Difference between revisions of "Measurement/Measurement kit"

 
(15 intermediate revisions by 3 users not shown)
Line 3: Line 3:
  
 
<div class="column full_size">
 
<div class="column full_size">
−
<h1>Measurement Resources</h1>
+
<h1>iGEM Measurement Kit</h1>
  
 
<p>
 
<p>
 
<span class="on_page"></span>
 
<span class="on_page"></span>
 
<b>On this page</b> you will find information on:
 
<b>On this page</b> you will find information on:
−
<a href="https://2020.igem.org/Measurement/Resources#measuring_fluorescence">Measuring Fluorescence</a>, <a href="https://2020.igem.org/Measurement/Resources#fluor_proteins">Selecting Fluorescent Proteins</a>, <a href="https://2020.igem.org/Measurement/Resources#microscopy">Using Microscopy</a>, and <a href="https://2020.igem.org/Measurement/Resources#plotting_data">Analyzing and Plotting Data</a>
+
The iGEM Measurement Kit
 
</p>
 
</p>
−
 
−
<p>Thinking about how your team might approach measurement? Check out some of the resources below to help get you started, some of which have been developed specifically for iGEM teams. We also encourage you to look at examples from past teams to get inspired.</p>
 
−
<div class="clear extra_space"></div>
 
  
 
<div class="highlight post_item gray">
 
<div class="highlight post_item gray">
 
<div class="details">
 
<div class="details">
−
<span class="icon notice"></span>
+
<span class="icon announcement"></span>
−
<div class="title">Have a resource to contribute?</div>
+
<div class="title">Measurement Kit Announcement</div>
 
</div>
 
</div>
−
<p>Please email the measurement committee at <i> measurement [AT] igem [DOT] org </i> and provide links to material with a short description. We’ll test out the material and if we believe it will be helpful, we’ll add it to this page!</p>
+
<p>As a result of the current pandemic, we will not be able to manufacture and ship out our 2020 DNA Distribution Kits to teams, which includes the Measurement Kits. This is due to our concern for the safety of our personnel, issues with shipping logistics, and our continued lack of lab access, which has resulted in an interrupted production schedule. We sincerely apologize for any inconvenience this may cause.
 +
<br><br>
 +
If you have any questions, please email us at <i>hq [AT] igem [DOT] org</i>.
 +
</p>
 
</div>
 
</div>
 
</div>
 
</div>
−
 
−
 
−
 
−
 
−
<div class="clear extra_space"></div>
 
−
<div class="line_divider"></div>
 
−
<div class="clear extra_space" id="measuring_fluorescence"></div>
 
  
−
<div class="column full_size">
+
<div class="clear extra_space"></div>
−
<h2>Measuring Fluorescence</h2>
+
<div class="line_divider"></div>
−
<p>Below are some tools that can help you transform arbitrary unit (AU) fluorescence measurements into standard comparable units.</p>
+
<div class="clear extra_space" id="About"></div>
−
</div>
+
  
−
<div class="clear extra_space"></div>
+
<div class="column full_size">
 +
<h2>Introducing the Measurement Kit</h2>
 +
<p>Every lab has different equipment with different settings, and measurements of fluorescence or absorbance are often reported using arbitrary units (AU). These AU values from different labs cannot be directly compared. The iGEM Measurement kit contains resources that allow calibration of plate readers for fluorescent intensity and cell density measurements. Once these calibration protocols have been performed, you’ll be able to convert the arbitrary units you produce during your project into standard units. This will make your results much more powerful by making them directly comparable with those of other teams who have also calibrated their equipment.
 +
<br/>
 +
Check out the <a href="">'What Is Measurement?'</a> page to learn more about why comparable data is important for your research.</p>
 +
<br/>
 +
<center><img src="https://static.igem.org/mediawiki/2020/2/29/Measurement_Kit_Graphic.png" style="width:70%;"></center>
 +
</div>
 +
 +
<div class="column quarter_size">
 +
 +
<img src="https://static.igem.org/mediawiki/2020/1/16/Measurement_Kit_Contents_Graphic.png">
 +
</div>
 +
 +
<div class="column three_quarter_size">
 +
<h3>Your Measurement Kit Contains:</h3>
 +
<table>
 +
 +
<tr>
 +
<th>Item</th>
 +
<th>Supplier</th>
 +
<th>Order Details</th>
 +
<th>Website</th>
 +
</tr>
 +
 +
<tr>
 +
<td>Microsphere Particles</td>
 +
<td>NanoCym</td>
 +
<td>950nM size particles</td>
 +
<td><a href="https://nanocym.com/product/silocym/">https://nanocym.com/product/silocym/</a></td>
 +
</tr>
 +
 +
 +
<tr>
 +
<td>Fluorescein sodium salt </td>
 +
<td>Sigma-Aldrich</td>
 +
<td>Product number: 46970</td>
 +
<td><a href="https://www.sigmaaldrich.com/catalog/product/sigma/46970">https://www.sigmaaldrich.com/catalog/product/sigma/46970</a></td>
 +
</tr>
 +
 +
 +
<tr>
 +
<td>Texas Red (Sulforhodamine 101 acid chloride)</td>
 +
<td>Sigma-Aldrich</td>
 +
<td>Product number: S3388</td>
 +
<td><a href="https://www.sigmaaldrich.com/catalog/product/sigma/s3388">https://www.sigmaaldrich.com/catalog/product/sigma/s3388</a></td>
 +
</tr>
 +
</table>
  
−
<div class="column third_size">
+
</div>
−
<img src="https://static.igem.org/mediawiki/parts/5/59/IGEMHQ_2019_MK.jpg">
+
−
<p class="image_caption">iGEM Measurement Kit</p>
+
−
</div>
+
  
−
<div class="column two_thirds_size">
+
<div class="clear extra_space"></div>
 +
<div class="line_divider"></div>
 +
<div class="clear extra_space" id="Cell_Density"></div>
  
−
<h3>Plate Reader</h3>
+
<div class="column full_size">
 +
<h2>Converting OD<sub>600</sub> to Absolute Units</h2>
 +
<p>Cell density (i.e. how many cells are in a culture) is normally measured and reported using optical density (OD) readings. Whilst this approach can be used to provide a good estimate of cell density, the units reported are arbitary and can vary between equipment and labs. The measurement kit provides silica microspheres/beads which are similar in size and optical properties as bacterial cells. Therefore, 1 microspheres is approximately equal to one bacterial cell. By creating a serial dilution with known numbers of silica microspheres, a standard curve of number of microspheres against OD readings can be produced. Using this standard curve, OD readings for bacterial cell cultures can then be converted to an approximate number of cells, which is an absolute unit and comparable between equipment and labs.
 +
</br>
 +
</br>
 +
The protocol for converting OD readings to absolute cell count measurements can be found <a href="https://2020.igem.org/Measurement/Protocols#kit_protocols">here</a>, along with spreadsheets to help with the required calculations.
 +
</p>
 +
<center><img src="https://static.igem.org/mediawiki/2020/3/33/Measurement_Kit_Graphic_OD.png" style="width:60%"/></center>
  
−
<p>The <a href="http://parts.igem.org/Help:2020_DNA_Distribution#Measurement_Kit">Measurement Kit </a>is included in your iGEM Distribution Kit and is intended to help your team measure green fluorescent protein (GFP) and red fluorescent protein (RFP) reliably in a plate reader. The kit includes: tubes of Fluorescein Sodium Salt, tubes of Texas Red, and a tube of silica beads.
+
</div>
−
<br>
+
−
<br>
+
−
Fluorescein has a similar range of excitation and emission as most GFPs. Because of the overlap of the excitation and emission spectrums, we can utilize fluorescein to create a standard curve to compare GFP measures against in plate readers. The same holds true for Texas Red and many RFPs. The silica beads are a suspension of silica particles in water that can be used for calibrating optical density (OD) measurements.
+
−
</p>
+
  
−
<ul>
+
<div class="clear extra_space"></div>
−
<li>Protocol for using the iGEM Measurement Kit to calibrate fluorescence and OD</li>
+
<div class="line_divider"></div>
−
<li>iGEM Measurement Kit calculation spreadsheet</li>
+
<div class="clear extra_space" id="Cell_Density"></div>
−
</ul>
+
  
−
<p><b>Storage</b>: The Measurement Kit should be stored at room temperature or at least higher than 4°C. </p>
+
<div class="column full_size">
 +
<h2>Converting Fluorescent Intensity for Green/Yellow Fluorescent Proteins to Absolute Units</h2>
 +
<p>Fluorescent intensity (FI) is a measure for the amount of fluorescent protein present in a sample. FI is an arbitary reading, which means that it can not be accurately compared between different equipment and labs. The Measurement kit provides resources to convert FI to an approximate concentration of green/yellow fluorescent protein in a sample. Fluorescein is a fluorescent compound which has a similar fluorescent profile to GFPs and YFPs. A serial dilution with known amounts of fluorescein can be prepared to calibrate a piece of equipment's arbitary FI readings and convert them to concentration, which results in data which can be compared between labs and equipment. The method described above to measure number of cells should also be used so that concentration of fluorescent protein per cell can be reported.
 +
</br>
 +
</br>
 +
The protocol for converting FI readings to absolute concentration measurements can be found <a href="https://2020.igem.org/Measurement/Protocols#kit_protocols">here</a>, along with spreadsheets to help with the required calculations.
 +
</p>
 +
<center><img src="https://static.igem.org/mediawiki/2020/1/18/Measurement_Kit_Graphic_GFP.png" style="width:60%"/></center>
  
−
</div>
+
</div>
  
 +
<div class="clear extra_space"></div>
 +
<div class="line_divider"></div>
 +
<div class="clear extra_space" id="Cell_Density"></div>
  
−
<div class="clear extra_space"></div>
+
<div class="column full_size">
 +
<h2>Converting Fluorescent Intensity for Red Fluorescent Proteins to Absolute Units</h2>
 +
<p>Fluorescent intensity (FI) is a measure for the amount of fluorescent protein present in a sample. FI is an arbitary reading, which means that it can not be accurately compared between different equipment and labs. The Measurement kit provides resources to convert FI to an approximate concentration of red fluorescent protein in a sample. Texas Red is a fluorescent dye which has a similar fluorescent profile to RFPs. A serial dilution with known amounts of Texas Red can be prepared to calibrate a piece of equipment's arbitary FI readings and convert them to concentration, which results in data which can be compared between labs and equipment. The method described above to measure number of cells should also be used so that concentration of fluorescent protein per cell can be reported.
 +
</br>
 +
</br>
 +
The protocol for converting FI readings to absolute concentration measurements can be found <a href="https://2020.igem.org/Measurement/Protocols#kit_protocols">here</a>, along with spreadsheets to help with the required calculations.
 +
</p>
 +
<center><img src="https://static.igem.org/mediawiki/2020/d/d1/Measurement_Kit_Graphic_RFP.png" style="width:60%"/></center>
  
 +
</div>
  
−
<div class="column third_size">
+
</div>
−
<img src="https://static.igem.org/mediawiki/2019/9/90/Flow_bead_example.png">
+
−
<p class="image_caption">Graph representing peak identification for SPHERO RCP-30-5A beads </p>
+
−
</div>
+
  
−
<div class="column two_thirds_size">
 
−
 
−
<h3>Flow Cytometry</h3>
 
−
<p>Flow cytometers allow high-throughput measurement of fluorescence from hundreds of thousands of individual cells. Calibration beads and appropriate controls allow you to turn raw “arbitrary unit” measurements into precise and replicable units.</p>
 
−
 
−
<p>Sources of calibration beads:</p>
 
−
<ul>
 
−
<li>SpheroTech Rainbow Calibration Particles (Recommended: URCP-38-2K) (<a href="https://www.spherotech.com/CalibrationParticles.htm">Product Link</a>)</li>
 
−
<li>ClonTech EGFP and mCherry Calibration Beads (<a href="https://www.clontech.com/US/Products/Fluorescent_Proteins_and_Reporters/Flow_Cytometer_Calibration_Beads/AcGFP_and_mCherry">Product Link</a>)</li>
 
−
</ul>
 
−
 
−
<p>Free and open data analysis software for calibrated flow cytometry:
 
−
<ul>
 
−
<li>TASBE Flow Analytics (Matlab/Octave library) (<a href="https://github.com/TASBE">TASBE link</a>)
 
−
<br><i>TASBE Flow Analytics is a software tool that analyzes flow cytometry data, including bead-based conversion to standard units.Experiment templates support automated processing, comparison, and plotting of data. TASBE Flow Analytics was developed as Matlab and Octave compatible software.</i></li>
 
−
 
−
<li>CytoFlow (Python library + graphical interface) (<a href="https://bpteague.github.io/cytoflow/">CytoFlow link</a>)
 
−
<br><i>CytoFlow is a collection of Python tools for quantitative, reproducible flow cytometry analysis, including bead-based conversion to standard units and a Jupyter notebook interface.</i></li>
 
−
 
−
<li>FlowCal (Python library + Excel interface) (<a href="https://taborlab.github.io/FlowCal/">FlowCal link</a>)
 
−
<br><i>FlowCal is a library for reading, analyzing, and calibrating flow cytometry data in Python, including bead-based conversion to standard units and an Excel worksheet interface for simple data entry.</i></li>
 
−
</ul>
 
−
 
−
</div>
 
−
 
−
 
−
<div class="clear extra_space"></div>
 
−
 
−
 
−
<div class="column third_size">
 
−
<img src="https://static.igem.org/mediawiki/2020/4/4f/Principles_of_fluorescence_microscopy_measurement_committee_2020.jpeg">
 
−
<p class="image_caption">Graph representing fundamental concepts underpinning fluorescence microscopy </p>
 
−
</div>
 
−
 
−
<div class="column two_thirds_size">
 
−
 
−
<h3>Using Microscopy</h3>
 
−
<p>Microscopes are powerful tools to measure fluorescence that produce images rich with spatial information. Absolute calibration for fluorescence microscopy is not straightforward, members of the Measurement Committee are working on a protocol, but there is not one currently available to recommend. Even uncalibrated, fluorescence microscopy is a powerful tool, but there are a number of pitfalls on the path from biological sample to data point. </p> <br>
 
−
 
−
<p>There is a group of microscopy specialists on the Measurement Committee, with experience in widefield and confocal fluorescence microscopy, image processing and analysis, and image data presentation. The article below provides good general practices, if you have specific questions don’t hesitate to email us. Starting an email subject line with ‘Microscopy –‘ will bring it to our attention and likely have a faster response.
 
−
</p><br>
 
−
<p> <a href="https://doi.org/10.1091/mbc.E17-05-0276">“A beginner’s guide to rigor and reproducibility in fluorescence imaging experiments”</a>
 
−
Free and open image visualization, processing and analysis software:
 
−
<a href="https://fiji.sc/"> Fiji:</a> Fiji, a distribution of ImageJ, is a powerful, free program that is widely used to explore, process, and analyze fluorescence microscopy data. With a scripting language and a large community of users, plugins exist to meet many image processing and analysis goals, and new extensions of the software can easily be written.
 
−
</p>
 
−
 
−
<br><br>
 
−
</div>
 
−
<div class="clear extra_space"></div>
 
−
<div class="line_divider"></div>
 
−
<div class="clear extra_space" id="fluor_proteins"></div>
 
−
 
−
<div class="column full_size">
 
−
<h2>Selecting Fluorescent Proteins</h2>
 
−
<p>With so many fluorescent proteins to choose, which ones should you use for your project? Fluorescent proteins have revolutionized experiments in synthetic biology. They are so useful that hundreds have been developed for many different uses. The Measurement Committee has recommendations aligned with NIST fluorescence calibration standards. </p>
 
−
</div>
 
−
 
−
<div class="column half_size">
 
−
 
−
<h3>Features of Fluorescent Proteins (FPs) to consider before starting your experiments</h3>
 
−
<p>
 
−
It is important to be aware of the properties of the fluorescent proteins you want to use and how these properties could influence results. We recommend that you use fluorescent proteins that are monomers, fold rapidly, and are pH stable.
 
−
<ul>
 
−
<li>Excitation and emission spectrums</li>
 
−
<li>pH stability (pKa) of the protein</li>
 
−
<li>Maturation time</li>
 
−
</ul>
 
−
If using multiple fluorescent proteins, you will need to consider bleed-through, host organism autofluorescence, and signal to background noise.
 
−
</p>
 
−
 
−
</div>
 
−
 
−
<div class="column half_size">
 
−
<h3>Instrumentation Properties</h3>
 
−
<p>
 
−
It is also very important to be aware of the properties of your instrumentation so you can determine the types of measurements you can take during your experiments. You should know this type of instrumentation when working with fluorescent measurements:
 
−
<ul>
 
−
<li>Excitation light source - laser, LED</li>
 
−
<li>Emission detectors - PMTs, sensitivity</li>
 
−
<li>Filter sets (if applicable)</li>
 
−
</ul>
 
−
</p>
 
−
</div>
 
−
 
−
<div class="clear"></div>
 
−
 
−
<div class="column full_size">
 
−
<h3>Fluorescent Protein Database</h3>
 
−
<p>FPbase is a free and open-source, community-editable database for fluorescent proteins (FPs) and their properties. FPbase was designed and created in 2018 by Talley Lambert at Harvard Medical School.</p>
 
−
<div class="button"> <a href="https://www.fpbase.org/about/"> FPbase.org </a> </div>
 
−
</div>
 
−
 
−
<div class="clear"></div>
 
−
 
−
<div class="column full_size">
 
−
<h3>Specific Fluorescent Protein Recommendations</h3>
 
−
<p>As a general recommendation from iGEM for a fluorescent reporter protein, it is important that the protein is a monomer, folds rapidly (min vs hours), is bright, and does not possess acid sensitivity.
 
−
</p>
 
−
 
−
<p><b>Green Fluorescent Proteins</b>
 
−
<br>
 
−
<ul>
 
−
<li><a href="http://parts.igem.org/Part:BBa_E0040">BBa_E0040</a>: GFPmut3 (500/513, brightness 35, maturation time 4.1 min, weak dimer) - more information can be found on <a href="https://www.fpbase.org/protein/gfpmut3/">FPBase</a></li>
 
−
</ul>
 
−
<br>
 
−
<b>Red Fluorescent Proteins</b>
 
−
<br>
 
−
<ul>
 
−
<li><a href="http://parts.igem.org/Part:BBa_J06504">BBa_J06504</a>: mCherry (587/610, brightness 16, maturation time 15 min, pKa 4.5 ) - more information can be found on <a href="https://www.fpbase.org/protein/mcherry/">FPbase</a></li>
 
−
<li>mKate2 (588/633, brightness 25, maturation time 20 min, pKa 5.4) - more information can be found on <a href="https://www.fpbase.org/protein/mkate2/">FPbase</a></li>
 
−
</ul>
 
−
 
−
<p>If a slow maturation time is acceptable, then we recommend these:
 
−
<ul>
 
−
<li><a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a>: mRFP1 (584/607, brightness 12.5, maturation time 60 min, pKa4.5) - more information can be found on <a href="https://www.fpbase.org/protein/mrfp1/">FPbase</a></li>
 
−
<li>mScarlet (569/594, brightness 70, maturation time 174 min, pKa 5.3) - more information can be found on <a href="https://www.fpbase.org/protein/mscarlet/">FPbase</a></li>
 
−
</ul>
 
−
<br>
 
−
 
−
<b>Red Organic Dyes</b>
 
−
<br>
 
−
The major organic dyes in this range include:
 
−
<ul>
 
−
<li>Nile Red (549/628) (part of the NIST fluorescence standards)</li>
 
−
<li>Texas Red (596/620)</li>
 
−
</ul>
 
−
<br><br>
 
−
 
−
<p>
 
−
<b>Blue Fluorescent Proteins</b>
 
−
<br>
 
−
Another consideration for blue fluorescent proteins can be damage from shorter wavelength light so moving to a cyan may be preferable depending on the experiment.
 
−
 
−
<ul>
 
−
<li><a href="http://parts.igem.org/Part:BBa_K592100">BBa_K592100</a>: TagBFP (402/457, brightness 33, maturation time 13 min, pKa 2.7) - more information can be found on  <a href="https://www.fpbase.org/protein/tagbfp/">FPbase</a></li>
 
−
</ul>
 
−
 
−
<p>
 
−
If a cyan is required with a longer maturation time then:
 
−
 
−
<ul>
 
−
<li>mCerulean3 (433/475, brightness 35, maturation time 70 min, pKa 3.2) - more information can be found on <a href="https://www.fpbase.org/protein/mcerulean3/">FPbase</a></li>
 
−
</ul>
 
−
 
−
The Coumarin 30 beads in the <a href="https://spherotech.com/Ultra%20Rainbow%20Quantitative%20Particle%20Kit.pdf">Spherotech Ultra Rainbow Quantitative Particle Kit</a> can be used to standardize the quantitation (these are the beads used by NIST and the previous iGEM InterLab studies).
 
−
</p>
 
−
</div>
 
−
 
−
 
−
<div class="clear extra_space"></div>
 
−
<div class="line_divider"></div>
 
−
<div class="clear extra_space"></div>
 
−
 
−
<div class="column full_size">
 
−
<h2>Analyzing and Plotting Data</h2>
 
−
<p>Below are some tools that can help you analyze your data and create useful plots to explain your results.</p>
 
−
</div>
 
−
 
−
<div class="clear extra_space"></div>
 
−
 
−
<div class="column third_size">
 
−
<img src="https://static.igem.org/mediawiki/2019/a/a0/DNAPlotLib.jpg">
 
−
<p class="image_caption">Image from DNAplotlib</p>
 
−
</div>
 
−
 
−
<div class="column two_thirds_size">
 
−
<a href="https://github.com/VoigtLab/dnaplotlib"><h3>DNAplotlib</h3></a>
 
−
 
−
<p>Visually integrating graphs of your data with a schematic representation of the parts and circuits which generated that data is an important aspect of scientific communication in synthetic biology. There are many ways to achieve this goal, but for teams with proficiency in the Python programming language, DNAplotlib is an excellent tool developed by the authors of Der and Glassey et al., 2016, <i>ACS Synthetic Biology</i> for this purpose. Even for teams without coding experience, we recommend looking at some of DNAplotlib’s sample graphs as an example of good data visualization practices in synthetic biology.</p>
 
−
</div>
 
−
 
−
<div class="clear extra_space"></div>
 
−
 
−
<div class="column third_size">
 
−
<img src="https://static.igem.org/mediawiki/2020/3/30/R_Logo.jpeg">
 
−
</div>
 
−
 
−
<div class="column two_thirds_size">
 
−
<a href="https://rstudio.com/"><h3>R and R Studio</h3></a>
 
−
 
−
<p>R studio is a free set of tools designed to let you use the programming language R in an easy and effective way. R is a programming language for statistical computing which can be used to analyse data from your experiments, and plot graphs for use on your wiki. As R is an opensource platform, scripts written to analyse and plot your data can be uploaded to iGEM wikis, which helps others better understand your data and hence more likely to use aspects of your project. A beginners tutorial for R can be found <a href="https://www.rforbiologists.org/">here</a>. </p>
 
−
</div>
 
−
 
−
<div class="clear extra_space"></div>
 
−
 
−
<div class="column third_size">
 
−
<img src="https://static.igem.org/mediawiki/2019/5/5b/WebPlot.jpg">
 
−
<p class="image_caption">Image from WebPlotDigitizer</p>
 
−
</div>
 
−
 
−
<div class="column two_thirds_size">
 
−
 
−
<a href="https://automeris.io/WebPlotDigitizer/"><h3>WebPlotDigitizer</h3></a>
 
−
 
−
<p>Often, published data (whether in scientific papers or in the BioBrick Registry) exists only in graphical form, which prevents you from being able to make quantitative comparisons between your results and existing work. WebPlotDigitizer, developed by Ankit Rohatgi, is an open-source web-based tool that solves this problem by allowing you to input an image of a graph or plot and returning numerical values for the data depicted in the image. No coding experience is required-- just upload an image, define values along the axes, and click on points within the graph to generate a table of data that you can analyze!</p>
 
−
</div>
 
−
 
−
</div>
 
−
 
 
</div>
 
</div>
  
 
</html>
 
</html>
 
{{Footer2020}}
 
{{Footer2020}}

Latest revision as of 15:56, 15 June 2021

CALENDAR
Meetups & Events
TEAMS
Team Information
Team List ⇗
Team Wikis ⇗
Team Parts ⇗
Community
Ambassadors
Podcast
Women in STEM
Global Meetup
Relive
Opening Weekend
Schedule
Sessions
Join
Mentorship
Dry Lab Impulse
Collaborations
iGEM Slack Channel
News Archive
Connect
COMPETITION
Results
Certificates and Awards
Surveys
Coronavirus Updates
New
Posters
Judging
Events
Giant Jamboree
Registration
Two Phase Projects
Promotion Video
Safety
Resources
2020 Requirements
Team Requirements
Team Registration
Team Fee
Handbook
Consent Forms
Rules of Conduct
Preventing Harm
Responsible Conduct
Deliverables
Team Wiki
Team Poster
Team Presentation
Promotion Video
Judging Form
Project Attribution
Safety Forms
Part Pages
Project Inspiration
Tracks
Diagnostics
Energy
Environment
Food and Nutrition
Foundational Advance
Hardware
Information Processing
Manufacturing
New Application
Open
Software
Therapeutics
Other Information
Join a Team ⇗
Title and Abstract
Distribution Kit
GIANT JAMBOREE
Individual Registration ⇗
My iGEM App ⇗
Team Registration
iGEM From Above
Watch Parties
Commentators
Volunteer
Program
Nov 13: Opening Ceremonies
Nov 14-16: Competition
Nov 17-20: Community Week
Nov 21: Contribution Day
Nov 22: Celebration!
JUDGING
Committee
For Judges
Application
Resources
Judging Handbook ⇗
For Teams
Judging Form
Rubric
Pages for Awards
Medals
Awards
Online Events
Office Hours
SAFETY
Requirements
Working Safely
Rules
Policies
Coronavirus
Do Not Release
Roles
Committee
Tools
Values and Risks Workshops
Risk Groups
White List
Risk Assessment Tool
Environmental Samples
Exemplary Projects
Forms
Safety Form
Check In Form
Animal Use Form
HUMAN PRACTICES
Introduction
How to Succeed
FAQ
Resources
Exemplary Projects
History
Committee
Online Events
Office Hours
MEASUREMENT
Introduction
How to Succeed
Measurement Kit
Resources
Protocols
Exemplary Projects
Committee
FAQ
Online Events
Webinars and Office Hours
DIVERSITY
Introduction
Gender Diversity
Committee
Recommendations
Examples
Black Lives Matter
Online Events
Office Hours
VIDEOS
Video Universe ⇗
Production
Submission
Examples
FAQ
RESOURCES
General
Video Technology
Video Universe ⇗
Submission Instructions
Production Resources
Examples
FAQ
SDGs
Start a Team
Navigating iGEM
FAQ
Funding
Wet and Dry Lab
Cloning Help
Transformations
Digests & Ligations
Parts Registry ⇗
Software Tools
Documentation
Telling your Story
Wiki Editing Help
HTML CSS Javascript
Template Documentation

iGEM Measurement Kit

On this page you will find information on: The iGEM Measurement Kit

Measurement Kit Announcement

As a result of the current pandemic, we will not be able to manufacture and ship out our 2020 DNA Distribution Kits to teams, which includes the Measurement Kits. This is due to our concern for the safety of our personnel, issues with shipping logistics, and our continued lack of lab access, which has resulted in an interrupted production schedule. We sincerely apologize for any inconvenience this may cause.

If you have any questions, please email us at hq [AT] igem [DOT] org.

Introducing the Measurement Kit

Every lab has different equipment with different settings, and measurements of fluorescence or absorbance are often reported using arbitrary units (AU). These AU values from different labs cannot be directly compared. The iGEM Measurement kit contains resources that allow calibration of plate readers for fluorescent intensity and cell density measurements. Once these calibration protocols have been performed, you’ll be able to convert the arbitrary units you produce during your project into standard units. This will make your results much more powerful by making them directly comparable with those of other teams who have also calibrated their equipment.
Check out the 'What Is Measurement?' page to learn more about why comparable data is important for your research.


Your Measurement Kit Contains:

Item Supplier Order Details Website
Microsphere Particles NanoCym 950nM size particles https://nanocym.com/product/silocym/
Fluorescein sodium salt Sigma-Aldrich Product number: 46970 https://www.sigmaaldrich.com/catalog/product/sigma/46970
Texas Red (Sulforhodamine 101 acid chloride) Sigma-Aldrich Product number: S3388 https://www.sigmaaldrich.com/catalog/product/sigma/s3388

Converting OD600 to Absolute Units

Cell density (i.e. how many cells are in a culture) is normally measured and reported using optical density (OD) readings. Whilst this approach can be used to provide a good estimate of cell density, the units reported are arbitary and can vary between equipment and labs. The measurement kit provides silica microspheres/beads which are similar in size and optical properties as bacterial cells. Therefore, 1 microspheres is approximately equal to one bacterial cell. By creating a serial dilution with known numbers of silica microspheres, a standard curve of number of microspheres against OD readings can be produced. Using this standard curve, OD readings for bacterial cell cultures can then be converted to an approximate number of cells, which is an absolute unit and comparable between equipment and labs.

The protocol for converting OD readings to absolute cell count measurements can be found here, along with spreadsheets to help with the required calculations.

Converting Fluorescent Intensity for Green/Yellow Fluorescent Proteins to Absolute Units

Fluorescent intensity (FI) is a measure for the amount of fluorescent protein present in a sample. FI is an arbitary reading, which means that it can not be accurately compared between different equipment and labs. The Measurement kit provides resources to convert FI to an approximate concentration of green/yellow fluorescent protein in a sample. Fluorescein is a fluorescent compound which has a similar fluorescent profile to GFPs and YFPs. A serial dilution with known amounts of fluorescein can be prepared to calibrate a piece of equipment's arbitary FI readings and convert them to concentration, which results in data which can be compared between labs and equipment. The method described above to measure number of cells should also be used so that concentration of fluorescent protein per cell can be reported.

The protocol for converting FI readings to absolute concentration measurements can be found here, along with spreadsheets to help with the required calculations.

Converting Fluorescent Intensity for Red Fluorescent Proteins to Absolute Units

Fluorescent intensity (FI) is a measure for the amount of fluorescent protein present in a sample. FI is an arbitary reading, which means that it can not be accurately compared between different equipment and labs. The Measurement kit provides resources to convert FI to an approximate concentration of red fluorescent protein in a sample. Texas Red is a fluorescent dye which has a similar fluorescent profile to RFPs. A serial dilution with known amounts of Texas Red can be prepared to calibrate a piece of equipment's arbitary FI readings and convert them to concentration, which results in data which can be compared between labs and equipment. The method described above to measure number of cells should also be used so that concentration of fluorescent protein per cell can be reported.

The protocol for converting FI readings to absolute concentration measurements can be found here, along with spreadsheets to help with the required calculations.

iGEM Foundation

The International Genetically Engineered Machine (iGEM) Foundation is an independent, non-profit organization dedicated to the advancement of synthetic biology, education and competition, and the development of an open community and collaboration. Visit us at igem.org

Contact


hq AT igem DOT org


45 Prospect Street
Cambridge, MA 02139


Due to COVID-19, iGEM staff are working remotely and cannot receive phone calls.
Please contact us by email.

Quicklinks

Main Page
News
Calendar
Events
Sponsor Offers
Collaborations
Sitemap

Keep in touch!



Stay up to date with our newsletter:

SUBSCRIBE