Difference between revisions of "Team:IISc-Bangalore/Hardware/Results"

 
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                 <li><a href="#calibration"> Calibration </a></li>
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                 <li><a href="#calibration"> Calibration <img src="https://static.igem.org/mediawiki/2017/6/68/T--IISc-Bangalore--navbar_bullet.png" /></a></li>
                 <li><a href="#growthcurves"> Growth Curves </a></li>
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                 <li><a href="#growthcurves"> Growth Curves <img src="https://static.igem.org/mediawiki/2017/6/68/T--IISc-Bangalore--navbar_bullet.png" /></a></li>
 
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<h1 id = "calibration">Calibration</h1>
 
<h1 id = "calibration">Calibration</h1>
  
<p>Calibration of Version1.1 was done using KMnO4 and milk solutions of different dilutions. V1.1 can hold a test tube containing the sample and measure its optical density (OD). The same electronic circuitry was used to measure the OD values of the sample placed in a cuvette. The OD values of the sample was taken using a spectrophotometer (actual) and the device-V1.1 (measured).
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<h2>Mini</h2>
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<p>Before we could try running growth curves with GCODe, we needed to check that its readings were linear. We calibrated the Mini (then called Version 1.1) against a spectrophotometer (Shimadzu UV-1800) at 648 nm by running serial dilutions of KMnO4 solutions and milk. We used a cuvette in the spectrophotometer, test tube in the Mini, and calbrated against cuvette reading with the Mini's electronic circuitry (the Cuvette Reading Graphs).
 
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</p>
  
<p>The Mini (then called Version 1.1) was calibrated against a spectrophotometer (Shimadzu UV-1800) at 648 nm by running serial dilutions of KMnO4 solutions and milk.  
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<p>Data for these graphs available <a href="https://static.igem.org/mediawiki/2017/7/78/T--IISc-Bangalore--HW-Results-Mini-GrowthCurveData.docx">here</a>.
 
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<img src="https://static.igem.org/mediawiki/2017/f/ff/T--IISc-Bangalore--ecoli2.jpg" align ="center">
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<center>
  
<p>There has to be a better way. That way is GCODe.</p>
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<img src="https://static.igem.org/mediawiki/2017/d/d6/T--IISc-Bangalore--HW-Results-Mini-KMnO4-2.jpg" align ="center" height = "300">
  
<p>GCODe (Growth Curve and Optical DEnsity) is an optical density measurement device that will not only take readings at pre-programmed intervals or continuously, but will also aerate and dilute the culture as required. It can even send you a message when the OD reaches a particular level, just in time to you to start the next stage of your experiment. Fundamentally, it automates the grunt work of growth curves, in a manner that allows you to walk away from the lab, secure in the knowledge that you will be alerted when your cells are ready for you.</p>
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<img src="https://static.igem.org/mediawiki/2017/a/ae/T--IISc-Bangalore--HW-Results-Mini-KMnO4-1.png" align ="center" height = "300">
  
<p>Our work stemmed from our own frustrations running growth curves night after night, and was also inspired by the OD meter built by the 2014 Aachen iGEM team. We worked incrementally, starting with a rudimentary box of wiring, designing version after version in response to feedback from the iGEM wetlab team, as they trialled our device, and other labs and professors from across IISc.</p>
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<img src="https://static.igem.org/mediawiki/2017/d/db/T--IISc-Bangalore--HW-Results-Mini-Milk-1.jpg" align ="center" height = "300">
  
<p>We currently have two versions of GCODe - The GCODE Mini and the GCODe Pro.
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<img src="https://static.igem.org/mediawiki/2017/f/f3/T--IISc-Bangalore--HW-Results-Mini-Milk-2.jpg" align ="center" height = "300">
  
The GCODe Mini has been developed, fabricated, tested and fully documented. The GCODe Pro is still under development and is in its third version.
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</center>
  
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<p>As you can see, the graphs are linear to within experimental error! </p>
  
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<h2>Pro</h2>
  
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<p>The Pro is still under development, so we do not have much data from it yet. However, we did manage to measure its dilution capabilities:</p>
  
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<center>
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<img src="https://static.igem.org/mediawiki/2017/e/ec/T--IISc-Bangalore--HW-Results-Pro-Dilution-linearity.png" align ="center" height = "400">
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<p>As you can see, the Pro dilutes quite linearly, (once we correct for the offset) with an r-squared value of 0.996 !</p>
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<p>(Data for this curve available at <a href = "https://static.igem.org/mediawiki/2017/c/ce/T--IISc-Bangalore--HW-Results-Pro-Dilution-linearity-Data.xlsx">HW-Results-Pro-Dilution-Linearity-Data.xlsx</a>)</p>
 
<h1 id="growthcurves">Growth Curves</h1>
 
<h1 id="growthcurves">Growth Curves</h1>
  
<h2>Intro</h2>
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<p>With the knowledge that the GCODe Mini's readings are linear, we are now ready to run growth curves! There is a slight hitch, however. Optical Density is calculated using Beer-Lambert's Law<sup>[1]</sup>, </p>
  
<p>The GCODe Mini is a sleek black acrylic cuboid that houses a test tube that will hold your culture. Connect the Mini to your laptop via USB, tell it what to do with our associated software, place it in the shaker-incubator, press Start … aaand you’re done !</p>
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<center>
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<p style="font-size: 2.0em">
  
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\[
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      \text{Absorbance} = \epsilon c l ,\\
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      \\
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      \text{ where}\\
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      \epsilon \text{ is Absorbance coefficient}\\
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      c \text{ is Concentration }\\
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      l \text{ is Path Length.}
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\]
  
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</p>
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</center>
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<p>But we realised that l and &epsilon; are different for our optical system as compared to the spectrophotometer. However since the concentration will still be the same in both cases, the OD will be proportional. We can then compare the graphs by normalizing, ie dividing by any one reading.</p>
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<p>Growth curves of <i>Salmonella typhimurium</i> (STMwt14028) and <i>Escherichia coli</i> were made. The optical densities were simultaneously measured using a spectrophotometer at wavelength 600nm [In the graph as OD600] and our device GCODe Mini. [In the graph as V1OD_N_12H and V1OD_N_24H]. In V1OD_N_12H, the proportionality factor was found by dividing with the 12H reading, and in V2OD_N_24H with the 24H reading.</p>
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<center>
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<img src="https://static.igem.org/mediawiki/2017/d/d4/T--IISc-Bangalore--hardware_gc1.jpg" align ="center" >
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<img src="https://static.igem.org/mediawiki/2017/3/3c/T--IISc-Bangalore--hardware_gc2.jpg" align ="center">
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</center>
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<p>As you can see, the normalized GCODe Mini readings (red and cyan) match the spectrophotometer readings (green) almost perfectly!</p>
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<center>
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<img src="https://static.igem.org/mediawiki/2017/f/f2/T--IISc-Bangalore--ecoli1.jpg" align ="center" >
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<img src="https://static.igem.org/mediawiki/2017/f/ff/T--IISc-Bangalore--ecoli2.jpg" align ="center" >
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<p>Notice that the <i>E.coli</i> readings from the Mini form an even smoother curve than those from the spectrophotometer! This is probably because the GCODe readings were taken instantaneously, while it takes time to load the cuvette into the spectrophotometer and take a reading, allowing lots more opportunities for human error, leading to fluctuations in the actual time intervals.</p>
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<p>At this point we were ready to try a growth curve with the GCODe all by its lonesome.</p>
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<center>
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<img src="https://static.igem.org/mediawiki/2017/9/98/T--IISc-Bangalore--HW-Results-Mini-ecoli-solo.png" align ="center" >
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</center>
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<p>It worked perfectly! We only used a spectrophotometer at the very end, so that we could calibrate the curve to 'normal' OD units. (Data for this curve available at <a href = "https://static.igem.org/mediawiki/2017/2/22/T--IISc-Bangalore--HW-Results-Mini-EcoliGrowthCurve-solo1.xlsx">Ecoli-GrowthCurve-Solo1.xlsx</a>)</p>
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<p>To find out more about how our project evolved through the problems we faced, user testing and user feedback, head over to <a href="https://2017.igem.org/Team:IISc-Bangalore/Hardware/Notebook">Hardware Evolution</a>.</p>
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<h2>References</h2>
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[1] IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997). XML on-line corrected version: http://goldbook.iupac.org (2006-) created by M. Nic, J. Jirat, B. Kosata; updates compiled by A. Jenkins. ISBN 0-9678550-9-8. https://doi.org/10.1351/goldbook.
  
 
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Latest revision as of 02:33, 2 November 2017

  1. Calibration
  2. Growth Curves

Calibration

Mini

Before we could try running growth curves with GCODe, we needed to check that its readings were linear. We calibrated the Mini (then called Version 1.1) against a spectrophotometer (Shimadzu UV-1800) at 648 nm by running serial dilutions of KMnO4 solutions and milk. We used a cuvette in the spectrophotometer, test tube in the Mini, and calbrated against cuvette reading with the Mini's electronic circuitry (the Cuvette Reading Graphs).

Data for these graphs available here.

As you can see, the graphs are linear to within experimental error!

Pro

The Pro is still under development, so we do not have much data from it yet. However, we did manage to measure its dilution capabilities:

As you can see, the Pro dilutes quite linearly, (once we correct for the offset) with an r-squared value of 0.996 !

(Data for this curve available at HW-Results-Pro-Dilution-Linearity-Data.xlsx)

Growth Curves

With the knowledge that the GCODe Mini's readings are linear, we are now ready to run growth curves! There is a slight hitch, however. Optical Density is calculated using Beer-Lambert's Law[1],

\[ \text{Absorbance} = \epsilon c l ,\\ \\ \text{ where}\\ \epsilon \text{ is Absorbance coefficient}\\ c \text{ is Concentration }\\ l \text{ is Path Length.} \]

But we realised that l and ε are different for our optical system as compared to the spectrophotometer. However since the concentration will still be the same in both cases, the OD will be proportional. We can then compare the graphs by normalizing, ie dividing by any one reading.

Growth curves of Salmonella typhimurium (STMwt14028) and Escherichia coli were made. The optical densities were simultaneously measured using a spectrophotometer at wavelength 600nm [In the graph as OD600] and our device GCODe Mini. [In the graph as V1OD_N_12H and V1OD_N_24H]. In V1OD_N_12H, the proportionality factor was found by dividing with the 12H reading, and in V2OD_N_24H with the 24H reading.

As you can see, the normalized GCODe Mini readings (red and cyan) match the spectrophotometer readings (green) almost perfectly!

Notice that the E.coli readings from the Mini form an even smoother curve than those from the spectrophotometer! This is probably because the GCODe readings were taken instantaneously, while it takes time to load the cuvette into the spectrophotometer and take a reading, allowing lots more opportunities for human error, leading to fluctuations in the actual time intervals.

At this point we were ready to try a growth curve with the GCODe all by its lonesome.

It worked perfectly! We only used a spectrophotometer at the very end, so that we could calibrate the curve to 'normal' OD units. (Data for this curve available at Ecoli-GrowthCurve-Solo1.xlsx)

To find out more about how our project evolved through the problems we faced, user testing and user feedback, head over to Hardware Evolution.

References

[1] IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997). XML on-line corrected version: http://goldbook.iupac.org (2006-) created by M. Nic, J. Jirat, B. Kosata; updates compiled by A. Jenkins. ISBN 0-9678550-9-8. https://doi.org/10.1351/goldbook.