Difference between revisions of "Team:Oxford/Proof"

Overview

A key goal of our project was to produce a functional proof of concept of our system, demonstrating that:

1. At least one of our promoter-driven devices was sensitive to copper.
2. Addition of copper to a bacterial population containing this device would induce measurable protein expression.

We have managed to achieve these goals with our part: pCopA MymT sfGFP with constitutive CueR (BBa_K1980012).

Experiments and Results

Our experimental aim was to determine whether GFP expression could be successfully induced over a range of copper concentrations in vitro. We chose to measure GFP expression rather than copper-binding, despite the presence of MymT in the device, as GFP fluorescence is more easily measurable than copper-binding.

The data described below successfully confirms that this does occur, meaning that our device acts as a functional proof of concept for our project.

Plate reader

In order to ensure our data was applicable to our project, we carried out our plate reader experiments at 37°C, gently shaking at 225 rpm. This simulated the temperature of the small intestine, in addition to the action of gastric movement. We also verified that the pH of our growth medium was pH 7.0, close to the pH of the intestinal fluid. Our negative and positive controls were comprised of the negative and positive control plasmids from the Interlab study.

For more information on the settings we used to set up the plate reader, can be found on our experiments page. This method allowed us to to measure the change in fluorescence over time and we used it to see the change in fluorescence, and hence in GFP expression, at different copper concentrations.

From this data, it is clear that there is an increase in GFP expression with increasing copper concentration, showing that our pCopA promoter is sensitive to copper, and that it stimulates protein expression.

Flow cytometry

Flow cytometry data was collected on pCopA MymT sfGFP with constitutive CueR to independently verify the plate reader. To ensure that the data was comparable, bacteria were Cu⁺ stimulated in LB media at 37°C, pH 7.0 and 225rpm. The bacteria were stimulated for 3-4 hrs until they reached the exponential growth phase. By measuring the fluorescence of individual cells, a fluorescence map of the entire population can be generated. For pCopA MymT sfGFP with constitutive CueR, as expected, the flow cytometry data and the plate reader data are in agreement thereby verifying both methods.

Microscopy

Our microscopy experiments were carried out to visually confirm the plate reader and flow cytometry experiments, and to check the cellular localization of the protein. To mimic stomach conditions, colonies were grown in 5ml overnight cultures at 37°C, 225 rpm, pH 7.0, and subsequently pipetted into fresh LB broth and grown until an OD of 0.4-0.6 was reached. Cells were then viewed.

GFP fluorescence is clearly observed at both 0.5mM and 2.0mM, apparently localised in the cytoplasm. This is consistent with the plate reader and flow cytometry experiments, in addition to confirming the literature information stating its localisation to the cytoplasm.

Additional information

Although this does not contribute specifically to our proof of concept, complementary data was provided in the form of FLIM analysis of MymT sfGFP (part of the pCopA MymT sfGFP with constitutive CueR device). This preliminary data suggests that MymT may show copper chelation activity in vivo. For a more in depth discussion of this data, please see our results page.