Model and manufacturer:
o Incubator – 2cm shaking diameter
o Spectrometer – 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).
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A dilution series was measured for phiLOV protein (Figure 2), converted to numerical readings (Table 2) and calibration curve (Figure 3) carried out to calibrate the Typhoon. | A dilution series was measured for phiLOV protein (Figure 2), converted to numerical readings (Table 2) and calibration curve (Figure 3) carried out to calibrate the Typhoon. | ||
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| + | <figcaption>Figure 2: Fluorescence readings of a dilution series of phiLOV. 67.5µg = 67.5µg phiLOV in 100µl PBS. Each concentration was carried out twice. | ||
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Revision as of 15:25, 8 September 2015
Glasglow
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.
Equipment
Equipment used to acquire measurements
Typhoon FLA 9500 calibration
A dilution series was measured for phiLOV protein (Figure 2), converted to numerical readings (Table 2) and calibration curve (Figure 3) carried out to calibrate the Typhoon.
Figure 2: Fluorescence readings of a dilution series of phiLOV. 67.5µg = 67.5µg phiLOV in 100µl PBS. Each concentration was carried out twice.
Methodology
Protocol for cloning devices
The devices, as shown in Table 1, were prepared using BioBrick assembly. Parts J23101, J23106, J23117, I13504, I20270 and R0040 were taken from the iGEM distribution plates and each transformed into TOP-10 competent cells. The promoters were digested with Pst1 and Spe1 and the GFP part, I13504, was digested with Xba1 and Pst1. The I13504 part was then ligated into each promoter plasmid and transformed into TOP-10 cells to create the three required devices in pSB1C3. Restreaks were carried out for one colony of each device and control and three colonies of each (labelled 1, 2 and 3) were picked and grown separately. Sequencing was carried out to check the correct devices had been created.
Protocol for measurements
The spectrometer was used to measure absorbance at 600nm of each sample. Samples were then diluted to 0.5 with PBS and rescanned. The Typhoon was used to measure the GFP fluorescence at 475nm of each device and control on a 96 well plate. These methods were repeated for each biological and technical replicate.
Protocol for calculating a conversion factor for absolute units
A dilution series of FAM labelled oligonucleotide was measured (Figure 4) and converted to numerical readings (Table 3) to enable absolute values for the devices to be calculated. The calibration curve (Figure 5) shows a y intercept of 4.797E+06x. Therefore the fluorescence readings of the devices will be divided by the conversion factor of 4,790,000 to give absolute fluorescence as equivalent to pmol of FAM labelled oligonucleotide. Absolute values should be comparable across different equipment and protocols.
Measurements
Direct Measurement (Raw Data)
The A600 of each device colony 1-3 and technical replicates were measured along with the controls (table 4).
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Protocol for calculating a conversion factor for absolute units
A dilution series of FAM labelled oligonucleotide was measured (Figure 4) and converted to numerical readings (Table 3) to enable absolute values for the devices to be calculated. The calibration curve (Figure 5) shows a y intercept of 4.797E+06x. Therefore the fluorescence readings of the devices will be divided by the conversion factor of 4,790,000 to give absolute fluorescence as equivalent to pmol of FAM labelled oligonucleotide. Absolute values should be comparable across different equipment and protocols.
Contamination
Future Considerations
Size
In hindsight, it was thought the toy may be slightly too large to fit comfortably on a child’s windowsill. If mass production were to be considered, a scaled down version of the toy would not affect its functionality as a pet, a light source or an educational tool, but would mean it would be more comfortable on a small ledge, as well as less material being consumed in its production and less broth being needed to fill it, bringing down costs. The larger model is, however, better for demonstrating and displaying the different components.
Alternatives
Sea monkey
“Sea Monkeys” were a very popular novelty pet during the 50s and 60s after their invention by Harold von Braunhut in 1957, and still sell well today. They are a hybrid of different brine shrimp within the Artemia species, named Artemia NYOS, which can exist as eggs in suspended animation for an extremely long time. Once poured into purified salt water they hatch “instantly”.
Pros
Sea Monkeys are large enough to see swimming around once they are fully grown, unlike our bacteria which would remain too small to be visible to the naked eye. They also only need to be feed once every few days, where our bacteria would need to be feed every day. In addition they do not pose any risk to the environment in the event of release, which is something that is theoretically true of our bacteria, however the small risk of mutation or other adverse effect will always be present.
Cons
Sea Monkeys do not require a UVA source, however they do not glow in the dark, which gives an extra element of functionality to our toy.
So yes… Sea Monkeys have a considerable degree of advantage over our product in terms of ease of use and maintenance. There is also the toss-up between whether they glow or are visible individually to the naked eye… but what if we combined them? A future iGEM team idea could be to create glow in the dark Sea Monkeys…!
Read More!
