Difference between revisions of "Team:Exeter/Proof"

 
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<h1>Human Practices - Silver Award</h1>
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<h1>Proof of Concept</h1>
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<h5>Education</h5>
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<h3>Continuous culture</h3>
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<h6>BioMech</h6>
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<p id="pp">The main focus of Project: Exepire in the lab was the robustness of kill switches in real world conditions. By looking at the effectiveness of the kill switches in continuous culture we have begun to show potential failure rates over time. By simulating a continuous culture that would take place on a much larger scale in industry, we have shown the potential failures that need to be addressed if kill switches are to replace traditional chemical and physical bio-containment. </p>
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<p id="pp">Synthetic Biology is severely under-taught at all stages of education, occasionally certain syllabi will lightly touch on certain concepts, but only to briefly move on without any real depth into the topic. One of the aims of our human practises is to address this by providing easily accessible synthetic biology learning resources such as a board game, open to teachers and students alike. This lack of focus on synthetic biology is not only seen in lower levels of education, but further up the age ladder at university. Very few universities offer modules in specifically synthetic biology which is why we have taken steps to implement a module in our own university. </p>
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<p id="pp">Before starting the project we spoke to Prof. Robert Beardmore EPSRC Leadership Fellow in the Mathematical Biosciences at Exeter University. Much of his research has been into antibiotic resistance. We discussed how high selection pressure is applied by prolonged use of antibiotics and how kill switches may be analogous to this. It is clear that cells which develop a mutation that inactivates the kill switch would be strongly selected for. It was estimated that functional loss of the kill switch would occur in a short amount of time as a result, and if this was the case, could have strong implications for kill switch longevity. To test this we decided to use a ministat to perform a continuous culture. The ministat was developed in the Dunham lab at the University of Washington (Miller <i>et al</i>, 2013). Each ministat chamber is fed from its own media container via a peristaltic pump, with the culture volume set by the height of the effluent needle in the chamber. Air is bubbled through flasks of water to hydrate it and then used to agitate the culture. Chambers were inoculated with freshly transformed <i>E. coli</i> BL21 (DE3) and samples taken to test if the kill switches were still viable. By simulating in miniature how a kill switch might behave in an industrial setting, the ministat provides a proof of concept for how a kill switch might be maintained in larger chemostats during a continuous culture. A protocol for running experiments in the ministat can be found <a href="#MinistatProt">here</a>
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<span class="caption">Media container used to feed a single ministat chamber.</span>
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<span class="caption">Peristaltic pump</span>
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<span class="caption">Ministat chambers in heatblock and 1 litre Duran bottle used to collect effluent</span>
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<p id="pp">BioMech is an educational synthetic biology board game, designed by the 2016 Exeter iGEM team for students aged 14-18. The concept of the board game was developed early on in the project, with the design informed by a year 10 work experience student. The board game was then tested with GCSE students from two schools, the feedback from which was used to tailor the game around the current knowledge of year 10 students. The intention is to introduce the concept of synthetic biology to a younger age group in a fun and engaging manner. In order to make the game widely accessible despite needing a physical copy BioMech has been uploaded to our wiki, and is able to download with printing instructions for free. We hope that BioMech will inspire students to further their own education independently given that there is currently very little taught on the topic prior to university level.</p>
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<p id="pp">You can read more about BioMech <a href="https://2016.igem.org/Team:Exeter/Human_Practices#section_2">here.</a></p>
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<h6>Synthetic Biology Module</h6>
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<p id="pp">Continuing with the theme of responding to a lack of education in synthetic biology, we also looked at university level education. Due to the lack of any type of formal synthetic biology teaching here at the University of Exeter, we have created a second year synthetic biology module to implement in the Biosciences department. During the design process we managed to talk to multiple academics about their thoughts on the process of implementing new modules into the syllabus, and how it could be made more efficient in the future. It will be available for second year Biosciences and Natural Sciences students to take from September 2017.</p>
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<p id="pp">You can read more about the module <a href="https://2016.igem.org/Team:Exeter/Human_Practices#section_3">here.</a></p>
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<div class="col-xs-6"><span class="caption" style="padding: 5px 30% 5px 30%;">Ministat running a preliminary experiment to calibrate parameters such as dilution rate and temperature of the heat block. 50 ml burettes used here to accurately measure effluent levels. 1 litre Duran bottles were used for effluent collection in the main experiment due to greater volumes of effluent. </span></div>
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<h5>Public Perception</h5>
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<h6>Desert Island... Science?</h6>
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<p id="pp">‘Desert Island… Science?’ is an interview series hosted on our YouTube channel and wiki, in which we spoke to a variety of academics and leading figures in science, and often more specifically, in synthetic biology. It is inspired by the similarly named ‘Desert Island Discs’ on BBC channel 4, and mixes fun, light questions with more interesting, complicated enquiries into their work, their opinions on topics like equality in science and need for education, etc.</p>
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<p id="pp">Our own growth curve was performed to determine the maximum specific growth rate of <i>E. coli </i> BL21 (DE3) in our lab, but could not be conducted for a sufficient length of time to be accurate. A maximum specific growth rate value of 1.730 was used (Cox, 2004). The ministat must be set to a flow rate at which dilution rate is less than maximum specific growth rate. This prevents the culture being washed out of the growth chambers. The dilution rate of the culture was calculated by measuring flow rate at a setting of 7.5 rpm on the peristaltic pump. For practical reasons the pump could not be run faster than this due to the amount of media needed. The dilution rate was set at 0.2 which produced cultures that grew at an average OD of 3.47 for KillerRed samples, 3.64 for KillerOrange samples and 3.17 for lysozyme samples. The ministat must be set to flow rate at which dilution rate is below the maximum specific growth rate. This prevents the culture being washed out of the chamber. OD was measured daily with a Bug Lab OD scanner. When the same sample was measured in a tecan infinite 200 pro plate reader the Bug Lab showed reading approximately three times higher. The difference between the samples was consistent regardless of the method used to measure OD.
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<p id="pp">The intention of ‘Desert Island… Science?’ is to humanise academics and experts to the public in light of the current anti-expert sentiment, and make the work of scientist more accessible and engaging to people not involved in the field. Whilst we recognise the perception of synthetic biology has improved greatly since several years ago, there is often still negativity and confusion associated with the general perception of the field, which is something we hope to negate by showing the positive impacts and implications of synthetic through our interviews.</p>
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<h6><u>Ministat experiment</u></h6>
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<p id="pp">All samples from the ministat were tested using the KillerRed, KillerOrange protocol found <a href="#KRKOProt">
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here</a>. Glycerol stocks were made from the samples taken at each time interval, testing was done using these glycerol stocks.</p>
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<p id="pp">The following graphs show the average number of colonies of samples taken at 0 h, 24 h, 120 h and 168 h of continuous culture and then tested in the light box. Values were
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averaged across three biological repeats. Colonies were not counted above 300 and so this is the maximum value given. All samples were induced to a final concentration of 0.2 nM IPTG. All samples were
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diluted 1000 times in a final volume of 4.5 ml liquid broth (LB). Error bars represent the standard error of the mean.</p>
  
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<p id="pp">You can read more about ‘Desert Island… Science?’ <a href="https://2016.igem.org/Team:Exeter/Human_Practices#section_4">here.</a></p>
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<h5>Public Outreach</h5>
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<p id="pp">The Big Bang South West science fair was the first science fair we attended, falling on 29th June 2016. It was aimed towards students of all ages, teachers and parents, and allowed to get a range of feedback on our board game, BioMech, which first premiered there. We also engaged students present with activities from William and Mary’s 2015 activities booklet, using the gene bracelet and sweetie DNA activities to grab attention and open dialogue, both of which worked effectively. We believed that the activities booklet supplemented our intentions of presenting synthetic biology in a friendly manner perfectly.</p>
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<p id="pp">The Britain Needs Scientists fair was a much smaller fair we attended on 6th July 2016, aimed primarily at A Level students looking at doing a degree in a science. Our primary focus here was highlighting synthetic biology as a multi-disciplinary subject, and engage in more challenging conversations with interested students. Due to the name; synthetic biology an assumption is quickly made that it is heavily biology based, when in fact is contains aspect of all STEM subjects.</p>
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            <span class="caption">Fig. 14. Comparison of CFUs formed by KillerRed exposed to light and kept in the dark for each sample taken from the ministat. The efficiency of the kill switch decreases over time as shown by the increasing number of CFUs.</span>
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<p id="pp">You can read more about our science fair visits <a href="https://2016.igem.org/Team:Exeter/Human_Practices#section_4">here.</a></p>
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            <span class="caption">Fig. 15. Comparison of CFUs formed by KillerOrange exposed to light and kept in the dark. The efficiency of the kill switch decreases over time as shown by the increasing number of CFUs. The effect is not as obvious in KillerOrange compared to KillerRed as the starting efficiency of KillerOrange is lower. </span>
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<h6>Discussion</h6>
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<p id="pp">The continuous culture of KillerRed showed a 15 fold increase in the percentage of viable cells after 168 hrs. A similar pattern is shown for KillerOrange but with around a two fold increase. Both KillerRed and KillerOrange show greater numbers of colonies forming over time (Fig. 14 & 15). This number approaches the amount produced in the dark condition by 168 hrs.
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The average fluorescence reading for 0 hr KillerRed samples was 506.3 A.U (recorded at an average OD of of 0.745). After 168
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hrs the average fluorescence reading  was 436 A.U (at an average OD of 0.96). It seems unlikely due to the readings being
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similar that a mutation has occurred in the kill switch itself. As fluorescence is proportional to the amount of ROS
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being produced, up regulation of native <i>E. coli</i> enzymes that mitigate the effects of ROS may be the cause of the increase
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in cell survival. Future transcriptome analysis could provide interesting data on the mechanism of this change, this was
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unfortunately beyond the scope of this project. This shows that there may be many ways for bacteria to circumvent the effects of a kill switch given the high selection pressure they pose.</p>
  
 
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Latest revision as of 14:46, 19 October 2016