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| − | <body data-spy="scroll" data-target=".col-md-4">
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| − | <div class="column">
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| − | <div class="row">
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| − | <div class="col-md-8">
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| − | <h2>Proof of Concept</h2>
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| − |
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| − | <div id="bio-bulb">
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| − | <h3>Electrically Induced 'Light Bulb'</h3>
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| − |
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| − | <p>We plan to clone <em>Escherichia coli</em> so that it fluoresces when a
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| − | current of around 400mA is passed through the broth that the <em>E. coli</em> are
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| − | growing in.</p>
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| − |
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| − | <p>Previous attempts at this experiment have used GFP to fluoresce with the
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| − | current. In terms of this experiment we shall use GFP to ensure the design
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| − | construct works. We have incorporated restriction sites within the construct so
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| − | that we are able to then switch the GFP with other constructs, such as luciferase
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| − | to emit light like an actual lightbulb.</p>
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| − |
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| − | <p>In order for the GFP to be activated by a current, a heat-shock induced
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| − | promoter must be present. Previous experiments have used sigma 32 as a heat shock
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| − | promoter which seems to have the desired effect, although we shall test this in
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| − | the lab. We have also inserted two restriction sites to allow us to either cut
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| − | out the sigma 32 promoter, or add an extra promoter. This will allow us to test
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| − | the effect that the sigma 32 has on the GFP production and whether it is induced
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| − | by heat-shock or not.</p>
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| − |
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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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| − |
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| − | <h4>Results</h4>
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| − |
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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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| − |
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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>
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| − |
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| − | <div id="bio-varistor">
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| − | <h3>Arabinose Controlled 'Variable Resistor'</h3>
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| − |
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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),
| |
| − | 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
| |
| − | (<a href="http://parts.igem.org/Part:BBa_K519010">BBa_K519010</a>). It has
| |
| − | previously been used in experiments for Cadmium (II) uptake, see <a href=
| |
| − | "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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| − |
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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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| − |
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| − | <h4>Results</h4>
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| − |
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| − | <p>We will be synthesising two variants of our construct, one with and one
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| − | without an ssRA degradation tag. This will allow us to see if we gain finer
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| − | control over the resistance by increasing the rate of protein degradation. Both
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| − | variants of the construct will include a polyhistidine-tag to allow for protein
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| − | purification from cultures.</p><img width="602" height="139" src=
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| − | "https://static.igem.org/mediawiki/2016/2/22/T--Newcastle--Varistor.png" border="0" />
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| − |
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| − | <p>BBa_R0080</p>
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| − |
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| − | <p>This is the AraC regulated promoter. The way this promoter behaves is that
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| − | transcription takes place in the presence of AraC or arabinose. Without arabinose
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| − | present there should be no transcription.</p>
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| − |
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| − | <p>BBa_B0034</p>
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| − |
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| − | <p>This is a standard RBS based that used in the construction of the
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| − | repressilator. (Elowitz, 1999). It has an efficiency of 1. Whilst not the most
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| − | efficient RBS it has a high efficiency and is widely used in iGEM projects. It is
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| − | also present in the 2016 distribution kit.</p>
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| − |
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| − | <h3>BBa_K519010</h3>
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| − |
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| − | <p>This is the coding sequence for SmtA originally from <em>Synechococcus
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| − | sp</em>, a cyanobacterial strain.</p>
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| − |
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| − | <h3>LVA-TAG</h3>
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| − |
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| − | <p>This is an ssRA protein degradation tag. Tagged proteins are degraded by the
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| − | proteases ClpXP or ClpAP. There are a number of tag sequences, variants of
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| − | AANDENYALAA, with the last three amino acids varying. The last three amino acids
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| − | determine the half-life of the protein. LVA is a fast protein degradation tag. We
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| − | use this to ensure that the resistance is reduced quickly after removal of
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| − | arabinose.</p>
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| − |
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| − | <h3>BBa_B1006</h3>
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| − |
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| − | <p>This is an artificial terminator part and was chosen because it has a high
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| − | forward efficiency of 0.99.</p>
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| − |
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| − | <h3>pSB1C3</h3>
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| − |
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| − | <p>We are using the standard BioBrick backbone part pSB1C3 as this will make it
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| − | easier to submit the part to the registry at a later date.</p>
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| − |
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| − | <p>We have included restriction sites around protein and tag so that it can be
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| − | replaced with a part without the tag to see if this has any effect on Zinc
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| − | uptake.</p>
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| − | </div>
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| − |
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| − | <div id="bio-ldr-red">
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| − | <h3>OmpR Controlled Red 'Light Dependent Resistor'</h3>
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| − |
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| − | <p>We plan to engineer <em>Escherichia coli</em> to behave like a light dependent
| |
| − | resistor. We aim to do this by using <em>E. coli</em> to vary the amount of free
| |
| − | ions in an electrolyte in response to light. Ion uptake will be controlled by the
| |
| − | expression of smtA. SmtA is a metallothionein that can bind to heavy metal ions
| |
| − | like cadmium (II), Zinc (II) and Copper (II).</p>
| |
| − |
| |
| − | <p>SmtA has been used in a number of iGEM projects and is in the registry
| |
| − | (<a href="http://parts.igem.org/Part:BBa_K519010">BBa_K519010</a>). It has
| |
| − | previously been used in experiments for Cadmium (II) uptake, see <a href=
| |
| − | "https://2011.igem.org/Team:Tokyo-NoKoGen/metallothionein">Tokyo-NokoGen 2011</a>
| |
| − | and for <a href="http://parts.igem.org/Part:BBa_K190021">accumulating Zinc (II)
| |
| − | intracellularly</a>. We will be examining firstly, the impact of smtA on Zinc
| |
| − | (II) concertation rather than Cadmium (II) and then the impact that this has on
| |
| − | the resistivity of the Zinc (II) containing media. In this instance we will be
| |
| − | using Zinc sulfate (ZnSO<sub>4</sub>) in solution where it disassociates into
| |
| − | Zn<sup>2+</sup> and SO<sub>4</sub><sup>2-</sup> ions. Various concentrations of
| |
| − | Zinc sulfate have <a href=
| |
| − | "http://sites.chem.colostate.edu/diverdi/all_courses/CRC%20reference%20data/electrical%20conductivity%20of%20aqueous%20solutions.pdf">
| |
| − | known Electrical conductivity</a> . When smtA is expressed it will render the
| |
| − | Zn<sup>2+</sup> unavailable and thereby reduce the conductivity of the
| |
| − | solution.</p>
| |
| − |
| |
| − | <p>For this LDR we will be using the red light detection system from the <a href=
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| − | "http://parts.igem.org/Coliroid">Colliroid project</a> ( <a href=
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| − | "http://www.nature.com/nature/journal/v438/n7067/full/nature04405.html#f1">paper</a>).
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| − | In this scheme the production of SmtA which affects the resistivity is placed
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| − | under the control of the OmpF upstream promoter (BBa_R0084). We propose to
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| − | engineer a system where this promoter is repressed in the dark and has increased
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| − | transcription in (red) light. This allows the device to mimic the behaviour of a
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| − | traditional electronic LDR whereby resistance is decreased in the light and
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| − | increased in the dark.</p>
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| − |
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| − | <p>The OmpF promoter is repressed by phosphorylated OmpR, OmpR-P. In normal
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| − | conditions the E. coli cell contains free OmpR which can be phosphorylated by
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| − | expression of a protein with an EnvZ domain. One such protein is the fusion
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| − | protein, Cph8 (BBa_I15010). In the dark this protein phosphorylates OmpR and so
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| − | prevents SmtA production, increasing resistance. In the light, the light
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| − | responsive domain Cph1 inhibits the activity of the EnvZ is prevented from
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| − | phosphorylating OmpR and therefore allows SmtA production and decreased
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| − | resistance.</p>
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| − |
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| − | <p>Note that this will only work in E. coli which are naturally deficient in
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| − | EnvZ.</p>
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| − |
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| − | <p>In order for the light responsive domain of the fusion protein cph8 to sense
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| − | red light the formation of a chromophore is required this is done by the
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| − | production of two proteins, ho1 and PcyA together with the cph8. In our system
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| − | these will be constitutively expressed to create the red light sensor.</p>
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| − |
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| − | <h2>Our Construct</h2>
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| − |
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| − | <p>There are two parts to our construct, the red light sensing component and the
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| − | SmtA production component. It is presented below as a one plasmid system with the
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| − | following parts.</p>
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| − |
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| − | <p><img width="602" height="113" src=
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| − | "https://static.igem.org/mediawiki/2016/4/43/T--Newcastle--RedLDR-BBNumbers.png"
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| − | border="0" /></p>
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| − |
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| − | <p align="center"><em>Figure 1: Red LDR.</em></p>
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| − |
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| − | <h3>BBa_J23100 - constitutive promoter</h3>
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| − |
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| − | <p>We will use <a href="http://parts.igem.org/Promoters/Catalog/Constitutive">a
| |
| − | σ<sup>70</sup> constitutive promoter</a> as this is the main <em>E.
| |
| − | coli</em> sigma factor. Consequently, there should be RNA polymerase present to
| |
| − | transcribe from this promoter at all stages during the bacterial growth cycle.
| |
| − | Specifically, we have chosen <a href=
| |
| − | "http://parts.igem.org/Part:BBa_J23100">BBa_J23100</a>, an artificial promoter
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| − | due to its widespread use, documentation and comparatively short sequence
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| − | (35bp).</p>
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| − |
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| − | <h3>BBa_I15008 - ho1</h3>
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| − |
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| − | <p>This is one of the proteins required for chromophore formation.</p>
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| − |
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| − | <h3>BBa_I15009 - PcyA</h3>
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| − |
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| − | <p>This is one of the proteins required for chromophore formation.</p>
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| − |
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| − | <h3>BBa_I15010 - cph8</h3>
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| − |
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| − | <p>This is a fusion protein consisting of a light receptor domain and EnvZ
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| − | domain. In the dark the EnvZ domain of this protein phosphorylates the free OmpR
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| − | in the cell which represses the OmpF promoter and induces the OmpC promoter.</p>
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| − |
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| − | <h3>BBa_R0084 - OmpR-P Promoter</h3>
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| − |
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| − | <p>This part is the promoter usually found upstream of OmpF. It is repressed by
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| − | phosphorylated OmpR.</p>
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| − |
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| − | <h3>BBa_K519010 - SmtA</h3>
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| − |
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| − | <p>This is the coding sequence for SmtA originally from <em>Synechococcus
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| − | sp</em>, a cyanobacterial strain.</p>
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| − |
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| − | <h3>BBa_B1006 - Terminator</h3>
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| − |
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| − | <p>This is an artificial terminator part and was chosen because it has a high
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| − | forward efficiency of 0.99.</p>
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| − |
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| − | <h3>pSB1C3 - Backbone</h3>
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| − |
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| − | <p>We are using the standard BioBrick backbone part pSB1C3 as this will make it
| |
| − | easier to submit the part to the registry at a later date.</p>
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| − |
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| − | <h2>Construction</h2>
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| − |
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| − | <h3>BioBrick Assembly</h3>
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| − |
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| − | <p>The fusion protein cph8 (BBa_I15010) is not available in the distribution kit,
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| − | it is however in stock at the registry and can be ordered. The remaining parts
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| − | are in the distribution so BioBrick assembly can be performed. However because of
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| − | the large number of parts, and there are no intermediaries in the registry or
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| − | distribution kit, this construct would be unwieldy to assemble in this
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| − | manner.</p>
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| − | </div>
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| − |
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| − | <div id="bio-ldr-blue">
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| − | <h3>YF1-FixJ Controlled Blue 'Light Dependent Resistor'</h3>
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| − |
| |
| − | <p>We plan to engineer <em>Escherichia coli</em> to behave like a light dependent
| |
| − | resistor. We aim to do this by using <em>E. coli</em> to vary the amount of free
| |
| − | ions in an electrolyte in response to light. Ion uptake will be controlled by the
| |
| − | expression of smtA. SmtA is a metallothionein that can bind to heavy metal ions
| |
| − | like cadmium (II), Zinc (II) and Copper (II).</p>
| |
| − |
| |
| − | <p>SmtA has been used in a number of iGEM projects and is in the registry
| |
| − | <a href="http://parts.igem.org/Part:BBa_K519010">BBa_K519010</a>. It has
| |
| − | previously been used in experiments for Cadmium (II) uptake, see <a href=
| |
| − | "https://2011.igem.org/Team:Tokyo-NoKoGen/metallothionein">Tokyo-NokoGen 2011</a>
| |
| − | and for <a href="http://parts.igem.org/Part:BBa_K190021">accumulating Zinc (II)
| |
| − | intracellularly</a>. We will be examining firstly, the impact of smtA of Zinc
| |
| − | (II) concertation rather than Cadmium (II) and then the impact that this has on
| |
| − | the resistivity of the Zinc (II) containing media. In this instance we will be
| |
| − | using Zinc sulfate (ZnSO<sub>4</sub>) in solution where it disassociates into
| |
| − | Zn<sup>2+</sup> and SO<sub>4</sub><sup>2-</sup> ions. Various concentrations of
| |
| − | Zinc sulfate have <a href=
| |
| − | "http://sites.chem.colostate.edu/diverdi/all_courses/CRC%20reference%20data/electrical%20conductivity%20of%20aqueous%20solutions.pdf">
| |
| − | known electrical conductivity</a> . When smtA is expressed it will render the
| |
| − | Zn<sup>2+</sup> unavailable and thereby reduce the conductivity of the
| |
| − | solution.</p>
| |
| − |
| |
| − | <table cellspacing="0" cellpadding="0">
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <td width="123" height="0"></td>
| |
| − | </tr>
| |
| − |
| |
| − | <tr>
| |
| − | <td></td>
| |
| − |
| |
| − | <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>
| |
| − | </tr>
| |
| − | </tbody>
| |
| − | </table>
| |
| − |
| |
| − | <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
| |
| − | 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
| |
| − | the downstream protein, in this case it is SmtA. Thus, in the presence of light
| |
| − | SmtA is not produced and so conductivity does not change, whilst in the absence
| |
| − | 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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| − |
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| − | <h4>Our Construct</h4>
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| − |
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| − | <p>The non-inverted construct is shown in Figure 1 and the inverted construct in
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| − | Figure 2. Currently our device is shown as a 1 plasmid system but there is no
| |
| − | reason that the two separate sub-components could not be split into a 2 plasmid
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| − | system for easier assembly.</p><img width="601" height="137" src=
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| − | "https://static.igem.org/mediawiki/2016/5/5e/T--Newcastle--BlueLDR.png" border="0" />
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| − |
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| − | <p align="center"><em>Figure 1: Standard Blue LDR construct.</em></p>
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| − |
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| − | <p><img width="601" height="110" src=
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| − | "https://static.igem.org/mediawiki/2016/6/67/T--Newcastle--BlueLDR-Inverted.png"
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| − | border="0" /></p>
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| − |
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| − | <p align="center"><em>Figure 2: Blue LDR with inverter.</em></p>
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| − |
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| − | <p>For the non-inverted construct the parts are as follows:</p>
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| − |
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| − | <h3>BBa_J23100 - constitutive promoter</h3>
| |
| − |
| |
| − | <p>We will use <a href="http://parts.igem.org/Promoters/Catalog/Constitutive">a
| |
| − | σ<sup>70</sup> constitutive promoter</a> as this is the main <em>E.
| |
| − | coli</em> sigma factor. Consequently, there should be RNA polymerase present to
| |
| − | transcribe from this promoter at all stages during the bacterial growth cycle.
| |
| − | Specifically, we have chosen <a href=
| |
| − | "http://parts.igem.org/Part:BBa_J23100">BBa_J23100</a>, an artificial promoter
| |
| − | due to its widespread use, documentation and comparatively short sequence
| |
| − | (35bp).</p>
| |
| − |
| |
| − | <h3>BBa_K592016 - FixJ & YF1 with RBSs</h3>
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| − |
| |
| − | <p>The YF1 and FixJ coding sequences are provided as a composite part together
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| − | with standard RBSs in part <a href=
| |
| − | "http://parts.igem.org/Part:BBa_K592016">BBa_K592016</a> which we have chosen for
| |
| − | ease of assembly, in the event that we or future teams wish to use the BioBrick
| |
| − | standard assembly to produce our part.</p>
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| − |
| |
| − | <h3>BBa_K592006 - FixK2</h3>
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| − |
| |
| − | <p>This is the wild-type promoter to which phosphorylated FixJ binds. It is
| |
| − | reported that this promoter has very little leaky activity in the absence of
| |
| − | FixJ.</p>
| |
| − |
| |
| − | <h3>BBa_K519010 - SmtA</h3>
| |
| − |
| |
| − | <p>This is the coding sequence for SmtA originally from <em>Synechococcus
| |
| − | sp</em>, a cyanobacterial strain.</p>
| |
| − |
| |
| − | <h3>BBa_B1006 - Terminator</h3>
| |
| − |
| |
| − | <p>This is an artificial terminator part and was chosen because it has a high
| |
| − | forward efficiency of 0.99.</p>
| |
| − |
| |
| − | <h3>pSB1C3 - Backbone</h3>
| |
| − |
| |
| − | <p>We are using the standard BioBrick backbone part pSB1C3 as this will make it
| |
| − | easier to submit the part to the registry at a later date.</p>
| |
| − |
| |
| − | <p>For the inverted part there are additional parts as follows:</p>
| |
| − |
| |
| − | <h3>BBa_C0051 - Lambda CI</h3>
| |
| − |
| |
| − | <p>This is the repressor protein from Lambda phage it represses the promoter
| |
| − | BBa_R0051.</p>
| |
| − |
| |
| − | <h3>BBa_B0010 and BBa_B0012 - Double stop terminators</h3>
| |
| − |
| |
| − | <p>These are the terminators used in the composite part <a href=
| |
| − | "http://parts.igem.org/Part:BBa_S04617">BBa_S04617</a> <u>which is replicated in
| |
| − | our construct.</u></p>
| |
| − |
| |
| − | <h3>BBa_R0051 - Lambda CI controlled promoter</h3>
| |
| − |
| |
| − | <p>This is a promoter from Lambda phage that is repressed by lambda Cl
| |
| − | (BBa_C0051).</p>
| |
| − |
| |
| − | <h2>Construction</h2>
| |
| − |
| |
| − | <h3>Synthesis</h3>
| |
| − |
| |
| − | <p>This construct can be sourced from IDT using our free allowance.</p>
| |
| − |
| |
| − | <h3>BioBrick Assembly</h3>
| |
| − |
| |
| − | <p>There exist a number of intermediate assembly components in the parts
| |
| − | distribution that can be used to assemble our part faster if we use BioBrick
| |
| − | assembly. Notably, <a href="http://parts.igem.org/Part:BBa_S04617">BBa_S04617</a>
| |
| − | contains the inverter,<a href=
| |
| − | "http://parts.igem.org/Part:BBa_K592016">BBa_K592016</a> <u>contains the FixJ and
| |
| − | YF1. The two devices can be constructed separately as follows.</u></p>
| |
| − |
| |
| − | <h4><em><u>Constitutive Production Device</u></em></h4>
| |
| − |
| |
| − | <ol>
| |
| − | <li>
| |
| − | <p>Cut the terminator BBa_B1006 with E & X.</p>
| |
| − | </li>
| |
| − |
| |
| − | <li>
| |
| − | <p>Cut BBa_K592016 with E & S.</p>
| |
| − | </li>
| |
| − |
| |
| − | <li>
| |
| − | <p>Mix & Ligate to form intermediate YF1FixJ+Terminator.</p>
| |
| − | </li>
| |
| − |
| |
| − | <li>
| |
| − | <p>Cut the intermediate part with E & X and the constitutive promoter
| |
| − | BBa_J23100 with E & S.</p>
| |
| − | </li>
| |
| − |
| |
| − | <li>
| |
| − | <p>Mix and Ligate to form the constitutive production device.</p>
| |
| − | </li>
| |
| − | </ol>
| |
| − |
| |
| − | <h4><em>SmtA Expression Device (inverted)</em></h4>
| |
| − |
| |
| − | <ol>
| |
| − | <li>
| |
| − | <p>Cut BBa_K519010 with E & X.</p>
| |
| − | </li>
| |
| − |
| |
| − | <li>
| |
| − | <p>Cut BBa_S04617 with E & S.</p>
| |
| − | </li>
| |
| − |
| |
| − | <li>
| |
| − | <p>Mix and ligate to produce intermediate part: inverted smtA production.</p>
| |
| − | </li>
| |
| − |
| |
| − | <li>
| |
| − | <p>Cut terminator with E & X.</p>
| |
| − | </li>
| |
| − |
| |
| − | <li>
| |
| − | <p>Cut SmtA production intermediate with E & S.</p>
| |
| − | </li>
| |
| − |
| |
| − | <li>
| |
| − | <p>Mix and ligate to produce SmtA expression device.</p>
| |
| − | </li>
| |
| − | </ol>
| |
| − |
| |
| − | <p>To produce the non-inverted device replace BBa_S04617 with the SmtA coding
| |
| − | sequence and use an additional step to join this to an RBS, we suggest the
| |
| − | standard RBS BBa_B0034 as this has good efficiency.</p>
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − | <table>
| |
| − | <tr>
| |
| − | <th>Part No.</th>
| |
| − |
| |
| − | <th>Name</th>
| |
| − |
| |
| − | <th>Purpose</th>
| |
| − | </tr>
| |
| − |
| |
| − | <tr>
| |
| − | <td>BBa_R0080</td>
| |
| − |
| |
| − | <td>L-Arabinose Promoter</td>
| |
| − |
| |
| − | <td>This part is an L-Arabinose inducible promoter with very low level
| |
| − | expression in the absence of L-Arabinose and AraC. Be aware, if the E. coli
| |
| − | strain used constitutively expresses AraC then this promoter will
| |
| − | ‘leak’. Check the <a href=
| |
| − | "http://openwetware.org/wiki/E._coli_genotypes">strain list</a> for
| |
| − | information.</td>
| |
| − | </tr>
| |
| − |
| |
| − | <tr>
| |
| − | <td>BBa_R0040</td>
| |
| − |
| |
| − | <td>TetR</td>
| |
| − |
| |
| − | <td>This is the coding sequence for TetR which represses BBa_R0040.</td>
| |
| − | </tr>
| |
| − |
| |
| − | <tr>
| |
| − | <td>BBa_R0040</td>
| |
| − |
| |
| − | <td>TetR Repressible Promoter</td>
| |
| − |
| |
| − | <td>This is a constitutively on promoter which can be repressed by TetR. Be
| |
| − | aware, if the strain used expresses TetR constitutively then this promoter
| |
| − | will be repressed. Check the <a href=
| |
| − | "http://openwetware.org/wiki/E._coli_genotypes">strain list</a> for
| |
| − | information.</td>
| |
| − | </tr>
| |
| − |
| |
| − | <tr>
| |
| − | <td>BBa_C0052</td>
| |
| − |
| |
| − | <td>434 Repressor</td>
| |
| − |
| |
| − | <td>Represses the output promoter.</td>
| |
| − | </tr>
| |
| − |
| |
| − | <tr>
| |
| − | <td>BBa_C0051</td>
| |
| − |
| |
| − | <td>Lambda Repressor</td>
| |
| − |
| |
| − | <td>Induces the output promoter.</td>
| |
| − | </tr>
| |
| − |
| |
| − | <tr>
| |
| − | <td>BBa_I12006</td>
| |
| − |
| |
| − | <td>Modified Promoter Part</td>
| |
| − |
| |
| − | <td>This is a modified promoter part, originally the lambda Prm promoter. The
| |
| − | modification allows it to be activated by lambda repressor and repressed by
| |
| − | 434 repressor.</td>
| |
| − | </tr>
| |
| − |
| |
| − | <tr>
| |
| − | <td>BBa_I746916</td>
| |
| − |
| |
| − | <td>Superfolder GFP</td>
| |
| − |
| |
| − | <td>This is the coding sequence for super folder GFP. We have chosen to use
| |
| − | this as our reporter because it can easily be quantified using a plate by
| |
| − | taking the OD600 measurement. This is harder to quantify with more visible
| |
| − | reporters like amilCP.</td>
| |
| − | </tr>
| |
| − |
| |
| − | <tr>
| |
| − | <td>BBa_B1006</td>
| |
| − |
| |
| − | <td>Standard Terminator</td>
| |
| − |
| |
| − | <td>We chose to use this promoter from the registry as it has a high forward
| |
| − | efficiency.</td>
| |
| − | </tr>
| |
| − |
| |
| − | <tr>
| |
| − | <td>BBa_B0034</td>
| |
| − |
| |
| − | <td>RBS</td>
| |
| − |
| |
| − | <td>We chose to use this RBS from the registry as it is efficient and widely
| |
| − | used in iGEM projects.</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | </div>
| |
| − | <div class="col-md-4" data-spy="affix-right">
| |
| − | <ul class="nav nav-pills nav-stacked" id="right-menu">
| |
| − | <li><a href="#bio-bulb">Light Bulb</a></li>
| |
| − | <li><a href="#bio-varistor">Arabinose Varistor</a></li>
| |
| − | <li><a href="#bio-ldr-red">LDR</a></li>
| |
| − | <li><a href="#bio-capacitor">Biological Capacitor</a></li>
| |
| − | <li><a href="#mfc-porin-overexpression">MFC with Porin Overexpression</a></li>
| |
| − | </ul>
| |
| − | </div>
| |
| − | </div>
| |
| − | <head>
| |
| | <style> | | <style> |
| − | #right-menu | + | #bodyContent{ |
| − | { | + | background-color: #EFE6C5; |
| − | position: fixed;
| + | } |
| | + | ol{ |
| | + | color:#000; |
| | } | | } |
| | </style> | | </style> |
| − | </head> | + | <body> |
| − | </html> | + | <br> |
| | + | <br> |
| | + | <div class="column"> |
| | + | <h2>Proof of Concept</h2> |
| | + | |
| | + | <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> |
| | + | <p>The functional proof of concept of our project was demonstrated by integrating three of our BioBricks in the following devices: </p> |
| | + | <ol> |
| | + | <li>Microbial Fuel Cell</li> |
| | + | <li>Heat Induced ‘Light Bulb’</li> |
| | + | </ol> |
| | + | <h3>Microbial Fuel Cell</h3> |
| | + | <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>. |
| | + | <figure><img alt="proof1" src="https://static.igem.org/mediawiki/2016/7/7c/T--Newcastle--proof1.png"></figure> |
| | + | <p><figcaption>Figure 1. The construction of our miniature microbial fuel cell component using a 3D printed mold and PDMS gel. |
| | + | </figcaption></p> |
| | + | <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> |
| | + | <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> |
| | + | <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> |
| | + | <figure><img alt="proof2" src="https://static.igem.org/mediawiki/2016/9/9c/T--Newcastle--proof2.png"></figure> |
| | + | <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> |
| | + | </figcaption></p> |
| | + | <h3>Heat Induced ‘Light Bulb’</h3> |
| | + | <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> |
| | + | <figure><img alt="proof3" src="https://static.igem.org/mediawiki/2016/d/d6/T--Newcastle--proof3.png"></figure> |
| | + | <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°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.</p> |
| | + | <figure><img alt="proof4" src="https://static.igem.org/mediawiki/2016/9/92/T--Newcastle--proof4.png"></figure> |
| | + | <p><figcaption>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).</figcaption></p> |
| | + | <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> |
| | + | </div> |
| | + | <br> |
