Difference between revisions of "Team:Glasgow/Interlab"
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<tr><td class="survivability">Equipment</td></tr> | <tr><td class="survivability">Equipment</td></tr> | ||
<tr><td class="conclusion">Methodology</td></tr> | <tr><td class="conclusion">Methodology</td></tr> | ||
− | <tr><td class="firstuse">Measurements</td></tr> | + | <tr><td class="firstuse">Sequencing</td></tr> |
+ | <tr><td class="discuss">Measurements</td></tr> | ||
<tr><td class="contamination">Conclusion</td></tr> | <tr><td class="contamination">Conclusion</td></tr> | ||
<tr><td class="top">Top</td></tr> | <tr><td class="top">Top</td></tr> | ||
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</figure></center> | </figure></center> | ||
<br> | <br> | ||
− | <center><figure> <img src="https://static.igem.org/mediawiki/2015/ | + | <center><figure> <img src="https://static.igem.org/mediawiki/2015/0/07/2015-Glasgow-interlab36.png"> |
<figcaption><b>Figure 1</b>: Spectrophotometer calibration curve | <figcaption><b>Figure 1</b>: Spectrophotometer calibration curve | ||
</figcaption> | </figcaption> | ||
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<div class="text"> | <div class="text"> | ||
</br> | </br> | ||
− | Overnight cultures of colony 1-3 of each device were set up (in Luria broth with chloramphenicol) to provide 1ml for measuring on a 96-well plate. As the | + | Overnight cultures of colony 1-3 of each device were set up (in Luria broth with chloramphenicol) to provide 1ml for measuring on a 96-well plate. As the lb broth gave noticeable background fluorescence samples were also prepared by spinning down cells, in the overnight cultures, to pellets and resuspending in PBS (phosphate buffered saline). It was determined the PBS method gave the most accurate measurements so readings were taken using this method for all three biological replicates and technical replicates. </br> |
</br> | </br> | ||
The recipe used for a 1 x solution of PBS was 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4 dissolved in 800ml of H2O, the pH adjusted to 7.4 and the final volume made up to 1 litre with distilled H2O. </div> | The recipe used for a 1 x solution of PBS was 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4 dissolved in 800ml of H2O, the pH adjusted to 7.4 and the final volume made up to 1 litre with distilled H2O. </div> | ||
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</br></br> | </br></br> | ||
− | + | <h2>Sequencing</h2> | |
<div class="box"> | <div class="box"> | ||
− | <h5> | + | <h5>J23101:I13504</h5> |
− | <div class="text"> | + | <div class="text"> |
+ | </br> | ||
+ | Plasmid Map: | ||
+ | </br> | ||
</figure></center> | </figure></center> | ||
<br> | <br> | ||
− | <center><figure> <img src="https://static.igem.org/mediawiki/2015/5/54/2015-Glasgow-interlab30.png" | + | <center><figure> <img src=" https://static.igem.org/mediawiki/2015/5/54/2015-Glasgow-interlab30.png"> |
+ | </br> | ||
+ | </br> | ||
+ | <a href="https://static.igem.org/mediawiki/2015/f/f0/2015-Glasgow-interlab34.pdf" target="_blank">Sequencing results can be found here</a> | ||
</div> | </div> | ||
− | |||
</div> | </div> | ||
</br> | </br> | ||
<div class="box"> | <div class="box"> | ||
− | <h5> | + | <h5>J23106:I13504</h5> |
<div class="text"> | <div class="text"> | ||
+ | </br> | ||
+ | Plasmid Map: | ||
</br> | </br> | ||
</figure></center> | </figure></center> | ||
<br> | <br> | ||
− | <center><figure> <img src="https://static.igem.org/mediawiki/2015/4/48/2015-Glasgow-interlab31.png" | + | <center><figure> <img src="https://static.igem.org/mediawiki/2015/4/48/2015-Glasgow-interlab31.png"> |
</br> | </br> | ||
+ | </br> | ||
+ | <a href="https://static.igem.org/mediawiki/2015/f/f0/2015-Glasgow-interlab34.pdf" target="_blank">Sequencing results can be found here</a> | ||
</div> | </div> | ||
− | |||
</div> | </div> | ||
</br> | </br> | ||
<div class="box"> | <div class="box"> | ||
− | <h5> | + | <h5>J23117:I13504</h5> |
− | <div class="text"> | + | <div class="text"> |
+ | </br> | ||
+ | Plasmid Map: | ||
</br> | </br> | ||
</figure></center> | </figure></center> | ||
<br> | <br> | ||
− | <center><figure> <img src="https://static.igem.org/mediawiki/2015/8/87/2015-Glasgow-interlab32.png" | + | <center><figure> <img src="https://static.igem.org/mediawiki/2015/8/87/2015-Glasgow-interlab32.png"> |
</br> | </br> | ||
+ | </br> | ||
+ | <a href="https://static.igem.org/mediawiki/2015/f/f0/2015-Glasgow-interlab34.pdf" target="_blank">Sequencing results can be found here</a> | ||
</div> | </div> | ||
− | |||
</div> | </div> | ||
− | |||
− | |||
</br> | </br> | ||
+ | <div class="scrolldiscuss"></div> | ||
+ | </br> | ||
<h2>Measurements</h2> | <h2>Measurements</h2> | ||
<div class="box"> | <div class="box"> | ||
− | <h5>Direct Measurement | + | <h5>Direct Measurement </h5> |
<div class="text"> | <div class="text"> | ||
</br> | </br> | ||
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</br> | </br> | ||
<div class="box"> | <div class="box"> | ||
− | <h5>Derived Measurements | + | <h5>Derived Measurements </h5> |
<div class="text"> | <div class="text"> | ||
</br> | </br> | ||
1. The average background absorbance was removed by subtracting the average of the empty wells with no PBS or sample (423,343.279). | 1. The average background absorbance was removed by subtracting the average of the empty wells with no PBS or sample (423,343.279). | ||
</br> | </br> | ||
− | 2. The average absorbance of control E.coli cells was removed by subtracting the average of the TOP 10 cells with R0040 (222,475). | + | 2. The average absorbance of control <i>E.coli</i> cells was removed by subtracting the average of the TOP 10 cells with R0040 (222,475). |
</br> | </br> | ||
3. These values were divided by the absorbance values at 600nm to give the fluorescence per OD 600 in arbitrary units (Table 6). | 3. These values were divided by the absorbance values at 600nm to give the fluorescence per OD 600 in arbitrary units (Table 6). | ||
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</div> | </div> | ||
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<h2>Conclusion</h2> | <h2>Conclusion</h2> | ||
− | + | <center> | |
+ | <p class="mainText"> | ||
+ | Our measurements have shown that promoters J23101 and J23106 produce more fluorescence with I13504 than the known GFP part I20270 and J23117 shows far less fluoresence, with measurements closer to the negative control R0040 (Figure 8). | ||
</br> | </br> | ||
− | < | + | <br> |
− | <img src="https://static.igem.org/mediawiki/2015/ | + | <center><figure><img src="https://static.igem.org/mediawiki/2015/0/0d/2015-Glasgow-interlab100.png"> |
− | <figcaption> <b>Figure 8</b> | + | <figcaption><b>Figure 8</b> Mean and standard deviation (error bars) for each device and sample.</figcaption></center> |
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</br> | </br> | ||
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<h2>References</h2> | <h2>References</h2> | ||
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<div style="visibility:hidden; height:0;width:0;" class="scrollSurvivability"></div> </p> | <div style="visibility:hidden; height:0;width:0;" class="scrollSurvivability"></div> </p> | ||
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<script> | <script> | ||
Latest revision as of 23:57, 13 November 2015
Interlab Study
Home > Measurement > Interlab Study
Overview
All 2015 iGEM teams have been invited to participate in the Second International InterLab Measurement Study in synthetic biology. Each lab will obtain fluorescence data for the same three GFP-coding devices with different promoters varying in strength. The objective is to assess the robustness of standard parts and the variability of measurements among different research groups using different lab techniques.
Introduction
This year iGEM Glasgow have participated in the InterLab study and Extra Credit. The three devices required were cloned, as specified, and using a plate reader measurements were obtained in absolute units in terms of moles of FAM labelled oligonucleotide.
Release
Individuals responsible for conducting InterLab study
Others who should be credited, e.g., in a publication based on this data
Dates of InterLab Study
Detailed Lab Book
Equipment
Equipment used to acquire measurements
o Incubator – 2cm shaking diameter o BioMate™ 3S Spectrophotometer: Life Science Analyzer – Used to measure absorbance at 600nm of each sample. o Typhoon FLA 9500: GE Healthcare Life Sciences - Wavelength used to excite cells - 475nm. Filter/channel used to capture the light emission from the cells - Filter BPB1 (530DF20).
Spectrophotometer calibration
![](https://static.igem.org/mediawiki/2015/0/07/2015-Glasgow-interlab36.png)
Typhoon FLA 9500 calibration
![](https://static.igem.org/mediawiki/2015/6/62/2015-Glasgow-interlab1.png)
![](https://static.igem.org/mediawiki/2015/a/af/2015-Glasgow-interlab3.png)
![](https://static.igem.org/mediawiki/2015/b/b8/2015-Glasgow-interlab2.png)
Methodology
Protocol for cloning devices
![](https://static.igem.org/mediawiki/2015/7/70/2015-Glasgow-interlab4.jpg)
![](https://static.igem.org/mediawiki/2015/2/21/2015-Glasgow-interlab5.png)
Preparation for measurements
Protocol for measurements
The controls
Protocol for calculating a conversion factor for absolute units
![](https://static.igem.org/mediawiki/2015/8/8d/2015-Glasgow-interlab6.png)
![](https://static.igem.org/mediawiki/2015/f/f2/2015-Glasgow-Interlab20.png)
![](https://static.igem.org/mediawiki/2015/0/0c/2015-Glasgow-interlab8.png)
Sequencing
J23101:I13504
![]( https://static.igem.org/mediawiki/2015/5/54/2015-Glasgow-interlab30.png)
J23106:I13504
![](https://static.igem.org/mediawiki/2015/4/48/2015-Glasgow-interlab31.png)
J23117:I13504
![](https://static.igem.org/mediawiki/2015/8/87/2015-Glasgow-interlab32.png)
Measurements
Direct Measurement
![](https://static.igem.org/mediawiki/2015/d/d0/2015-Glasgow-interlab9.png)
The fluorescence was also measured (figure 7) and the resulting images converted to numerical readings (table 5).
![](https://static.igem.org/mediawiki/2015/7/75/2015-Glasgow-interlab10.png)
![](https://static.igem.org/mediawiki/2015/9/9d/2015-Glasgow-interlab23.png)
Derived Measurements
![](https://static.igem.org/mediawiki/2015/f/ff/2015-Glasgow-interlab21.png)
Estimation of absolute number of GFP molecules per cell
![](https://static.igem.org/mediawiki/2015/9/98/2015-Glasgow-Interlab24.png)
In order to estimate the absolute number of GFP molecules per cell the following calculations were carried out:
ilov stock = 1.35 mg/ml = 1.35 g/l
MW = approx. 150x110 = 16500
ilov stock = 82uM
Avogadro’s number = 6.02x10^23
⇒ Diluted stock 2 fold and used 100 ul in well = 1/20000 litre = 4.1 nmoles
⇒ 4.1 nmoles of phiLOV gave a reading of 490,000,000
J23101:I13504
- So 200 ul cells equivalent to 4.1 *1000/490 = 8.4 nmoles iLOV
- So 8.4 nmoles iLOV gives equivalent fluorescence to 8.4/11.5 = 0.73 nmoles of GFP per 200 ul cells
- So 200 ul cells contains 0.73x10^-9 x (Avogadro’ s number 6.02x10^23) = 4.4x 10^14 molecules
- So 1 cell contains 4.4 x 10^14 / 4 x10^8 = 1 million copies of GFP
- 27,000 x 1.7 x 10^-18 = 4.7 x 10^-14 g = 47 femto grams
- So approximately half of all cellular protein is GFP
J23106:I13504
- So 200 ul cells equivalent to 4.1 *25/49 = 2.09 nmoles iLOV
- so 0.209 nmoles iLOV gives equivalent fluorescence to 2.09/11.5 = 0.181 nmoles of GFP per 200 ul cells so 200 ul cells contains 0.181x10^-9 x (Avogadro’ s number 6.02x10^23) = 1.08x 10^14 molecules
- So 1 cell contains 1.08x10^14 / 4 x10^8 = 272,405 = 270,000 copies of GFP
- 27,000 x 4.48x10^-19 = 1.2 x 10^-14 g = 12 femto grams
- so 12% of cellular protein is GFP
J23117:I13504
- So 200 ul cells equivalent to 4.1 *25/4900 = 0.0206 nmoles iLOV
- so 0.0206 nmoles iLOV gives equivalent fluorescence to 0.0206/11.5 = 1.79x10^-3 nmoles of GFP per 200 ul cells
- so 200 ul cells contains 1.79x10^-12 x (Avogadro’ s number 6.02x10^23) = 1.08x 10^12 molecules
- So 1 cell contains 1.08 x 10^12 / 4 x10^8 = 2,700 copies of GFP
- 27,000 x 4.48x10^-21 = 1.2 x 10^-16 g = 0.12 femto grams
- so 1.2x10^-3 % of all cellular protein is GFP
Conclusion
Our measurements have shown that promoters J23101 and J23106 produce more fluorescence with I13504 than the known GFP part I20270 and J23117 shows far less fluoresence, with measurements closer to the negative control R0040 (Figure 8).
![](https://static.igem.org/mediawiki/2015/0/0d/2015-Glasgow-interlab100.png)
References
Buckley, A. Petersen, J. Roe, A. Douce, G. Christie, J. (2015). LOV-based reporters for fluorescence imaging. Current Opinion in Chemical Biology. 27 (1), p39–45.