Difference between revisions of "Team:Newcastle/Proof"

 
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      <h2>Proof of Concept</h2>
 
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      <div id="bio-bulb">
 
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        <h3>Electrically Induced 'Light Bulb'</h3>
 
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        <p>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.</p>
 
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        <p> Our 96 well plate was set up as seen below in Diagram 1.</p>
 
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        <p><b> INSERT DIAGRAM (see google drive) </p></b>
 
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        <p>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.  </p>
 
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        <p>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. </p>
 
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        <p>The results from the experiment can be seen below. </p>
 
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        <h4>Results</h4>
 
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        <p>INSERT GRAPHS</p>
 
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        <p>INSERT GRAPHS</p>
 
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        <h3>Arabinose Controlled 'Variable Resistor'</h3>
 
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        <p>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). </p>
 
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        <p>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. </p>
 
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        <p>INSERT DIAGRAM OF THE PLATE LAYOUT</p>
 
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        <p>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. </p>
 
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        <p>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.</p>
 
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        <p>Arabinose (XXmM) was then added to each well correspondingly. </p>
 
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        <p>The results from the experiment can be seen below. </p>
 
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        <h4>Results</h4>
 
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      <p> INSERT GRAPHS </p>
 
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        <h3>Battery</h3>
 
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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. Neoprene gaskets were provided to prevent leaks from the cell.</p>
 
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<p><img src="https://static.igem.org/mediawiki/2016/3/32/T--Newcastle--MFC-Construction-A.jpg" /></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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<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>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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          <h4>Results</h4>
 
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          <p> INSERT GRAPHS </p>
 
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<h2>Proof of Concept</h2>
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<p>In order to fulfill the proof of concept gold medal criteria we tested our devices (<a href="http://parts.igem.org/Part:BBa_K1895000">BBa_K1895000</a>, <a href="http://parts.igem.org/Part:BBa_K1895006">BBa_K1895006</a>, <a href="http://parts.igem.org/Part:BBa_K1895004">BBa_K1895004</a>) in components compatible with our modular breadboard.</p>
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<p>The functional proof of concept of our project was demonstrated by integrating three of our BioBricks in the following devices: </p>
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<li>Microbial Fuel Cell</li>
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<li>Heat Induced ‘Light Bulb’</li>
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<h3>Microbial Fuel Cell</h3>
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<p>We designed a miniature fuel cell part compatible with the modular design of our breadboard circuit system which we had spent the summer designing.<a href="https://2016.igem.org/Team:Newcastle/Hardware"> Our design procedure can be seen here</a>.
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<figure><img alt="proof1" src="https://static.igem.org/mediawiki/2016/7/7c/T--Newcastle--proof1.png"></figure>
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<p><figcaption>Figure 1. The construction of our miniature microbial fuel cell component using a 3D printed mold and PDMS gel.
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</figcaption></p>
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<p>This miniature device allowed us to test our construct <a href="http://parts.igem.org/Part:BBa_K1895004">BBa_K1895004</a> under the real world conditions in which it would be used. The miniature device was made using a 3D printed mold made of Poly Lactic Acid (PLA) designed on TinkerCad and cast using Poly Dimethyl Siloxane (PDMS) gel. This device can be attached to our modular breadboard kit using magnets, which will also allow electrical flow.</p>
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<p>In order to test this miniature device, the protocol previously used to test our constructs in the Reading microbial fuel cell, had to be edited. Our new protocol was appropriately scaled down and the same buffers were used.<a href="https://2016.igem.org/Team:Newcastle/Protocols">The full version can be seen here.</a></p>
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<p>We successfully measured a voltage output from the miniature fuel cell containing <i>E. coli</i> with our BioBrick device (<a href="http://parts.igem.org/Part:BBa_K1895004">BBa_K1895004</a>) inserted. The results can be seen below. <a href="https://2016.igem.org/Team:Newcastle/Proof/MFC">You can also view all results concerning this part here.</a><p>
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<figure><img alt="proof2" src="https://static.igem.org/mediawiki/2016/9/9c/T--Newcastle--proof2.png"></figure>
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<p><figcaption>Figure 2. Output of our microfluidic microbial fuel cell (mean±SE, mV) using the <a href="http://parts.igem.org/Part:BBa_K1895004">BBa_K1895004</a> construct undergoing porin expression. Solutions were made up as per the larger fuel cell, thoroughly mixed and injected by syringe to fill each chamber following insertion of the cation exchange membrane. Voltages were measured every 3 minutes via digital voltmeter and the experiment stopped after 60 minutes. For more information on how we designed the miniature fuel cell, <a href="https://2016.igem.org/Team:Newcastle/Hardware">please see our hardware design page</a>
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</figcaption></p>
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<h3>Heat Induced ‘Light Bulb’</h3>
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<p>Similarly to the battery constructs, we planned to test our constructs (<a href="http://parts.igem.org/Part:BBa_K1895000">BBa_K1895000</a> and <a href="http://parts.igem.org/Part:BBa_K1895006">BBa_K1895006</a>) using a microfluidics style device that will be integrated into our modular breadboard using custom built components.</p>
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<figure><img alt="proof3" src="https://static.igem.org/mediawiki/2016/d/d6/T--Newcastle--proof3.png"></figure>
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<p><figcaption>Figure 3. The modular light bulb component compatible with our breadboard.</figcaption></p><p>We have previously shown that both of our ‘light bulb’ constructs can be induced with a temperature of 37&deg;C but this induction is intensified with an even higher temperature of 42&deg;C. In order to prove our concept we first started by attempting to create a heating effect on LB broth within our microfluidics chamber using an electrical current. We timed how long it took to cause a 15&deg;C change in the LB media, enough to induce the promoters in both of our ‘light bulb’ constructs. We tested times at varying currents from 8 to 20 mA; the times were relatively short with the heating taking less than 60s on many occasions.</p>
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<figure><img alt="proof4" src="https://static.igem.org/mediawiki/2016/9/92/T--Newcastle--proof4.png"></figure>
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<p><figcaption>Figure 4. Time taken in seconds to cause a 15&deg;C increase in temperature of 250&mu;l of LB broth in our microfluidic light bulb component with varying currents(mA).</figcaption></p>
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<p>This result along with the previous data collected regarding the effect of temperature on <i>E. coli</i> containing our construct (<a href="https://2016.igem.org/Team:Newcastle/Proof/ElectricallyInducedLightBulb">seen here</a>) demonstrates our proof of concept nicely.</p>
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Latest revision as of 23:58, 19 October 2016



Proof of Concept

In order to fulfill the proof of concept gold medal criteria we tested our devices (BBa_K1895000, BBa_K1895006, BBa_K1895004) in components compatible with our modular breadboard.

The functional proof of concept of our project was demonstrated by integrating three of our BioBricks in the following devices:

  1. Microbial Fuel Cell
  2. Heat Induced ‘Light Bulb’

Microbial Fuel Cell

We designed a miniature fuel cell part compatible with the modular design of our breadboard circuit system which we had spent the summer designing. Our design procedure can be seen here.

proof1

Figure 1. The construction of our miniature microbial fuel cell component using a 3D printed mold and PDMS gel.

This miniature device allowed us to test our construct BBa_K1895004 under the real world conditions in which it would be used. The miniature device was made using a 3D printed mold made of Poly Lactic Acid (PLA) designed on TinkerCad and cast using Poly Dimethyl Siloxane (PDMS) gel. This device can be attached to our modular breadboard kit using magnets, which will also allow electrical flow.

In order to test this miniature device, the protocol previously used to test our constructs in the Reading microbial fuel cell, had to be edited. Our new protocol was appropriately scaled down and the same buffers were used.The full version can be seen here.

We successfully measured a voltage output from the miniature fuel cell containing E. coli with our BioBrick device (BBa_K1895004) inserted. The results can be seen below. You can also view all results concerning this part here.

proof2

Figure 2. Output of our microfluidic microbial fuel cell (mean±SE, mV) using the BBa_K1895004 construct undergoing porin expression. Solutions were made up as per the larger fuel cell, thoroughly mixed and injected by syringe to fill each chamber following insertion of the cation exchange membrane. Voltages were measured every 3 minutes via digital voltmeter and the experiment stopped after 60 minutes. For more information on how we designed the miniature fuel cell, please see our hardware design page

Heat Induced ‘Light Bulb’

Similarly to the battery constructs, we planned to test our constructs (BBa_K1895000 and BBa_K1895006) using a microfluidics style device that will be integrated into our modular breadboard using custom built components.

proof3

Figure 3. The modular light bulb component compatible with our breadboard.

We have previously shown that both of our ‘light bulb’ constructs can be induced with a temperature of 37°C but this induction is intensified with an even higher temperature of 42°C. In order to prove our concept we first started by attempting to create a heating effect on LB broth within our microfluidics chamber using an electrical current. We timed how long it took to cause a 15°C change in the LB media, enough to induce the promoters in both of our ‘light bulb’ constructs. We tested times at varying currents from 8 to 20 mA; the times were relatively short with the heating taking less than 60s on many occasions.

proof4

Figure 4. Time taken in seconds to cause a 15°C increase in temperature of 250μl of LB broth in our microfluidic light bulb component with varying currents(mA).

This result along with the previous data collected regarding the effect of temperature on E. coli containing our construct (seen here) demonstrates our proof of concept nicely.