Difference between revisions of "Team:Mingdao/InterLab"

 
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           <div class="m-text-area">
−
             <h1>Interlab</h1>
+
             <h1>Interlab Study</h1>
 
             <div id="model-intro" class="m-block" >
 
             <div id="model-intro" class="m-block" >
−
                <h2 class="m-subtitle">Introduction</h2>
 
−
                <img src="https://static.igem.org/mediawiki/2017/0/06/Csmuxnchu_model_line_green.png" style="width: 60%; transform: translate(35%, -150%);">
 
  
−
                 <p>Reliable and repeatable measurement is a key component to all engineering disciplines. The same
+
<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
 
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,
 
ability to repeat measurements in different labs has been difficult. The Measurement Committee,
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fluorescent protein (GFP) over the last several years. We chose GFP as the measurement marker
 
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
 
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.  
+
laboratories are equipped to measure this protein."
 
<p>
 
<p>
−
The aim to improve the measurement tools available to both the iGEM community and the synthetic
 
−
biology community as a whole. One of the big challenges in synthetic biology measurement has
 
−
been that fluorescence data usually cannot be compared because it has been reported in different
 
−
units or because different groups process data in different ways. Many have tried to work around
 
−
this using “relative expression” comparisons; however, being unable to directly compare
 
−
measurements makes it harder to debug engineered biological constructs, harder to effectively
 
−
share constructs between labs, and harder even to just interpret your experimental controls.
 
−
<p>
 
−
The InterLab protocol aims to address these issues by providing researchers with a detailed
 
−
protocol and data analysis form that yields absolute units for measurements of GFP in a plate
 
−
reader.<br></p>
 
  
  
−
            </div>
+
</ br>
−
             <div id="parts-Material" class="Goal for the Fifth InterLab" >
+
</ br></ br></p>
−
                 <h2 class="m-subtitle">Goal for the Fifth InterLab</h2>
+
 
−
                <img src="https://static.igem.org/mediawiki/2017/0/06/Csmuxnchu_model_line_green.png" style="width: 60%; transform: translate(35%, -150%);">
+
             
−
                 <p>The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability
+
         
 +
             <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
 
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
 
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
 
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.
+
labs will not be able to reliably build upon others’ work."
 
<p>
 
<p>
−
In the previous interlab studies, it was shown that by measuring GFP expression in absolute
+
 
−
fluorescence units calibrated against a known concentration of fluorescent molecule can greatly
+
"This year, teams participating in the interlab study helped iGEM to answer the following
−
reduce the variability in measurements between labs. However, when taking bulk measurements of
+
question: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to
−
a population of cells (such as with a plate reader), there is still a large source of variability in these
+
absolute cell count or colony-forming units (CFUs) instead of OD?"
−
measurements: the number of cells in the sample.
+
 
<p>
 
<p>
−
Because the fluorescence value measured by a plate reader is an aggregate measurement of an
+
 
−
entire population of cells, we need to divide the total fluorescence by the number of cells in order to
+
 
−
determine the mean expression level of GFP per cell. Usually this is done by measuring the
+
           
−
absorbance of light at 600nm, from which the “optical density (OD)” of the sample is computed as
+
            </div>
−
an approximation of the number of cells. OD measurements are subject to high variability between
+
                <h3>Calibration Reference</h3>
−
labs, however, and it is unclear how good of an approximation an OD measurement actually is. If a
+
               
−
more direct method is used to determine the cell count in each sample, then potentially another
+
                <div id="model-calibration1" class="m-block" >
−
source of variability can be removed from the measurements.
+
                <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>
−
This year, teams participating in the interlab study helped iGEM to answer the following
+
<P>1ml LUDOX CL-X
−
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>
 
<p>
−
In order to compute the cell count in the different teams samples, two orthogonal approaches were
 
−
be used:
 
 
<p>
 
<p>
−
1. Converting between absorbance of cells to absorbance of a known concentration of beads.
+
ddH2O
 
<p>
 
<p>
−
Absorbance measurements use the way that a sample of cells in liquid scatter light in order
 
−
to approximate the concentration of cells in the sample. In this year’s Measurement Kit,
 
−
teams were provided with a sample containing silica beads that are roughly the same size
 
−
and shape as a typical E. coli cell, so that it should scatter light in a similar way. Because the
 
−
concentration of the beads is known, each lab’s absorbance measurements can be
 
−
converted into a universal, standard “equivalent concentration of beads” measurement.
 
 
<p>
 
<p>
−
2. Counting colony-forming units (CFUs) from the sample.
+
96 well Black Clear Bottom Plate
 +
<p>
 +
<p>
 +
</P>
 
<p>
 
<p>
−
A simple way to determine the number of cells in a sample of liquid media is to pour some out
 
−
on a plate and see how many colonies grow on the plate. Since each colony begins as a
 
  
−
single cell (for cells that do not stick together), we can determine how many live cells were in
+
<p><span style="background-color: #ccffff;"><strong>Method</strong></span></p>
−
the volume of media that we plated out and obtain a cell concentration for our sample as a
+
−
whole. Each team will have to determine the number of CFUs in positive and negative control
+
−
samples in order to compute a conversion factor from absorbance to CFU.
+
 
<p>
 
<p>
−
By using these two approaches, Interlab Measurement Study will be able to determine how much
+
<P>
−
they agree with each other, and whether using one (or both) can help to reduce lab-to-lab variability
+
&#8595; Add 100 μl LUDOX into wells A1, B1, C1, D1
−
in measurements. If it can, then together we will have brought synthetic biology one step closer to
+
<p>
−
becoming a true, reliable engineering discipline.
+
<p>
−
<br></p>
+
&#8595; Add 100 μl of ddH2 O into wells A2,B2,C2,D2
−
 
+
<p>
−
 
+
<p>
−
            </div>
+
&#8595; Measure absorbance at 600 nm
−
            <div id="parts-Result" class="Calibration Reference" >
+
<p>
−
                <h2 class="m-subtitle">Calibration Reference</h2>
+
<p>
−
                <img src="https://static.igem.org/mediawiki/2017/0/06/Csmuxnchu_model_line_green.png" style="width: 60%; transform: translate(35%, -150%);">
+
&#8595; Record the data <p>
−
 
+
<p>
−
 
+
</p>
−
                <br>
+
−
                <img src="https://static.igem.org/mediawiki/2017/5/50/T--CSMU_NCHU_Taiwan--MSMEG5998od0-4.png" alt="" style="width: 100%" >
+
−
                <br><br>
+
−
 
+
−
              <p><strong>Fig. I</strong> The growth curve of BL21 induced by IPTG from 0 to 4 hours. The concentration of BL21 reached stationary phase at 4 hours.</p>
+
−
                <br>
+
−
                <br>
+
−
                <img src="https://static.igem.org/mediawiki/2017/d/d0/T--CSMU_NCHU_Taiwan--MSMEG5998od0-8.png" alt="" style="width:100%" >
+
−
                <br>
+
−
              <p><strong>Fig. II</strong> The growth curve of BL21 from 0 to 8 hr. The concentration of BL21 reached stationary phase at 4 hours and then declined slightly.</p>
+
−
                <br>
+
−
 
+
−
                <img src="https://static.igem.org/mediawiki/2017/a/a5/T--CSMU_NCHU_Taiwan--MSMEG5998western0-8.png" alt="" style="width:100%" >
+
−
                <br>
+
−
                <img src="https://static.igem.org/mediawiki/2017/c/c2/T--CSMU_NCHU_Taiwan--MSMEG5998western0-4.png" alt="" style="width:100%" >
+
−
                <br>
+
−
              <p><strong>Fig. III</strong> Cell lysates from E. coli BL21 with Synthetic MSMEG5998 from 0 to 8 hours and 0 to 4 hours were analyzed by Western blot. The amount of Synthetic MSMEG5998 increased consistently with time.</p>
+
−
                <br>
+
  
 +
<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=""
 +
style="width:80%">
 +
<p>
 +
<p><span style="background-color: #ccffff;"><strong>Result</strong></span></p>
 +
<p>
 +
<p><em><strong>Fluorescence Raw Reading</strong></em></p>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/2/2b/T--Mingdao--Interlab13.jpg"alt=""
 +
style="width:80%">
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/6/60/T--Mingdao--Interlab14.jpg"alt=""
 +
style="width:80%">
 +
<p><em><strong>Abs600 Raw Reading</strong></em></p>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/2/2d/T--Mingdao--Interlab15.jpg"
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/4/45/T--Mingdao--Interlab16.jpg">
 +
<div id="model-protocol" class="m-block" >
  
 +
<h3>Colony Forming Units per E. coli cultures at OD600=0.1 </h3>
 +
<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>
 +
<p>
 +
&#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>
 +
<p>
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/0/06/T--Mingdao--Interlab20.jpg"alt=""
 +
style="width:80%">
 +
<img class="center" src="https://static.igem.org/mediawiki/2018/3/36/T--Mingdao--Interlab21.jpg"alt=""
 +
style="width:80%">
  
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                <h2 class="m-subtitle">Discussion</h2>
 
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                <p>1. According to the data shown above, the growth curve of E.coli BL21 with Synthetic MSMEG_5998 reached the ceiling when the O.D. value was approximately at 2 while the  amount of Synthetic MSMEG_5998 were still increasing.</p>
 
−
                <p>2. Though the amount of Synthetic MSMEG_5998 increased consistently with time, we could not jump to conclusions that it was proper to incubate E.coli as long as possible. Another consideration was the time it would take. Just as our expected, it growed fast at the first 2.5 hours. That’s why we also chose 2.5hr after induced by IPTG when we  extracted Synthetic MSMEG_5998 from total cell lysate in other experiments.</p>
 
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                <p>3. Based on previous experience, if the E.coli was incubated over 4 hours, the protein that it expressed may be degraded or mis-folded, leading to malfunction. As a result, it was also an important issue for this modeling. However, because of the lack of F420, we did not have the chance to check the enzyme activity of each time spot.  It was still unknown whether the titer of the Synthetic MSMEG_5998 would change or not and awaited further research.</p>
 
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           <a href="https://2017.igem.org/Team:CSMU_NCHU_Taiwan/Model" style="color:black;font-size:18px"></a>
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           <p class="tag">Interlab Study</p>
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           <li id="intro-btn" class="tag-btn">- Introduction</li>
 
           <li id="intro-btn" class="tag-btn">- Introduction</li>
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           <li id="Material-btn" class="tag-btn">- Goal for the Fifth InterLab</li>
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           <li id="Result-btn" class="tag-btn">- Calibration Reference</li>
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           <li id="calibration1-btn" class="tag-btn">- Calibration 1</li>
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           <li id="Discussion-btn"class="tag-btn">- Discussion</li>
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           <li id="calibration2-btn" class="tag-btn">- Calibration 2</li>
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          <li id="calibration3-btn" class="tag-btn">- Calibration 3</li>
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          <li id="cell-btn" class="tag-btn">- Cell Measurement</li>
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          <li id="protocol-btn" class="tag-btn">- Protocol</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