Team:Leicester/Measurement

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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 - This is where there are different biological material in each replicate

  • Technical Replicate - This is where the biological material of each replicate is the same. For example looking at a plate consisting of a particular device, the colonies that are very close to one another are assumed to be made up of the same biological material such they are inoculated three times.

Methodology

In order to conudct the measurement study we had to use a range of different methods. This included:

  • Transformations: Transform genetic material into competent bacteria. In our case we chemically induced the bacteria with CaCl2
  • Minipreps: Required in isolating the required plasmid, for example the plamsids consisting of ligated parts. It is hoped that using this method will allow us to obtain the largest concentration of plasmid DNA, which can be used when doing digests and gels
  • Nanodrops: Allows to determine the amount of DNA present in a given solution, the higher present will
  • Ligations: Use of ligase to ligate particular parts together
  • Digestions: Digesting parts from plasmid backbone and of ligated parts with the use of restriction enzymes. Enzymes include Pst1, EcoR1, Spe1 and Xba1
  • Gel electrophoresis: To determine the fragments produced from restriction digests of the ligated parts to ensure that the parts we require were ligated together.

When conducting the Interlab study we worked to ensure that we followed the methods outlined by iGEM and other protocols known to work for measurements done. However when conducting the repeats for Device 3 we used different intermediates for example use of TGE buffer and Luria broth.

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:

Device 1

Device 1 Graph

Mean of BR/TR - 48965

Standard Deviation of BR - 37511

Standard Deviation of TR - 2670


Device 2

Device 2 Graph

Mean of BR/TR - 711

Standard Deviation of BR - 730

Standard Deviation of TR - 70


Device 3

Device 3 Graph

Mean of BR/TR - 87

Standard Deviation of BR - 7.24

Standard Deviation of TR - 7.63

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 but also from the data that we have obtained. 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 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 EcoR1 and Pst1 and then done gel electrophoresis. From the figure to the left, you can faintly see a fragment which is of about 900bp showing that the GFP and promoter are ligated together 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.