• Interlab Study: Measurement
• Methodology
• Results
• Discussion/ Difficulties

Overview

As a team we decided that we would do some extra work during our time doing the iGEM project this such that we entered to take part in the Interlab Measurement study. The study requires us to look at the fluorescence of given gene circuits made up of parts found in the 2015 Distribution Kits.

The study requires producing 3 devices each consisting of a promoter sequence (depicted as different coloured arrows on Figure 1) and Green Fluorescence Protein (GFP) intermediate (I13504). Each of the devices are shown below:

• Device 1 - J23101 + I13504


• Device 2 - J23106 + I13504


• Device 3 - J23117 + I13504

As part of the Interlab study, we then did extra credit where we had get biological and technical replicates of each device. They have been defined as such:

• Biological Replicate -


• Technical Replicate -

Methodology

In order to get the

Results

Fluorescence results for the Interlab study were obtained using the BMG LABTECH FLUOstar Omega Microplate Reader. We placed all the replicates (technical and biological) on a black based 96 well plate. Results were obtained in the format of arbitrary fluorescence unit/OD 600

Microplate Reader Settings

• Emission - EMS20
• Excitation - 485-12nm
• No. of flashes per well- 20

All results from each device were then placed in the format of graphs as such:

Discussion and Difficulties

From the results obtained it can be seen that Device 1 had the highest fluorescence value, this was also confirmed by visually looking at the bacteria that consisted of Device 1. This is excluding the lower fluorescence value of the biological replicate 2, which may just be an anomaly.

However, Device 3 was found to have very minute fluorescence, this was observed visually but also from the data that we obtained from the fluorescence readings. One reason there is a very minute fluorescence could be due to background fluorescence from the TBE buffer that we used rather than actual fluorescence. Another is the possibility that there was no device in the bacteria. To analytically prove that the Device 3 that we had made from ligating the parts was accurate we had done restriction digests of Device 3 using enzymes …………… and then done gel electrophoresis. From the figure below you can see that both parts were present such we can conclude that the promoter must be very weak and such cannot be detected. If more time was on hand we would have been able to have sequenced the plasmid itself.

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