Difference between revisions of "Team:Newcastle/Notebook/Lab"

 
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<h1 id="teams">Lab Book</h1>
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<h2 id="20/06/16">20/06/16</h2>
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<p>After the interlab study, we made streak plates from the colonies we had grown. We regrew all the samples on LB agar with 1 in 1000 dilution of Chloramphenicol. We did this to isolate a pure strain of the transformed interlab E. coli, therefore allowing us to grow up a new, genetically-identical plate. Our lab supervisor, Matthew Peake, showed us the correct streaking technique as the Computer Science students had not learnt this technique before.
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<h2 id="22/06/16">22/06/16</h2>
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<p>After analysing the trial interlab results, we decided to re-plate up the positive control to ensure that we would have enough colonies to carry out the interlab study again.
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<h2 id="28/06/16">28/06/16</h2>
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<p>Today we made a microbial fuel cell by following the Reading University’s protocol, see below.
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<p>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. We also sourced electric wires with crocodile clips and a multimeter from the Engineering Departments.
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<p>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.
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<p><u>Phosphate Buffer</u></p>
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<p>To start we made a stock solution of the two constituents compounds and then we diluted them down.</p>
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<p><i>1M Potassium Hydrogen Phosphate Stock Solution</i></p>
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<p>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.</p>
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<p><i>1M Potassium Dihydrogen Phosphate Stock Solution</i></p>
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<p>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.</p>
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<p><i>0.01M Potassium Phosphate Buffer, pH7.0</i></p>
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<p>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.</p>
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<u><p>10mM of Methylene Blue</u></p>
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<p>For the methylene blue, we dissolved 1.87g in 500ml of the potassium phosphate buffer. </p>
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<u><p>0.02M Potassium hexacyanoferrate (III)</u></p>
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<p>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.
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<u><p>1M Glucose Solution</u></p>
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<p>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.
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<p>The four <i>Perspex</i>® components of the fuel cell were then bolted together to make the two compartments of the fuel cell, Figure 1. Neoprene gaskets were provided to prevent leaks from the cell.</p>
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<p>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. </p>
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<p>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 <i>Perspex</i>®.</p>
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<p>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.</p>
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<p>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.</p>
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<h3>Lab Notes</h3>
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<p>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.</p>
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<p> In the links presented below are the lab diaries for each individual team.</p>
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<p>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.</p>
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<h2><a href="https://2016.igem.org/Team:Newcastle/Notebook/Lab/AlternativeConstructs">Alternative Constructs Diary</a></h2>
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<p>Today, we also grew up some liquid cultures for the interlab study, which we then left to incubate over-night at 37&deg;C at 220rpm.</p>
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<h2><a href="https://2016.igem.org/Team:Newcastle/Notebook/Lab/Lightbulb">Lightbulb Diary</a></h2>
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<h2 id="29/06/16">29/06/16</h2>
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<h2><a href="https://2016.igem.org/Team:Newcastle/Notebook/Lab/Microfluidics">Microfluidics Diary</a></h2>
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<h2><a href="https://2016.igem.org/Team:Newcastle/Notebook/Lab/Component">Measuring GFP Expression using off-the-shelf Hardware Write-up</a></h2>
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<p>The interlab was carried out on the 29th of July. This was a practise run as our Sample 2 had not transformed well. We believe this may have been due to the fact that the competent cells had been carried over from one building to another and not been on dried ice. After a lot of confusion with the protocol, we managed to get the interlab up and running. It was good to have this practise run as we now know what to do for the final run. For example, we were confused by having to dilute down to an OD600 of 0.02, we now know to do this quickly and have a rough idea of what dilution to make.</p>
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<h2 id="30/06/16">30/06/16</h2>
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<p>Liquid cultures were regrown overnight, until they were required again for the interlab study.</p>
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<p>The interlab was carried out again. This time, we used the iGEM interlab protocol exactly, as well as using a new plate reader that our lab had on loan. The ThermoScientific Varioskan Lux Plate Reader had the ability to shake and incubate, so we were able to run for the full six hours without interrupting the cycle. </p>
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Latest revision as of 03:20, 20 October 2016

Lab Notes

In the links presented below are the lab diaries for each individual team.

Alternative Constructs Diary

Lightbulb Diary

Microfluidics Diary

Measuring GFP Expression using off-the-shelf Hardware Write-up