Difference between revisions of "Team:Newcastle/Proof"

Electrically Induced 'Light Bulb'

In order to prove that our initial aims were correct; i.e. to induce GFP production by heat shocking the E. coli, we used a plate reader to measure cell growth and fluorescence over a 24 hour period.

Our 96 well plate was set up as seen below in Diagram 1.

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All BioBrick samples were placed in the same pSB1C3 backbone and BL21? cells. The cells were grown up in liquid culture of LB broth with chloramphenicol overnight at 37°C. The following day, the cells were diluted down to an optical density of 0.05 at 600nm using LB broth with chloramphenicol. The cells were then laid out accordingly with Diagram 1. The cells were laid out in this manner, with a border of LB broth, to allow for any inaccuracies that may occur because the plate reader heats the plate from the outside in.

The plate reader was then set at either 30°C, 37°C and 42°C and measured for growth using OD600 and fluorescence of GFP using 485nm excitation wavelength and 520nm emission wavelength. The cells were left to grow for 24 hours and measured every five minutes by the plate reader. In between measurements, the plate reader was programmed to shake to ensure the cells didn’t clump together.

The results from the experiment can be seen below.

Results

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Arabinose Controlled 'Variable Resistor'

We plan to engineer Escherichia coli to behave like a variable resistor. We aim to do this by using E. coli to vary the amount of free ions in an electrolyte. Ion uptake will be controlled by the expression of smtA. SmtA is a metallothionein that can bind to heavy metal ions like cadmium (II), Zinc (II) and Copper (II).

SmtA has been used in a number of iGEM projects and is in the registry (BBa_K519010). It has previously been used in experiments for Cadmium (II) uptake, see Tokyo-NokoGen 2011. We will be examining firstly, the impact of smtA of Zinc (II) concertation rather than Cadmium (II) and then the impact that this has on the resistivity of the Zinc (II) containing media. In this instance we will be using Zinc sulfate (ZnSO₄) in solution where it disassociates into Zn²⁺ and SO₄^2- ions. Various concentrations of Zinc sulfate have known electrical conductivity . When smtA is expressed it will render the Zn²⁺ unavailable and thereby reduce the conductivity of the solution.

We will be placing smtA under the control of an AraC regulated promoter allowing the expression of smtA to be controlled by the addition or removal of arabinose.

Results

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Battery

Results

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