Difference between revisions of "Team:Newcastle/Proof"

 
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<h2>Proof of Concept</h2>
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        <h3><a href="https://2016.igem.org/Team:Newcastle/Proof/ElectricallyInducedLightBulb">Electrically Induced 'Light Bulb'</a></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 (<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>) 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><a href="https://2016.igem.org/Team:Newcastle/Proof/VariableResistor">Arabinose Controlled Variable Resistor</a></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>We aimed to create a biological “variable resistor” by modifying the E. coli’s natural systems to allow for controlled ion uptake. In order to do so, we looked at the work carried out by the Tokyo-NokoGen iGEM Team in 2011 who used SmtA gene from Cyanobacteria and inserted it into a strain of E. coli. SmtA is thought to play a role in preventing heavy metal toxicity by binding excess heavy metal ions such as Cadmium (II), shown by Tokyo-NokoGen, or Zinc (II). </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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        <p>We took the SmtA part (BBa_K519010) and put it under the control of a pBAD promoter, induced by the presence of L-arabinose. This should allow us to control the uptake of Zinc ions by adding or removing L-arabinose, resulting in control over the resistance of the LB media.</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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        <h3><a href="https://2016.igem.org/Team:Newcastle/Proof/MFC">Microbial Fuel Cell</a></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>We aimed to look at different ways of improving the voltage output of a microbial fuel cell. At first we looked at yeast microbial fuel cells with the help of Dr Ed Milner, Dr Paniz Izadi and Professor Ian Head, but after talking with PEALS (link to the PEALS talk) we decided to move away from using yeast and looked at working with E. coli instead. </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>For inspiration we looked at the Bielefeld 2013 iGEM Team (link to their MFC page). One of the issues we noticed with their design was that their porin overexpression protein was taken from Pseudomonas fluorescens and so the pores size was too large for the E. coli to handle. We changed this by overexpressing E. coli’s natural porin producing genes, OmpF. Bielefeld also had issues with cell growth due to the metabolic stress of using a T7 promoter. To improve this part we used a pBAD promoter to allow the cell population to grow before inducing the porin overexpression. JOSH CAN YOU LOOK AT THIS!!!</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.