Difference between revisions of "Team:US AFRL CarrollHS/InterLab"

 
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<div class="row"><p>This year, we complete the Interlab along with the Flow Cytometry portion to qualify for the bronze medal. Below you can find our data including the results of our competent cell test kit, background fluorescence information from out samples, and raw data. This year we were also able to utilize our Flow Cytometer as well to submit our flow cytometry data to iGEM. We hope that by submitting our data to iGEM, we can help standardize measuring protocols for reproducibility within experiments.</p>
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<p>In this year’s InterLab, our team transformed E. coli DH5α with different plasmids expressing Green Fluorescent Protein (GFP). A plate reader was then used to measure fluorescence and absorbance values from the transformed strains. </p></div>
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<div class="row"><p>This year, we completed the Interlab along with the Flow Cytometry portion to qualify for the bronze medal. Below you can find our data including the results of our competent cell test kit, background fluorescence information from out samples, and raw data. This year we were also able to utilize our Flow Cytometer as well to submit our flow cytometry data to iGEM. We hope that by submitting our data to iGEM, we can help standardize measuring protocols for reproducibility within experiments.</p>
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<p>In this year’s InterLab, our team transformed <i>E. coli</i> DH5α with different plasmids expressing Green Fluorescent Protein (GFP). A plate reader was then used to measure fluorescence and absorbance values from the transformed strains. </p></div>
 
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<div class="row"><h1>Competent Cells Test</h1></div>
 
<div class="row"><h1>Competent Cells Test</h1></div>
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<div class="row"><p>We performed the competent cells test and submitted the data to iGEM.</p></div>
 
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  <div class="col-sm-6 text-center"><img src="https://static.igem.org/mediawiki/2018/4/48/T--US_AFRL_CarrollHS--CompCells1.jpg" style="width:90%; position:relative; top:120px;"></div>
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<div class="col-sm-12 text-center"><img src="https://static.igem.org/mediawiki/2018/4/48/T--US_AFRL_CarrollHS--CompCells1.jpg" style="width:45%;"></div>
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  <div class="col-sm-6 text-center"><img src="https://static.igem.org/mediawiki/2018/4/41/T--US_AFRL_CarrollHS--CompCells2.jpg" width="80%"></div>
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<div class="col-sm-12 label"><p class="text-center">Figure 1. Plates from the competent cell test</p></div>
 
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<div class="row"><h2>Calibration 1: OD600 Reference point with LUDOX Protocol</h2></div>
 
<div class="row"><h2>Calibration 1: OD600 Reference point with LUDOX Protocol</h2></div>
 
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   <div class="col-sm-12 text-center"><img src="https://static.igem.org/mediawiki/2018/f/f9/T--US_AFRL_CarrollHS--LudoxMeasurements.png" style="width:50%;"></div>
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<div class="col-sm-12"><p class="text-center">Table 1. OD600/Abs600 measurements, showing a correction factor of 2.895.</p></div>
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<div class="col-sm-12 label"><p class="text-center">Table 1. OD600/Abs600 measurements, showing a correction factor of 2.895</p></div>
 
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<div class="row"><h2>Calibration 2: Particle Standard Curve with Microsphere Protocol</h2></div>
 
<div class="row"><h2>Calibration 2: Particle Standard Curve with Microsphere Protocol</h2></div>
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<div class="row"><p>After following the Microsphere Protocol, we were able to produce a Particle Standard Curves in order to observe the relationship between Abs600 and amount of particles present. </p>
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<div class="row"><p>After following the Microsphere Protocol, we were able to produce a Particle Standard Curves in order to observe the relationship between Abs600 and amount of particles present. </p></div>
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  <div class="col-md-6 text-center"><img src="https://static.igem.org/mediawiki/2018/9/94/T--US_AFRL_CarrollHS--ParticleStandardCurve.png" width="100%"></div>
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  <div class="col-md-6 text-center"><img src="https://static.igem.org/mediawiki/2018/0/09/T--US_AFRL_CarrollHS--ParticleStandardCurveLog.png" width="100%"></div>
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<div class="col-sm-6 label"><p class="text-center">Figure 2. Particle Standard Curve</p></div>
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<div class="col-sm-6 label"><p class="text-center">Figure 3. Particle Standard Curve (log scale)</p></div>
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<p>From our data, we observed a linear relationship between Abs600 and particle count. </p>
 
<p>From our data, we observed a linear relationship between Abs600 and particle count. </p>
 
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  <div class="col-md-12 text-center"><img src="https://static.igem.org/mediawiki/2018/2/23/T--US_AFRL_CarrollHS--ParticleTable.png" width="90%"></div></div>
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<div class="col-sm-12 label"><p class="text-center">Table 2. Tabularized Data of Our Results</p></div>
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<div class="row"><h2>Calibration 3: Fluorescence standard curve with Fluorescence Protocol</h2></div>
 
<div class="row"><h2>Calibration 3: Fluorescence standard curve with Fluorescence Protocol</h2></div>
 
<div class="row"><p>Next we were able to utilize the Microsphere Protocol in order to obtain a standard curve for our fluorescence values. </p></div>
 
<div class="row"><p>Next we were able to utilize the Microsphere Protocol in order to obtain a standard curve for our fluorescence values. </p></div>
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  <div class="col-md-6 text-center"><img src="https://static.igem.org/mediawiki/2018/5/52/T--US_AFRL_CarrollHS--FluoresceinStandardCurve.png" width="100%"></div>
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<div class="col-sm-6 label"><p class="text-center">Figure 4. Fluorescence Standard Curve</p></div>
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<div class="col-sm-6 label"><p class="text-center">Figure 5. FluorescenceStandard Curve (log scale)</p></div>
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  <div class="col-md-12 text-center"><img src="https://static.igem.org/mediawiki/2018/9/91/T--US_AFRL_CarrollHS--FluorescenceTable.png" width="90%"></div></div>
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<div class="col-sm-12 label"><p class="text-center">Table 3. Tabularized data of Fluorescence Results</p></div>
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<div class="row"><h1>Cell Measurement Protocol</h1></div>
 
<div class="row"><h1>Cell Measurement Protocol</h1></div>
 
<div class="row"><h2>Day One</h2></div>
 
<div class="row"><h2>Day One</h2></div>
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<div class="row"><p>All cells were transformed into E. coli DH5a based on well locations in kit provided by iGEM.</p></div>
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<div class="row"><p>All cells were transformed into <i>E. coli</i> DH5a based on well locations in kit provided by iGEM.</p></div>
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<div class="row"><h2>Day Two</h2></div>
 
<div class="row"><h2>Day Two</h2></div>
 
<div class="row"><p>Colonies from the transformation plates were then picked and inoculated in 10.0 mL LB broth + Chloramphenicol resistance. The cells were then incubated overnight at 37°C and shaking at 220 rpm.</p></div>
 
<div class="row"><p>Colonies from the transformation plates were then picked and inoculated in 10.0 mL LB broth + Chloramphenicol resistance. The cells were then incubated overnight at 37°C and shaking at 220 rpm.</p></div>
 
<div class="row"><h2>Day Three</h2></div>
 
<div class="row"><h2>Day Three</h2></div>
 
<div class="row"><p>Followed all protocols provided by iGEM, with extra care during serial dilution steps. </p></div>
 
<div class="row"><p>Followed all protocols provided by iGEM, with extra care during serial dilution steps. </p></div>
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<div class="col-sm-12 label"><p class="text-center">Table 4. Raw Plate Reader Measurements</p></div>
 
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  <div class="col-md-12 text-center"><img src="https://static.igem.org/mediawiki/2018/a/a6/T--US_AFRL_CarrollHS--FluorescencePerOD.png" width="90%"></div></div>
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<div class="col-sm-12 label"><p class="text-center">Table 5. Fluorescence Per OD</p></div>
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<div class="col-sm-12 label"><p class="text-center">Table 6. Fluorescence Per Particle</p></div>
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Latest revision as of 03:31, 18 October 2018

Interlab

This year, we completed the Interlab along with the Flow Cytometry portion to qualify for the bronze medal. Below you can find our data including the results of our competent cell test kit, background fluorescence information from out samples, and raw data. This year we were also able to utilize our Flow Cytometer as well to submit our flow cytometry data to iGEM. We hope that by submitting our data to iGEM, we can help standardize measuring protocols for reproducibility within experiments.

In this year’s InterLab, our team transformed E. coli DH5α with different plasmids expressing Green Fluorescent Protein (GFP). A plate reader was then used to measure fluorescence and absorbance values from the transformed strains.

Competent Cells Test

We performed the competent cells test and submitted the data to iGEM.

Figure 1. Plates from the competent cell test

Results

Calibration 1: OD600 Reference point with LUDOX Protocol

Table 1. OD600/Abs600 measurements, showing a correction factor of 2.895

Calibration 2: Particle Standard Curve with Microsphere Protocol

After following the Microsphere Protocol, we were able to produce a Particle Standard Curves in order to observe the relationship between Abs600 and amount of particles present.

Figure 2. Particle Standard Curve

Figure 3. Particle Standard Curve (log scale)

From our data, we observed a linear relationship between Abs600 and particle count.

Table 2. Tabularized Data of Our Results

Calibration 3: Fluorescence standard curve with Fluorescence Protocol

Next we were able to utilize the Microsphere Protocol in order to obtain a standard curve for our fluorescence values.

Figure 4. Fluorescence Standard Curve

Figure 5. FluorescenceStandard Curve (log scale)

Table 3. Tabularized data of Fluorescence Results

Cell Measurement Protocol

Day One

All cells were transformed into E. coli DH5a based on well locations in kit provided by iGEM.

Day Two

Colonies from the transformation plates were then picked and inoculated in 10.0 mL LB broth + Chloramphenicol resistance. The cells were then incubated overnight at 37°C and shaking at 220 rpm.

Day Three

Followed all protocols provided by iGEM, with extra care during serial dilution steps.

Table 4. Raw Plate Reader Measurements

Table 5. Fluorescence Per OD

Table 6. Fluorescence Per Particle

Contact us @iGEMLabPats or iGEM@carrollhs.org