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 </p></b>
 
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        <p>The design has a natural ribosome binding site, which we will be adding to the
 
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        registry (BBa_K1895001). This is to ensure that the ribosome does bind to the DNA
 
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        and synthesise the protein correctly, however we will make variants of this DNA
 
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        with two different medium bicistronic rbs. The medium bicistronic rbs will avoid
 
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        the problem of placing too high a translational burden on the cell. We will then
 
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        test all three variants to determine which is the best rbs to use in the final
 
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        design.</p>
 
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        <h4>Results</h4>
 
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        <p><img src=
 
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        "https://static.igem.org/mediawiki/2016/4/40/T--Newcastle--Bulb-Natural-RBS.png" width=
 
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        "100%" /></p>
 
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        <p><img src=
 
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        "https://static.igem.org/mediawiki/2016/8/80/T--Newcastle--Bulb-Artifical-RBS.png"
 
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        width="100%" /></p>
 
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      <div id="bio-varistor">
 
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        <h3>Arabinose Controlled 'Variable Resistor'</h3>
 
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        <p>We plan to engineer <em>Escherichia coli</em> to behave like a variable
 
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        resistor. We aim to do this by using <em>E. coli</em> to vary the amount of free
 
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        ions in an electrolyte. Ion uptake will be controlled by the expression of smtA.
 
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        SmtA is a metallothionein that can bind to heavy metal ions like cadmium (II),
 
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        Zinc (II) and Copper (II).</p>
 
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        <p>SmtA has been used in a number of iGEM projects and is in the registry
 
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        (<a href="http://parts.igem.org/Part:BBa_K519010">BBa_K519010</a>). It has
 
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        previously been used in experiments for Cadmium (II) uptake, see <a href=
 
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        "https://2011.igem.org/Team:Tokyo-NoKoGen/metallothionein">Tokyo-NokoGen 2011</a>.
 
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        We will be examining firstly, the impact of smtA of Zinc (II) concertation rather
 
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        than Cadmium (II) and then the impact that this has on the resistivity of the
 
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        Zinc (II) containing media. In this instance we will be using Zinc sulfate
 
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        (ZnSO<sub>4</sub>) in solution where it disassociates into Zn<sup>2+</sup> and
 
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        SO<sub>4</sub><sup>2-</sup> ions. Various concentrations of Zinc sulfate have
 
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        <a href=
 
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        "http://sites.chem.colostate.edu/diverdi/all_courses/CRC%20reference%20data/electrical%20conductivity%20of%20aqueous%20solutions.pdf">
 
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        known electrical conductivity</a> . When smtA is expressed it will render the
 
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        Zn<sup>2+</sup> unavailable and thereby reduce the conductivity of the
 
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        solution.</p>
 
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        <p>We will be placing smtA under the control of an AraC regulated promoter
 
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        allowing the expression of smtA to be controlled by the addition or removal of
 
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        arabinose.</p>
 
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        <h4>Results</h4>
 
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              <td></td>
 
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              <td><img width="355" height="193" src=
 
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              "https://static.igem.org/mediawiki/2016/7/7f/T--Newcastle--YFP_FIXJ.png" /></td>
 
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        <p><br clear="all" />
 
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        We will be placing smtA under the control of a FixJ-P (phosphorylated FixJ)
 
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        promoter. This allows it to be regulated by blue light through a series of
 
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        reactions with its response regulator protein YF1 (below).</p>
 
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        <p>In the absence of light, YF1 undergoes autophosphorylation to produce YF1-P
 
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        which can then phosphorylate FixJ. This in turn activates the transcription of
 
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        the downstream protein, in this case it is SmtA. Thus, in the presence of light
 
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        SmtA is not produced and so conductivity does not change, whilst in the absence
 
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        of light SmtA is produced resulting in a decrease in resistance.</p>
 
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        <p>Clearly, this behaviour is the inverse of an electrical light dependent
 
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        resistor where resistance increases with light intensity. To mimic this behaviour
 
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        using biological circuits we would place an inverter before the FixK2 promoter
 
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        (which is activated by FixJ-P). The inverter is constructed by placing the
 
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        desired output, here SmtA, under the control of a lambda cl regulated promoter
 
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        (BBa_R0051). As lambda cl represses the promoter having this produced under
 
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        control of FixK2 promoter inverts the system so that SmtA is produced in the
 
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        presence of light rather than the absence thereof. <a href=
 
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        "http://parts.igem.org/Part:BBa_K592020">BBa_K592020</a> is an example of a part
 
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        that uses this technique.</p>
 
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        <h3>Battery</h3>
 
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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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<ol>
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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.