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<h6>Our methods are innovative, as they utilize normally harmful PAHs as sources of carbon to allow bacteria to undergo cellular respiration. In an ironic twist of fate, we used the growth of one organism to measure another compound’s degradation. Our methods are logical and apply basic processes of science to better the world around us. </h6>
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<h6>For consistency of results, we used E.Coli k-12 DH5 Alpha for all of our transformations. We also conducted 4 trials to generate 4 replicates on the final sampling plate. <br><br>
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To measure the cell-based assays, we started by transforming 8 plates of E. coli with the 8 samples provided in Kit 7. After allowing the colonies to form on the plates, each plate had 2 colonies selected to be grown in 5-10 mL LB medium + Chloramphenicol overnight (16-18 hours) at 37°C and 220 rpm.
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<br><h6>Figure 3: In order from left to right and top to bottom: Negative Control, Device 1, Device 2, Device 3, Device 4, Device 5, Device 6 and Puc19 serving as a plasmid indicator.</h6>
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<h6>The plate readers were configured to measure OD<sub>600</sub> and the overnight cultures were measured once to help calibrate the concentrations to the desired OD<sub>600</sub> of 0.02. The samples were once again incubated at 37°and 220 rpm. At the 0, 2, 4 and 6 hour time points during the incubation, 500 µL of each sample from both colonies were taken and measured for fluorescence.</h6>
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<br><h6>Figure 4: The overnight cultures show indications of varying fluorescence even before the formal testing period.</h6>
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<h6>For each time point, we had a 96 well plate that was organized according to the diagram provided by iGEM with each of our 8 devices over 4 replicates and an LB blank. Finally, the data we gathered was corrected and converted into the standardized units that the interlab uses to compare data across all the participating labs.
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Latest revision as of 03:42, 2 November 2017

Measurement

CCA_San_Diego created a method for measuring the effective nature of PAH degradation by substituting the media’s standard carbon source for PAH compounds, therefore rendering the bacteria without an energy supply unless the pathway of PAH degradation succeeds and efficiently leads to the creation of two substrates: salicylate and phthalate (which supply carbon for the Krebs Cycle in E. coli). By doing this, growth curves can be effectively used to monitor the degradation efficiency and progression over time as bacterial colonies grow.


Figure 1. Time course biotransformation experiments using crude oil samples from Pennsylvania (PA), Saudi Arabia, and Ecuador, measuring absorbance at 600nm of E.coli BL21 recombinant cultures containing the fluorene and phenanthrene catabolic pathways or control vectors. MM=M9 minimal medium.

Results show growth in oil or isolated PAH is equivalent to easy to access carbon source (Glucose).

Carbon is the building block of organic molecules and makes up a large portion of the structures in life forms. Thus to grow and reproduce, a method of breaking down and accessing carbon is necessary. In this context, using a singular carbon source in a growth setup for bacteria allows us to isolate if the bacteria degrade and process complex hydrocarbons to form molecules capable of facilitating life and growth. This measurement technique is very useful in testing bioremediation projects that involve degradation

In our tests, Fluorene or Phenanthrene was used a singular carbon source. Since bacteria need some source of carbon to grow, they must be using the only carbon source present when growing. The growth curves indicate that the bacteria with the pathways inserted are growing at rates comparable with a common carbon source, Glucose. This means the bacteria are degrading the PAHs and consuming the substrate products.

Our methods are innovative, as they utilize normally harmful PAHs as sources of carbon to allow bacteria to undergo cellular respiration. In an ironic twist of fate, we used the growth of one organism to measure another compound’s degradation. Our methods are logical and apply basic processes of science to better the world around us.

email igemcca@gmail.com
Canyon Crest Academy iGEM 2017 CC;