Team:NRP-UEA-Norwich/Project

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             <a href="https://2014.igem.org/Team:NRP-UEA-Norwich"/><img src="https://static.igem.org/mediawiki/2014/e/e0/Uea_logo.png" class="logo"/></a>
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               <a href="https://2014.igem.org/Main_Page"/ target="_blank"><img src="https://static.igem.org/mediawiki/2014/a/a3/Uea_igem-logo.png"/></a>
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                   <li><a href="https://2014.igem.org/Team:NRP-UEA-Norwich/Project_System">System</a></li>
                   <li><a href="https://2014.igem.org/Team:NRP-UEA-Norwich/Project_System">System</a></li>
                   <li><a href="https://2014.igem.org/Team:NRP-UEA-Norwich/Project_Parts">Parts</a></li>
                   <li><a href="https://2014.igem.org/Team:NRP-UEA-Norwich/Project_Parts">Parts</a></li>
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                   <li><a href="https://2014.igem.org/Team:NRP-UEA-Norwich/Project_GG-Cloning">GoldenGate cloning</a></li>
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                   <li><a href="https://2014.igem.org/Team:NRP-UEA-Norwich/Project_GG-Cloning">Golden Gate cloning</a></li>
                   <li><a href="https://2014.igem.org/Team:NRP-UEA-Norwich/Project_Mo-Flipper">Golden Gate Modular Flipper</a></li>
                   <li><a href="https://2014.igem.org/Team:NRP-UEA-Norwich/Project_Mo-Flipper">Golden Gate Modular Flipper</a></li>
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                  <li><a href="https://2014.igem.org/Team:NRP-UEA-Norwich/HP_Collaborations">Collaborations</a></li>
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                  <li><a href="https://2014.igem.org/Team:NRP-UEA-Norwich/Judging_Criteria">Judging Criteria</a></li>
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                 <a data-toggle="dropdown" class="dropdown-toggle" href="#">Human Practices <span class="caret"></span></a>
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                 <a data-toggle="dropdown" class="dropdown-toggle" href="#">Policy and Practices<span class="caret"></span></a>
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                   <li><a href="https://2014.igem.org/Team:NRP-UEA-Norwich/HP">Overview</a></li>
                   <li><a href="https://2014.igem.org/Team:NRP-UEA-Norwich/HP">Overview</a></li>
                   <li><a href="https://2014.igem.org/Team:NRP-UEA-Norwich/HP_CUT">The CUT event</a></li>
                   <li><a href="https://2014.igem.org/Team:NRP-UEA-Norwich/HP_CUT">The CUT event</a></li>
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                   <li><a href="https://2014.igem.org/Team:NRP-UEA-Norwich/HP_School-Events">The Hewett School</a></li>
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                  <li><a href="https://2014.igem.org/Team:NRP-UEA-Norwich/HP_Collaborations">Collaborations</a></li>
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                 <a data-toggle="dropdown" class="dropdown-toggle" href="#">Safety <span class="caret"></span></a>
                 <a data-toggle="dropdown" class="dropdown-toggle" href="#">Safety <span class="caret"></span></a>
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                   <li><a href="https://2014.igem.org/Team:NRP-UEA-Norwich/Safety_Forms">Safety Forms</a></li>
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                   <li><a href="https://2014.igem.org/Team:NRP-UEA-Norwich/Safety">Safety Overview</a></li>
                   <li><a href="https://2014.igem.org/Team:NRP-UEA-Norwich/Safety_RA">Risk Assessments</a></li>
                   <li><a href="https://2014.igem.org/Team:NRP-UEA-Norwich/Safety_RA">Risk Assessments</a></li>
                   <li><a href="https://2014.igem.org/Team:NRP-UEA-Norwich/Safety_UEA">UEA Safety</a></li>
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         <h1>Project Overview</h1>
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         <h1>Green Canary</h1>
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<h2>Green Canary - A biosensor to induce chromoprotein signal in response to plant pathogen infection</h2> <img src="https://static.igem.org/mediawiki/2014/e/e0/Uea_logo.png" class="logo"/>
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<img src="https://static.igem.org/mediawiki/2014/c/c7/HeaderGCLogo.jpg" width=800/>
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Food security is a prominent health challenge faced by the increasing global population, which is exacerbated by high loss of crop yields to pests and diseases. Applying synthetic biology approaches, we aim to produce proof of concept, sentinel plants that will diagnose the presence of two pathogens, Xanthomonas oryzae and Xanthomonas campestris. The plant sentinels will produce a chromoprotein output, observable by the human eye, within 48 hours of pathogen infection. The sentinels would allow growers to apply appropriate agrochemical application before the diseases progress to symptomatic pathogenesis in neighbouring crops. This approach will reduce crop losses whilst decreasing the necessity for continual use of agrochemicals. Furthermore, we are constructing a series of BioBricks that will allow Golden Gate assembly to assist cloning of transcriptional units within the iGEM standard. These important developments will aid future iGEM teams to work with plant chassis’ as well as utilise Golden Gate technology.</p>
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<h2> What is Green Canary? </h2>
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A sentinel plant which warns of the presence of plant pathogens by producing a visible signal. </h1>
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<h2> Abstract </h2>
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Food security is a prominent challenge faced by the increasing global population. Currently  about 40% of crop losses are due to pests and diseases. Our aim is to reduce crop losses whilst decreasing the use of agrochemicals, contributing to more sustainable and less environmentally damaging farming practices. Applying synthetic biology approaches, we aim to produce proof-of-concept, sentinel plants that will diagnose the presence of two pathogens, <i>Xanthomonas oryzae</i> and <i>Xanthomonas campestris</i>. These <b>Green Canaries</b> will produce a signal, visible to the naked eye output, within 48 hours of detecting the pathogen. This will allow growers to make appropriate agrochemical application before the diseases progress to symptomatic pathogenesis in neighbouring crops. <b>Green Canaries</b> will also allow scientists to gather epidemiological data about plant diseases. Furthermore, we are constructing a series of BioBricks that will allow Golden Gate assembly to assist cloning of transcriptional units within the iGEM standard. These important developments will aid future iGEM teams to work with plant chassis as well as utilise the <a href="https://2014.igem.org/Team:NRP-UEA-Norwich/Project_GG-Cloning">Golden Gate</a> parallel assembly method.
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            <h2>Mischa Spiegel</h2>
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            <h3>Course</h3>
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<h2>The Experiments</h2>
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            <p>Molecular Biology and Genetics</p>
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Initially we selected several promoters known to respond to plant pathogens. We chose PDF1.2, a promoter from <i>Arabidopsis thaliana</i> that is induced by the hormone methyl jasmonate, produced naturally by plants in response to various biotic stress. We also chose PR1, which responds to salicylic acid (another plant hormone produced in response to infection). Lastly, we identified two promoters from capsicum and rice plants that are induced by TALES (<b>T</b>ranscriptional <b>A</b>ctivator <b>L</b>ike <b>E</b>ffectors</b>). Effectors are small molecules secreted by specific plant pathogens, <i>Xanthomonas oryzae</i> and '<i>Xanthomonas campestris</i> that enable the pathogen to invade. They bind to a very small region of their cognate promoter, inducing expression.  
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            <h3>Why iGEM?</h3>
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            <p>iGEM appealed to me as the idea of using genetics and engineering to create a novel system that would be of benefit to society excited me. The fact that the project is quite student led giving an aspect of laboratory and general scientific independence is beneficial to understanding research and science generally.</p>
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We then selected several coding sequences that, when expressed in plants, might produce a visible signal. First we chose <i>BaxI</i> from mice, which is known to induce cell death. We also chose genes that would make the plants go white (de-green) by breaking down chlorophyll and chromoproteins to colour the leaves canary-yellow or deep-blue. Ultimately, we planned to make a plant that would de-green in the presence of any pathogen and then re-colour to a new colour that identified the specific pathogen it had sensed.<br><br> <img src= "https://static.igem.org/mediawiki/2014/c/c0/NRPUEA_iGEM_Leaf.png" width=400/><br><br>  
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            <h3>Future career ideas</h3>
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            <p>I would like to be a medical doctor specialising in Oncological Genetics</p>
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We planned to test our disease-responsive promoters by using them to drive expression of GFP, which we know is easy to detect in the leaves of our chassis. To test our visible signals, we used a well-characterised constitutive promoter. To express our circuits in plants we assembled them in <i>E.coli</i> then transferred them to a second chassis, <a href="https://2014.igem.org/Team:NRP-UEA-Norwich/Project_System"> <i>Agrobacterium tumefaciens</i></a>, which has the ability to transfer DNA into plant cells. We then injected the leaves of our plant chassis with cultures of <i>A. tumefaciens</i> and monitored the plants for the expected signal. To avoid working directly with plant pathogens that require special permits, we painted the leaves with the appropriate plant hormone, (or simultaneously expressed the TALE protein) to test our inducible promoters.  
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            <h3>Interests/Hobbies</h3>
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            <p>I work part time with youth, in various roles. I also enjoy partaking in various aspects of church life including directing a gospel choir at my university. I love to dance, especially ballet and swim.</p>
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<h2>Materials and Methods</h2>
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            <h3>Main project roles</h3>
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We used <a href="https://2014.igem.org/Team:NRP-UEA-Norwich/Project_GG-Cloning">GoldenGate cloning </a>to assembly our constructs. This was very efficient as it allowed us to assemble a whole transcriptional unit (promoter, coding sequence and terminator) in a single step. We could then combine several transcriptional units into a multi-gene contract in a second step.
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            <p>Human Practices Queen</p>
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We used <a href="https://2014.igem.org/Team:NRP-UEA-Norwich/Project_System"><i>Nicotiana benthamina</i> </a> as the plant chassis for testing our plant circuits. ''N. benthamiana'' is a widely used experimental plant from the solanaceous group of flowering plants that includes tomatoes, potatoes and capsicums. We chose it because it is possible to obtain high-levels of transient expression in just a few days. Although this transient expression only lasts about a week, it is much quicker that making a stably integrating genes into a plant genome, which takes months! <br><br>
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Because we had to submit our parts to the registry in the iGEM shipping backbone, we also made some <a href="https://2014.igem.org/Team:NRP-UEA-Norwich/Project_Mo-Flipper">"flipper" plasmids</a>. These plasmids "flip" Golden Gate MoClo parts into standard biobricks.
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            <h2>Jessica Gray</h2>
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            <h3>Course</h3>
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The methods that we used for cloning and transfecting as well as other other useful protocols are given on our <a href="https://2014.igem.org/Team:NRP-UEA-Norwich/Notebook_Protocols">lab protocols </a>page.
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            <p>Biological Sciences with a year in North America</p>
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            <h3>Why iGEM?</h3>
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            <p>I was really keen on the idea of working as a team, making new friends with similar passions and interests whilst having a fun and productive summer. I believed iGEM would be a great opportunity to improve my lab skills and provide the chance to try new things including Golden Gate cloning and html coding for the wiki.</p>
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<h2>Results</h2>
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            <h3>Future career ideas</h3>
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The promoters that respond to plant hormones were successfully able to drive expression of GFP. Expression was strongly up-regulated in response to the hormone but a background expression level was also observed. This may have been caused by our transient delivery method, <i>A. tumefaciens</i>, which the plant recognises as a pathogen, even though the strain that we used is not capable of causing disease.<br><br>
-
            <p>I have a specific passion for Microbiology and pathogenesis which I believe is rooted in my summer placement at Rothamsted Research Institute. I hope to pursue my interest in Microbiology through further study and potentially as a career.</p>
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<div class="container">
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            <h3>Interests/Hobbies</h3>
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<div class="row">
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            <p>In my free time I write a science blog, read fiction novels and watch repeats of orange is the new black, dexter and game of thrones!</h3>
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<img src="https://static.igem.org/mediawiki/2014/8/87/Pdf1image2.jpg"/>
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            <p>Wiki Master, Golden Gate Girl</p> 
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<div class="row">
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<img src="https://static.igem.org/mediawiki/2014/7/75/PR1IMAGE.jpg"/>
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            <img src="https://static.igem.org/mediawiki/2014/2/22/Uea_steven.JPG" class="img-responsive"/>
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</div>
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            <h2>Steven Monsey</h2>
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            <h3>Course</h3>
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            <p>Biological Sciences</p>
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The promoters from <i>Xanthomonas oryzae</i> and <i>Xanthomonas campestris</i>, however, only induced expression of GFP in the presence of the corresponding TALE, indicating that expression from this promoter was very tightly regulated.<br><br>
-
            <h3>Why iGEM?</h3>
+
 
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            <p>I’ve been fascinated about biology for as long as I can remember, having been influenced by my Grandfather’s passion for the subject from a young age. University opened my mind to a wide range of biological subjects and I have taken interest in the microbial, genetic, and evolutionary disciplines. Having these interests naturally drew me to the iGEM project, which I saw as an ideal opportunity to not only broaden my mind on a wide range of innovative practices, but also to experience the day to day world of biological research. </p>
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<div class="row">
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            <h3>Future career ideas</h3>
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<img src="https://static.igem.org/mediawiki/2014/c/c7/AVRBS3IMAGE.jpg"/>
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            <p>If the right opportunity arises after my degree I would relish the chance to enter into biological research myself; however, the education of children in science is a subject close to my heart and I would love to pursue a career in teaching.</p>
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</div>
-
            <h3>Interests/Hobbies</h3>
+
 
-
            <p>Outside of biology and university I am a passionate musician and football fan.</p>
+
We were able to induce cell death using the coding sequence from the <i>Bax1</i> gene from mice. This produced a lesion on the plant leaf where the <i>A. tumefaciens</i>, carrying our circuit, had been delivered. We were unable to see expression of chromoproteins in our plant chassis, even when expressed from a known strong, constitutive promoter. It may be that the colour was masked by the green chlorophyll. The next step would be to attempt to express the chromoproteins simultaneously with a de-greening circuit that would remove the chlorophyll or to express them in the roots, which are devoid of chlorophyll.<br><br>
-
            <h3>Main project roles</h3>
+
 
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            <p>Human Practices King</p>
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<div class="row">
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<img src="https://static.igem.org/mediawiki/2014/b/b8/BAXIMAGE.jpg"/>
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<h2>Conclusions and Future Work</h2>
-
            <h2>Alistair Middlemiss</h2>
+
 
-
            <h3>Course</h3>
+
We were able to express circuits in plants that were switched on or up-regulated in response to signals of pathogen invasion. We were able to demonstrate promoters that respond to general stress signals that could be used even when the sentinel was unable to diagnose the specific pathogen. We were also able to demonstrate very tightly regulated expression in response to a particular pathogens.<br><br>
-
            <p>Microbiology with integrated Msc</p>
+
 
-
            <h3>Why iGEM?</h3>
+
We conclude that our project is viable and that we would continue with our original plan to produce plants that induce a mild readout (e.g. de-green) in the presence of any pathogen. The second step would be to use the pathogen-specific promoters to induce a second signal to diagnose the specific pathogen. Once these circuits had been tested in the transient system, we would make stably transformed plants that could be tested with the actual pathogens in the laboratory and, if successful and permits were granted, in field conditions.<br><br>
-
            <p>I applied to the iGEM team as I wanted to develop my scientific skills as well as challenge myself in areas that I had no expertise in for example, the Wiki, and Human Practices.  I have been interested in science since I was a child and as I grew, I developed an interest in microbiology; our project utilises microbiology which is what grabbed me.  The iGEM competition itself is very well regarded and of a high academic standard which will allow me to excel in my future scientific career.  </p>
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            <h3>Future career ideas</h3>
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Finally, we would investigate the switches that would shut off the circuit as presently the <b>Green Canaries</b> are single use, unable to return to green once they have encountered a pathogen.
-
            <p>I am fascinated by bacterial pathogenesis and infection and immunity so I hope to spend my career in that field of microbiology.  I have spent some time in hospitals in the past and wish to eventually work in the health sector so I can give back some of the fantastic treatment I received whilst in hospital.</p>
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            <h3>Interests/Hobbies</h3>
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            <p>When I’m not sitting on the sofa or at my part-time job, my spare time is usually spent playing Ultimate Frisbee!</p>
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            <h3>Main project roles</h3>
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            <p>Infiltration Prince, Lab Clown</p>
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            <img src="https://static.igem.org/mediawiki/2014/8/8d/Uea_jack.JPG" class="img-responsive"/>
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            <h2>Jack Day</h2>
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            <h3>Course</h3>
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            <p>Biological and Medicinal Chemistry</p>
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            <h3>Why iGEM?</h3>
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            <p>I got involved in iGEM as I just could not let this opportunity go to waste. The ability to work in a team on a project of our own design is an experience not many get the chance to pursue. iGEM gives participants the chance to see and explore research not otherwise taught during a standard degree and how the inner workings of a research project is undertaken.</p>
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            <h3>Future career ideas</h3>
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            <p>Whilst still being on the fence about continuing my education and studying for a Phd after I graduate, I would like to work in the field of understanding and developing treatments for neurodegenerative disease. </p>
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            <h3>Interests/Hobbies</h3>
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            <p>I have played cricket for the vast majority of my life and is my bread and butter. I also dabble in the art of juggling. Also I have a keen interest in social and behavioural psychology as a side interest to biochemistry.</p>
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            <h3>Main project roles</h3>
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            <p>Funding Guru, Lab Safety Emperor</p>
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          </div>
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            <img src="https://static.igem.org/mediawiki/2014/c/cb/Uea_cara.JPG" class="img-responsive"/>
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            <h2>Cara Deal</h2>
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            <h3>Course</h3>
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            <p>Biomedicine</p>
+
-
            <h3>Why iGEM?</h3>
+
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            <p>The iGEM competition appealed to me as it allowed us to design our own project and work within a team to achieve our aims.  I wanted to get some experience working in a lab and learning the techniques used in synthetic biology as well as developing skills in communicating science to a wide variety of people.</p>
+
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            <h3>Future career ideas</h3>
+
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            <p>After completing my degree in Biomedicine I would like to work in a Research lab, specialising in Biochemistry.</p>
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            <h3>Interests/Hobbies</h3>
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            <p>I enjoy playing sports such as football and hockey and have been dancing for 15 years.  I have also completed my Gold Duke of Edinburgh’s award which I really enjoyed doing.</p>
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            <h3>Main project roles</h3>
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            <p>Lab Queen Bee, Life Organiser, Golden Gate Girl</p>
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            <img src="https://static.igem.org/mediawiki/2014/b/bd/Uea_bowater.jpg" class="img-responsive advisors"/>
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            <h2>Dr Richard Bowater</h2>
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            <p>Richard is a senior lecturer in the School of Biological Sciences at UEA with a particular interest in biochemistry. He and his research group have many achievements including the characterisation of DNA repair processes from a variety of organisms. This research then feeds into mechanisms that influence genome stability. Furthermore, he is on the editorial advisory panel of the journal of biochemistry and the scientific world journal.</p>
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          </div>
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            <img src="https://static.igem.org/mediawiki/2014/6/68/Uea_thomas.jpg" class="img-responsive advisors"/>
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            <h2>Dr Colwyn Thomas</h2>
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            <p>Colwyn is a senior lecturer in the School of Biological Sciences at UEA, particularly interested in plant molecular genetics. His research is concentrated in determining the genetic and molecular basis of resistance or susceptibility to pathogen infection in plants. Furthermore, he is a mentor for the Gatsby Plant Science undergraduate programme. His proudest achievement is being a member of one of the first groups in the world to isolate and characterise a plant disease resistance gene – tomato Cf-9 at The Sainsbury Laboratory.
+
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Some of his interests outside of the lab are: Sport, particularly football, swimming and rowing. He is a member of Norwich Rowing Club and is currently working towards a rowing coach’s qualification.</p>
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            <img src="https://static.igem.org/mediawiki/2014/a/a3/Uea_patron.jpg" class="img-responsive advisors"/>
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            <h2>Dr Nicola Patron</h2>
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            <p>Nicola is the head of Synthetic Biology and a senior support specialist at The Sainsbury Laboratory. Her research is focused on developing molecular tools and methods for the precise modification of plant genomes, efficient high-order DNA assembly and the controlled delivery of molecular tools into plant cells.</p>
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            <h2>Dr Anna Smajdor</h2>
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            <p>Anna is lecturer and researcher in biomedical ethics. Her research interests are focused on the ethical implications of innovation and research in all areas of the biosciences, including: new reproductive technologies; research ethics and governance; justice and resource allocation. She has been vice chair of the Riverside Research Ethics Committee for a number of years as well as being a member of clinical ethics committees in London and Norwich.</p>
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            <img src="https://static.igem.org/mediawiki/2014/a/a3/Uea_banfield.jpg" class="img-responsive advisors"/>
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            <h2>Dr Mark Banfield</h2>
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            <p>Mark is a Biological Chemistry project leader at the John Innes Centre (JIC). Mark’s research is aimed at the study of structure/function relationships in effector proteins from pathogens of mammals and plants. He also studies the host side of plant immunity, through the use of biochemical, biophysical and structural studies. Furthermore, many of his studies are aimed at engineering plants to better resist infection by pathogens, improving performance in agriculture with a reduced need for chemical interventions.</p>
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Latest revision as of 21:50, 17 October 2014

NRP UEA Norwich iGEM 2014

Green Canary

What is Green Canary?

A sentinel plant which warns of the presence of plant pathogens by producing a visible signal.

Abstract

Food security is a prominent challenge faced by the increasing global population. Currently about 40% of crop losses are due to pests and diseases. Our aim is to reduce crop losses whilst decreasing the use of agrochemicals, contributing to more sustainable and less environmentally damaging farming practices. Applying synthetic biology approaches, we aim to produce proof-of-concept, sentinel plants that will diagnose the presence of two pathogens, Xanthomonas oryzae and Xanthomonas campestris. These Green Canaries will produce a signal, visible to the naked eye output, within 48 hours of detecting the pathogen. This will allow growers to make appropriate agrochemical application before the diseases progress to symptomatic pathogenesis in neighbouring crops. Green Canaries will also allow scientists to gather epidemiological data about plant diseases. Furthermore, we are constructing a series of BioBricks that will allow Golden Gate assembly to assist cloning of transcriptional units within the iGEM standard. These important developments will aid future iGEM teams to work with plant chassis as well as utilise the Golden Gate parallel assembly method.

The Experiments

Initially we selected several promoters known to respond to plant pathogens. We chose PDF1.2, a promoter from Arabidopsis thaliana that is induced by the hormone methyl jasmonate, produced naturally by plants in response to various biotic stress. We also chose PR1, which responds to salicylic acid (another plant hormone produced in response to infection). Lastly, we identified two promoters from capsicum and rice plants that are induced by TALES (Transcriptional Activator Like Effectors). Effectors are small molecules secreted by specific plant pathogens, Xanthomonas oryzae and 'Xanthomonas campestris that enable the pathogen to invade. They bind to a very small region of their cognate promoter, inducing expression. We then selected several coding sequences that, when expressed in plants, might produce a visible signal. First we chose BaxI from mice, which is known to induce cell death. We also chose genes that would make the plants go white (de-green) by breaking down chlorophyll and chromoproteins to colour the leaves canary-yellow or deep-blue. Ultimately, we planned to make a plant that would de-green in the presence of any pathogen and then re-colour to a new colour that identified the specific pathogen it had sensed.



We planned to test our disease-responsive promoters by using them to drive expression of GFP, which we know is easy to detect in the leaves of our chassis. To test our visible signals, we used a well-characterised constitutive promoter. To express our circuits in plants we assembled them in E.coli then transferred them to a second chassis, Agrobacterium tumefaciens, which has the ability to transfer DNA into plant cells. We then injected the leaves of our plant chassis with cultures of A. tumefaciens and monitored the plants for the expected signal. To avoid working directly with plant pathogens that require special permits, we painted the leaves with the appropriate plant hormone, (or simultaneously expressed the TALE protein) to test our inducible promoters.

Materials and Methods

We used GoldenGate cloning to assembly our constructs. This was very efficient as it allowed us to assemble a whole transcriptional unit (promoter, coding sequence and terminator) in a single step. We could then combine several transcriptional units into a multi-gene contract in a second step. We used Nicotiana benthamina as the plant chassis for testing our plant circuits. ''N. benthamiana'' is a widely used experimental plant from the solanaceous group of flowering plants that includes tomatoes, potatoes and capsicums. We chose it because it is possible to obtain high-levels of transient expression in just a few days. Although this transient expression only lasts about a week, it is much quicker that making a stably integrating genes into a plant genome, which takes months!

Because we had to submit our parts to the registry in the iGEM shipping backbone, we also made some "flipper" plasmids. These plasmids "flip" Golden Gate MoClo parts into standard biobricks. The methods that we used for cloning and transfecting as well as other other useful protocols are given on our lab protocols page.

Results

The promoters that respond to plant hormones were successfully able to drive expression of GFP. Expression was strongly up-regulated in response to the hormone but a background expression level was also observed. This may have been caused by our transient delivery method, A. tumefaciens, which the plant recognises as a pathogen, even though the strain that we used is not capable of causing disease.

The promoters from Xanthomonas oryzae and Xanthomonas campestris, however, only induced expression of GFP in the presence of the corresponding TALE, indicating that expression from this promoter was very tightly regulated.

We were able to induce cell death using the coding sequence from the Bax1 gene from mice. This produced a lesion on the plant leaf where the A. tumefaciens, carrying our circuit, had been delivered. We were unable to see expression of chromoproteins in our plant chassis, even when expressed from a known strong, constitutive promoter. It may be that the colour was masked by the green chlorophyll. The next step would be to attempt to express the chromoproteins simultaneously with a de-greening circuit that would remove the chlorophyll or to express them in the roots, which are devoid of chlorophyll.

Conclusions and Future Work

We were able to express circuits in plants that were switched on or up-regulated in response to signals of pathogen invasion. We were able to demonstrate promoters that respond to general stress signals that could be used even when the sentinel was unable to diagnose the specific pathogen. We were also able to demonstrate very tightly regulated expression in response to a particular pathogens.

We conclude that our project is viable and that we would continue with our original plan to produce plants that induce a mild readout (e.g. de-green) in the presence of any pathogen. The second step would be to use the pathogen-specific promoters to induce a second signal to diagnose the specific pathogen. Once these circuits had been tested in the transient system, we would make stably transformed plants that could be tested with the actual pathogens in the laboratory and, if successful and permits were granted, in field conditions.

Finally, we would investigate the switches that would shut off the circuit as presently the Green Canaries are single use, unable to return to green once they have encountered a pathogen.
A big thank you to our sponsors