Difference between revisions of "Team:Glasgow/Interlab"
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− | Sean Colloms - Supervisor for the InterLab study. Helped with cloning devices, taking measurements of the devices and editing the wiki page. | + | o Sean Colloms - Supervisor for the InterLab study. Helped with cloning devices, taking measurements of the devices and editing the wiki page. |
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− | Vilija Lomeikaite - Set up overnight cultures of the devices | + | o Vilija Lomeikaite - Set up overnight cultures of the devices |
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− | Ye Yang - Designed and formatted the Interlab wiki page | + | o Ye Yang - Designed and formatted the Interlab wiki page |
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− | Andrey Filipov - Carried out the calibration measurements for the spectrophotometer | + | o Andrey Filipov - Carried out the calibration measurements for the spectrophotometer |
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− | James Provan - Sent cloned devices for sequencing </div> | + | o James Provan - Sent cloned devices for sequencing </div> |
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− | 17/08/2015 (Day 1) - Made TOP-10 competent cells using overnights. Got the parts J23101, J23106, J23117, I13504, I20270 and R0040 off the iGEM distributions plates. Transformed the resuspended DNA into the competent cells, plated them with the appropriate antibiotic and grew them overnight. | + | o 17/08/2015 (Day 1) - Made TOP-10 competent cells using overnights. Got the parts J23101, J23106, J23117, I13504, I20270 and R0040 off the iGEM distributions plates. Transformed the resuspended DNA into the competent cells, plated them with the appropriate antibiotic and grew them overnight. |
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− | 18/08/2015 (Day 2) - Picked a single colony of each part, inoculated broth and grew over night. | + | o 18/08/2015 (Day 2) - Picked a single colony of each part, inoculated broth and grew over night. |
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− | 19/08/2015 (Day 3) - Carried out mini preps of each part from overnights and made glycerol stocks. Digested promoters J23101, J23106 and J23117 with Pst1 and Spe1 and I13504 with Xba1 and Pst1. J23106 and I13504 didn't cut correctly therefore set up more overnights to repeat digestion. | + | o 19/08/2015 (Day 3) - Carried out mini preps of each part from overnights and made glycerol stocks. Digested promoters J23101, J23106 and J23117 with Pst1 and Spe1 and I13504 with Xba1 and Pst1. J23106 and I13504 didn't cut correctly therefore set up more overnights to repeat digestion. |
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− | 20/08/2015 (Day 4) - Carried out mini preps I13504 and J23106 and re digested each part. Carried out gel extractions, purifications and ligated each promoter to the GFP part I13504. Set up over nights of TOP-10. | + | o 20/08/2015 (Day 4) - Carried out mini preps I13504 and J23106 and re digested each part. Carried out gel extractions, purifications and ligated each promoter to the GFP part I13504. Set up over nights of TOP-10. |
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− | 21/08/2015 (Day 5) - Transformed TOP-10 cells with each device (I13504:J23101, I13504:J23106 and I13504:J23117) and positive/negative controls and plated them. | + | o 21/08/2015 (Day 5) - Transformed TOP-10 cells with each device (I13504:J23101, I13504:J23106 and I13504:J23117) and positive/negative controls and plated them. |
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− | 24/08/2015 (Day 6) - Set up overnights and restreaks of 1 colony of each device. | + | o 24/08/2015 (Day 6) - Set up overnights and restreaks of 1 colony of each device. |
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− | 25/08/2015 (Day 7) - Prepared samples of each device and controls for measurement by two methods; measuring the A600 of devices in broth and diluting to 0.5 and diluting samples of devices in PBS and measuring their A600 to determine to most accurate method. | + | o 25/08/2015 (Day 7) - Prepared samples of each device and controls for measurement by two methods; measuring the A600 of devices in broth and diluting to 0.5 and diluting samples of devices in PBS and measuring their A600 to determine to most accurate method. |
</br> | </br> | ||
− | 26/08/2015 (Day 8) - Set up overnights of colony 1,2 and 3 of each device, positive and negative control and technical replicants. | + | o 26/08/2015 (Day 8) - Set up overnights of colony 1,2 and 3 of each device, positive and negative control and technical replicants. |
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− | 27/08/2015 (Day 9) - Carried out mini preps of colony 1 of each device and controls and sent for sequencing. Prepared samples of each device (colony1-3) for measurement along with two technical replicants using the PBS method. Carried out GFP fluorescence readings on a 96 well plate of each device, positive and negative control and a dilution series of LOV proteins and FAM labelled oligonucleoutide. | + | o 27/08/2015 (Day 9) - Carried out mini preps of colony 1 of each device and controls and sent for sequencing. Prepared samples of each device (colony1-3) for measurement along with two technical replicants using the PBS method. Carried out GFP fluorescence readings on a 96 well plate of each device, positive and negative control and a dilution series of LOV proteins and FAM labelled oligonucleoutide. |
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− | 28/08/2015 (Day 10) - Calculated absolute values of fluorescence of GFP and submitted the completed InterLab Worksheet and Protocol forms. | + | o 28/08/2015 (Day 10) - Calculated absolute values of fluorescence of GFP and submitted the completed InterLab Worksheet and Protocol forms. |
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− | 31/08/2015 -13/08/2015 – Completed wiki page. | + | o 31/08/2015 -13/08/2015 – Completed wiki page. |
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Revision as of 12:24, 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 Life Science Analyzer: BioMate™ 3S Spectrophotometer – 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)
Measurements
Direct Measurement (Raw Data)
![](https://static.igem.org/mediawiki/2015/d/d0/2015-Glasgow-interlab9.png)
![](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 (Conversion to Absolute units)
![](https://static.igem.org/mediawiki/2015/f/ff/2015-Glasgow-interlab21.png)
![](https://static.igem.org/mediawiki/2015/7/78/2015-Glasgow-Interlab25.png)
Estimation of absolute number of GFP molecules per cell
![](https://static.igem.org/mediawiki/2015/9/98/2015-Glasgow-Interlab24.png)
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
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.