Difference between revisions of "Team:Newcastle/Description"

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<h2>Our Project </h2>
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<h2>Results</h2>
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        <h3><a href="https://2016.igem.org/Team:Newcastle/Proof/ElectricallyInducedLightBulb">Heat Induced 'Light Bulb'</a></h3>
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<img alt="IDT" src="https://static.igem.org/mediawiki/2016/5/50/T--Newcastle--Project.jpeg"/>
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<p> The concept of BioBricks was first introduced by Tom Knight at MIT in 2003. His vision was to standardise synthetic biological parts in a similar way to Lego bricks that interlock to form larger constructs. In turn, this has enabled research groups to engineer novel biological systems. Our team has been inspired by Tom Knight’s “Lego-like” approach to synthetic biology.
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<p>However, we wondered how we could mix electronic devices with synthetic biological devices. Our project therefore involves us replacing some of the traditional electronic components in a circuit with biological alternatives. Like Lego, the circuit will allow synthetic biologists to mix and match bacterial and electronic components to create electro-biological circuits. This approach will represent a foundational advance in the way synthetic biological circuits are designed and implemented.</p>
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<p> We aimed to engineer <i>Escherichia coli</i> so that it increases expression of a fluorescent protein (sfGFP) when an electrical current is passed through the growth medium, via the use of inducible promoters that respond to the heat-stress response created by the conversion of electrical energy to waste heat.</p>
  
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<p>We believe that by merging biology, electronic engineering and computer science, our project also holds fantastic opportunities for education. There is a common misconception that all bugs are bad, but our project will demonstrate the benefits of bacteria in a fun and safe manner. </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><a href="http://parts.igem.org/Part:BBa_K1895000">BBa_K1895000</a> contains the <em>E. coli</em> <i>htpG</i> promoter. RNA polymerase requires the stress response sigma-factor (&sigma;<sup>32</sup>) to initiate transcription of genes downstream of this promoter. &sigma;<sup>32</sup> is produced by cells when under different forms of stress, one of which is heat. This composite part also contains a modified BioBrick compatible &sigma;<sup>32</sup> coding region (the gene <em>rpoH</em>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1895001">BBa_K1895001</a>) which will create a positive feedback loop to the P<em><sub>htpG</em></sub> promoter, therefore increasing the expression of the downstream reporter gene <em>sfGFP</em> and the  fluorescence of the cell.</li>
  
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<p>Synthetic biology is an exciting, and unique field that we want to make accessible to children worldwide. We want the next generation to have a better understanding of what synthetic biology is, and inspire new ways to apply synthetic biology to real world applications. Perhaps our circuits will alter the perception of Genetically Modified Organisms, inspire a new way to generate sustainable electricity, or even work towards creating a computer made entirely from synthetic organisms. </p>
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<li><a href="http://parts.igem.org/Part:BBa_K1895006">BBa_K1895006</a> contains the <em>dnaK</em> promoter which, like P<em><sub>htpG</em></sub>, is transcribed via binding of RNA polymerase by &sigma;<sup>32</sup>. P<em><sub>dnaK</sub></em> is placed upstream of the BBa_0034 RBS and BBa_I746916 sfGFP.</li>
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<h2>Design of Hardware</h2>
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<p>Having decided on an electrical theme, we were inspired by educational electronics kits to build a standardised breadboard-style testing system for our constructs that could also be used for demonstrations. This was a great chance to reach out to the local community for help and advice, as Newcastle has its own local maker group We would go on to work with lasercutters and 3D printers to produce parts to fulfil our own specifications.</p>  
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<p>Initially we worked with FabLab, a local fabrication workshop based in Sunderland to come up with our initial designs, building prototypes and investigating the best ways to join parts together. Later we moved our base of operations to OpenLab within Newcastle University where we were assigned a design intern who helped us bring our final designs to life. We opted for a system that used magnets which allow the user to freely interchange parts and pass electricity through our microfluidic devices.</p>
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<p>To see our results <a href="https://2016.igem.org/Team:Newcastle/Proof/ElectricallyInducedLightBulb">click here </a></p>
  
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<p>This was a fantastic learning opportunity for us, as none of us had any previous design experience, <a href="https://2016.igem.org/Team:Newcastle/Design">and you can read more about our design process here</a></p>
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<h2>Design of Parts</h2>
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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>We aimed to create a biological “variable resistor” by modifying the <i>E. coli’s</i> natural systems to allow for controlled ion uptake. In order to do so, we looked at the work carried out by the <a href="https://2011.igem.org/Team:Tokyo-NoKoGen/metallothionein">2011 Tokyo-NokoGen iGEM team</a> who used the <i>smtA</i> gene from Cyanobacteria and inserted it into a strain of <i>E. coli</i>. SmtA is thought to play a role in preventing heavy metal toxicity by binding excess heavy metal ions such as Cadmium (II), as characterised by <a href="https://2011.igem.org/Team:Tokyo-NoKoGen/metallothionein">Tokyo-NokoGen</a>, or Zinc (II). </p>
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<img alt="IDT" src="https://static.igem.org/mediawiki/2016/4/40/T--Newcastle--Bulb-Natural-RBS.png"/>
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<p>Our goal of creating biological analogues of electronic devices offered us a huge range of potential biobricks. At the planning stage, we designed theoretical versions of many standard components, including lightbulbs, batteries, variable resistors and even a capacitor!</p>  
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<p>Our parts rely on electrical heating for activation, allowing integration into existing electrical systems. We have created functional versions of our lightbulb and variable resistor designs, along with an improved efficiency microbial fuel cell based on a design proposed by Team Bielefield in 2013. We also developed a simulator that predicts the output of our devices, whether alone or integrated into more complex systems.</p>
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        <p>We took the <i>smtA</i> gene, (<a href="http://parts.igem.org/Part:BBa_K519010">BBa_K519010</a>), and put it under the control of a P<em><sub>BAD</em></sub> promoter, induced by the presence of L-arabinose, making our BioBrick <a href="http://parts.igem.org/Part:BBa_K1895999">BBa_K1895999</a>. 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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<p>Being able to work on such a range of different devices has allowed us to learn a great deal about cellular mechanisms, enhancing our general biological knowledge. It was really exciting to try and combine the two different disciplines of electrical and genetic engineering.<a href="https://2016.igem.org/Team:Newcastle/Parts">You can read more about our parts here</a></p>
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<p>To see our results <a href="https://2016.igem.org/Team:Newcastle/Proof/VariableResistor">click here </a></p>
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<h2>Design of Human Practices</h2>
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<img alt="IDT" src="https://static.igem.org/mediawiki/2016/b/bc/T--Newcastle--drsimonwoods.png"/>
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<p> As we're competing in the Foundational Advance track we felt obligated to consider, at great length, the ethical issues that could be raised by future development in the new field of science we're striving to lay the foundation for. After discussing with experts from PEALS (The Policy, Ethics and Life Sciences Research Centre at our University) it became apparent that due to the wide range of potential applications our research could contribute towards - our end Human Practices output had to take a holistic view. </p>
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<p> After considering this and looking at the strengths of our team, we decided to utilize the skills of the three computer scientists on the team and program a simulator to act as a thought piece on the future implications of Bio-electrics. The simulator features 5 levels which provide different scenarios, each raising potential ethical concerns. Each level carries an inspiration from our interactions with others - whether it be our discussions with PEALS, talking to a space expert who inspired the the Mars Level or the sixth formers day we ran, from which one student's experiences with a Kidney dialysis machine forms the narrative behind another level. <a href="https://2016.igem.org/Team:Newcastle/HP/Background">You can read more about our design process for Human Practices here </a></p>
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<h2>Modelling</h2>
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<p>Our teams goal is to show how our technology could work in practice. Consequently, we used modelling to explore potential uses of our technology beyond the summer, and to inform our design process. We used a number of different modelling tools, both for designing our biological constructs as well as our physical hardware. For our phsyical hardware we made use of the COMSOL software to perform multiphysics modelling whist for the biological constructs we usd RuleBender and rule based modelling as well as our own ordinary differential equation modelling.</p>
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<p>All of these gave us valuable insights into our design process. For instance, after we conducted multiphysics modelling of our original chamber designs we found they were too large. Our modelling showed us that we needed to switch to microfluidics scale hardware. Modelling also gave us new directions to explore, for example during the modelling of our resistor constructs we came upon the idea of a cell-free system. Finally, modelling gives us a way to showcase how bacteria and electronics might be integrated through our ‘simulator’ which models our constructs in circuits users can build themselves.</p>
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<h2>Results</h2>
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<img alt="IDT" src="https://static.igem.org/mediawiki/2016/5/57/T--Newcastle--Proof.jpg"/>
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<p>Over the course of the summer, we have performed a large number of experiments, both to inform further research and to help with characterising our parts. The earlier tests were vital in directing the flow of project, indicating the level of electrical current we would need and allowing us to refine the design of our breadboard around integration of the microfluidic devices.</p>  
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<p><a href="https://2016.igem.org/Team:Newcastle/Proof">Our results are covered in detail here</a></p>
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<h2>Proof of Concept</h2>
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<img alt="IDT" src="https://static.igem.org/mediawiki/2016/9/9f/NCLtinker1.png"/>
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<p>In order to achieve a gold medal, iGEM teams can demonstrate their constructs working under real world conditions. With this in mind, we have used our breadboard kit hardware to electrically heat transformant bacteria and activate our 'lightbulb' biobricks.</p>  
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<p>Electrical current is transferred through the combination of magnets and conductive tape in the breadboard, through to the electrodes in the microfluidic device. The test detailed illustrates how GFP production in our 'lightbulb' bacteria can be activated in simulated real-world conditions using standard equipment. </p>
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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>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 <a href="http://www.ncl.ac.uk/ceg/role/profile/edmilner.html#publications">Dr Ed Milner</a>, <a href="https://www.researchgate.net/profile/Paniz_Izadi">Dr Paniz Izadi</a> and <a href="http://www.ncl.ac.uk/ceg/role/profile/ianhead.html#background">Professor Ian Head</a>, but after <a href="https://2016.igem.org/Team:Newcastle/HP/Gold">talking with PEALS</a> we decided to move away from using yeast and looked at working with <i>E. coli</i> instead. </p>
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<p>For inspiration we looked at the <a href="https://2013.igem.org/Team:Bielefeld-Germany/Project/MFC">Bielefeld 2013 iGEM Team </a>. One of the issues we noticed with their design was that their porin overexpression protein was taken from <i>Pseudomonas fluorescens</i> and so the pores size was too large for the <i>E. coli</i> to handle. We changed this by overexpressing <i>E. coli’s</i> natural porin producing genes <i>ompF</i>,<a href="http://parts.igem.org/Part:BBa_K1895004 "> BBa_K1895004</a>. Bielefeld also had issues with cell growth due to the metabolic stress of using a T7 promoter. To improve this part we used a P<sub><i>BAD</i></sub> promoter to allow the cell population to grow before inducing the porin over-expression, <a href="http://parts.igem.org/Part:BBa_K1895005 ">BBa_K1895005.</a></p>
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<p>To see our results <a href="https://2016.igem.org/Team:Newcastle/Proof/MFC">click here </a></p>
  
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<p><a href="https://2016.igem.org/Team:Newcastle/Demonstrate">See how we demonstrated our work here</a></p>
 
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<h2>Protocols</h2>
 
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<p>We have used a wide variety of protocols throughout our project, including previously designed standards such as the University of Reading's fuel cell preparation guide, alongside those we have written ourselves, for example creation of our microfluidic microbial fuel cells.</p>
 
  
  
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<p> <a href="https://2016.igem.org/Team:Newcastle/Protocols">See how we demonstrated our work here</a></p>
 
 
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Latest revision as of 02:05, 20 October 2016


Results

Heat Induced 'Light Bulb'

We aimed to engineer Escherichia coli so that it increases expression of a fluorescent protein (sfGFP) when an electrical current is passed through the growth medium, via the use of inducible promoters that respond to the heat-stress response created by the conversion of electrical energy to waste heat.

We designed two parts (BBa_K1895000 and BBa_K1895006) which respond to the heat-stress in two different ways:

  1. BBa_K1895000 contains the E. coli htpG promoter. RNA polymerase requires the stress response sigma-factor (σ32) to initiate transcription of genes downstream of this promoter. σ32 is produced by cells when under different forms of stress, one of which is heat. This composite part also contains a modified BioBrick compatible σ32 coding region (the gene rpoH, BBa_K1895001) which will create a positive feedback loop to the PhtpG promoter, therefore increasing the expression of the downstream reporter gene sfGFP and the fluorescence of the cell.
  2. BBa_K1895006 contains the dnaK promoter which, like PhtpG, is transcribed via binding of RNA polymerase by σ32. PdnaK is placed upstream of the BBa_0034 RBS and BBa_I746916 sfGFP.

To see our results click here


Arabinose Controlled Variable Resistor

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 2011 Tokyo-NokoGen iGEM team who used the 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), as characterised by Tokyo-NokoGen, or Zinc (II).

We took the smtA gene, (BBa_K519010), and put it under the control of a PBAD promoter, induced by the presence of L-arabinose, making our BioBrick BBa_K1895999. 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.

To see our results click here


Microbial Fuel Cell

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 we decided to move away from using yeast and looked at working with E. coli instead.

For inspiration we looked at the Bielefeld 2013 iGEM Team . 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, BBa_K1895004. 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 over-expression, BBa_K1895005.

To see our results click here