Difference between revisions of "Team:Mingdao/InterLab"

 
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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2018.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2018.igem.org/Judging/Awards"> award listed below</a>. </p>
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2018.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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<h1>InterLab</h1>
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<h3>Bronze Medal Criterion #4</h3>
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  }
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<p><b>Standard Tracks:</b> Participate in the Interlab Measurement Study and/or obtain new, high quality experimental characterization data for an existing BioBrick Part or Device and enter this information on that part's Main Page in the Registry. The part that you are characterizing must NOT be from a 2018 part number range.
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For teams participating in the <a href="https://2018.igem.org/Measurement/InterLab">InterLab study</a>, all work must be shown on this page.  
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    <div class="bg-container" style="max-height:none;">
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      <img class="top-picture" src="https://static.igem.org/mediawiki/2018/4/45/T--Mingdao--Phil13-8.png" style="width:100%">
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      <div class="my-main-container">
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        <div class="main-content">
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          <div class="m-text-area">
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            <h1>Interlab Study</h1>
 +
            <div id="model-intro" class="m-block" >
 +
 +
<h3>Note</h3>
 +
<p>Description: the goal and main contents were quoted from iGEM International InterLab Measurement Study <p>
 +
Methods: the protocol was provided by iGEM InterLab Committee and described briefly in here <p>
 +
Results: the experiment and data presented here were all made by members of team Mingdao <p>
 +
Reference: <a href="https://2018.igem.org/Measurement/InterLab">Fifth International InterLab Measurement Study@iGEM</a>
 +
 +
</br></br>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2018/8/8b/T--Mingdao--Interlablastday1.jpeg" alt="" style="width:49%">
 +
<img src="https://static.igem.org/mediawiki/2018/9/9f/T--Mingdao--Interlablastday2.jpeg" alt="" style="width:49%"></center><br />
 +
<center><img src="https://static.igem.org/mediawiki/2018/7/75/T--Mingdao--Interlablastday3.jpeg" alt="" style="width:49%">
 +
<img src="https://static.igem.org/mediawiki/2018/e/ef/T--Mingdao--Interlablastday4.jpeg" alt="" style="width:49%">
 +
</center></br>
 +
 +
<h3>Instrument</h3>
 +
<p>The machine in the Biolab of Mingdao High School: Synergy H1 Hybrid Multi-Mode Microplate Reader
 +
<p><img class="center" src="https://static.igem.org/mediawiki/2018/e/e6/T--Mingdao--Interlab0.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
</br></br>
 +
 +
 +
                <h3>Introduction</h3>
 +
                <p>"Reliable and repeatable measurement is a key component to all engineering disciplines. The same
 +
holds true for synthetic biology, which has also been called engineering biology. However, the
 +
ability to repeat measurements in different labs has been difficult. The Measurement Committee,
 +
through the InterLab study, has been developing a robust measurement procedure for green
 +
fluorescent protein (GFP) over the last several years. We chose GFP as the measurement marker
 +
for this study since it's one of the most used markers in synthetic biology and, as a result, most
 +
laboratories are equipped to measure this protein."
 +
<p>
 +
 +
 +
</ br>
 +
</ br></ br></p>
 +
 +
             
 +
         
 +
            <div id="model-goal" class="m-block" >
 +
                <h3>Goal for the Fifth InterLab</h3>
 +
 +
                <p>"The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability
 +
in synthetic biology measurements, so that eventually, measurements that are taken in different
 +
labs will be no more variable than measurements taken within the same lab. Until we reach this
 +
point, synthetic biology will not be able to achieve its full potential as an engineering discipline, as
 +
labs will not be able to reliably build upon others’ work."
 +
<p>
 +
 +
"This year, teams participating in the interlab study helped iGEM to answer the following
 +
question: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to
 +
absolute cell count or colony-forming units (CFUs) instead of OD?"
 +
<p>
 +
 +
 +
           
 +
            </div>
 +
                <h3>Calibration Reference</h3>
 +
               
 +
                <div id="model-calibration1" class="m-block" >
 +
                <h2 class="m-subtitle">Calibration 1:OD600 Reference point - LUDOX Protocol</h2>
 +
               
 +
<p><span style="background-color: #ccffff;"><strong>Materials</strong></span></p>
 +
<p>
 +
<P>1ml LUDOX CL-X
 +
<p>
 +
<p>
 +
ddH2O
 +
<p>
 +
<p>
 +
96 well Black Clear Bottom Plate
 +
<p>
 +
<p>
 +
</P>
 +
<p>
 +
 +
<p><span style="background-color: #ccffff;"><strong>Method</strong></span></p>
 +
<p>
 +
<P>
 +
&#8595; Add 100 μl LUDOX into wells A1, B1, C1, D1
 +
<p>
 +
<p>
 +
&#8595; Add 100 μl of ddH2 O into wells A2,B2,C2,D2
 +
<p>
 +
<p>
 +
&#8595; Measure absorbance at 600 nm
 +
<p>
 +
<p>
 +
&#8595; Record the data <p>
 +
<p>
 
</p>
 
</p>
−
</div>
 
  
 +
<p>
  
 +
<p><span style="background-color: #ccffff;"><strong>Result</strong></span></p>
 +
<P>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/9/9a/T--Mingdao--Modeling--Chart%28img45%29.jpg"alt=""
 +
style="width:80%">
 +
<p>
  
 +
<p>The table shows the OD600 measured by a spectrophotometer (see table above) and plate
 +
reader data for H2O and LUDOX corresponding to the expected results. The corrected
 +
Abs600 is calculated by subtracting the mean H2O reading. The reference OD600 is defined
 +
as that measured by the reference spectrophotometer. The correction factor to convert
 +
measured Abs600 to OD600 is thus the reference OD600 divided by Abs600. All cell density
 +
readings using this instrument with the same settings and volume can be converted to
 +
OD600 by multiplying by 4.200.</p>
 +
<p>
  
  
 +
<div id="model-calibration2" class="m-block" >
 +
<h2 class="m-subtitle">Calibration 2: Particle Standard Curve - Microsphere Protocol</h2>
 +
<p>
  
 +
<p><span style="background-color: #ccffff;"><strong>Materials</strong></span></p>
 +
<p>
 +
300 μL silica beads Microsphere suspension
 +
<p>
 +
<p>
 +
ddH2O
 +
<p>
 +
<p>
 +
96 well Black Clear Bottom Plate
 +
<p>
 +
<p>
 +
</p>
 +
<p>
 +
<p><span style="background-color: #ccffff;"><strong>Method</strong></span></p>
 +
<p>
 +
<p><em><strong>Preparation of the Microsphere stock solution:</strong></em></p>
 +
<p>
 +
<p>
 +
&#8595; Obtain Silica Beads
 +
<p>
 +
&#8595; Pipet 96 μL beads into an eppendorf
 +
<p>
 +
<p>
 +
&#8595; Add 904 μL of ddH2O to the microspheres
 +
<p>
 +
<p>
 +
&#8595; Vortex well to obtain stock Microsphere Solution.
 +
</p>
 +
<p>
 +
&#8595; Preparation of microsphere serial dilutions as follows
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/b/b0/T--Mingdao--Modeling--SerialDelution%28img47%29.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
&#8595; Measure Abs 600
 +
<p>
 +
&#8595; Record the data
 +
<p>
 +
<p><span style="background-color: #ccffff;"><strong>Result</strong></span></p>
 +
<p>
 +
<p><em><strong>Raw Data</strong></em></p>
 +
<p>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/5/56/T--Mingdao--Modeling--RawData%28img50%29.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
<p><em><strong>Particle Standard Curve</strong></em></p>
 +
<p>
 +
<p>
 +
<img class="center"src="https://static.igem.org/mediawiki/2018/0/04/T--Mingdao--Interlab4.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
<p><em><strong>Particle Standard Curve(log scale)</strong></em></p>
 +
<p>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/a/ac/T--Mingdao--interlab5.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
<div id="model-calibration3" class="m-block" >
 +
<h2 class="m-subtitle">Calibration 3: Fluorescence standard curve - Fluorescein Protocol</h2>
 +
<p>
 +
<p><span style="background-color: #ccffff;"><strong>Materials</strong></span></p>
 +
<p>
 +
<p>
 +
Fluorescein (provided in kit)
 +
<p>
 +
<p>
 +
10ml 1xPBS pH 7.4-7.6 (phosphate buffered saline; provided by team)
 +
<p>
 +
<p>
 +
96 well Black Clear Bottom Plate
 +
<p></p>
 +
<p>
 +
<p>
 +
<p><span style="background-color: #ccffff;"><strong>Method</strong></span></p>
 +
<p>
 +
&#8595; Spin down fluorescein kit tube to make sure pellet is at the bottom of tube.
 +
&#8595; Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS.
 +
<p>
 +
&#8595; Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration of 10 μM
 +
<p>
 +
&#8595; Prepare the serial dilutions of fluorescein as follows:
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/0/0b/T--Mingdao--Interlab6.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
&#8595; Measure fluorescence of all samples in instrument
 +
<p>
 +
&#8595; Record the data
 +
<p>
 +
 +
<p><span style="background-color: #ccffff;"><strong>Result</strong></span></p>
 +
<p>
 +
<p><em><strong>Raw Data</strong></em></p>
 +
<p>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/3/3c/T--Mingdao--Interlab7.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
<p><em><strong>Fluorescein Standard Curves</strong></em></p>
 +
<p>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/f/f2/T--Mingdao--Interlab8.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
<p><em><strong>Fluorescein Standard Curves(log scale)</strong></em></p>
 +
<p>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/6/69/T--Mingdao--Interlab9.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
<div id="model-cell" class="m-block" >
 +
<h3>Cell Measurement</h3>
 +
<p>
 +
<p><span style="background-color: #ccffff;"><strong>Materials</strong></span></p>
 +
<p> Competent cells ( Escherichia coli strain DH5 )
 +
<p>
 +
 LB (Luria Bertani) media
 +
<p>
 +
 Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
 +
<p>
 +
 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)
 +
<p>
 +
 Incubator at 37°C
 +
<p>
 +
 1.5 ml eppendorf tubes for sample storage
 +
<p>
 +
 Ice bucket with ice
 +
<p>
 +
 Micropipettes and tips
 +
<p>
 +
 96 well Black Clear Bottom Plate
 +
<p></p>
 +
<p>
 +
<p><span style="background-color: #ccffff;"><strong>Workflow</strong></span></p>
 +
<p>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/2/22/T--Mingdao--Interlab10.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
<p><span style="background-color: #ccffff;"><strong>Method</strong></span></p>
 +
<p>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/c/c6/T--Mingdao--Interlab11.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
<p><em><strong>Day1</strong></em></p>
 +
<p>
 +
&#8595; Transform Escherichia coli DH5 with these plasmids
 +
<p>
 +
<p><em><strong>Day2</strong></em></p>
 +
<p>
 +
&#8595; Pick 2 colonies from each group
 +
<p>
 +
&#8595; Inoculate in 5-10 mL LB medium + Cm
 +
<p>
 +
&#8595; Grow the cells overnight (16-18 hours) at 37°C and shake at 220 rpm.
 +
<p>
 +
<p><em><strong>Day 3</strong></em></p>
 +
<p>
 +
&#8595; Make a 1:10 dilution of each overnight culture in LB + Cm by putting 0.5mL of culture into 4.5mL of LB + Cm
 +
<p>
 +
&#8595; Measure Abs 600 of these 1:10 diluted cultures
 +
<p>
 +
&#8595; Record the data
 +
<p>
 +
&#8595; Dilute the cultures further to a target Abs6 00 of 0.02 in a final volume of 12 ml LB medium + Cm in 50 mL tube
 +
<p>
 +
&#8595; Incubate the cultures at 37°C and shake at 220 rpm for 6 hours.
 +
<p>
 +
&#8595; Measure your samples for Abs600 and fluorescence
 +
<p>
 +
&#8595; Record data in your notebook
 +
<p>
 +
<center> Layout for Abs 600 and fluorescence measurement </center>
 +
<p></p>
 +
<p>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/1/1a/T--Mingdao--Interlab12.jpg"alt=""
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style="width:80%">
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<p>
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<p><span style="background-color: #ccffff;"><strong>Result</strong></span></p>
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<p>
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<p><em><strong>Fluorescence Raw Reading</strong></em></p>
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/2/2b/T--Mingdao--Interlab13.jpg"alt=""
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style="width:80%">
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/6/60/T--Mingdao--Interlab14.jpg"alt=""
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style="width:80%">
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<p><em><strong>Abs600 Raw Reading</strong></em></p>
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/2/2d/T--Mingdao--Interlab15.jpg"
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/4/45/T--Mingdao--Interlab16.jpg">
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<div id="model-protocol" class="m-block" >
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<h3>Colony Forming Units per E. coli cultures at OD600=0.1 </h3>
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<p>
 +
&#8595; Measure the OD600 of your cell cultures
 +
<p>
 +
&#8595; Dilute your overnight culture to OD600 = 0.1 in 1mL of LB + Cm media. Do this in triplicate.
 +
<p>
 +
&#8595; Make the following serial dilutions for your triplicates
 +
<p><p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/8/8a/T--Mingdao--Interlab19.jpg"alt=""
 +
style="width:80%">
 +
<p>
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<p>
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&#8595; Aseptically spread plate with 100 μL of the dilutions
 +
<p>
 +
&#8595; Incubate at 37°C overnight
 +
<p>
 +
&#8595; Count colonies after 18-20 hours of growth.
 +
<p>
 +
<p>
 +
 +
<p><span style="background-color: #ccffff;"><strong>Result</strong></span></p>
 +
<p>
 +
<p>Colony Forming Units per o.1 OD600 E.coli cultures</p>
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<p>
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<img class="center" src="https://static.igem.org/mediawiki/2018/0/06/T--Mingdao--Interlab20.jpg"alt=""
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style="width:80%">
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<img class="center" src="https://static.igem.org/mediawiki/2018/3/36/T--Mingdao--Interlab21.jpg"alt=""
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style="width:80%">
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      <div class="path-tags">
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          <p class="tag">Interlab Study</p>
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          <li id="intro-btn" class="tag-btn">- Introduction</li>
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          <li id="goal-btn" class="tag-btn">- Goal </li>
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          <li id="calibration1-btn" class="tag-btn">- Calibration 1</li>
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          <li id="calibration3-btn" class="tag-btn">- Calibration 3</li>
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{{:Team:Mingdao/test6}}

Latest revision as of 02:13, 18 October 2018

HOME
Model

Interlab Study

Note

Description: the goal and main contents were quoted from iGEM International InterLab Measurement Study

Methods: the protocol was provided by iGEM InterLab Committee and described briefly in here

Results: the experiment and data presented here were all made by members of team Mingdao

Reference: Fifth International InterLab Measurement Study@iGEM



Instrument

The machine in the Biolab of Mingdao High School: Synergy H1 Hybrid Multi-Mode Microplate Reader



Introduction

"Reliable and repeatable measurement is a key component to all engineering disciplines. The same holds true for synthetic biology, which has also been called engineering biology. However, the ability to repeat measurements in different labs has been difficult. The Measurement Committee, through the InterLab study, has been developing a robust measurement procedure for green fluorescent protein (GFP) over the last several years. We chose GFP as the measurement marker for this study since it's one of the most used markers in synthetic biology and, as a result, most laboratories are equipped to measure this protein."

Goal for the Fifth InterLab

"The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements, so that eventually, measurements that are taken in different labs will be no more variable than measurements taken within the same lab. Until we reach this point, synthetic biology will not be able to achieve its full potential as an engineering discipline, as labs will not be able to reliably build upon others’ work."

"This year, teams participating in the interlab study helped iGEM to answer the following question: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?"

Calibration Reference

Calibration 1:OD600 Reference point - LUDOX Protocol

Materials

1ml LUDOX CL-X

ddH2O

96 well Black Clear Bottom Plate

Method

↓ Add 100 μl LUDOX into wells A1, B1, C1, D1

↓ Add 100 μl of ddH2 O into wells A2,B2,C2,D2

↓ Measure absorbance at 600 nm

↓ Record the data

Result

The table shows the OD600 measured by a spectrophotometer (see table above) and plate reader data for H2O and LUDOX corresponding to the expected results. The corrected Abs600 is calculated by subtracting the mean H2O reading. The reference OD600 is defined as that measured by the reference spectrophotometer. The correction factor to convert measured Abs600 to OD600 is thus the reference OD600 divided by Abs600. All cell density readings using this instrument with the same settings and volume can be converted to OD600 by multiplying by 4.200.

Calibration 2: Particle Standard Curve - Microsphere Protocol

Materials

300 μL silica beads Microsphere suspension

ddH2O

96 well Black Clear Bottom Plate

Method

Preparation of the Microsphere stock solution:

↓ Obtain Silica Beads

↓ Pipet 96 μL beads into an eppendorf

↓ Add 904 μL of ddH2O to the microspheres

↓ Vortex well to obtain stock Microsphere Solution.

↓ Preparation of microsphere serial dilutions as follows

↓ Measure Abs 600

↓ Record the data

Result

Raw Data

Particle Standard Curve

Particle Standard Curve(log scale)

Calibration 3: Fluorescence standard curve - Fluorescein Protocol

Materials

Fluorescein (provided in kit)

10ml 1xPBS pH 7.4-7.6 (phosphate buffered saline; provided by team)

96 well Black Clear Bottom Plate

Method

↓ Spin down fluorescein kit tube to make sure pellet is at the bottom of tube. ↓ Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS.

↓ Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration of 10 μM

↓ Prepare the serial dilutions of fluorescein as follows:

↓ Measure fluorescence of all samples in instrument

↓ Record the data

Result

Raw Data

Fluorescein Standard Curves

Fluorescein Standard Curves(log scale)

Cell Measurement

Materials

 Competent cells ( Escherichia coli strain DH5 )

 LB (Luria Bertani) media

 Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)

 50 ml Falcon tube (or equivalent, preferably amber or covered in foil to block light)

 Incubator at 37°C

 1.5 ml eppendorf tubes for sample storage

 Ice bucket with ice

 Micropipettes and tips

 96 well Black Clear Bottom Plate

Workflow

Method

Day1

↓ Transform Escherichia coli DH5 with these plasmids

Day2

↓ Pick 2 colonies from each group

↓ Inoculate in 5-10 mL LB medium + Cm

↓ Grow the cells overnight (16-18 hours) at 37°C and shake at 220 rpm.

Day 3

↓ Make a 1:10 dilution of each overnight culture in LB + Cm by putting 0.5mL of culture into 4.5mL of LB + Cm

↓ Measure Abs 600 of these 1:10 diluted cultures

↓ Record the data

↓ Dilute the cultures further to a target Abs6 00 of 0.02 in a final volume of 12 ml LB medium + Cm in 50 mL tube

↓ Incubate the cultures at 37°C and shake at 220 rpm for 6 hours.

↓ Measure your samples for Abs600 and fluorescence

↓ Record data in your notebook

Layout for Abs 600 and fluorescence measurement

Result

Fluorescence Raw Reading

Abs600 Raw Reading

Colony Forming Units per E. coli cultures at OD600=0.1

↓ Measure the OD600 of your cell cultures

↓ Dilute your overnight culture to OD600 = 0.1 in 1mL of LB + Cm media. Do this in triplicate.

↓ Make the following serial dilutions for your triplicates

↓ Aseptically spread plate with 100 μL of the dilutions

↓ Incubate at 37°C overnight

↓ Count colonies after 18-20 hours of growth.

Result

Colony Forming Units per o.1 OD600 E.coli cultures

    Interlab Study

  • - Introduction
  • - Goal
  • - Calibration 1
  • - Calibration 2
  • - Calibration 3
  • - Cell Measurement
  • - Protocol