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 BioBricks 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. We then pipetted 100µl of the diluted cells into the corresponding wells, 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'

Our aim was to prove that the Arabinose Controlled ‘Variable Resistor’ grew in zinc (II) chloride? When arabinose is present, therefore showing that the SmtA is being produced and binding to the zinc (II).

For this experiment, we used a plate reader and measured cell survival at various concentrations of zinc (II) chloride?, with and without arabinose present. The plate was set out as seen in Diagram 2.

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The DH10(α)? cells with the pSB1C3 cells were grown up in liquid culture of LB broth with chloramphenicol overnight at 37°C. The cells were then diluted, using LB broth with chloramphenicol, to an optical density of 0.05 at 600nm.

The zinc chloride was created using XXX of zinc diluted in XXXX? A serial dilution was then made of the zinc chloride in each of the corresponding wells. The cells were then placed in the correct wells.

Arabinose (XXmM) was then added to each well correspondingly.

The results from the experiment can be seen below.

Results

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Battery

To make a microbial fuel cell we followed the Reading University’s protocol, see below.

We sourced the material such as the neoprene gaskets, carbon fibre electrode material, cation-exchange membrane, J-cloth from Professor Ian Head, Dr. Ed Milner and Paniz Izadi from the School of Civil Engineering and Geosciences.

First, we prepared the 1M glucose solution, 0.02M potassium hexacyanoferrate (III) solution, 10mM methylene blue solution, these were made up in a 0.1M potassium phosphate buffer.

Phosphate Buffer

To start we made a stock solution of the two constituents compounds and then we diluted them down.

1M Potassium Hydrogen Phosphate Stock Solution

We dissolved 87.09g of potassium hydrogen phosphate (K2HPO4) in 400ml of distilled water. Once dissolved, this was made up to 500ml with distilled water.

1M Potassium Dihydrogen Phosphate Stock Solution

For the stock solution we dissolved 68.05g of potassium dihydrogen phosphate (KH2PO4) in 400ml of distilled water. This was again, once dissolved, made up to 500ml with distilled water.

0.01M Potassium Phosphate Buffer, pH7.0

For the final potassium phosphate buffer, we mixed 61.5ml of the 1M K2HPO4 stock solution with 38.5ml of the 1M KH2PO4 stock solution. We then added 900ml of distilled water to make up to 1 litre. This buffer was then used to make up the rest of the solutions required for the fuel cell, see below.

10mM of Methylene Blue

For the methylene blue, we dissolved 1.87g in 500ml of the potassium phosphate buffer.

0.02M Potassium hexacyanoferrate (III)

3.39g of potassium hexacyanoferrate (III) was dissolved in 500ml of potassium phosphate buffer. It was then stored in a labelled bottle and wrapped in tin foil.

1M Glucose Solution

First we dissolved 9g of glucose in 50ml of the potassium phosphate buffer. This solution had to be used immediately because it wasn’t sterile and supported the growth of microorganisms, because of this it was the last solution we made.

The four Perspex® components of the fuel cell were then bolted together to make the two compartments of the fuel cell. Neoprene gaskets were provided to prevent leaks from the cell.

Before we start to assemble the fuel cell, we rehydrated the 2.5g dried Baker’s yeast in 5ml of the potassium phosphate buffer. Next, 5ml of the 1M glucose solution was added to the yeast and mixed well.

We then cut out and folded two carbon fibre electrodes, as seen in Figure 2. One electrode was then inserted into each of the chambers made from the Perspex®.

Two pieces of J-cloth were then cut out and placed into each chamber of the fuel cell, on top of the electrodes. This is to prevent the electrodes from touching the cation exchange membrane.

A neoprene gasket was placed on each half of the fuel cell. The two halves were then placed together with the cation exchange membrane sandwiched between them. The two halves were then tightened by passing four bolts and tightened with the wing nuts. Although we were warned not to over-tighten the nuts as it would distort the cell and allow liquid to weep out. We did find that a lot of our liquid leaked out of the cell and we believe it may be due to the over tightening of the nuts.

We added 5ml of 10mM methylene blue solution to the yeast suspension. After stirring the mixture, we used a clean syringe to add the yeast mixture to one half of the fuel cell. In the other half of the fuel cell, we syringed around 10ml of the 0.02M potassium hexacyanoferrate (III) solution. The multimeter was then connected to the electrode terminals using the wires and crocodile clips.

Our results showed that we had an overall voltage of 397mV. Although this result was really impressive, it would not be enough to power our light bulb component of the board. Therefore, we shall now work on improving this part and seeing if we can increase the voltage.

Results

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