Difference between revisions of "Team:Glasgow/Interlab"
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<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"> | ||
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+ | <a href="https://static.igem.org/mediawiki/2015/f/f0/2015-Glasgow-interlab34.pdf" target="_blank">Sequencing results can be found here</a> | ||
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Revision as of 20:33, 18 September 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/2015/2/24/2015_InterLab_13.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
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
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.