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> We aimed to engineer Escherichia coli that it increases fluoresce when an electrical current is passed through the growth medium, via the use of inducible promoters that respond to the heat-stress created by an electrical current.</p>
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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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<p> We designed two parts (BBa_K1895000 and BBa_K1895006) which respond to the heat-stress in two different ways:</p>
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<li>BBa_K1895000 contains a HtpG promoter which is induced by a sigma-factor (&sigma;<sup>32</sup>). This sigma factor is produced by cells when under different forms of stress. This part also contains a &sigma;<sup>32</sup> coding region which should create a positive feedback loop and therefore increase fluorescence.</li>
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<li>Microbial Fuel Cell</li>
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<li>BBa_K1895006 contains a DnaK promoter which is induced by dnaK, a product of other stress related responses within the 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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      <div id="bio-varistor">
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</figcaption></p>
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        <h3>Arabinose Controlled 'Variable Resistor'</h3>
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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>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>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>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><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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        <p>INSERT DIAGRAM OF THE PLATE LAYOUT</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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        <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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<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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        <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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<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>Arabinose (XXmM) was then added to each well correspondingly. </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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        <p>The results from the experiment can be seen below. </p>
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        <h4>Results</h4>
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<p>To make a microbial fuel cell we followed <a href="https://2016.igem.org/Team:Newcastle/Notebook/Lab/Protocols/#Microbial-fuel-cell">the Reading University’s protocol</a> </p>
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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.</p>
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          <h4>Results</h4>
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          <p> INSERT GRAPHS </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.