http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=500&target=Joyehicks&year=&month=2012.igem.org - User contributions [en]2024-03-29T06:17:03ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:NRP-UEA-Norwich/TeamTeam:NRP-UEA-Norwich/Team2012-09-26T23:11:40Z<p>Joyehicks: /* The Team Members */</p>
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= '''The NRP UEA iGEM Team 2012''' = <br />
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[[File:FinalDraftPurple.png|right|300px|The NRP-UEA iGEM2012 Logo]] <br />
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The iGEM 2012 team for the Norwich Research Park and University of East Anglia (NRP UEA) are a close knit group of seven biology-based undergraduate students working in the labs within UEA’s BIO building. They are the second iGEM team that the NRP has produced and truly relish the opportunity to compete in a competition that has placed itself in such an innovative and exciting field of biology. <br />
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The NRP is one of Europe's largest concentrations of researchers in the life sciences and the environment, the UEA being a core constituent of the park. Known for its abundance of rabbits, its Ziggurats (as seen in various album covers) and its world-leading research. Despite having a olympic sized swimming pool on campus, the team prefer taking leisurely swimming in the river Yare that flows through the campus, a site of special scientific interest. It is frequented by cuckoos, sedge warblers, great crested grebes and, if you're lucky, you will see the odd flock of students too! <br />
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The iGEM team really are spoiled by both the natural beauty of their surroundings and the teaching excellence. Despite only having two main official academic advisers, practically everyone in the UEA School of Biology has very kindly imparted some of their wisdom to the student participance, as well as members of the John Innes Centre and the rest of the NRP, making the project a real representation of UEAs best and brightest!<br />
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==The Team Members==<br />
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<b><font size="3">Joy Edwards-Hicks</font><br><font size="2">Biomedicine Undergraduate</font></b><br><br><br />
Joy is going into her third year and is looking forward to a future career working within cell biology; preferably working in a lab out in the jungle somewhere! She has done a fantastic job of managing the lab, as well as the human practices side of the NRP UEA iGEM team, liasing with places such as STAR Radio and the Norwich Forum to score the team some brilliant opportunities to present the project. In her spare time Joy is normally found in the shops, until they close, when she can be found surfing eBay adding to her already-impressive clothes and shoes collection. <br />
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<b><font size="3">Khadija Ouadi</font><br><font size="2">Molecular Biology & Genetics Undergraduate</font></b><br><br><br />
Khadija anticipates beginning her third year of studies in September, and as someone that finds the intricacy of molecular biology amazing, she hopes to earn a living in academic research. iGEM has proved to be an experience she has relished, educating her both in what life in academia entails and in her own skill sets. Currently, her interests include tricking Rebecca into pegging herself in the lab groups many Peg Wars, failing to teach herself to play the guitar and using OligoAnalyser to make pretty shapes out of DNA constructs!<br />
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<b><font size="3">Lukas Harnisch</font><br><font size="2">Biomedicine Undergraduate</font></b><br><br><br />
Lukas is entering his 3rd year of studying Biomedicine. For him iGEM is a great opportunity to dive into the world of synthetic biology, the possibility to learn new skills and broaden his understanding of this field of research, and to meet and collaborate with students from all over the world. This summer he is looking forward to spending lots of time with his team members both inside the lab and outside. In his free time he enjoys photography, cooking and cycling, as well as travelling, and is therefore especially excited to go to this years jamboree in Amsterdam.<br />
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<b><font size="3">Pascoe Harvey</font><br><font size="2">Biological Sciences Undergraduate</font></b><br><br><br />
Pascoe is a Biological Sciences undergraduate, about to enter his final year. The iGEM experience has been invaluable to him and he is now strongly considering a future in academia specialising in synthetic biology. The project has fortified his presentational skills and blue sky thinking. It has also further developed his research and practical lab skills within a team environment from his previous work experience involving research into curculionoidea morphology in the Natural History Museum. He has a diverse range of interests from sports to the arts to politics. He is the only member who can ride a unicycle and is currently teaching his fellow iGEMers his self taught circus skills.<br><br> <br />
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<b><font size="3">Rachel Dobson</font><br><font size="2">Molecular Biology & Genetics Undergraduate</font></b><br><br><br />
Rachel is a smiley, enthusiastic second year student, studying Molecular Biology and Genetics at UEA. Over the last two years at UEA, she has also developed an interest in cell biology and is keen to continue further studies in this area. She has really enjoyed her summer, from getting to know the other iGEMers while chilling and playing games by the lake, to listening to a wide variety of music while carrying out aspects of wet lab. Rachel likes the variation of tasks that she has been able to undertake as a consequence of participating in this rewarding competition, both the practical and research side, as well as taking part with the elements of human outreach. When Rachel is not in the lab she is usually filling her time being very active, for example she supervises a weekly cooking class for a local charity, and more recently she has been improving her badminton skills.<br><br><br />
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<b><font size="3">Rebecca Lo</font><br><font size="2">Molecular Biology & Genetics Undergraduate</font></b><br><br><br />
Rebecca is about to enter her final year at UEA. After university she plans to pursue a career in academia with a particular interest in plant genetics. She greatly anticipates an iGEM filled summer. Applying for iGEM was an easy decision for her as it brought about the opportunity to meet students with similar interests to her, gain research experience, and of course have 10 weeks of awesomeness! Currently, her greatest joy would be to get revenge on Khadija in Peg Wars. She loves KPop, especially songs that blows your brains out, only difference is you do you the shooting. She also loves playing team sports but can be rather competitive; someone always gets hurt! She has a long list of accidental injuries, of which none are to herself, so you have been warned…<br />
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<b><font size="3">Russell Gritton</font><br><font size="2">Biomedicine Undergraduate</font></b><br><br><br />
Russell is a third-year Biomedicine student looking forward to a career in cell biology research. He sees iGEM as a fantastic opportunity to become a part of a new, innovative branch of science with many real-world applications for the future, and is therefore excited to be a part of this year’s NRP UEA team. He particularly likes the creative side of the project and is the main user of the team’s Twitter, as well as having designed the Wiki and many of the graphics on it. In his free time Russell enjoys good music and good company, and is currently trying to learn the ukulele.<br />
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<b><font size="3">Richard Bowater</font><br><font size="2">Advisor - Senior Lectuer</font></b><br><br><br />
Richard is a senior lecturer at the University of East Anglia, in the School of Biological Sciences. He specialises in Biochemistry, and is currently leading a research team at UEA. The work carried out within his lab studies the macromolecular interactions of DNA repair mechanisms in bacteria, in particular focusing on DNA ligases. Not only is he the founder of the team, but he also the spiritual and academic advisor of the team, therefore consistently keeping the team and their project on track.<br />
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<b><font size="3">Richard Kelwick</font><br><font size="2">Advisor - PhD Student</font></b><br><br><br />
Richard Kelwick is a doctoral Student at UEA, focusing his studies in Cancer Research. His research is centered around understanding the role of proteases in breast cancer and is interested in elucidating the tumour suppressor functions of several metalloproteinases, particularly ADAMTS15. Richard is THE KING of all things technological. Among the iGEM team he is renowned for his tweeting as he tweets more than Stephen Fry, as well as introducing google hangout to the UK team meet up. Richard is fueled by Twix's, giving him the energy that makes him super efficient and always the guy to save the day. It is Richards great advice, organisation of events and elevating the team spirit higher than a giraffe on stilts makes the NRP-UEA iGEM team the team that it is- #Thank you Richard and thank god for Twix's.<br><br><br />
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<b><font size="3">Amy Congdon</font><br><font size="2">NRP UEA iGEM Artist</font></b><br><br><br />
Amy Congdon completed her BA in Contemporary Textile Practices, at Norwich University College of Arts, and then went on to do her masters in Textile Futures, based at Central Saint Martins in London. Amy has played an important role as the team’s artist, since she got in touch with the team a few months before they started on their project. She is fascinated between the links of design, craft and science, and has used her interest to help the team to engage with the public. The team are very thankful to have her on board for her creative input, as well as her dedication and time to the team’s project. Amy has been very active within our human outreach program; from designing the team’s eye catching logo, to directing and editing their film. <br />
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==Team Spirit==<br />
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The iGEM competition 2012 was an amazing time for the whole NRP UEA team. At the beginning of the 12 weeks that our project would span, we didn't imagine the amount of knowledge and experience we would gain and the fun we would have. Not only did we work with many very interesting researchers at our university, we also became part of the great community that iGEM is and met other students that were just as drawn in by synthetic biology as we were. A highlight for this was definitely the UK team meetup that we hosted in London. <br />
All this made us grow close together as a team and the summer flew by. We celebrated our results and rejoiced when hard work payed off. But not only did we enjoy our time in the lab, we also made the most of our free time. We threw birthday parties, traveled together, enjoyed culture and music and in general just had a good time.<br />
To be honest, not everything was pleasant and in the lab especially first tries often did not succeed. We soon realised that research can confront you with setbacks that press down hard on moral. Everyone who has experienced this will know that team spirit is very important. Be it a failed gel purification or the realisation that you spend the last 3 days working with the wrong plasmid, it is never pleasant to see a team member disappointed. We were lucky to have an easy cure for this. We passed the time with fun games that sprung our imagination or were inspired by funny realisations or activities. Pegging, Dress-Up Thursday and mini prep queen/king, to name only things that caused lots of laughter and joy. But the game Ninjas was always the highlight be it during late nights of research, sunny lunch breaks or during the UK meetup where we played with lots of other teams.<br />
Team spirit has been high the whole time of iGEM 2012 and was one of the things that made this summer so rememberable. Read on and be inspired by the fun!<br />
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When 7 UEA undergraduates collide, we come together and have lots and lots of fun. Thanks to this awesome opportunity that iGEM has provided, the last few months have been AMAZING. We worked really hard but we also played hard and we would like to take you through some of our experiences and share the Joy - yes pun intended, with you.<br />
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This was our 10 weeks in, shortened to 6 minutes. It has been crazy experience and scary how fast it flew by. Below are some activities that we played more than a lot. We changed some of the rules around a little but we hope this gives others an idea of how to enjoy the project all the more.<br />
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For all iGEMers and researchers out there, you all know that there are times when during experiments you have some free time but not much or you just need a good pick me up, well these activities are perfect for you. They bring the team together, keep you alert and can make you laugh so hard that any stress of failed cloning or unexpected breakdowns become a thing of the past.<br />
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===Ninja's===<br />
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Contrary to the name of this game, you do not need to dress up in black or as turtles and throw shurikens, instead you just need to be swift, fluid and tactical. For this game you need to ideally have a large group of people but more than 3 is fine too. The aim of the game is to be the last man or woman standing. <br />
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====How to====<br />
If the video demonstration is not clear enough, here is a written version of the rules. There are actually quite a different set of rules for this game but we like to keep things fast, fun and unexpected by using this set of rules (i.e. we made up bits)<br />
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1) Get into a circle and everyone put your hands to the centre and have your finger tips pretty much touching.<br />
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2) Wiggle your fingers and say "EEEERRRRRRRRRRR Ninjas!", on the word of "Ninjas", everyone jumps back from the circle and gets into a ninja-like stance. What kind of stance you get into will probably depend on what films you watch.<br />
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3) One person then starts, they pick one person who is immediately to their left or right and in one fluid motion moves to hit their hand. In the meanwhile, the person who has been targetted is also allowed to move in one fluid continual movement.<br />
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4) If the person targetted has their hand hit they are out, and the next person in their direction continues the game. If the person evades being hit, then they move, they are also allowed to choose whichever direction to move in and hence you you target.<br />
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5) This game continues till only 2 people are left. These two then stop and get back to back and on the count of three jump away and make a VERY cool ninja pose. The coolest/hardest pose is the person who starts first. The winner is the last one remaining.<br />
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====Rules====<br />
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1) Only hitting the person's hand counts, that means no arms! However, it does not matter whether it is the palm or back of the hand you hit.<br />
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2) When the defending person moves, if they hit the next person in the hand in the process, that person who is hit, is out. This is a sneaky move but it makes the game more interesting.<br />
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3) Some people play this game so that you are only allowed to move in one direction, but moving in both makes it more unexpected.<br />
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4) When you move, it is like a robotic movement. If you move you arm forward, you may not retract your hand in the same move. Check out the dos and don't of this in the video.<br />
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5) Defense or approach movements are allowed. For defense, it may be that you know you are very close to being hit so you purposely jump back to evade being hit. You can also jump towards a person if you are too far away. <br />
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That's it, that's all the rules of Ninjas. If you think we are crazy for playing this, we are not the only ones. At the UK meet up, we got many many people involved. Don't believe me? Check out our team spirit video. This game is a great ice breaker. You just have to get past the bit, that some people hit really hard.<br />
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===Pegging Assassin===<br />
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This is a long game and takes more organisation but is very simple. It is literally pegging someone. The aim of the game is to have as many pegs as you can by the end of the game.<br />
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====How to====<br />
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1) At the start of the game, we write every participants name on pegs and put them into a bag or envelope, anything opaque is fine. <br />
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2) The team then take out a peg each, if one person gets the peg with their own name on it, then all pegs are put back into the bag and a reselection is called.<br />
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3) Once everyone has different pegs then the pegging commences. <br />
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====Rules====<br />
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1) The how to is really simple, now for some ground rules. When we play in lab, we make sure that when anyone is working, that includes talking to someone about work, dry lab work or wet lab, they are not pegged. <br />
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2) You can peg that person (e.g hair) or any of the clothes that person is wearing so that means no bags. However due to 1 minute rule, you can peg clothes they are not wearing and the minute rule commences when they put that item of clothing on. <br />
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3) The one minute rule means that if you peg that person and if for a minute they do not remove the peg, then you have successfully pegged them, if not then you have to retrieve the peg and continue.<br />
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4) You can trick the person to peg themselves. Rebecca found this out the hard way and the rule stayed.<br />
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5) Once someone has been pegged, then the pegger tells the pegged and the pegged surrenders all their pegs to the pegger. Therefore, if the pegged has 4 pegs, then the pegger is very lucky indeed.<br />
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6) If you get a peg with your own name on it, then that means you can peg anyone with that peg. This seemingly messes the game up but it actually makes the game rather interesting.<br />
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7) When there are only two people left and you can't seem to successfully peg each other, then there is a tie breaker. The tie breaker is to peg someone the first. This is decided by the team. For us, it was anyone who is extremely hard to peg. To make things harder, you must peg them and remove the peg after a minute, without them noticing.<br />
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This is a good game to get advisors into too. Not just the 7 of us undergraduates played this, we roped in Richard Kelwick and Richard Bowater. Pegging rules are quite relaxed so have fun and make up and change some rules, these are rules that we kept to but some of the best games are ones that you create so create some of your own too.</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/Human_OutreachTeam:NRP-UEA-Norwich/Human Outreach2012-09-26T23:01:06Z<p>Joyehicks: /* The team presented at the John Innes Centre Synthetic Biology conference */</p>
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=Human Practices=<br />
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The UEA NRP team really went up and beyond this year, carrying out a wide selection of activity's to inform the public of both synthetic biology and iGEM, aiming to raise awareness of this branch of science. We used many methods of communication in order to cater for a range of audiences, including a radio show, newspaper articles, pubic events and talks as well as collaborating with all the other UK teams, in order to organize the UK team meet up and steam it live on the internet. <br />
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== iGEM collaborations==<br />
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===The iGEM UK team meet up, hosted by the NRP-UEA team at Google campus London (Friday 17th August) The most open and publically accessible UK team meetup EVER===<br />
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The NRPUEA iGEM team organised and hosted the UK team meet up at Google campus in London. The NRPUEA iGEM team greeted the UK teams, provided refreshments and a lovely buffet lunch, as well as chairing the speeches and making sure the day ran smoothly. There was a great atmosphere at the meet up, and the event was a huge success.<br />
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The day consisted of a number of guest speakers, including advice and tips for iGEM success from Dr. Tom Ellis (advisor of the 2009 winning iGEM project E.chromi) and an interactive talk on synthetic biology and science communication from the BBC science presenter, Nature editor and Guardian writer, Dr. Adam Rutherford. The UK iGEM teams presented a poster and a 15 minute presentation about their project. This gave the members of each team the chance to practice their presentation skills before the European team meet up, as well as a chance for others to ask questions and see what other project routes the other teams have taken. <br />
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The event was also available '''LIVE on the internet''' via Google+ hangout, to allow the public and those that were unable to attend to still watch the presentations. Despite there being a few technical difficulties with the quality of the image, figures have shown that over 600 people watched the event.<br />
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[https://dl.dropbox.com/u/9957127/UK%20iGEM%20Team%20Hangout%2017th%20Aug%202012%20Programme.pdf '''Click here for programme PDF''']<br />
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=== Communicating with other iGEM teams===<br />
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As part of our efforts to collaborate with the other iGEM teams this year we've made an effort to keep in contact with an array of iGEM teams. Here are some highlights:<br />
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- We communicated with all of the UK iGEM teams via Twitter, Email, Facebook and Google+ before, during and after the UK team meet up.<br />
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- We tweeted with many other iGEM teams from around the world and closely followed the iGEM community.<br />
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- We video called the St Andrews iGEM team via Google+ Hang-out to discuss our projects further as well as to seek advice about the arrangements for the UK team meet-up.<br />
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- Our advisor Richard Kelwick was interviewed by the Edinburgh iGEM team via Skype about our teams perspectives on public awareness and understanding of synthetic biology.<br />
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- We visited the Cambridge team near the beginning of our project and they were kind enough to give us a tour of their labs. <br />
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- Finally, the British Columbia team sent us a questionnaire about intellectual property that we happily filled out.<br />
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==Public engagement==<br />
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===The Future of Science event held at the forum, Norwich (Sunday 19th August)===<br />
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The Forum is located at the heart of the city of Norwich, and hosts a wide range of free events and exhibits to be enjoyed by the whole community. The venue is packed with restaurants, cafes and shops, as well as being home to Norwich library and BBC East Anglia. <br />
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The NRPUEA iGEM team were fortunate enough to hold an interactive day at the Forum, giving the public a chance to engage within the world of synthetic biology. Before the date of the event, the team increased awareness via a range of techniques, including posters, leaflets and newspaper and internet adverts, including the Forums website. This resulted with a large turnout at the event, including the local press. <br />
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At the Future of Science event, the team presented a selection of informative posters, revealed their new film on a large screen, as well as showing examples of work carried out by the students during iGEM. For example, the public could view ''E.coli'' which had been transformed with GFP within a UV light box, as well as carrying out a counting colonies exercise. The NRPUEA team also created a range of synthetic biology educational material for the event, to inform the public of the concepts and terminology of synthetic biology. The different activities were designed to interest a selection of different ages. The educational resources include a range of quizzes and worksheets such as crosswords and matching exercises.<br />
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For younger children we designed a colourful jigsaw plasmid (as seen in image). The idea of this activity was for the participants to choose jigsaw pieces that represented the genes that code for certain characteristics, such as green or red hair, and piece them together to form a complete plasmid. Once they had designed their plasmid they used a biscuit base, icing of different colours and various sweets to create a "biscuit creature" that corresponded to the plasmid they had created. The learning outcome of this activity was to give the children an understanding that a different selection of genes, results in different characteristics, and that editing the DNA can result in a change in the characteristics. The activity was very popular and most of the participants seemed to understand this concept, as well as having great fun.<br />
<br />
The event attracted a range of ages, as well as mixed levels of previous interest into synthetic biology. It also became clear that many members of the public knew very little about synthetic biology, and therefore were very interested in finding out more about the new branch of biology and the iGEM competition. The NRPUEA iGEM team also took the opportunity to talk to the public and listen to their opinions about the work the iGEM teams are carrying out, which demonstrated how mixed the view of the public were. The day was a huge success.<br />
<br />
'''Synthetic Biology activity sheets'''<br />
<br />
[[File:Word_Search_of_Synthetic_Biology.pdf]],<br />
[[File:Decode_Disney.pdf]],<br />
[[File:Questions_comprehensive.pdf]],[[File:Decode_Music_Artists_and_Films.pdf]],[[File:Word_Search_of_Synthetic_Biology.pdf]]<br />
<br />
=== Creation of a futuristic company, Quanticare and production of a thought-provoking film===<br />
<br />
<br />
[[File:Tattoo1.png |300px]][[File:QuanticareLogo_(2).png |300px]][[File:Tattoo2.png |300px]]<br />
<br />
<br />
As part of our human practices we decided to look into an artistic approach to bringing synthetic biology to a wider audience. As part of this we created "Quanticare", a fictitious and futuristic company forming on the back of a rise to prominence of synthetic biology over the next few years, as well as the results of our own projects on nitric oxide sensing and the comparator circuit. The fictitious company's latest development is to introduce a visual bio sensors in to the human body in the form of a tattoo-based health monitor, ''Cura''. <br />
<br />
<br />
Not only did the development of the fictitious company allow the team to explore potential future applications of synthetic biology and bio sensors, but also via the creation of a film , fuel a range of ethical questions. The team released the video on the day of the Wiki Freeze as well as bringing transferable tattoo samples of ''Cura'' to the European Jamboree as a form of marketing for the video and concept as a whole. <br />
<br />
<br />
The team also produced a film with Amy Congdon exploring ''Cura'' itself and its many applications. They decided to angle the film as a product launch in order to capture the imagination and bring Quanticare and ''Cura'' to the public as a realistic future involving synthetic biology. The film is available to view below, we thank the Biochemical Society for their outreach grant to fund this film.<br />
<br />
<html><center><table width=100% cellpadding=0 cellspacing=0 align=center><tr><td valign=absmiddle align=center><iframe width="560" height="315" src="http://www.youtube.com/embed/StNFePmymbc" frameborder="0" allowfullscreen></iframe></td></tr></table></center></html><br />
<br />
<br />
<html><font size=3pt><b>Please check out out <a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Quanticare">Quanticare</a> page for more details!</b></font></html><br />
<br />
<br><br />
<br />
Some initial feedback, the Quanticare video was launched less than 24 before the wiki freeze:<br />
<br />
-Blogged about on Biochemical Society blog<br />
-On UEA news pages <br />
-Mentioned in tweet from the BBSRC<br />
-Many likes and tweets<br />
<br />
-Some quotes <br />
<br />
"Inspiring and Innovative" Global Strategist and Author @DeanneLawrence <br />
"Great project. Good luck!" @SocratesLogos Synthetic Biologist<br />
"Sooooooo cool!" Kimberley Hirst Jones UEA-JIC_Norwich iGEM 2011 team member<br />
<br />
==Social Media==<br />
The NRPUEA iGEM team has been VERY active on a range of social media over the summer.Any one is welcome to follow the team on Facebook or twitter. The team kept everyone updated on what was going on, both in the lab and in our spare time. This has been a great method of connecting with the public, companies,researchers, as well as getting in touch with other iGEM teams. The NRPUEA iGEM team believe human out reach via social media is important since it allows the public to follow the teams achievements and lab results, as well as providing an opportunity for members of the public to get to know the personality of the team members. The human stories behind science are crucial, informative and a powerful way to promote science in a positive light. Please follow us on twitter @NRPiGEM or UEA iGEM 2012 page on Facebook.<br />
<br />
===Twitter===<br />
<br />
[[File:Twitter stats NRP iGEM.png]]<br />
<br />
[[File:TweetReach_nrpigem.pdf| '''Download a PDF of our twitter statistics''']]<br />
<br />
===Facebook===<br />
<br />
Creating social media pages also allowed us to access information about who was checking our page out, showing interesting information, showing that social media techniques such as facebook attracted mostly 18-24 years olds interest, and that it attracted no attention from those above 65. Therefore the team thought it was important to promote our project, not only through the internet, but also other techniques such as newspaper articles and radio shows. <br />
<br />
The figure bellow shows the age ranges and gender of people who liked any posts from the NRP UEA iGEM team<br />
[[File:Likes.png |600px| centre]]<br />
<br />
Below is a list of the international locations that people liked our teams status updates from. This highlighted that whilst the majority of followers were located in Norwich, we also had followers from a range of places over the world, highlighting the importance of using such social media to reach these followers. <br />
[[File:Location_of_likes.png |600px| centre]]<br />
<br />
As mentioned the NRP UEA iGEM team created facebook statuses and tweets very regularly and as the graph below shows, there was a consistent increase in action and interest in the NRPUEA iGEM team throughout the summer. <br />
<br />
[[File:Posts_ratings.png |600px| centre]]<br />
<br />
===Google+===<br />
<br />
We championed the use of Google+, a new social network from Google. We encouraged the UK iGEM teams to utilise Google+ to help make the 2012 UK team meetup as open and accessible as possible.<br />
<br />
==Media appearances==<br />
<br />
===STAR Radio===<br />
<br />
[http://www.star107.co.uk/farming-show.php STAR 107.9 FM's Farming Show]<br />
<br />
[[Image:NRPSTARRadio.jpg | 300px | right]]<br />
<br />
[http://www.star107.co.uk/podcasts/the-farming-show/show-23.mp3 Listen To Our Radio Show Appearence!]<br />
<br />
In early July the team travelled to Cambridge to appear on STAR Radio's program: "The Farming Show". We were interviewed by Mark Peters (one of the presenters) about synthetic biology, iGEM and our project. The interview went really well as Pascoe and Khadija got across all of the information that was needed in a clear and concise way, and the presenter was extremely helpful in making sure all of the salient points of the project were brought out. <br />
<br />
The team were able to give a brief description of what synthetic biology is before talking about the future applications of synthetic biology for medicine, agriculture and business. We were also able to mention our event at the Norwich Forum in late August in order to help promote it!<br />
<br />
A big thanks goes out to all at STAR Radio 107.9 for their help with the project in setting up the interview and allowing us air time to discuss iGEM and synthetic biology. Please visit the link above to hear the radio show.<br />
<br />
<br />
<br />
[[File:Paper.jpg|100px|left]]<br />
<br />
===Newspapers===<br />
<br />
The team featured on the university website a few times through the summer, as well as a brief article in the Eastern daily press newspaper.This was great, as allowed the locals of Norwich to become aware not only of the team and iGEM, but also introduce many to the scientific branch of synthetic biology.<br />
<br />
<br />
<br />
<br />
<br />
==Engagement with the wider scientific community==<br />
<br />
=== The team presented at the John Innes Centre Synthetic Biology conference ===<br />
[[File:JIC.png|500px|right]]<br />
The John Innes Center (JIC) is a world leading center of plant research, the home of UEAs 2011 team, and part of the Norwich Research Park (NRP). Researchers at the JIC recognized that synthetic biology was an important field and therefore hosted a synthetic biology conference. The aim of the day was to enhance local researchers' knowledge of what synthetic biology is, and highlight projects in which synthetic biology is currently being used. When the team was invited to give a talk to the JIC researchers we were extremely excited. At the talk we gave an over view of what iGEM is, our project and how it developed over the summer, as well informing them about aspects of human practice. The team had carried out over the summer. the team took also showed them a clip of our futuristic applications film. We enjoyed presenting our work and results, as well having an opportunity to improve our presentation skills before the European jamboree. There seemed to be a real interest by the researchers in to our project, and the advice given was encouraging giving encouraging advice. The researchers also seemed very interested to hear more about our human practices elements of iGEM, wanting to know what we had discovered about the public's opinion our research. More than anything, researchers at the John Innes Centre couldn't believe that we were undergraduate students who had been working together for 10 weeks!<br />
<br />
The NRP iGEM teams are helping to foster collaborations between scientists across the NRP and have begun to create a sense of community amongst NRP synthetic biologists.<br />
<br />
===SGM microbiology synthetic biology (September 3rd -5th)===<br />
<br />
The society of general microbiology held a 3 day synthetic biology conference at the University of Warwick. Three members of the UEA NRP iGEM team were lucky enough to go and enjoy talks on streptococcus, molecular motors, and the dynamics of the genome and designer microbes. The event also gave the team member the opportunity to meet other synthetic biology scientists as well as informing them of the current advances in the field and inspiring them with the future possibilities. The event also allowed the team members to present a poster and therefore engage with the scientific community, both informing them more about the iGEM competition and our project . The team believed it was important for the scientists in the synthetic biology world to have the opportunity to meet undergraduate iGEM members, allowing them to express the skills it has allowed them to develop and to encourage further interest in to lab groups forming teams, and funding iGEM projects.<br />
<br />
=== The Biochemical Society kindly mentioned us in their blog post ===<br />
<br />
Not only did the Biochemical Society generously fund our project, they also were kind enough to feature our teams efforts in an encouraging ethical debate around the subject of synthetic biology (via our Quanticare video) in their blog.<br />
<br />
[http://biochemicalsociety.wordpress.com/2012/09/26/exploring-ethical-dilemmas-of-synthetic-biology-with-the-nrp-uea-norwich-igem-team/ '''Click here to view the blog post''']<br />
<br />
This post was also seen by the BBSRC, who very kindly tweeted about it!<br />
<br />
[[File:BBSRC.png | centre | 600px]]</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/Human_OutreachTeam:NRP-UEA-Norwich/Human Outreach2012-09-26T23:00:29Z<p>Joyehicks: /* The team presented at the John Innes Centre Synthetic Biology conference */</p>
<hr />
<div>{{UEANRP}}<br />
<br />
=Human Practices=<br />
<br />
<br />
The UEA NRP team really went up and beyond this year, carrying out a wide selection of activity's to inform the public of both synthetic biology and iGEM, aiming to raise awareness of this branch of science. We used many methods of communication in order to cater for a range of audiences, including a radio show, newspaper articles, pubic events and talks as well as collaborating with all the other UK teams, in order to organize the UK team meet up and steam it live on the internet. <br />
<br />
== iGEM collaborations==<br />
<br />
===The iGEM UK team meet up, hosted by the NRP-UEA team at Google campus London (Friday 17th August) The most open and publically accessible UK team meetup EVER===<br />
<br />
[[File:Hangout stats.png | center]]<br />
<br />
[[File:UKiGEMteams.jpg | 300px | right]]<br />
<br />
The NRPUEA iGEM team organised and hosted the UK team meet up at Google campus in London. The NRPUEA iGEM team greeted the UK teams, provided refreshments and a lovely buffet lunch, as well as chairing the speeches and making sure the day ran smoothly. There was a great atmosphere at the meet up, and the event was a huge success.<br />
<br />
The day consisted of a number of guest speakers, including advice and tips for iGEM success from Dr. Tom Ellis (advisor of the 2009 winning iGEM project E.chromi) and an interactive talk on synthetic biology and science communication from the BBC science presenter, Nature editor and Guardian writer, Dr. Adam Rutherford. The UK iGEM teams presented a poster and a 15 minute presentation about their project. This gave the members of each team the chance to practice their presentation skills before the European team meet up, as well as a chance for others to ask questions and see what other project routes the other teams have taken. <br />
<br />
The event was also available '''LIVE on the internet''' via Google+ hangout, to allow the public and those that were unable to attend to still watch the presentations. Despite there being a few technical difficulties with the quality of the image, figures have shown that over 600 people watched the event.<br />
<br />
[https://dl.dropbox.com/u/9957127/UK%20iGEM%20Team%20Hangout%2017th%20Aug%202012%20Programme.pdf '''Click here for programme PDF''']<br />
<br />
<br><br />
<br />
<html><align=center><br />
<table align=center width=100% cellpadding=0 cellspacing=0><br />
<tr><br />
<td valign=absmiddle align=center><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/Kv_Z14OIrKc" frameborder="0" allowfullscreen></iframe><br />
</td><br />
</tr><br />
</table><br />
</align><br />
</html><br />
<br />
<br><br />
<br />
=== Communicating with other iGEM teams===<br />
<br />
As part of our efforts to collaborate with the other iGEM teams this year we've made an effort to keep in contact with an array of iGEM teams. Here are some highlights:<br />
<br />
- We communicated with all of the UK iGEM teams via Twitter, Email, Facebook and Google+ before, during and after the UK team meet up.<br />
<br />
- We tweeted with many other iGEM teams from around the world and closely followed the iGEM community.<br />
<br />
- We video called the St Andrews iGEM team via Google+ Hang-out to discuss our projects further as well as to seek advice about the arrangements for the UK team meet-up.<br />
<br />
- Our advisor Richard Kelwick was interviewed by the Edinburgh iGEM team via Skype about our teams perspectives on public awareness and understanding of synthetic biology.<br />
<br />
- We visited the Cambridge team near the beginning of our project and they were kind enough to give us a tour of their labs. <br />
<br />
- Finally, the British Columbia team sent us a questionnaire about intellectual property that we happily filled out.<br />
<br />
==Public engagement==<br />
<br />
===The Future of Science event held at the forum, Norwich (Sunday 19th August)===<br />
<br />
<br />
<br />
[[File:Forum_team.JPG | 300px| right]]<br />
<br />
<br />
The Forum is located at the heart of the city of Norwich, and hosts a wide range of free events and exhibits to be enjoyed by the whole community. The venue is packed with restaurants, cafes and shops, as well as being home to Norwich library and BBC East Anglia. <br />
<br />
[[File:Poster for Forum.jpg | 250px | left]]<br />
<br />
The NRPUEA iGEM team were fortunate enough to hold an interactive day at the Forum, giving the public a chance to engage within the world of synthetic biology. Before the date of the event, the team increased awareness via a range of techniques, including posters, leaflets and newspaper and internet adverts, including the Forums website. This resulted with a large turnout at the event, including the local press. <br />
<br />
At the Future of Science event, the team presented a selection of informative posters, revealed their new film on a large screen, as well as showing examples of work carried out by the students during iGEM. For example, the public could view ''E.coli'' which had been transformed with GFP within a UV light box, as well as carrying out a counting colonies exercise. The NRPUEA team also created a range of synthetic biology educational material for the event, to inform the public of the concepts and terminology of synthetic biology. The different activities were designed to interest a selection of different ages. The educational resources include a range of quizzes and worksheets such as crosswords and matching exercises.<br />
<br />
[[File:Child_making_plasmid.jpg |300px | right]]<br />
<br />
For younger children we designed a colourful jigsaw plasmid (as seen in image). The idea of this activity was for the participants to choose jigsaw pieces that represented the genes that code for certain characteristics, such as green or red hair, and piece them together to form a complete plasmid. Once they had designed their plasmid they used a biscuit base, icing of different colours and various sweets to create a "biscuit creature" that corresponded to the plasmid they had created. The learning outcome of this activity was to give the children an understanding that a different selection of genes, results in different characteristics, and that editing the DNA can result in a change in the characteristics. The activity was very popular and most of the participants seemed to understand this concept, as well as having great fun.<br />
<br />
The event attracted a range of ages, as well as mixed levels of previous interest into synthetic biology. It also became clear that many members of the public knew very little about synthetic biology, and therefore were very interested in finding out more about the new branch of biology and the iGEM competition. The NRPUEA iGEM team also took the opportunity to talk to the public and listen to their opinions about the work the iGEM teams are carrying out, which demonstrated how mixed the view of the public were. The day was a huge success.<br />
<br />
'''Synthetic Biology activity sheets'''<br />
<br />
[[File:Word_Search_of_Synthetic_Biology.pdf]],<br />
[[File:Decode_Disney.pdf]],<br />
[[File:Questions_comprehensive.pdf]],[[File:Decode_Music_Artists_and_Films.pdf]],[[File:Word_Search_of_Synthetic_Biology.pdf]]<br />
<br />
=== Creation of a futuristic company, Quanticare and production of a thought-provoking film===<br />
<br />
<br />
[[File:Tattoo1.png |300px]][[File:QuanticareLogo_(2).png |300px]][[File:Tattoo2.png |300px]]<br />
<br />
<br />
As part of our human practices we decided to look into an artistic approach to bringing synthetic biology to a wider audience. As part of this we created "Quanticare", a fictitious and futuristic company forming on the back of a rise to prominence of synthetic biology over the next few years, as well as the results of our own projects on nitric oxide sensing and the comparator circuit. The fictitious company's latest development is to introduce a visual bio sensors in to the human body in the form of a tattoo-based health monitor, ''Cura''. <br />
<br />
<br />
Not only did the development of the fictitious company allow the team to explore potential future applications of synthetic biology and bio sensors, but also via the creation of a film , fuel a range of ethical questions. The team released the video on the day of the Wiki Freeze as well as bringing transferable tattoo samples of ''Cura'' to the European Jamboree as a form of marketing for the video and concept as a whole. <br />
<br />
<br />
The team also produced a film with Amy Congdon exploring ''Cura'' itself and its many applications. They decided to angle the film as a product launch in order to capture the imagination and bring Quanticare and ''Cura'' to the public as a realistic future involving synthetic biology. The film is available to view below, we thank the Biochemical Society for their outreach grant to fund this film.<br />
<br />
<html><center><table width=100% cellpadding=0 cellspacing=0 align=center><tr><td valign=absmiddle align=center><iframe width="560" height="315" src="http://www.youtube.com/embed/StNFePmymbc" frameborder="0" allowfullscreen></iframe></td></tr></table></center></html><br />
<br />
<br />
<html><font size=3pt><b>Please check out out <a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Quanticare">Quanticare</a> page for more details!</b></font></html><br />
<br />
<br><br />
<br />
Some initial feedback, the Quanticare video was launched less than 24 before the wiki freeze:<br />
<br />
-Blogged about on Biochemical Society blog<br />
-On UEA news pages <br />
-Mentioned in tweet from the BBSRC<br />
-Many likes and tweets<br />
<br />
-Some quotes <br />
<br />
"Inspiring and Innovative" Global Strategist and Author @DeanneLawrence <br />
"Great project. Good luck!" @SocratesLogos Synthetic Biologist<br />
"Sooooooo cool!" Kimberley Hirst Jones UEA-JIC_Norwich iGEM 2011 team member<br />
<br />
==Social Media==<br />
The NRPUEA iGEM team has been VERY active on a range of social media over the summer.Any one is welcome to follow the team on Facebook or twitter. The team kept everyone updated on what was going on, both in the lab and in our spare time. This has been a great method of connecting with the public, companies,researchers, as well as getting in touch with other iGEM teams. The NRPUEA iGEM team believe human out reach via social media is important since it allows the public to follow the teams achievements and lab results, as well as providing an opportunity for members of the public to get to know the personality of the team members. The human stories behind science are crucial, informative and a powerful way to promote science in a positive light. Please follow us on twitter @NRPiGEM or UEA iGEM 2012 page on Facebook.<br />
<br />
===Twitter===<br />
<br />
[[File:Twitter stats NRP iGEM.png]]<br />
<br />
[[File:TweetReach_nrpigem.pdf| '''Download a PDF of our twitter statistics''']]<br />
<br />
===Facebook===<br />
<br />
Creating social media pages also allowed us to access information about who was checking our page out, showing interesting information, showing that social media techniques such as facebook attracted mostly 18-24 years olds interest, and that it attracted no attention from those above 65. Therefore the team thought it was important to promote our project, not only through the internet, but also other techniques such as newspaper articles and radio shows. <br />
<br />
The figure bellow shows the age ranges and gender of people who liked any posts from the NRP UEA iGEM team<br />
[[File:Likes.png |600px| centre]]<br />
<br />
Below is a list of the international locations that people liked our teams status updates from. This highlighted that whilst the majority of followers were located in Norwich, we also had followers from a range of places over the world, highlighting the importance of using such social media to reach these followers. <br />
[[File:Location_of_likes.png |600px| centre]]<br />
<br />
As mentioned the NRP UEA iGEM team created facebook statuses and tweets very regularly and as the graph below shows, there was a consistent increase in action and interest in the NRPUEA iGEM team throughout the summer. <br />
<br />
[[File:Posts_ratings.png |600px| centre]]<br />
<br />
===Google+===<br />
<br />
We championed the use of Google+, a new social network from Google. We encouraged the UK iGEM teams to utilise Google+ to help make the 2012 UK team meetup as open and accessible as possible.<br />
<br />
==Media appearances==<br />
<br />
===STAR Radio===<br />
<br />
[http://www.star107.co.uk/farming-show.php STAR 107.9 FM's Farming Show]<br />
<br />
[[Image:NRPSTARRadio.jpg | 300px | right]]<br />
<br />
[http://www.star107.co.uk/podcasts/the-farming-show/show-23.mp3 Listen To Our Radio Show Appearence!]<br />
<br />
In early July the team travelled to Cambridge to appear on STAR Radio's program: "The Farming Show". We were interviewed by Mark Peters (one of the presenters) about synthetic biology, iGEM and our project. The interview went really well as Pascoe and Khadija got across all of the information that was needed in a clear and concise way, and the presenter was extremely helpful in making sure all of the salient points of the project were brought out. <br />
<br />
The team were able to give a brief description of what synthetic biology is before talking about the future applications of synthetic biology for medicine, agriculture and business. We were also able to mention our event at the Norwich Forum in late August in order to help promote it!<br />
<br />
A big thanks goes out to all at STAR Radio 107.9 for their help with the project in setting up the interview and allowing us air time to discuss iGEM and synthetic biology. Please visit the link above to hear the radio show.<br />
<br />
<br />
<br />
[[File:Paper.jpg|100px|left]]<br />
<br />
===Newspapers===<br />
<br />
The team featured on the university website a few times through the summer, as well as a brief article in the Eastern daily press newspaper.This was great, as allowed the locals of Norwich to become aware not only of the team and iGEM, but also introduce many to the scientific branch of synthetic biology.<br />
<br />
<br />
<br />
<br />
<br />
==Engagement with the wider scientific community==<br />
<br />
=== The team presented at the John Innes Centre Synthetic Biology conference ===<br />
[[File:JIC.png|400px|right]]<br />
The John Innes Center (JIC) is a world leading center of plant research, the home of UEAs 2011 team, and part of the Norwich Research Park (NRP). Researchers at the JIC recognized that synthetic biology was an important field and therefore hosted a synthetic biology conference. The aim of the day was to enhance local researchers' knowledge of what synthetic biology is, and highlight projects in which synthetic biology is currently being used. When the team was invited to give a talk to the JIC researchers we were extremely excited. At the talk we gave an over view of what iGEM is, our project and how it developed over the summer, as well informing them about aspects of human practice. The team had carried out over the summer. the team took also showed them a clip of our futuristic applications film. We enjoyed presenting our work and results, as well having an opportunity to improve our presentation skills before the European jamboree. There seemed to be a real interest by the researchers in to our project, and the advice given was encouraging giving encouraging advice. The researchers also seemed very interested to hear more about our human practices elements of iGEM, wanting to know what we had discovered about the public's opinion our research. More than anything, researchers at the John Innes Centre couldn't believe that we were undergraduate students who had been working together for 10 weeks!<br />
<br />
The NRP iGEM teams are helping to foster collaborations between scientists across the NRP and have begun to create a sense of community amongst NRP synthetic biologists.<br />
<br />
===SGM microbiology synthetic biology (September 3rd -5th)===<br />
<br />
The society of general microbiology held a 3 day synthetic biology conference at the University of Warwick. Three members of the UEA NRP iGEM team were lucky enough to go and enjoy talks on streptococcus, molecular motors, and the dynamics of the genome and designer microbes. The event also gave the team member the opportunity to meet other synthetic biology scientists as well as informing them of the current advances in the field and inspiring them with the future possibilities. The event also allowed the team members to present a poster and therefore engage with the scientific community, both informing them more about the iGEM competition and our project . The team believed it was important for the scientists in the synthetic biology world to have the opportunity to meet undergraduate iGEM members, allowing them to express the skills it has allowed them to develop and to encourage further interest in to lab groups forming teams, and funding iGEM projects.<br />
<br />
=== The Biochemical Society kindly mentioned us in their blog post ===<br />
<br />
Not only did the Biochemical Society generously fund our project, they also were kind enough to feature our teams efforts in an encouraging ethical debate around the subject of synthetic biology (via our Quanticare video) in their blog.<br />
<br />
[http://biochemicalsociety.wordpress.com/2012/09/26/exploring-ethical-dilemmas-of-synthetic-biology-with-the-nrp-uea-norwich-igem-team/ '''Click here to view the blog post''']<br />
<br />
This post was also seen by the BBSRC, who very kindly tweeted about it!<br />
<br />
[[File:BBSRC.png | centre | 600px]]</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/Human_OutreachTeam:NRP-UEA-Norwich/Human Outreach2012-09-26T22:58:56Z<p>Joyehicks: /* The team presented at the John Innes Centre Synthetic Biology conference */</p>
<hr />
<div>{{UEANRP}}<br />
<br />
=Human Practices=<br />
<br />
<br />
The UEA NRP team really went up and beyond this year, carrying out a wide selection of activity's to inform the public of both synthetic biology and iGEM, aiming to raise awareness of this branch of science. We used many methods of communication in order to cater for a range of audiences, including a radio show, newspaper articles, pubic events and talks as well as collaborating with all the other UK teams, in order to organize the UK team meet up and steam it live on the internet. <br />
<br />
== iGEM collaborations==<br />
<br />
===The iGEM UK team meet up, hosted by the NRP-UEA team at Google campus London (Friday 17th August) The most open and publically accessible UK team meetup EVER===<br />
<br />
[[File:Hangout stats.png | center]]<br />
<br />
[[File:UKiGEMteams.jpg | 300px | right]]<br />
<br />
The NRPUEA iGEM team organised and hosted the UK team meet up at Google campus in London. The NRPUEA iGEM team greeted the UK teams, provided refreshments and a lovely buffet lunch, as well as chairing the speeches and making sure the day ran smoothly. There was a great atmosphere at the meet up, and the event was a huge success.<br />
<br />
The day consisted of a number of guest speakers, including advice and tips for iGEM success from Dr. Tom Ellis (advisor of the 2009 winning iGEM project E.chromi) and an interactive talk on synthetic biology and science communication from the BBC science presenter, Nature editor and Guardian writer, Dr. Adam Rutherford. The UK iGEM teams presented a poster and a 15 minute presentation about their project. This gave the members of each team the chance to practice their presentation skills before the European team meet up, as well as a chance for others to ask questions and see what other project routes the other teams have taken. <br />
<br />
The event was also available '''LIVE on the internet''' via Google+ hangout, to allow the public and those that were unable to attend to still watch the presentations. Despite there being a few technical difficulties with the quality of the image, figures have shown that over 600 people watched the event.<br />
<br />
[https://dl.dropbox.com/u/9957127/UK%20iGEM%20Team%20Hangout%2017th%20Aug%202012%20Programme.pdf '''Click here for programme PDF''']<br />
<br />
<br><br />
<br />
<html><align=center><br />
<table align=center width=100% cellpadding=0 cellspacing=0><br />
<tr><br />
<td valign=absmiddle align=center><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/Kv_Z14OIrKc" frameborder="0" allowfullscreen></iframe><br />
</td><br />
</tr><br />
</table><br />
</align><br />
</html><br />
<br />
<br><br />
<br />
=== Communicating with other iGEM teams===<br />
<br />
As part of our efforts to collaborate with the other iGEM teams this year we've made an effort to keep in contact with an array of iGEM teams. Here are some highlights:<br />
<br />
- We communicated with all of the UK iGEM teams via Twitter, Email, Facebook and Google+ before, during and after the UK team meet up.<br />
<br />
- We tweeted with many other iGEM teams from around the world and closely followed the iGEM community.<br />
<br />
- We video called the St Andrews iGEM team via Google+ Hang-out to discuss our projects further as well as to seek advice about the arrangements for the UK team meet-up.<br />
<br />
- Our advisor Richard Kelwick was interviewed by the Edinburgh iGEM team via Skype about our teams perspectives on public awareness and understanding of synthetic biology.<br />
<br />
- We visited the Cambridge team near the beginning of our project and they were kind enough to give us a tour of their labs. <br />
<br />
- Finally, the British Columbia team sent us a questionnaire about intellectual property that we happily filled out.<br />
<br />
==Public engagement==<br />
<br />
===The Future of Science event held at the forum, Norwich (Sunday 19th August)===<br />
<br />
<br />
<br />
[[File:Forum_team.JPG | 300px| right]]<br />
<br />
<br />
The Forum is located at the heart of the city of Norwich, and hosts a wide range of free events and exhibits to be enjoyed by the whole community. The venue is packed with restaurants, cafes and shops, as well as being home to Norwich library and BBC East Anglia. <br />
<br />
[[File:Poster for Forum.jpg | 250px | left]]<br />
<br />
The NRPUEA iGEM team were fortunate enough to hold an interactive day at the Forum, giving the public a chance to engage within the world of synthetic biology. Before the date of the event, the team increased awareness via a range of techniques, including posters, leaflets and newspaper and internet adverts, including the Forums website. This resulted with a large turnout at the event, including the local press. <br />
<br />
At the Future of Science event, the team presented a selection of informative posters, revealed their new film on a large screen, as well as showing examples of work carried out by the students during iGEM. For example, the public could view ''E.coli'' which had been transformed with GFP within a UV light box, as well as carrying out a counting colonies exercise. The NRPUEA team also created a range of synthetic biology educational material for the event, to inform the public of the concepts and terminology of synthetic biology. The different activities were designed to interest a selection of different ages. The educational resources include a range of quizzes and worksheets such as crosswords and matching exercises.<br />
<br />
[[File:Child_making_plasmid.jpg |300px | right]]<br />
<br />
For younger children we designed a colourful jigsaw plasmid (as seen in image). The idea of this activity was for the participants to choose jigsaw pieces that represented the genes that code for certain characteristics, such as green or red hair, and piece them together to form a complete plasmid. Once they had designed their plasmid they used a biscuit base, icing of different colours and various sweets to create a "biscuit creature" that corresponded to the plasmid they had created. The learning outcome of this activity was to give the children an understanding that a different selection of genes, results in different characteristics, and that editing the DNA can result in a change in the characteristics. The activity was very popular and most of the participants seemed to understand this concept, as well as having great fun.<br />
<br />
The event attracted a range of ages, as well as mixed levels of previous interest into synthetic biology. It also became clear that many members of the public knew very little about synthetic biology, and therefore were very interested in finding out more about the new branch of biology and the iGEM competition. The NRPUEA iGEM team also took the opportunity to talk to the public and listen to their opinions about the work the iGEM teams are carrying out, which demonstrated how mixed the view of the public were. The day was a huge success.<br />
<br />
'''Synthetic Biology activity sheets'''<br />
<br />
[[File:Word_Search_of_Synthetic_Biology.pdf]],<br />
[[File:Decode_Disney.pdf]],<br />
[[File:Questions_comprehensive.pdf]],[[File:Decode_Music_Artists_and_Films.pdf]],[[File:Word_Search_of_Synthetic_Biology.pdf]]<br />
<br />
=== Creation of a futuristic company, Quanticare and production of a thought-provoking film===<br />
<br />
<br />
[[File:Tattoo1.png |300px]][[File:QuanticareLogo_(2).png |300px]][[File:Tattoo2.png |300px]]<br />
<br />
<br />
As part of our human practices we decided to look into an artistic approach to bringing synthetic biology to a wider audience. As part of this we created "Quanticare", a fictitious and futuristic company forming on the back of a rise to prominence of synthetic biology over the next few years, as well as the results of our own projects on nitric oxide sensing and the comparator circuit. The fictitious company's latest development is to introduce a visual bio sensors in to the human body in the form of a tattoo-based health monitor, ''Cura''. <br />
<br />
<br />
Not only did the development of the fictitious company allow the team to explore potential future applications of synthetic biology and bio sensors, but also via the creation of a film , fuel a range of ethical questions. The team released the video on the day of the Wiki Freeze as well as bringing transferable tattoo samples of ''Cura'' to the European Jamboree as a form of marketing for the video and concept as a whole. <br />
<br />
<br />
The team also produced a film with Amy Congdon exploring ''Cura'' itself and its many applications. They decided to angle the film as a product launch in order to capture the imagination and bring Quanticare and ''Cura'' to the public as a realistic future involving synthetic biology. The film is available to view below, we thank the Biochemical Society for their outreach grant to fund this film.<br />
<br />
<html><center><table width=100% cellpadding=0 cellspacing=0 align=center><tr><td valign=absmiddle align=center><iframe width="560" height="315" src="http://www.youtube.com/embed/StNFePmymbc" frameborder="0" allowfullscreen></iframe></td></tr></table></center></html><br />
<br />
<br />
<html><font size=3pt><b>Please check out out <a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Quanticare">Quanticare</a> page for more details!</b></font></html><br />
<br />
<br><br />
<br />
Some initial feedback, the Quanticare video was launched less than 24 before the wiki freeze:<br />
<br />
-Blogged about on Biochemical Society blog<br />
-On UEA news pages <br />
-Mentioned in tweet from the BBSRC<br />
-Many likes and tweets<br />
<br />
-Some quotes <br />
<br />
"Inspiring and Innovative" Global Strategist and Author @DeanneLawrence <br />
"Great project. Good luck!" @SocratesLogos Synthetic Biologist<br />
"Sooooooo cool!" Kimberley Hirst Jones UEA-JIC_Norwich iGEM 2011 team member<br />
<br />
==Social Media==<br />
The NRPUEA iGEM team has been VERY active on a range of social media over the summer.Any one is welcome to follow the team on Facebook or twitter. The team kept everyone updated on what was going on, both in the lab and in our spare time. This has been a great method of connecting with the public, companies,researchers, as well as getting in touch with other iGEM teams. The NRPUEA iGEM team believe human out reach via social media is important since it allows the public to follow the teams achievements and lab results, as well as providing an opportunity for members of the public to get to know the personality of the team members. The human stories behind science are crucial, informative and a powerful way to promote science in a positive light. Please follow us on twitter @NRPiGEM or UEA iGEM 2012 page on Facebook.<br />
<br />
===Twitter===<br />
<br />
[[File:Twitter stats NRP iGEM.png]]<br />
<br />
[[File:TweetReach_nrpigem.pdf| '''Download a PDF of our twitter statistics''']]<br />
<br />
===Facebook===<br />
<br />
Creating social media pages also allowed us to access information about who was checking our page out, showing interesting information, showing that social media techniques such as facebook attracted mostly 18-24 years olds interest, and that it attracted no attention from those above 65. Therefore the team thought it was important to promote our project, not only through the internet, but also other techniques such as newspaper articles and radio shows. <br />
<br />
The figure bellow shows the age ranges and gender of people who liked any posts from the NRP UEA iGEM team<br />
[[File:Likes.png |600px| centre]]<br />
<br />
Below is a list of the international locations that people liked our teams status updates from. This highlighted that whilst the majority of followers were located in Norwich, we also had followers from a range of places over the world, highlighting the importance of using such social media to reach these followers. <br />
[[File:Location_of_likes.png |600px| centre]]<br />
<br />
As mentioned the NRP UEA iGEM team created facebook statuses and tweets very regularly and as the graph below shows, there was a consistent increase in action and interest in the NRPUEA iGEM team throughout the summer. <br />
<br />
[[File:Posts_ratings.png |600px| centre]]<br />
<br />
===Google+===<br />
<br />
We championed the use of Google+, a new social network from Google. We encouraged the UK iGEM teams to utilise Google+ to help make the 2012 UK team meetup as open and accessible as possible.<br />
<br />
==Media appearances==<br />
<br />
===STAR Radio===<br />
<br />
[http://www.star107.co.uk/farming-show.php STAR 107.9 FM's Farming Show]<br />
<br />
[[Image:NRPSTARRadio.jpg | 300px | right]]<br />
<br />
[http://www.star107.co.uk/podcasts/the-farming-show/show-23.mp3 Listen To Our Radio Show Appearence!]<br />
<br />
In early July the team travelled to Cambridge to appear on STAR Radio's program: "The Farming Show". We were interviewed by Mark Peters (one of the presenters) about synthetic biology, iGEM and our project. The interview went really well as Pascoe and Khadija got across all of the information that was needed in a clear and concise way, and the presenter was extremely helpful in making sure all of the salient points of the project were brought out. <br />
<br />
The team were able to give a brief description of what synthetic biology is before talking about the future applications of synthetic biology for medicine, agriculture and business. We were also able to mention our event at the Norwich Forum in late August in order to help promote it!<br />
<br />
A big thanks goes out to all at STAR Radio 107.9 for their help with the project in setting up the interview and allowing us air time to discuss iGEM and synthetic biology. Please visit the link above to hear the radio show.<br />
<br />
<br />
<br />
[[File:Paper.jpg|100px|left]]<br />
<br />
===Newspapers===<br />
<br />
The team featured on the university website a few times through the summer, as well as a brief article in the Eastern daily press newspaper.This was great, as allowed the locals of Norwich to become aware not only of the team and iGEM, but also introduce many to the scientific branch of synthetic biology.<br />
<br />
<br />
<br />
<br />
<br />
==Engagement with the wider scientific community==<br />
<br />
=== The team presented at the John Innes Centre Synthetic Biology conference ===<br />
<br />
The John Innes Center (JIC) is a world leading center of plant research, the home of UEAs 2011 team, and part of the Norwich Research Park (NRP). Researchers at the JIC recognized that synthetic biology was an important field and therefore hosted a synthetic biology conference. The aim of the day was to enhance local researchers' knowledge of what synthetic biology is, and highlight projects in which synthetic biology is currently being used. When the team was invited to give a talk to the JIC researchers we were extremely excited. At the talk we gave an over view of what iGEM is, our project and how it developed over the summer, as well informing them about aspects of human practice. The team had carried out over the summer. the team took also showed them a clip of our futuristic applications film. We enjoyed presenting our work and results, as well having an opportunity to improve our presentation skills before the European jamboree. There seemed to be a real interest by the researchers in to our project, and the advice given was encouraging giving encouraging advice. The researchers also seemed very interested to hear more about our human practices elements of iGEM, wanting to know what we had discovered about the public's opinion our research. More than anything, researchers at the John Innes Centre couldn't believe that we were undergraduate students who had been working together for 10 weeks!<br />
<br />
The NRP iGEM teams are helping to foster collaborations between scientists across the NRP and have begun to create a sense of community amongst NRP synthetic biologists.<br />
[[File:JIC.png|right|thumbnail| Programme of events]]<br />
<br />
===SGM microbiology synthetic biology (September 3rd -5th)===<br />
<br />
The society of general microbiology held a 3 day synthetic biology conference at the University of Warwick. Three members of the UEA NRP iGEM team were lucky enough to go and enjoy talks on streptococcus, molecular motors, and the dynamics of the genome and designer microbes. The event also gave the team member the opportunity to meet other synthetic biology scientists as well as informing them of the current advances in the field and inspiring them with the future possibilities. The event also allowed the team members to present a poster and therefore engage with the scientific community, both informing them more about the iGEM competition and our project . The team believed it was important for the scientists in the synthetic biology world to have the opportunity to meet undergraduate iGEM members, allowing them to express the skills it has allowed them to develop and to encourage further interest in to lab groups forming teams, and funding iGEM projects.<br />
<br />
=== The Biochemical Society kindly mentioned us in their blog post ===<br />
<br />
Not only did the Biochemical Society generously fund our project, they also were kind enough to feature our teams efforts in an encouraging ethical debate around the subject of synthetic biology (via our Quanticare video) in their blog.<br />
<br />
[http://biochemicalsociety.wordpress.com/2012/09/26/exploring-ethical-dilemmas-of-synthetic-biology-with-the-nrp-uea-norwich-igem-team/ '''Click here to view the blog post''']<br />
<br />
This post was also seen by the BBSRC, who very kindly tweeted about it!<br />
<br />
[[File:BBSRC.png | centre | 600px]]</div>Joyehickshttp://2012.igem.org/File:JIC.pngFile:JIC.png2012-09-26T22:57:53Z<p>Joyehicks: </p>
<hr />
<div></div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/Human_OutreachTeam:NRP-UEA-Norwich/Human Outreach2012-09-26T22:54:02Z<p>Joyehicks: /* The team presented at the John Innes Centre Synthetic Biology conference */</p>
<hr />
<div>{{UEANRP}}<br />
<br />
=Human Practices=<br />
<br />
<br />
The UEA NRP team really went up and beyond this year, carrying out a wide selection of activity's to inform the public of both synthetic biology and iGEM, aiming to raise awareness of this branch of science. We used many methods of communication in order to cater for a range of audiences, including a radio show, newspaper articles, pubic events and talks as well as collaborating with all the other UK teams, in order to organize the UK team meet up and steam it live on the internet. <br />
<br />
== iGEM collaborations==<br />
<br />
===The iGEM UK team meet up, hosted by the NRP-UEA team at Google campus London (Friday 17th August) The most open and publically accessible UK team meetup EVER===<br />
<br />
[[File:Hangout stats.png | center]]<br />
<br />
[[File:UKiGEMteams.jpg | 300px | right]]<br />
<br />
The NRPUEA iGEM team organised and hosted the UK team meet up at Google campus in London. The NRPUEA iGEM team greeted the UK teams, provided refreshments and a lovely buffet lunch, as well as chairing the speeches and making sure the day ran smoothly. There was a great atmosphere at the meet up, and the event was a huge success.<br />
<br />
The day consisted of a number of guest speakers, including advice and tips for iGEM success from Dr. Tom Ellis (advisor of the 2009 winning iGEM project E.chromi) and an interactive talk on synthetic biology and science communication from the BBC science presenter, Nature editor and Guardian writer, Dr. Adam Rutherford. The UK iGEM teams presented a poster and a 15 minute presentation about their project. This gave the members of each team the chance to practice their presentation skills before the European team meet up, as well as a chance for others to ask questions and see what other project routes the other teams have taken. <br />
<br />
The event was also available '''LIVE on the internet''' via Google+ hangout, to allow the public and those that were unable to attend to still watch the presentations. Despite there being a few technical difficulties with the quality of the image, figures have shown that over 600 people watched the event.<br />
<br />
[https://dl.dropbox.com/u/9957127/UK%20iGEM%20Team%20Hangout%2017th%20Aug%202012%20Programme.pdf '''Click here for programme PDF''']<br />
<br />
<br><br />
<br />
<html><align=center><br />
<table align=center width=100% cellpadding=0 cellspacing=0><br />
<tr><br />
<td valign=absmiddle align=center><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/Kv_Z14OIrKc" frameborder="0" allowfullscreen></iframe><br />
</td><br />
</tr><br />
</table><br />
</align><br />
</html><br />
<br />
<br><br />
<br />
=== Communicating with other iGEM teams===<br />
<br />
As part of our efforts to collaborate with the other iGEM teams this year we've made an effort to keep in contact with an array of iGEM teams. Here are some highlights:<br />
<br />
- We communicated with all of the UK iGEM teams via Twitter, Email, Facebook and Google+ before, during and after the UK team meet up.<br />
<br />
- We tweeted with many other iGEM teams from around the world and closely followed the iGEM community.<br />
<br />
- We video called the St Andrews iGEM team via Google+ Hang-out to discuss our projects further as well as to seek advice about the arrangements for the UK team meet-up.<br />
<br />
- Our advisor Richard Kelwick was interviewed by the Edinburgh iGEM team via Skype about our teams perspectives on public awareness and understanding of synthetic biology.<br />
<br />
- We visited the Cambridge team near the beginning of our project and they were kind enough to give us a tour of their labs. <br />
<br />
- Finally, the British Columbia team sent us a questionnaire about intellectual property that we happily filled out.<br />
<br />
==Public engagement==<br />
<br />
===The Future of Science event held at the forum, Norwich (Sunday 19th August)===<br />
<br />
<br />
<br />
[[File:Forum_team.JPG | 300px| right]]<br />
<br />
<br />
The Forum is located at the heart of the city of Norwich, and hosts a wide range of free events and exhibits to be enjoyed by the whole community. The venue is packed with restaurants, cafes and shops, as well as being home to Norwich library and BBC East Anglia. <br />
<br />
[[File:Poster for Forum.jpg | 250px | left]]<br />
<br />
The NRPUEA iGEM team were fortunate enough to hold an interactive day at the Forum, giving the public a chance to engage within the world of synthetic biology. Before the date of the event, the team increased awareness via a range of techniques, including posters, leaflets and newspaper and internet adverts, including the Forums website. This resulted with a large turnout at the event, including the local press. <br />
<br />
At the Future of Science event, the team presented a selection of informative posters, revealed their new film on a large screen, as well as showing examples of work carried out by the students during iGEM. For example, the public could view ''E.coli'' which had been transformed with GFP within a UV light box, as well as carrying out a counting colonies exercise. The NRPUEA team also created a range of synthetic biology educational material for the event, to inform the public of the concepts and terminology of synthetic biology. The different activities were designed to interest a selection of different ages. The educational resources include a range of quizzes and worksheets such as crosswords and matching exercises.<br />
<br />
[[File:Child_making_plasmid.jpg |300px | right]]<br />
<br />
For younger children we designed a colourful jigsaw plasmid (as seen in image). The idea of this activity was for the participants to choose jigsaw pieces that represented the genes that code for certain characteristics, such as green or red hair, and piece them together to form a complete plasmid. Once they had designed their plasmid they used a biscuit base, icing of different colours and various sweets to create a "biscuit creature" that corresponded to the plasmid they had created. The learning outcome of this activity was to give the children an understanding that a different selection of genes, results in different characteristics, and that editing the DNA can result in a change in the characteristics. The activity was very popular and most of the participants seemed to understand this concept, as well as having great fun.<br />
<br />
The event attracted a range of ages, as well as mixed levels of previous interest into synthetic biology. It also became clear that many members of the public knew very little about synthetic biology, and therefore were very interested in finding out more about the new branch of biology and the iGEM competition. The NRPUEA iGEM team also took the opportunity to talk to the public and listen to their opinions about the work the iGEM teams are carrying out, which demonstrated how mixed the view of the public were. The day was a huge success.<br />
<br />
'''Synthetic Biology activity sheets'''<br />
<br />
[[File:Word_Search_of_Synthetic_Biology.pdf]],<br />
[[File:Decode_Disney.pdf]],<br />
[[File:Questions_comprehensive.pdf]],[[File:Decode_Music_Artists_and_Films.pdf]],[[File:Word_Search_of_Synthetic_Biology.pdf]]<br />
<br />
=== Creation of a futuristic company, Quanticare and production of a thought-provoking film===<br />
<br />
<br />
[[File:Tattoo1.png |300px]][[File:QuanticareLogo_(2).png |300px]][[File:Tattoo2.png |300px]]<br />
<br />
<br />
As part of our human practices we decided to look into an artistic approach to bringing synthetic biology to a wider audience. As part of this we created "Quanticare", a fictitious and futuristic company forming on the back of a rise to prominence of synthetic biology over the next few years, as well as the results of our own projects on nitric oxide sensing and the comparator circuit. The fictitious company's latest development is to introduce a visual bio sensors in to the human body in the form of a tattoo-based health monitor, ''Cura''. <br />
<br />
<br />
Not only did the development of the fictitious company allow the team to explore potential future applications of synthetic biology and bio sensors, but also via the creation of a film , fuel a range of ethical questions. The team released the video on the day of the Wiki Freeze as well as bringing transferable tattoo samples of ''Cura'' to the European Jamboree as a form of marketing for the video and concept as a whole. <br />
<br />
<br />
The team also produced a film with Amy Congdon exploring ''Cura'' itself and its many applications. They decided to angle the film as a product launch in order to capture the imagination and bring Quanticare and ''Cura'' to the public as a realistic future involving synthetic biology. The film is available to view below, we thank the Biochemical Society for their outreach grant to fund this film.<br />
<br />
<html><center><table width=100% cellpadding=0 cellspacing=0 align=center><tr><td valign=absmiddle align=center><iframe width="560" height="315" src="http://www.youtube.com/embed/StNFePmymbc" frameborder="0" allowfullscreen></iframe></td></tr></table></center></html><br />
<br />
<br />
<html><font size=3pt><b>Please check out out <a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Quanticare">Quanticare</a> page for more details!</b></font></html><br />
<br />
<br><br />
<br />
Some initial feedback, the Quanticare video was launched less than 24 before the wiki freeze:<br />
<br />
-Blogged about on Biochemical Society blog<br />
-On UEA news pages <br />
-Mentioned in tweet from the BBSRC<br />
-Many likes and tweets<br />
<br />
-Some quotes <br />
<br />
"Inspiring and Innovative" Global Strategist and Author @DeanneLawrence <br />
"Great project. Good luck!" @SocratesLogos Synthetic Biologist<br />
"Sooooooo cool!" Kimberley Hirst Jones UEA-JIC_Norwich iGEM 2011 team member<br />
<br />
==Social Media==<br />
The NRPUEA iGEM team has been VERY active on a range of social media over the summer.Any one is welcome to follow the team on Facebook or twitter. The team kept everyone updated on what was going on, both in the lab and in our spare time. This has been a great method of connecting with the public, companies,researchers, as well as getting in touch with other iGEM teams. The NRPUEA iGEM team believe human out reach via social media is important since it allows the public to follow the teams achievements and lab results, as well as providing an opportunity for members of the public to get to know the personality of the team members. The human stories behind science are crucial, informative and a powerful way to promote science in a positive light. Please follow us on twitter @NRPiGEM or UEA iGEM 2012 page on Facebook.<br />
<br />
===Twitter===<br />
<br />
[[File:Twitter stats NRP iGEM.png]]<br />
<br />
[[File:TweetReach_nrpigem.pdf| '''Download a PDF of our twitter statistics''']]<br />
<br />
===Facebook===<br />
<br />
Creating social media pages also allowed us to access information about who was checking our page out, showing interesting information, showing that social media techniques such as facebook attracted mostly 18-24 years olds interest, and that it attracted no attention from those above 65. Therefore the team thought it was important to promote our project, not only through the internet, but also other techniques such as newspaper articles and radio shows. <br />
<br />
The figure bellow shows the age ranges and gender of people who liked any posts from the NRP UEA iGEM team<br />
[[File:Likes.png |600px| centre]]<br />
<br />
Below is a list of the international locations that people liked our teams status updates from. This highlighted that whilst the majority of followers were located in Norwich, we also had followers from a range of places over the world, highlighting the importance of using such social media to reach these followers. <br />
[[File:Location_of_likes.png |600px| centre]]<br />
<br />
As mentioned the NRP UEA iGEM team created facebook statuses and tweets very regularly and as the graph below shows, there was a consistent increase in action and interest in the NRPUEA iGEM team throughout the summer. <br />
<br />
[[File:Posts_ratings.png |600px| centre]]<br />
<br />
===Google+===<br />
<br />
We championed the use of Google+, a new social network from Google. We encouraged the UK iGEM teams to utilise Google+ to help make the 2012 UK team meetup as open and accessible as possible.<br />
<br />
==Media appearances==<br />
<br />
===STAR Radio===<br />
<br />
[http://www.star107.co.uk/farming-show.php STAR 107.9 FM's Farming Show]<br />
<br />
[[Image:NRPSTARRadio.jpg | 300px | right]]<br />
<br />
[http://www.star107.co.uk/podcasts/the-farming-show/show-23.mp3 Listen To Our Radio Show Appearence!]<br />
<br />
In early July the team travelled to Cambridge to appear on STAR Radio's program: "The Farming Show". We were interviewed by Mark Peters (one of the presenters) about synthetic biology, iGEM and our project. The interview went really well as Pascoe and Khadija got across all of the information that was needed in a clear and concise way, and the presenter was extremely helpful in making sure all of the salient points of the project were brought out. <br />
<br />
The team were able to give a brief description of what synthetic biology is before talking about the future applications of synthetic biology for medicine, agriculture and business. We were also able to mention our event at the Norwich Forum in late August in order to help promote it!<br />
<br />
A big thanks goes out to all at STAR Radio 107.9 for their help with the project in setting up the interview and allowing us air time to discuss iGEM and synthetic biology. Please visit the link above to hear the radio show.<br />
<br />
<br />
<br />
[[File:Paper.jpg|100px|left]]<br />
<br />
===Newspapers===<br />
<br />
The team featured on the university website a few times through the summer, as well as a brief article in the Eastern daily press newspaper.This was great, as allowed the locals of Norwich to become aware not only of the team and iGEM, but also introduce many to the scientific branch of synthetic biology.<br />
<br />
<br />
<br />
<br />
<br />
==Engagement with the wider scientific community==<br />
<br />
=== The team presented at the John Innes Centre Synthetic Biology conference ===<br />
<br />
The John Innes Center (JIC) is a world leading center of plant research, the home of UEAs 2011 team, and part of the Norwich Research Park (NRP). Researchers at the JIC recognized that synthetic biology was an important field and therefore hosted a synthetic biology conference. The aim of the day was to enhance local researchers' knowledge of what synthetic biology is, and highlight projects in which synthetic biology is currently being used. When the team was invited to give a talk to the JIC researchers we were extremely excited. At the talk we gave an over view of what iGEM is, our project and how it developed over the summer, as well informing them about aspects of human practice. The team had carried out over the summer. the team took also showed them a clip of our futuristic applications film. We enjoyed presenting our work and results, as well having an opportunity to improve our presentation skills before the European jamboree. There seemed to be a real interest by the researchers in to our project, and the advice given was encouraging giving encouraging advice. The researchers also seemed very interested to hear more about our human practices elements of iGEM, wanting to know what we had discovered about the public's opinion our research. More than anything, researchers at the John Innes Centre couldn't believe that we were undergraduate students who had been working together for 10 weeks!<br />
[[File:JIC_Synthetic_Biology_Workshop.pdf]]<br />
<br />
===SGM microbiology synthetic biology (September 3rd -5th)===<br />
<br />
The society of general microbiology held a 3 day synthetic biology conference at the University of Warwick. Three members of the UEA NRP iGEM team were lucky enough to go and enjoy talks on streptococcus, molecular motors, and the dynamics of the genome and designer microbes. The event also gave the team member the opportunity to meet other synthetic biology scientists as well as informing them of the current advances in the field and inspiring them with the future possibilities. The event also allowed the team members to present a poster and therefore engage with the scientific community, both informing them more about the iGEM competition and our project . The team believed it was important for the scientists in the synthetic biology world to have the opportunity to meet undergraduate iGEM members, allowing them to express the skills it has allowed them to develop and to encourage further interest in to lab groups forming teams, and funding iGEM projects.<br />
<br />
=== The Biochemical Society kindly mentioned us in their blog post ===<br />
<br />
Not only did the Biochemical Society generously fund our project, they also were kind enough to feature our teams efforts in encouraging ethical debate around the subject of synthetic biology (via our Quanticare video) in their blog.<br />
<br />
[http://biochemicalsociety.wordpress.com/2012/09/26/exploring-ethical-dilemmas-of-synthetic-biology-with-the-nrp-uea-norwich-igem-team/ '''Click here to view the blog post''']<br />
<br />
This post was also seen by the BBSRC, who very kindly tweeted about it!<br />
<br />
[[File:BBSRC.png | centre | 600px]]</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/Human_OutreachTeam:NRP-UEA-Norwich/Human Outreach2012-09-26T22:38:43Z<p>Joyehicks: /* STAR Radio */</p>
<hr />
<div>{{UEANRP}}<br />
<br />
=Human Practices=<br />
<br />
<br />
The UEA NRP team really went up and beyond this year, carrying out a wide selection of activity's to inform the public of both synthetic biology and iGEM, aiming to raise awareness of this branch of science. We used many methods of communication in order to cater for a range of audiences, including a radio show, newspaper articles, pubic events and talks as well as collaborating with all the other UK teams, in order to organize the UK team meet up and steam it live on the internet. <br />
<br />
== iGEM collaborations==<br />
<br />
===The iGEM UK team meet up, hosted by the NRP-UEA team at Google campus London (Friday 17th August) The most open and publically accessible UK team meetup EVER===<br />
<br />
[[File:Hangout stats.png | center]]<br />
<br />
[[File:UKiGEMteams.jpg | 300px | right]]<br />
<br />
The NRPUEA iGEM team organised and hosted the UK team meet up at Google campus in London. The NRPUEA iGEM team greeted the UK teams, provided refreshments and a lovely buffet lunch, as well as chairing the speeches and making sure the day ran smoothly. There was a great atmosphere at the meet up, and the event was a huge success.<br />
<br />
The day consisted of a number of guest speakers, including advice and tips for iGEM success from Dr. Tom Ellis (advisor of the 2009 winning iGEM project E.chromi) and an interactive talk on synthetic biology and science communication from the BBC science presenter, Nature editor and Guardian writer, Dr. Adam Rutherford. The UK iGEM teams presented a poster and a 15 minute presentation about their project. This gave the members of each team the chance to practice their presentation skills before the European team meet up, as well as a chance for others to ask questions and see what other project routes the other teams have taken. <br />
<br />
The event was also available '''LIVE on the internet''' via Google+ hangout, to allow the public and those that were unable to attend to still watch the presentations. Despite there being a few technical difficulties with the quality of the image, figures have shown that over 600 people watched the event.<br />
<br />
[https://dl.dropbox.com/u/9957127/UK%20iGEM%20Team%20Hangout%2017th%20Aug%202012%20Programme.pdf '''Click here for programme PDF''']<br />
<br />
<br><br />
<br />
<html><align=center><br />
<table align=center width=100% cellpadding=0 cellspacing=0><br />
<tr><br />
<td valign=absmiddle align=center><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/Kv_Z14OIrKc" frameborder="0" allowfullscreen></iframe><br />
</td><br />
</tr><br />
</table><br />
</align><br />
</html><br />
<br />
<br><br />
<br />
=== Communicating with other iGEM teams===<br />
<br />
As part of our efforts to collaborate with the other iGEM teams this year we've made an effort to keep in contact with an array of iGEM teams. Here are some highlights:<br />
<br />
- We communicated with all of the UK iGEM teams via Twitter, Email, Facebook and Google+ before, during and after the UK team meet up.<br />
<br />
- We tweeted with many other iGEM teams from around the world and closely followed the iGEM community.<br />
<br />
- We video called the St Andrews iGEM team via Google+ Hang-out to discuss our projects further as well as to seek advice about the arrangements for the UK team meet-up.<br />
<br />
- Our advisor Richard Kelwick was interviewed by the Edinburgh iGEM team via Skype about our teams perspectives on public awareness and understanding of synthetic biology.<br />
<br />
- We visited the Cambridge team near the beginning of our project and they were kind enough to give us a tour of their labs. <br />
<br />
- Finally, the British Columbia team sent us a questionnaire about intellectual property that we happily filled out.<br />
<br />
==Public engagement==<br />
<br />
===The Future of Science event held at the forum, Norwich (Sunday 19th August)===<br />
<br />
<br />
<br />
[[File:Forum_team.JPG | 300px| right]]<br />
<br />
<br />
The Forum is located at the heart of the city of Norwich, and hosts a wide range of free events and exhibits to be enjoyed by the whole community. The venue is packed with restaurants, cafes and shops, as well as being home to Norwich library and BBC East Anglia. <br />
<br />
[[File:Poster for Forum.jpg | 250px | left]]<br />
<br />
The NRPUEA iGEM team were fortunate enough to hold an interactive day at the Forum, giving the public a chance to engage within the world of synthetic biology. Before the date of the event, the team increased awareness via a range of techniques, including posters, leaflets and newspaper and internet adverts, including the Forums website. This resulted with a large turnout at the event, including the local press. <br />
<br />
At the Future of Science event, the team presented a selection of informative posters, revealed their new film on a large screen, as well as showing examples of work carried out by the students during iGEM. For example, the public could view ''E.coli'' which had been transformed with GFP within a UV light box, as well as carrying out a counting colonies exercise. The NRPUEA team also created a range of synthetic biology educational material for the event, to inform the public of the concepts and terminology of synthetic biology. The different activities were designed to interest a selection of different ages. The educational resources include a range of quizzes and worksheets such as crosswords and matching exercises.<br />
<br />
[[File:Child_making_plasmid.jpg |300px | right]]<br />
<br />
For younger children we designed a colourful jigsaw plasmid (as seen in image). The idea of this activity was for the participants to choose jigsaw pieces that represented the genes that code for certain characteristics, such as green or red hair, and piece them together to form a complete plasmid. Once they had designed their plasmid they used a biscuit base, icing of different colours and various sweets to create a "biscuit creature" that corresponded to the plasmid they had created. The learning outcome of this activity was to give the children an understanding that a different selection of genes, results in different characteristics, and that editing the DNA can result in a change in the characteristics. The activity was very popular and most of the participants seemed to understand this concept, as well as having great fun.<br />
<br />
The event attracted a range of ages, as well as mixed levels of previous interest into synthetic biology. It also became clear that many members of the public knew very little about synthetic biology, and therefore were very interested in finding out more about the new branch of biology and the iGEM competition. The NRPUEA iGEM team also took the opportunity to talk to the public and listen to their opinions about the work the iGEM teams are carrying out, which demonstrated how mixed the view of the public were. The day was a huge success.<br />
<br />
'''Synthetic Biology activity sheets'''<br />
<br />
[[File:Word_Search_of_Synthetic_Biology.pdf]],<br />
[[File:Decode_Disney.pdf]],<br />
[[File:Questions_comprehensive.pdf]],[[File:Decode_Music_Artists_and_Films.pdf]],[[File:Word_Search_of_Synthetic_Biology.pdf]]<br />
<br />
=== Creation of futuristic company, Quanticare and production of a though-provoking film===<br />
<br />
<br />
[[File:Tattoo1.png |300px]][[File:QuanticareLogo_(2).png |300px]][[File:Tattoo2.png |300px]]<br />
<br />
<br />
As part of our human practices we decided to look into an artistic approach to bringing synthetic biology to a wider audience. As part of this we created "Quanticare", a fictitious and futuristic company forming on the back of a rise to prominence of synthetic biology over the next few years, as well as the results of our own projects on nitric oxide sensing and the comparator circuit. The fictitious company's latest development is to introduce a visual bio sensors in to the human body in the form of a tattoo-based health monitor, ''Cura''. <br />
<br />
<br />
Not only did the development of the fictitious company allow the team to explore potential future applications of synthetic biology and bio sensors, but also via the creation of a film , fuel a range of ethical questions. The team released the video on the day of the Wiki Freeze as well as bringing transferable tattoo samples of ''Cura'' to the European Jamboree as a form of marketing for the video and concept as a whole. <br />
<br />
<br />
The team also produced a film with Amy Congdon exploring ''Cura'' itself and its many applications. They decided to angle the film as a product launch in order to capture the imagination and bring Quanticare and ''Cura'' to the public as a realistic future involving synthetic biology. The film is available to view below, we thank the Biochemical Society for their outreach grant to fund this film.<br />
<br />
<html><center><table width=100% cellpadding=0 cellspacing=0 align=center><tr><td valign=absmiddle align=center><iframe width="560" height="315" src="http://www.youtube.com/embed/StNFePmymbc" frameborder="0" allowfullscreen></iframe></td></tr></table></center></html><br />
<br />
<br />
<html><font size=3pt><b>Please check out out <a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Quanticare">Quanticare</a> page for more details!</b></font></html><br />
<br />
==Social Media==<br />
The NRPUEA iGEM team has been VERY active on a range of social media over the summer.Any one is welcome to follow the team on Facebook or twitter. The team keep everyone updated on what is going on, both in the lab and in our spare time. This has been a great method of connecting with the public, companies,researchers, as well as getting in touch with other iGEM teams. The NRPUEA iGEM team believe human out reach via social media is important since it allows the public to follow the teams achievements and lab results, as well as get to know the personality of the team members. please follow us on twitter @NRPiGEM or UEA iGEM 2012 page on Facebook.<br />
<br />
===Twitter===<br />
<br />
[[File:Twitter stats NRP iGEM.png]]<br />
<br />
[[File:TweetReach_nrpigem.pdf| '''Download a PDF of our twitter statistics''']]<br />
<br />
===Facebook===<br />
<br />
Creating social media pages also allowed us to access information about who was checking our page out, showing interesting information, showing that social media techniques such as facebook attracted mostly 18-24 years olds interest, and that it attracted no attention from those above 65. Therefore the team though it was important to promote our project, not only through the internet, but also other techniques such as newspaper articles and radio shows. <br />
<br />
The figure bellow shows the age ranges and gender of people who liked any posts from the NRP UEA iGEM team<br />
[[File:Likes.png |600px| centre]]<br />
<br />
Below is a list of the location that people liked the teams statuses from. This highlighted that whilst the majority of followers were located in Norwich, we also had followers from a range of places over the world, highlighting the importance of using such social media to reach these followers. <br />
[[File:Location_of_likes.png |600px| centre]]<br />
<br />
As mentioned the NRP UEA iGEM team created facebook statuses and tweets very regularly and as the graph below shows, there was a consistent increase in action and interest in the NRPUEA iGEM team throughout the summer. <br />
<br />
[[File:Posts_ratings.png |600px| centre]]<br />
<br />
===Google+===<br />
<br />
We championed the use of Google+, a new social network from Google. We encouraged the UK iGEM teams to utilise Google+ to help make the 2012 UK team meetup as open and accessible as possible.<br />
<br />
==Media appearances==<br />
<br />
===STAR Radio===<br />
<br />
[http://www.star107.co.uk/farming-show.php STAR 107.9 FM's Farming Show]<br />
<br />
[[Image:NRPSTARRadio.jpg | 300px | right]]<br />
<br />
[http://www.star107.co.uk/podcasts/the-farming-show/show-23.mp3 Listen To Our Radio Show Appearence!]<br />
<br />
In early July the team travelled to Cambridge to appear on STAR Radio's program: "The Farming Show". We were interviewed by Mark Peters (one of the presenters) about synthetic biology, iGEM and our project. The interview went really well as Pascoe and Khadija got across all of the information that was needed in a clear and concise way, and the presenter was extremely helpful in making sure all of the salient points of the project were brought out. <br />
<br />
The team were able to give a brief description of what synthetic biology is before talking about the future applications of synthetic biology for medicine, agriculture and business. We were also able to mention our event at the Norwich Forum in late August in order to help promote it!<br />
<br />
A big thanks goes out to all at STAR Radio 107.9 for their help with the project in setting up the interview and allowing us air time to discuss iGEM and synthetic biology. Please visit the link above to hear the radio show.<br />
<br />
<br />
<br />
[[File:Paper.jpg|100px|left]]<br />
<br />
===Newspapers===<br />
<br />
The team featured on the university website a few times through the summer, as well as a brief article in the Eastern daily press newspaper.This was great, as allowed the locals of Norwich to become aware not only of the team and iGEM, but also introduce many to the scientific branch of synthetic biology.<br />
<br />
<br />
<br />
<br />
<br />
==Engagement with the wider scientific community==<br />
<br />
=== The team presented at the John Innes Centre Synthetic Biology conference ===<br />
<br />
The john innes center (JIC) is a world leading center of plant research, the home of UEAs 2011 team, and is part of the Norwich research part. Researchers at the JIC recognized that synthetic biology was an important field and therefore hosted a synthetic biology confrere, with the aim of the day to enhance their reserches knowledge of what synthetic biology is, and projects in which synthetic biology is being used. when the NRP-UEA was invited to give a talk to the JIC researchers we were extremely excited, giving them an over view of what iGEM was, our project and how it developed over the summer, as well informing them about aspects of human practice the NRP- UEA team had carried out over the summer. the team took also showed them a clip of our futuristic applications film. The team enjoyed presenting their work and results, as well as using it as an opportunity to improve their presentation skills before the European jamboree. There seemed to be a real interest by their researchers in to our project, giving encouraging advice, with many asking further questions after the presentation. The researchers also seemed very interested to hear more about our human practices elements after the presentation, wanting to know what the public's opinion of the research carried out at Norwich research park was, as well as synthetic biology as a whole.<br />
[[File:JIC_Synthetic_Biology_Workshop.pdf]]<br />
<br />
===SGM microbiology synthetic biology (September 3rd -5th)===<br />
<br />
The society of general microbiology held a 3 day synthetic biology conference at the University of Warwick. Three members of the UEA NRP iGEM team were lucky enough to go and enjoy talks on streptococcus, molecular motors, and the dynamics of the genome and designer microbes. The event also gave the team member the opportunity to meet other synthetic biology scientists as well as informing them of the current advances in the field and inspiring them with the future possibilities. The event also allowed the team members to present a poster and therefore engage with the scientific community, both informing them more about the iGEM competition and our project . The team believed it was important for the scientists in the synthetic biology world to have the opportunity to meet undergraduate iGEM members, allowing them to express the skills it has allowed them to develop and to encourage further interest in to lab groups forming teams, and funding iGEM projects.<br />
<br />
=== The Biochemical Society kindly mentioned us in their blog post ===<br />
<br />
Not only did the Biochemical Society generously fund our project, they also were kind enough to feature our teams efforts in encouraging ethical debate around the subject of synthetic biology (via our Quanticare video) in their blog.<br />
<br />
[http://biochemicalsociety.wordpress.com/2012/09/26/exploring-ethical-dilemmas-of-synthetic-biology-with-the-nrp-uea-norwich-igem-team/ '''Click here to view the blog post''']<br />
<br />
This post was also seen by the BBSRC, who very kindly tweeted about it!<br />
<br />
[[File:BBSRC.png | centre | 600px]]</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/Human_OutreachTeam:NRP-UEA-Norwich/Human Outreach2012-09-26T22:32:11Z<p>Joyehicks: /* Facebook */</p>
<hr />
<div>{{UEANRP}}<br />
<br />
=Human Practices=<br />
<br />
<br />
The UEA NRP team really went up and beyond this year, carrying out a wide selection of activity's to inform the public of both synthetic biology and iGEM, aiming to raise awareness of this branch of science. We used many methods of communication in order to cater for a range of audiences, including a radio show, newspaper articles, pubic events and talks as well as collaborating with all the other UK teams, in order to organize the UK team meet up and steam it live on the internet. <br />
<br />
== iGEM collaborations==<br />
<br />
===The iGEM UK team meet up, hosted by the NRP-UEA team at Google campus London (Friday 17th August) The most open and publically accessible UK team meetup EVER===<br />
<br />
[[File:Hangout stats.png | center]]<br />
<br />
[[File:UKiGEMteams.jpg | 300px | right]]<br />
<br />
The NRPUEA iGEM team organised and hosted the UK team meet up at Google campus in London. The NRPUEA iGEM team greeted the UK teams, provided refreshments and a lovely buffet lunch, as well as chairing the speeches and making sure the day ran smoothly. There was a great atmosphere at the meet up, and the event was a huge success.<br />
<br />
The day consisted of a number of guest speakers, including advice and tips for iGEM success from Dr. Tom Ellis (advisor of the 2009 winning iGEM project E.chromi) and an interactive talk on synthetic biology and science communication from the BBC science presenter, Nature editor and Guardian writer, Dr. Adam Rutherford. The UK iGEM teams presented a poster and a 15 minute presentation about their project. This gave the members of each team the chance to practice their presentation skills before the European team meet up, as well as a chance for others to ask questions and see what other project routes the other teams have taken. <br />
<br />
The event was also available '''LIVE on the internet''' via Google+ hangout, to allow the public and those that were unable to attend to still watch the presentations. Despite there being a few technical difficulties with the quality of the image, figures have shown that over 600 people watched the event.<br />
<br />
[https://dl.dropbox.com/u/9957127/UK%20iGEM%20Team%20Hangout%2017th%20Aug%202012%20Programme.pdf '''Click here for programme PDF''']<br />
<br />
<br><br />
<br />
<html><align=center><br />
<table align=center width=100% cellpadding=0 cellspacing=0><br />
<tr><br />
<td valign=absmiddle align=center><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/Kv_Z14OIrKc" frameborder="0" allowfullscreen></iframe><br />
</td><br />
</tr><br />
</table><br />
</align><br />
</html><br />
<br />
<br><br />
<br />
=== Conversing with others team===<br />
<br />
As part of our efforts to collaborate with the other iGEM teams this year we've made an effort to contact various teams in addition to the UK team meet-up.<br />
<br />
- We have have had talks with the Edinburgh iGEM team via Google Hang-out to discuss our projects further as well as arrangements for the UK team meet-up.<br />
<br />
- We visited the Cambridge team near the beginning of our project and they were kind enough to give us a tour of their labs. <br />
<br />
- Finally, the British Columbia team sent us a questionnaire that we happily filled out.<br />
<br />
==Public engagement==<br />
<br />
===The Future of Science event held at the forum, Norwich (Sunday 19th August)===<br />
<br />
<br />
<br />
[[File:Forum_team.JPG | 300px| right]]<br />
<br />
<br />
The Forum is located at the heart of the city of Norwich, and hosts a wide range of free events and exhibits to be enjoyed by the whole community. The venue is packed with restaurants, cafes and shops, as well as being home to Norwich library and BBC East Anglia. <br />
<br />
[[File:Poster for Forum.jpg | 250px | left]]<br />
<br />
The NRPUEA iGEM team were fortunate enough to hold an interactive day at the Forum, giving the public a chance to engage within the world of synthetic biology. Before the date of the event, the team increased awareness via a range of techniques, including posters, leaflets and newspaper and internet adverts, including the Forums website. This resulted with a large turnout at the event, including the local press. <br />
<br />
At the Future of Science event, the team presented a selection of informative posters, revealed their new film on a large screen, as well as showing examples of work carried out by the students during iGEM. For example, the public could view ''E.coli'' which had been transformed with GFP within a UV light box, as well as carrying out a counting colonies exercise. The NRPUEA team also created a range of synthetic biology educational material for the event, to inform the public of the concepts and terminology of synthetic biology. The different activities were designed to interest a selection of different ages. The educational resources include a range of quizzes and worksheets such as crosswords and matching exercises.<br />
<br />
[[File:Child_making_plasmid.jpg |300px | right]]<br />
<br />
For younger children we designed a colourful jigsaw plasmid (as seen in image). The idea of this activity was for the participants to choose jigsaw pieces that represented the genes that code for certain characteristics, such as green or red hair, and piece them together to form a complete plasmid. Once they had designed their plasmid they used a biscuit base, icing of different colours and various sweets to create a "biscuit creature" that corresponded to the plasmid they had created. The learning outcome of this activity was to give the children an understanding that a different selection of genes, results in different characteristics, and that editing the DNA can result in a change in the characteristics. The activity was very popular and most of the participants seemed to understand this concept, as well as having great fun.<br />
<br />
The event attracted a range of ages, as well as mixed levels of previous interest into synthetic biology. It also became clear that many members of the public knew very little about synthetic biology, and therefore were very interested in finding out more about the new branch of biology and the iGEM competition. The NRPUEA iGEM team also took the opportunity to talk to the public and listen to their opinions about the work the iGEM teams are carrying out, which demonstrated how mixed the view of the public were. The day was a huge success.<br />
<br />
'''Synthetic Biology activity sheets'''<br />
<br />
[[File:Word_Search_of_Synthetic_Biology.pdf]],<br />
[[File:Decode_Disney.pdf]],<br />
[[File:Questions_comprehensive.pdf]],[[File:Decode_Music_Artists_and_Films.pdf]],[[File:Word_Search_of_Synthetic_Biology.pdf]]<br />
<br />
=== Creation of futuristic company, Quanticare and production of a though-provoking film===<br />
<br />
<br />
[[File:Tattoo1.png |300px]][[File:QuanticareLogo_(2).png |300px]][[File:Tattoo2.png |300px]]<br />
<br />
<br />
As part of our human practices we decided to look into an artistic approach to bringing synthetic biology to a wider audience. As part of this we created "Quanticare", a fictitious and futuristic company forming on the back of a rise to prominence of synthetic biology over the next few years, as well as the results of our own projects on nitric oxide sensing and the comparator circuit. The fictitious company's latest development is to introduce a visual bio sensors in to the human body in the form of a tattoo-based health monitor, ''Cura''. <br />
<br />
<br />
Not only did the development of the fictitious company allow the team to explore potential future applications of synthetic biology and bio sensors, but also via the creation of a film , fuel a range of ethical questions. The team released the video on the day of the Wiki Freeze as well as bringing transferable tattoo samples of ''Cura'' to the European Jamboree as a form of marketing for the video and concept as a whole. <br />
<br />
<br />
The team also produced a film with Amy Congdon exploring ''Cura'' itself and its many applications. They decided to angle the film as a product launch in order to capture the imagination and bring Quanticare and ''Cura'' to the public as a realistic future involving synthetic biology. The film is available to view below, we thank the Biochemical Society for their outreach grant to fund this film.<br />
<br />
<html><center><table width=100% cellpadding=0 cellspacing=0 align=center><tr><td valign=absmiddle align=center><iframe width="560" height="315" src="http://www.youtube.com/embed/StNFePmymbc" frameborder="0" allowfullscreen></iframe></td></tr></table></center></html><br />
<br />
<br />
<html><font size=3pt><b>Please check out out <a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Quanticare">Quanticare</a> page for more details!</b></font></html><br />
<br />
==Social Media==<br />
The NRPUEA iGEM team has been VERY active on a range of social media over the summer.Any one is welcome to follow the team on Facebook or twitter. The team keep everyone updated on what is going on, both in the lab and in our spare time. This has been a great method of connecting with the public, companies,researchers, as well as getting in touch with other iGEM teams. The NRPUEA iGEM team believe human out reach via social media is important since it allows the public to follow the teams achievements and lab results, as well as get to know the personality of the team members. please follow us on twitter @NRPiGEM or UEA iGEM 2012 page on Facebook.<br />
<br />
===Twitter===<br />
<br />
[[File:Twitter stats NRP iGEM.png]]<br />
<br />
[[File:TweetReach_nrpigem.pdf| '''Download a PDF of our twitter statistics''']]<br />
<br />
===Facebook===<br />
<br />
Creating social media pages also allowed us to access information about who was checking our page out, showing interesting information, showing that social media techniques such as facebook attracted mostly 18-24 years olds interest, and that it attracted no attention from those above 65. Therefore the team though it was important to promote our project, not only through the internet, but also other techniques such as newspaper articles and radio shows. <br />
<br />
The figure bellow shows the age ranges and gender of people who liked any posts from the NRP UEA iGEM team<br />
[[File:Likes.png |600px| centre]]<br />
<br />
Below is a list of the location that people liked the teams statuses from. This highlighted that whilst the majority of followers were located in Norwich, we also had followers from a range of places over the world, highlighting the importance of using such social media to reach these followers. <br />
[[File:Location_of_likes.png |600px| centre]]<br />
<br />
As mentioned the NRP UEA iGEM team created facebook statuses and tweets very regularly and as the graph below shows, there was a consistent increase in action and interest in the NRPUEA iGEM team throughout the summer. <br />
<br />
[[File:Posts_ratings.png |600px| centre]]<br />
<br />
===Google+===<br />
<br />
We championed the use of Google+, a new social network from Google. We encouraged the UK iGEM teams to utilise Google+ to help make the 2012 UK team meetup as open and accessible as possible.<br />
<br />
==Media appearances==<br />
<br />
===STAR Radio===<br />
<br />
[http://www.star107.co.uk/farming-show.php STAR 107.9 FM's Farming Show]<br />
<br />
[[Image:NRPSTARRadio.jpg | 300px | right]]<br />
<br />
[http://www.star107.co.uk/podcasts/the-farming-show/show-23.mp3 Listen To Our Radio Show Appearence!]<br />
<br />
In early July the NRP UEA team travelled to Cambridge to appear on STAR Radio's "The Business Hub" and "The Farming Show". They were interviewed by Mark, one of the presenters, about synthetic biology, iGEM, and our project. The interview went really well as Pascoe and Khadija got across all the information that was needed in a clear and concise way, and Mark was extremely helpful in making sure all the salient points of the project were brought out. <br />
<br />
The team were able to give a brief description of what synthetic biology is before talking about the future applications of synthetic biology for medicine, agriculture and business. We were also able to mention our event at the Norwich Forum in late August in order to help promote it!<br />
<br />
A big thanks goes out to all at STAR Radio 107.9 for their help with the project in setting up the interview and allowing us air time to discuss iGEM and synthetic biology. Please visit the link above to hear the radio show.<br />
<br />
<br />
<br />
[[File:Paper.jpg|100px|left]]<br />
<br />
===Newspapers===<br />
<br />
The team featured on the university website a few times through the summer, as well as a brief article in the Eastern daily press newspaper.This was great, as allowed the locals of Norwich to become aware not only of the team and iGEM, but also introduce many to the scientific branch of synthetic biology.<br />
<br />
<br />
<br />
<br />
<br />
==Engagement with the wider scientific community==<br />
<br />
=== The team presented at the John Innes Centre Synthetic Biology conference ===<br />
<br />
The john innes center (JIC) is a world leading center of plant research, the home of UEAs 2011 team, and is part of the Norwich research part. Researchers at the JIC recognized that synthetic biology was an important field and therefore hosted a synthetic biology confrere, with the aim of the day to enhance their reserches knowledge of what synthetic biology is, and projects in which synthetic biology is being used. when the NRP-UEA was invited to give a talk to the JIC researchers we were extremely excited, giving them an over view of what iGEM was, our project and how it developed over the summer, as well informing them about aspects of human practice the NRP- UEA team had carried out over the summer. the team took also showed them a clip of our futuristic applications film. The team enjoyed presenting their work and results, as well as using it as an opportunity to improve their presentation skills before the European jamboree. There seemed to be a real interest by their researchers in to our project, giving encouraging advice, with many asking further questions after the presentation. The researchers also seemed very interested to hear more about our human practices elements after the presentation, wanting to know what the public's opinion of the research carried out at Norwich research park was, as well as synthetic biology as a whole.<br />
[[File:JIC_Synthetic_Biology_Workshop.pdf]]<br />
<br />
===SGM microbiology synthetic biology (September 3rd -5th)===<br />
<br />
The society of general microbiology held a 3 day synthetic biology conference at the University of Warwick. Three members of the UEA NRP iGEM team were lucky enough to go and enjoy talks on streptococcus, molecular motors, and the dynamics of the genome and designer microbes. The event also gave the team member the opportunity to meet other synthetic biology scientists as well as informing them of the current advances in the field and inspiring them with the future possibilities. The event also allowed the team members to present a poster and therefore engage with the scientific community, both informing them more about the iGEM competition and our project . The team believed it was important for the scientists in the synthetic biology world to have the opportunity to meet undergraduate iGEM members, allowing them to express the skills it has allowed them to develop and to encourage further interest in to lab groups forming teams, and funding iGEM projects.<br />
<br />
=== The Biochemical Society kindly mentioned us in their blog post ===<br />
<br />
Not only did the Biochemical Society generously fund our project, they also were kind enough to feature our teams efforts in encouraging ethical debate around the subject of synthetic biology (via our Quanticare video) in their blog.<br />
<br />
[http://biochemicalsociety.wordpress.com/2012/09/26/exploring-ethical-dilemmas-of-synthetic-biology-with-the-nrp-uea-norwich-igem-team/ '''Click here to view the blog post''']<br />
<br />
This post was also seen by the BBSRC, who very kindly tweeted about it!<br />
<br />
[[File:BBSRC.png | centre | 600px]]</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/PartsTeam:NRP-UEA-Norwich/Parts2012-09-26T22:21:49Z<p>Joyehicks: /* Parts to characterise in the future */</p>
<hr />
<div>{{UEANRP}}<br />
<br />
==Our Parts==<br />
<br />
We truly believe in the iGEM ethos of maintaining a Registry of Parts of constantly improving BioBricks and feel that, while creating new entirely new BioBricks is required for the registry to grow, the further characterisation and improvement of BioBricks that already exist is the key to maintaining the registry's credibility.<br />
<br />
<br />
<groupparts>iGEM012 NRP-UEA-Norwich</groupparts><br />
<br />
<br><br><br />
<br />
== Characterisation of other parts ==<br />
<br />
1) Part:BBa_K216005:Please see experience page<br />
[http://partsregistry.org/Part:BBa_K216005:Experience] (Group: iGEM09_Edinburgh (2009-09-25))<br />
[[File:PYEAR_page.png|800px]] <br />
<br />
<br><br><br />
2) Part:BBa_K381001:Please see experience page [http://partsregistry.org/Part:BBa_K381001:Experience] (Group: iGEM10_BCCS-Bristol (2010-10-05))<br />
[[File:K381001.png|800px]]<br />
<br><br><br />
<br />
== Characterisation of our parts ==<br />
1)Bacterial-Mammalian Promoter, Part:BBa_K774000[http://partsregistry.org/wiki/index.php?title=Part:BBa_K774000]<br />
[[File:4000.png|800px]]<br />
<br><br><br />
2)Mammalian-Bacterial Promoter, Part:BBa_K774001[http://partsregistry.org/wiki/index.php?title=Part:BBa_K774001]<br />
[[File:001.png|800px]]<br />
<br><br><br />
3)Bacterial-Mammalian promoter with eCFP reporter, Part:BBa_K774004[http://partsregistry.org/wiki/index.php?title=Part:BBa_K774004]<br />
[[File:004.png|800px]]<br />
<br><br><br />
4)Bacterial-Mammalian promoter with RFP reporter,Part:BBa_K774005[http://partsregistry.org/wiki/index.php?title=Part:BBa_K774005]<br />
[[File:005.png|800px]]<br />
<br><br><br />
5)Mammalian-Bacterial promoter with eCFP reporter, Part:BBa_K774006[http://partsregistry.org/wiki/index.php?title=Part:BBa_K774006]<br />
[[File:006.png|800px]]<br />
<br><br><br />
6)Mammalian-Bacterial promoter with RFP reporter, Part:BBa_K774007[http://partsregistry.org/wiki/index.php?title=Part:BBa_K774007]<br />
[[File:007.png|800px]]<br />
<br />
== Parts to characterise in the future ==<br />
1)Comparator Circuit Part 1, Part:BBa_K774002<br />
<br><br><br />
2)Comparator Circuit Part 2, Part:BBa_K774003<br />
<br><br><br />
Lack of specificity is a problem which many of the parts in the parts registry suffer from, and certainly a challenge which we faced when trying to detect nitrogenous species. From this potential problem spawned a potential solution; the Comparator Circuit. The two biobricks form a pair which are designed to specifically bind to each other while ligated to two different promoters of overlapping specificity. This results in an integrating of the conflicting outputs of the two opposing gene systems.<br />
<br />
Our system relies on two constructs that interact via complimentary base pair sequences both before and after the ribosome binding site of the reporter protein. The idea being that, when both transcripts are present in the chassis, they would bind together, inhibiting the translation of the reporter proteins.<br />
Any imbalance of transcription due to the presence of the substrate of interest results in free mRNA of the gene system that detects that substrate. Crucially, if both promoters detect the same substrates but differ with one extra substrate being detected by one of the promoters, it is this substrate and this substrate only that our system will be able to detect in a simple and quantitative way.<br />
<br />
Our team have constructed a countercurrent comparator circuit in which the reporter proteins are at the same end of the complimentary region, although a contracurrent system has been theorised. Both systems share a crucial subtractive nature comparable to an analogue computer. We envisage that, should the system be fine-tuned and expanded on, a variety of different business sectors from agriculture to spinoff pharmaceutical companies (such as the fictious QuantaCare which you can read about on our wiki) could capitalise on this novel genetic technology.<br />
What we have produced is a biobrick pair that work in harmony, when ligated to promoters of interest and genes of interest, to sequester translation when both mRNA transcripts are present in the cell. The use of quantative tuners with these biobricks is encouraged to ensure that the transcription rate both gene constructs are equal when both promoters are transcribing at their optimal rate. Although the parts have been submitted to the registry and theoretically characterised, time constraints have meant that further lab-based characterisation could not occur.<br />
However, we hope to utilise any free time in our timetables during the next semester to characterise the biobricks further (please see our project proposal), and hope that we will be given a chance to present our further findings at MIT!<br />
<br />
To conclude, what we have created is a pair of antagonistic BioBricks that turned the pair of mRNAs in which they reside into translational repressor molecules when both are transcribed in tandum within a specific chassis of interest, a new application for mRNA complimentary base pairing within the registry and a project we feel could go very far indeed.</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/PartsTeam:NRP-UEA-Norwich/Parts2012-09-26T22:21:21Z<p>Joyehicks: /* Parts to characterise in the future */</p>
<hr />
<div>{{UEANRP}}<br />
<br />
==Our Parts==<br />
<br />
We truly believe in the iGEM ethos of maintaining a Registry of Parts of constantly improving BioBricks and feel that, while creating new entirely new BioBricks is required for the registry to grow, the further characterisation and improvement of BioBricks that already exist is the key to maintaining the registry's credibility.<br />
<br />
<br />
<groupparts>iGEM012 NRP-UEA-Norwich</groupparts><br />
<br />
<br><br><br />
<br />
== Characterisation of other parts ==<br />
<br />
1) Part:BBa_K216005:Please see experience page<br />
[http://partsregistry.org/Part:BBa_K216005:Experience] (Group: iGEM09_Edinburgh (2009-09-25))<br />
[[File:PYEAR_page.png|800px]] <br />
<br />
<br><br><br />
2) Part:BBa_K381001:Please see experience page [http://partsregistry.org/Part:BBa_K381001:Experience] (Group: iGEM10_BCCS-Bristol (2010-10-05))<br />
[[File:K381001.png|800px]]<br />
<br><br><br />
<br />
== Characterisation of our parts ==<br />
1)Bacterial-Mammalian Promoter, Part:BBa_K774000[http://partsregistry.org/wiki/index.php?title=Part:BBa_K774000]<br />
[[File:4000.png|800px]]<br />
<br><br><br />
2)Mammalian-Bacterial Promoter, Part:BBa_K774001[http://partsregistry.org/wiki/index.php?title=Part:BBa_K774001]<br />
[[File:001.png|800px]]<br />
<br><br><br />
3)Bacterial-Mammalian promoter with eCFP reporter, Part:BBa_K774004[http://partsregistry.org/wiki/index.php?title=Part:BBa_K774004]<br />
[[File:004.png|800px]]<br />
<br><br><br />
4)Bacterial-Mammalian promoter with RFP reporter,Part:BBa_K774005[http://partsregistry.org/wiki/index.php?title=Part:BBa_K774005]<br />
[[File:005.png|800px]]<br />
<br><br><br />
5)Mammalian-Bacterial promoter with eCFP reporter, Part:BBa_K774006[http://partsregistry.org/wiki/index.php?title=Part:BBa_K774006]<br />
[[File:006.png|800px]]<br />
<br><br><br />
6)Mammalian-Bacterial promoter with RFP reporter, Part:BBa_K774007[http://partsregistry.org/wiki/index.php?title=Part:BBa_K774007]<br />
[[File:007.png|800px]]<br />
<br />
== Parts to characterise in the future ==<br />
1)Comparator Circuit Part 1, Part:BBa_K774002<br />
<br><br><br />
2)Comparator Circuit Part 2, Part:BBa_K774003<br />
<br><br><br />
Lack of specificity is a problem which many of the parts in the parts registry suffer from, and certainly a challenge which we faced when trying to detect nitrogenous species. From this potential problem spawned a potential solution; the Comparator Circuit. The two biobricks form a pair which are designed to specifically bind to each other while ligated to two different promoters of overlapping specificity. This results in an integrating of the conflicting outputs of the two opposing gene systems.<br />
<br />
Our system relies on two constructs that interact via complimentary base pair sequences both before and after the ribosome binding site of the reporter protein. The idea being that, when both transcripts are present in the chassis, they would bind together, inhibiting the translation of the reporter proteins.<br />
Any imbalance of transcription due to the presence of the substrate of interest results in free mRNA of the gene system that detects that substrate. Crucially, if both promoters detect the same substrates but differ with one extra substrate being detected by one of the promoters, it is this substrate and this substrate only that our system will be able to detect in a simple and quantitative way.<br />
Our team have constructed a countercurrent comparator circuit in which the reporter proteins are at the same end of the complimentary region, although a contracurrent system has been theorised. Both systems share a crucial subtractive nature comparable to an analogue computer. We envisage that, should the system be fine-tuned and expanded on, a variety of different business sectors from agriculture to spinoff pharmaceutical companies (such as the fictious QuantaCare which you can read about on our wiki) could capitalise on this novel genetic technology.<br />
What we have produced is a biobrick pair that work in harmony, when ligated to promoters of interest and genes of interest, to sequester translation when both mRNA transcripts are present in the cell. The use of quantative tuners with these biobricks is encouraged to ensure that the transcription rate both gene constructs are equal when both promoters are transcribing at their optimal rate. Although the parts have been submitted to the registry and theoretically characterised, time constraints have meant that further lab-based characterisation could not occur.<br />
However, we hope to utilise any free time in our timetables during the next semester to characterise the biobricks further (please see our project proposal), and hope that we will be given a chance to present our further findings at MIT!<br />
To conclude, what we have created is a pair of antagonistic BioBricks that turned the pair of mRNAs in which they reside into translational repressor molecules when both are transcribed in tandum within a specific chassis of interest, a new application for mRNA complimentary base pairing within the registry and a project we feel could go very far indeed.</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/AttributionsTeam:NRP-UEA-Norwich/Attributions2012-09-26T22:10:47Z<p>Joyehicks: </p>
<hr />
<div>{{UEANRP}}<br />
<br><br><br />
<br />
'''Advisory Team:''' '''[http://www.uea.ac.uk/bio/People/Academic/Matthew+Hutchings Dr Matthew Hutchings]''', '''[http://www.uea.ac.uk/bio/People/Academic/Colwyn+Thomas Dr. Colwyn Thomas]''', '''[http://www.uea.ac.uk/bio/People/Academic/Kay+Yeoman Dr. Kay Yeomen]''', '''[http://www.uea.ac.uk/bio/People/Academic/Gary+Rowley Dr Gary Rowley]'''<br />
<br />
<br />
'''[http://www.amycongdon.com/#!mainPage Amy Congdon]''' has acted as our team artist and has designed our logos as well as helping with the design and production of our video and various pieces of equipment for our human outreach work.<br />
<br />
<br />
'''[http://www.biochemistry.org The Biochemical Society]''' were kind enough to provide funding to go towards videos that have proven to be central to the human practices aspect of our project.<br />
<br />
<br />
'''[http://www.bioline.com/h_uk.asp Bioline]''' and their representative, Josh Wright, have been extremely supportive in offering us a discount from their catalogued products as well as donating plasmid isolation mini kits, PCR gel clean-up kits and enzymes.<br />
<br />
<br />
'''[http://www.uea.ac.uk/bio/People/Research+Associate/Paul+Thomas Dr. Paul Thomas]''' for all of his help with imaging mammalian cells, and for providing the SNAP used for producing nitric oxide.<br />
<br />
<br />
'''[http://www.promega.com Promega]''' have been extremely supportive in offering us a discount from their catalogued products as well as donating PCR gel clean-up kits and DNA purification mini-preps.<br />
<br />
<br />
'''Sebastian Runkel''' , '''Anke Arkenberg''' , '''Colin Lockwood''' and '''Sean McManus''' are PhD students at the University of East Anglia whose research involve nitric oxide. They gave in-depth presentations on nitric oxide and how to work with it. '''Dan Tromans''' is a PhD student based at the UEA and the John Innes Centre in Norwich. <br />
<br />
The experience and advice of all of these PhD students has proven invaluable to our own project.<br />
<br />
<br />
'''[http://www.star107.co.uk/ STAR Radio 107.9/1 FM]''' allowed us interview time on their radio show to discuss the iGEM competition, synthetic biology, and the future applications of this branch of science. They gave us a lengthy slot on the show and helped us promote the competition and synthetic biology as a whole.<br />
<br />
<br />
'''[http://www.uea.ac.uk/bio/People/Academic/Tom+Clarke Dr. Tom Clarke]''' helped us with all of our fluorometer studies, and we are very grateful for him taking the time to teach us how to use the equipment. <br />
<br />
<br />
'''[http://www.wellcome.ac.uk/ The Wellcome Trust]''' kindly provided us with stipends that have allowed the team to work on the project for the ten week period allotted. <br />
<br />
<br />
The '''UEA Annual Fund''' provided us with funds to cover some of the costs assoicated with participating in the iGEM competition. In particular, funds were provided for essential consumables, without which the lab work performed could not have been performed.<br />
<br />
[[File:FCS_banner_logo.gif]]<br />
<br />
'''[http://www.flowcytometryservices.co.uk UEA Flow Cytometry Services]''' kindly provided us free usage of a flow cytometer and their expertise. <br />
<br />
<br />
'''[http://www.uea.ac.uk/che/people/techstaff UEA Technical Staff]''' we would like to particularly say a big thank you to all of the support that has been given to us by UEA technicians over the summer, especially at the start of our project when we were all new to independent lab work!<br />
<br />
<br />
'''[http://www.uea.ac.uk/bio/eande/eandeclub The UEA Enterprise and Engagement Club]''', the '''[http://www.uea.ac.uk/bio School of Biological Sciences at UEA]''' and the '''John & Pamela Salter Trust Fund''' were able to provide vital funding towards travel expenses, lab consumables and other necessities for the project.<br />
<br><br><br />
[[File:Thankyou.png|centre]]</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/AttributionsTeam:NRP-UEA-Norwich/Attributions2012-09-26T22:03:23Z<p>Joyehicks: </p>
<hr />
<div>{{UEANRP}}<br />
<br><br><br />
<br />
'''Advisory Team:''' '''[http://www.uea.ac.uk/bio/People/Academic/Matthew+Hutchings Dr Matthew Hutchings]''', '''[http://www.uea.ac.uk/bio/People/Academic/Colwyn+Thomas Dr. Colwyn Thomas]''', '''[http://www.uea.ac.uk/bio/People/Academic/Kay+Yeoman Dr. Kay Yeomen]''', '''[http://www.uea.ac.uk/bio/People/Academic/Gary+Rowley Dr Gary Rowley]'''<br />
<br />
'''[http://www.amycongdon.com/#!mainPage Amy Congdon]''' has acted as our team artist and has designed our logos as well as helping with the design and production of our video and various pieces of equipment for our human outreach work.<br />
<br />
<br />
'''[http://www.biochemistry.org The Biochemical Society]''' were kind enough to provide funding to go towards videos that have proven to be central to the human practices aspect of our project.<br />
<br />
<br />
'''[http://www.bioline.com/h_uk.asp Bioline]''' and their representative, Josh Wright, have been extremely supportive in offering us a discount from their catalogued products as well as donating plasmid isolation mini kits, PCR gel clean-up kits and enzymes.<br />
<br />
'''[http://www.uea.ac.uk/bio/People/Research+Associate/Paul+Thomas Dr. Paul Thomas]''' for all of his help with imaging mammalian cells, and for providing the SNAP used for producing nitric oxide.<br />
<br />
<br />
'''[http://www.promega.com Promega]''' have been extremely supportive in offering us a discount from their catalogued products as well as donating PCR gel clean-up kits and DNA purification mini-preps.<br />
<br />
<br />
'''Sebastian Runkel''' , '''Anke Arkenberg''' , '''Colin Lockwood''' and '''Sean McManus''' are PhD students at the University of East Anglia whose research involve nitric oxide. They gave in-depth presentations on nitric oxide and how to work with it. '''Dan Tromans''' is a PhD student based at the UEA and the John Innes Centre in Norwich. <br />
<br />
The experience and advice of all of these PhD students has proven invaluable to our own project.<br />
<br />
<br />
'''[http://www.star107.co.uk/ STAR Radio 107.9/1 FM]''' allowed us interview time on their radio show to discuss the iGEM competition, synthetic biology, and the future applications of this branch of science. They gave us a lengthy slot on the show and helped us promote the competition and synthetic biology as a whole.<br />
<br />
'''[http://www.uea.ac.uk/bio/People/Academic/Tom+Clarke Dr. Tom Clarke]''' helped us with all of our fluorometer studies, and we are very grateful for him taking the time to teach us how to use the equipment. <br />
<br />
<br />
'''[http://www.wellcome.ac.uk/ The Wellcome Trust]''' kindly provided us with stipends that have allowed the team to work on the project for the ten week period allotted. <br />
<br />
<br />
The '''UEA Annual Fund''' provided us with funds to cover some of the costs assoicated with participating in the iGEM competition. In particular, funds were provided for essential consumables, without which the lab work performed could not have been performed.<br />
<br />
[[File:FCS_banner_logo.gif]]<br />
<br />
'''[http://www.flowcytometryservices.co.uk UEA Flow Cytometry Services]''' kindly provided us free usage of a flow cytometer and their expertise. <br />
<br />
'''[http://www.uea.ac.uk/bio/eande/eandeclub The UEA Enterprise and Engagement Club]''', the '''[http://www.uea.ac.uk/bio School of Biological Sciences at UEA]''' and the '''John & Pamela Salter Trust Fund''' were able to provide vital funding towards travel expenses, lab consumables and other necessities for the project.<br />
<br><br><br />
[[File:Thankyou.png|centre]]</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/FutureTeam:NRP-UEA-Norwich/Future2012-09-26T21:41:34Z<p>Joyehicks: /* Construction and Buildings Emissions */</p>
<hr />
<div>{{UEANRP}}<br />
<br />
Nitric oxide (NO) is produced in different environments, be that within humans or by bacteria in soil. It is a nitrogenous species that is involved in the nitrogen cycle. With further research, development and integration into various systems, the future of medicine and agriculture could involve NO sensors.<br />
<br />
=='''NO sensing'''==<br />
<br />
Many NO sensors exist, but usually have problems with specific and quantitative NO detection (Bedioui, F .et al.,2002). To do this in a mlore specific manner, a comparator circuit system can be put into place. The circuit could involve multiple sensors that sense different reactive oxygen and nitrogenous species. Within this system, short interfering RNA (siRNA) could be implemented to inhibit the expression of certain enzymes and proteins. This would produce an overall output that corresponds only to NO levels. To give a visual quantitative output, the use of bioluminescence can be augmented. The brightness would relate to the amount of NO in cells environment.<br />
<br />
Furthermore, as mentioned previously, the E.chromi project can be incorporated. In addition to its application in disease communication relative to NO levels, different sensors can be incorporated into the system to accurately measure all nitrogenous compounds. The levels of these can be qualitatively measured through the resulting coloured product.<br />
<br />
[[File:Future applications.png|950px]]<br />
<br />
=='''Agriculture'''==<br />
<br />
NO can be produced by nitrifying bacteria during oxidation of ammonium (Lipschultz, F. et al. 1981). These gases can be released into the atmosphere. NO is also converted back to atmospheric nitrogen in a reductive process called denitrification. The differences in the bacterial species that compose soil flora produce and reduce nitric oxide lead to different levels of NO in soil, which can affect soil fertility and thus agricultural yield. Besides soil flora, soil composition, temperature, water levels (Davidson, E. 2012), tilling and fertiliser quantities (Civerolo, K.L. et al. 1998) can all affect the levels of NO. This is particularly significant now as the world population is ever increasing; the higher the agricultural yield the more people that can be supported. An accurate NO sensor can allow a farmer to be able to better utilise their resources and to accurately apply fertiliser, water and needed farming techniques to maximise the crop yield.<br />
<br />
=='''Medicine'''==<br />
<br />
[[File:JAMES_CAVALLINI_SCIENCE_PHOTO_LIBRARY.jpg| 200px | right | thumbnail | <html><u>This photograph is the property of James Cavallini/ Science Photo Library.</u></html>]]<br />
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Nitric oxide is an important physiological signalling molecule within the human body. This highly reactive molecule is produced through the action of enzymes called nitic oxide synthases (NOS). There are three main types of NOS in the body, which each produce NO for a different physiological role: inducible (iNOS), endothelial (eNOS) and neuronal (nNOS) (Xu, W. et al. 2002). NO in the blood, causes vasodilation through increase of cGMP, a second messenger which activates many receptors and processes (Ferreira, L.F. et al 2010). As NO signalling is so widespread within the body there are many applications NO sensors can have in medicine.<br />
<br><br>Whilst NO triggers several defence mechanisms within the innate immune response, it has been found that some bacteria, such as the food-borne pathogen ''Listeria monocytogenes'', are able to use increased NO levels (produced by nitric oxide synthase-2) to promote their colonisation of host cells; culminating in a condition known as listeriosis. ''L. monoctogenes'' is absorbed from donor to recipient cell within vacuoles, and as NO decreases the rate at which the immune response destroys these vacuoles the bacteria is increasingly successful (Cole, C. et al. 2012). A system which is able to both detect and decrease levels of nitric oxide would thus be highly advantageous in therapeutic strategies to treat ''L. monocytogenes'' infection.<br />
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=='''Cancer Research'''==<br />
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[[File:Warrior_cell.png| 200px| left | thumbnail |This image is a logo which represents a future application of how our biobricks could be used to produce a bacteria capable of acting as a cancer thereapeutic.]]<br />
All the isoforms of NO synthases aforementioned are involved in the promotion or inhibition of the tumour cells (Xu, W. et al. 2002). Research has shown that high levels of NO can be cytotoxic /or cytostatic to tumour cells; however, at the low levels produced by tumour cells, NO signals angiogenic factors such as VEGF leading to angiogenesis and enlargement of the tumour (Xu, W. et al. 2002). Macrophages, as part of the innate immune system, produce NO to kill off the tumour. Research by Xu et al., shows that NO has greater activity when the tumour is less<br />
differentiated and hence a higher grade.<br />
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Using NO as a chemoattractant, the low and high levels of NO produced by cancer cells and macrophages, respectively can be perceived by the sensor which can target drugs directly to the tumour. An alternative method is to use oxygen as a chemorepellent so modified cells can specifically target hypoxic cells. Specificity can be increased through the use of targeting using specific peptide sequences which home onto receptors or glycoproteins which are specific to tumour cells such as the NGR peptide (Pasqualini, R. et al. 2000).<br />
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=='''Diagnostics'''==<br />
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[[File:E.chromi.jpg| 200px| left | thumbnail |This image is from the Cambridge E.chromi project, showing the coloured faecal matter as a form of diagnostic which has potential for identifying damage to the gut.]]NO is produced throughout the mammalian body in a range of cells, including endothelial cells (Bedioi.F et,al., 2002). NO has a range of functions within the body, from being a secondary messenger in signalling pathways to being a relaxing factor within the cardiovascular system (Pang, et,al.,2003). Abnormal levels of NO can be an indication of diseases and disorders such as hypertension, impotence and obesity (Bedioui.F et,al.,2002). A NO sensor can be augmented into two ways to aid diagnosis and treatment. For example, measuring the NO level of externally exhaled air can be an important and non-invasive diagnostic for airways inflammation. This is due to endothelial cells producing NO synthase (eNOS), both constitutive and inducible (iNOS). The iNOS is induced by pro inflammatory cytokines, as well as there being an increase in enzyme expression after exposure to oxidants. Therefore, measuring the NO level produced by eNOS has led to finding that there are higher levels of epithelial nitric oxide when asthma is untreated, and that there is a fall after anti-inflammatory treatment (Narang ,et al., 2002).<br />
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Alternatively,Building upon the Cambridge 2009 iGEM E.Chromi project, the NO sensor can be incorporated to perceive abnormal levels of NO in the gut flora or damage to the gut, which can visually be seen in the form of colours in faecal matter (Cambridge iGEM team , 2009). For example NO levels increase during damage of the gut, potentially due to its role in functional repair ( Miller et al, 1993). Therefore,an NO sensor would produce particular colours that could correspond to different levels of NO expected for certain diseases and disorders.<br />
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Another use is through the cells being stimulated to create a protein coat for resistance and persistence within the human body. These cells could be mass produced and contain vaccines which can be taken at birth. These cells can be used to target pathogens or other abnormalities that arise in the body.<br />
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=='''Understanding Pathogens'''==<br />
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[[File:Sensing.jpg| 200px| left | thumbnail |(Henares, et, al., 2012). The process used by bacteria in which NO course quorum sensing through lux U within V.harveyi.]]<br />
Pathogens have many different mechanisms and strategies of invasion and attack. For example some pathogens produce biofilms. To create biofilms, bacteria signal to other bacteria to assess the population size and density (Tsou, et al., 2009). When numbers are sufficient, they switch on gene expression which leads to aggregation and adhesion. This is an example of quorum sensing, as has been demonstrated through Nitric Oxide responsive quorum sensing circuits though lux U within Vibrio harveyi(Henares, et al., 2012). A greater understanding of NO can be applied to research into pathogenic diseases which could lead to advances in the creation of cures and vaccines.<br />
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=='''Construction and Buildings Emissions'''==<br />
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In 2008 the Modern Buildings Services released an article detailing recommendations from the Buildings Regulation Establishment Environmental Assessment Method (BREEAM) encouraging construction and buildings companies to monitor levels of nitric oxide emissions. They detail nitric oxide formation through combustion reactions involved in certain construction methods and even household applicances (e.g. boilers) and the consequences of excess atmospheric nitric oxide, including the production of smog and acid rain. Production of a way to accurately sense nitric oxide levels in the form of the hybrid promoter would be an extremely useful tool in detailing these levels; further to this combining the hybrid promoter and the comparator circuit could see bacteria that could not only sense nitric oxide being released by these appliances etc., but could also express an enzyme to safely break nitric oxide down and prevent the formation of harmful compounds.<br />
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==References==<br />
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Bedioui,F., Villeneuue, N.(2002)‘ Electrochemical Nitric Oxide Sensors for biological samples- Principle, selected examples and applications’, Electroanalysis, 15; 5-18. <br />
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Cambridge iGEM team (2009) Available at: https://2009.igem.org/Team:Cambridge [Accessed on: 21/09.2012] <br />
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Civerolo, K.L. and Dickerson, R.R. (1998) ‘Nitric oxide soil emissions from tilled and untilled corn®elds’, Agricultural and Forest Meteorology, 90; 307-311. <br />
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Cole, C., Thomas, S., Filak, H., Henson, P., Lenz, L. (2012) ‘Nitric Oxide Increases Susceptibility of Toll-like Receptor-Activated Macrophages to Spreading Listeria monocytogenes’, Immunity, 36; 885.<br />
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Davidson, E. (2012) Sources of Nitric Oxide and Nitrous Oxide following Wetting of Dry Soil, Soil Sci. Soc. Am. J. 56; 95–102<br />
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Ferreira, L.F. and Behnke, B.J. (2010) ‘A toast to health and performance! Beetroot juice lowers blood pressure and the O2 cost of exercise’, Journal of Applied Physiology, 110; 585-586.<br />
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Henares, B.M., Higgins, K.E., Boon, E.M. (2012)'Discovery of a Nitric Oxide Responsive Quorum Sensing Circuit in Vibrio harveyi',ACS chemical biology,17;1331-6.<br />
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Lipschultz, F., Zafiriou, O.C. Wofsy, S.C., Elroy, M.B., Valois, F.W. and Watson, S.W. (1981) ‘Production of NO and N2O by soil nitrifying bacteria’, Macmillan Journals, 294; 641-643.<br />
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Miller, M.J., Zhang, X.J., Sadowska-Krowicka, H., Chotinaruemol ,S., McIntyre ,J.A., Clark ,D.A. and Bustamante ,S.A. (1993) 'Nitric oxide release in response to gut injury',scandinavian journal of gastroenterology,28; 149-54.<br />
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Modern Building Services, (2008), ''Understanding NOx emissions [online]'', Available at: [http://www.modbs.co.uk/news/archivestory.php/aid/5380/Understanding_NOx_emissions.html http://www.modbs.co.uk/news/archivestory.php/aid/5380/Understanding_NOx_emissions.html], accessed on 26/09/2012<br />
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Narang, I., Ersu,R., Wilson, N.M., Bush. A. (2002) 'Nitric oxide in chronic airway inflammation in children: diagnostic use and pathophysiological significance', Thorax ,57;586-589.<br />
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Pang.J, Fan.C, Liv.X, Chem.T, Li.G (2003)'A nitric oxide biosensor or based on the multi-assembly of hemoglobin, montmorillonite/ polyvinyl alcohol at pyrolytic graphite electrode.', Bio sensors and bioelectronics, 19: 441-445. <br />
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Pasqualini, R., Koivunen, E., Kain, R., Lahdenranta, J., Sakamoto, M., Stryhn, A., Ashmun, R.A., Shapiro, L.H., Arap, W. And Ruoslahti, E. (2000) ‘Aminopeptidase N is a receptor for tumour-homing peptides and a target for inhibiting angiogenesis’, The Journal of Cancer Research, 60; 722-727.<br />
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Tsou,A.M., Cai,T., Liu,Z.,Zhu,J., and Kulkarni, R.V.,(2009)'Regulatory targets of quorum sensing in Vibrio cholerae: evidence for two distinct HapR-binding motifs',Nucleic Acids Res, 37; 2747–2756.<br />
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Xu, W., Liu, L.Z., Loizidou, M., Ahmed, M. And Charles, I.G. (2002) ‘The role of nitric oxide in cancer’, Cell Research, 12; 311-320</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/QuanticareTeam:NRP-UEA-Norwich/Quanticare2012-09-26T14:34:34Z<p>Joyehicks: /* Quanticare & The Cura Tattoo */</p>
<hr />
<div>{{UEANRPQuanticare}}<br />
<br />
<html><center><table width=100% cellpadding=0 cellspacing=0 align=center><tr><td valign=absmiddle align=center><iframe width="560" height="315" src="http://www.youtube.com/embed/StNFePmymbc" frameborder="0" allowfullscreen></iframe></td></tr></table><br><b>The NRP UEA iGEM team would like to thank the Biochemical Society for funding of our film, and Amy Congdon for direction and production of our film.</b><br />
</center></html><br />
<br />
==Quanticare & The ''Cura'' Tattoo==<br />
<br />
As part of our human practices we have produced a film in conjunction with our artist [http://www.amycongdon.com/#!mainPage Amy Congdon] which explores a futuristic world where synthetic biology is an aspect of everyday life. As we explored the idea of synthetic biology in the future we developed the idea of "Quanticare", a company created on the back of synthetic biology's rise to prominence. As the project within the labs continued and the idea of the [https://2012.igem.org/Team:NRP-UEA-Norwich/ComparatorCircuit Comparator Circuit] and the [https://2012.igem.org/Team:NRP-UEA-Norwich/NOSensing Flexible Bacterial/Mammalian Hybrid Promoter] came about we looked at how this new idea of cellular computing could be used within the Quanticare concept, leading to the idea of the living tattoo, ''Cura''.<br />
<br />
<br />
We wanted to produce something that would spark the imagination of the wider public and truly show the future power and potential of synthetic biology, and we believe that ''Cura'' and Quanticare have achieved those aims. With ''Cura'' we have developed an original idea of a tattoo not drawn with ink, but instead drawn with immortalised mammalian cells containing different BioBricks. The concept involves each shape of the tattoo containing cells with specific BioBricks that sense a particular chemical within the body, and report on any particular changes on that chemical's levels. <br />
<br />
[[File:QuanticareComparator.png | right | 250 px | thumbnail | '''Figure 1.''' A graphic showing the result of the comparator circuit; mRNAs transcribed following activation of two promoters have cancelled one another out, leaving only the overhang representative to the imbalance in substrate chemicals to be translated and expressed.]]<br />
<br />
<br />
The comparator circuit project involves multiple promoters that are sensitive to similar chemicals, a different reporter/effector enzyme for each promoter, and a range of specially designed DNA leaders that go between the promoter and reporter of each gene system. The idea involves the specially designed DNA leaders being complementary to one another and resulting in DNA annealing which covers the ribosome binding site; this means that mRNAs transcribed following activation of the promoters will cancel one another out and fail to be translated. If there is an imbalance of substrates one set of mRNAs would be produced in excess of the others as this set would not anneal to one another, therefore resulting in translation and expression of the associated protein. This is demonstrated in ''Figure 1.'' where two sets of mRNA have been produced, one with an RFP reporter and one with a CFP reporter; due to an imbalance in substrates four lots of the RFP-associated mRNA have been produced, while only two lots of the CFP-associated mRNAs have been produced. As the image shows the two CFP-associated mRNAs have annealed with two of the RFP-associated mRNAs, blocking the RBS and preventing translation, however due to the imbalance there are two RFP-associated mRNAs left to be translated and thus RFP would be expressed in this situation.<br />
<br />
<br />
Similar comparator circuit set-ups would be used in the ''Cura'' tattoo. For instance in the case of Type I Diabetes one promoter would sense blood sugar while another would sense insulin; if the blood sugar outweighed the insulin, more of the mRNA with the blood sugar promoter would be produced and thus an effector enzyme for the production of insulin could be included in this mRNA in order to increase insulin production. If insulin production becomes too high the imbalance would shift so more of the mRNA with the insulin-sensing promoter was produced, and an effector enzyme to break down excess insulin could be translated from this mRNA strand in order to normalise insulin levels. A system such as this, as well as others (which simply report changes through fluorescent proteins), are explored in Figure 2.<br />
<br />
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[[File:NRPCura.png | center | 950 px | thumbnail | '''Figure 2.''' ''Cura'' is Quanticare's revolutionary healthcare solution, a tattoo made of immortalised mammalian cells containing different biobricks that have been specifically tailored to monitor and respond to changes in the body. Different areas of the tattoo correspond to different areas of the body, and using biological technology developed by the Norwich Research Park and University of East Anglia 2012 iGEM Team can react automatically to many unwanted changes, as well as fluorescing to alert the user of any problems.]]</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/QuanticareTeam:NRP-UEA-Norwich/Quanticare2012-09-26T14:28:19Z<p>Joyehicks: /* Quanticare & The Cura Tattoo */</p>
<hr />
<div>{{UEANRPQuanticare}}<br />
<br />
<html><center><table width=100% cellpadding=0 cellspacing=0 align=center><tr><td valign=absmiddle align=center><iframe width="560" height="315" src="http://www.youtube.com/embed/StNFePmymbc" frameborder="0" allowfullscreen></iframe></td></tr></table><br><b>The NRP UEA iGEM team would like to thank the Biochemical Society for funding of our film, and Amy Congdon for direction and production of our film.</b><br />
</center></html><br />
<br />
==Quanticare & The ''Cura'' Tattoo==<br />
<br />
As part of our human practices we have produced a film in conjunction with our artist [http://www.amycongdon.com/#!mainPage Amy Congdon] which explores a futuristic world where synthetic biology is an aspect of everyday life. As we explored the idea of synthetic biology in the future we developed the idea of "Quanticare", a company created on the back of synthetic biology's rise to prominence. As the project within the labs continued and the idea of the [https://2012.igem.org/Team:NRP-UEA-Norwich/ComparatorCircuit Comparator Circuit] and the [https://2012.igem.org/Team:NRP-UEA-Norwich/NOSensing Flexible Bacterial/Mammalian Hybrid Promoter] came about we looked at how this new idea of cellular computing could be used within the Quanticare concept, leading to the idea of the living tattoo, ''Cura''.<br />
<br />
<br />
We wanted to produce something that would spark the imagination of the wider public and truly show the future power and potential of synthetic biology, and we believe that ''Cura'' and Quanticare have achieved those aims. With ''Cura'' we have developed an original idea of a tattoo not drawn with ink, but instead drawn with immortalised mammalian cells containing different BioBricks. The concept involves each shape of the tattoo containing cells with specific BioBricks that sense a particular chemical within the body, and report on any particular changes on that chemical's levels. <br />
<br />
[[File:QuanticareComparator.png | right | 250 px | thumbnail | '''Figure 1.''' A graphic showing the result of the comparator circuit; mRNAs transcribed following activation of two promoters have cancelled one another out, leaving only the overhang representative to the imbalance in substrate chemicals to be translated and expressed.]]<br />
<br />
<br />
The comparator circuit project involves multiple promoters that are sensitive to similar chemicals, a different reporter/effector enzyme for each promoter, and a range of specially designed DNA leaders that go between the promoter and reporter of each gene system. The idea involves the specially designed DNA leaders being complementary to one another and resulting in DNA annealing which covers the ribosome binding site; this means that mRNAs transcribed following activation of the promoters will cancel one another out and fail to be translated. If there is an imbalance of substrates one set of mRNAs would be produced in excess of the others and would not anneal to one another, therefore resulting in translation and expression of the associated protein. This is demonstrated in ''Figure 1.'' where two sets of mRNA have been produced, one with an RFP reporter and one with a CFP reporter; due to an imbalance in substrates four lots of the RFP-associated mRNA have been produced, while only two lots of the CFP-associated mRNAs have been produced. As the image shows the two CFP-associated mRNAs have annealed with two of the RFP-associated mRNAs, blocking the RBS and preventing translation, however due to the imbalance there are two RFP-associated mRNAs left to be translated and thus RFP would be expressed in this situation.<br />
<br />
<br />
Similar comparator circuit set-ups would be used in the ''Cura'' tattoo. For instance in the case of Type I Diabetes one promoter would sense blood sugar while another would sense insulin; if the blood sugar outweighed the insulin, more of the mRNA with the blood sugar promoter would be produced and thus an effector enzyme for the production of insulin could be included in this mRNA in order to increase insulin production. If insulin production becomes too high the imbalance would shift so more of the mRNA with the insulin-sensing promoter was produced, and an effector enzyme to break down excess insulin could be translated from this mRNA strand in order to normalise insulin levels. A system such as this, as well as others (which simply report changes through fluorescent proteins), are explored in Figure 2.<br />
<br />
<br />
[[File:NRPCura.png | center | 950 px | thumbnail | '''Figure 2.''' ''Cura'' is Quanticare's revolutionary healthcare solution, a tattoo made of immortalised mammalian cells containing different biobricks that have been specifically tailored to monitor and respond to changes in the body. Different areas of the tattoo correspond to different areas of the body, and using biological technology developed by the Norwich Research Park and University of East Anglia 2012 iGEM Team can react automatically to many unwanted changes, as well as fluorescing to alert the user of any problems.]]</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/Human_OutreachTeam:NRP-UEA-Norwich/Human Outreach2012-09-26T13:28:52Z<p>Joyehicks: </p>
<hr />
<div>{{UEANRP}}<br />
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=Human Practices=<br />
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The UEA NRP team really went up and beyond this year, caring out a wide selection of activity's to inform the public of both synthetic biology and iGEM, aiming to raise awareness of this branch of science. We used many methods of communication in order to cater for a range of audiences, including a radio show, newspaper articles, pubic events and talks as well as collaborating with all the other UK teams, in order to organize the UK team meet up and steam it live on the internet. <br />
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== iGEM collaborations==<br />
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===The iGEM UK team meet up, hosted by the NRP-UEA team at Google campus London (Friday 17th August) The most open and publically accessible UK team meetup EVER===<br />
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[[File:Hangout stats.png | center]]<br />
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[[File:UKiGEMteams.jpg | 300px | right]]<br />
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The NRPUEA iGEM team organised and hosted the UK team meet up at Google campus in London. The NRPUEA iGEM team greeted the UK teams, provided refreshments and a lovely buffet lunch, as well as chairing the speeches and making sure the day ran smoothly. There was a great atmosphere at the meet up, and the event was a huge success.<br />
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The day consisted of a number of guest speakers, including advice and tips for iGEM success from Tom Ellis (advisor of the 2009 winning iGEM project E.chromi) and an interactive talk on synthetic biology and science communication from the BBC science presenter, Nature editor and Guardian writer,Adam Rutherford. The UK iGEM teams presented a poster and a 15 minute presentation about their project. This gave the members of each team the chance to practice their presentation skills before the European team meet up, as well as a chance for others to ask questions and see what other project routes the other teams have taken. <br />
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The event was also available '''LIVE on the internet''' via Google+ hangout, to allow the public and those that were unable to attend to still watch the presentations. Despite there being a few technical difficulties with the quality of the image, figures have shown that over 600 people watched the event.<br />
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[https://dl.dropbox.com/u/9957127/UK%20iGEM%20Team%20Hangout%2017th%20Aug%202012%20Programme.pdf '''Click here for programme PDF''']<br />
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==Public engagement==<br />
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===The Future of Science event held at the forum, Norwich (Sunday 19th August)===<br />
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[[File:Forum_team.JPG | 300px| right]]<br />
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The Forum is located at the heart of the city of Norwich, and hosts a wide range of free events and exhibits to be enjoyed by the whole community. The venue is packed with restaurants, cafes and shops, as well as being home to Norwich library and BBC East Anglia. <br />
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The NRPUEA iGEM team were fortunate enough to hold an interactive day at the Forum, giving the public a chance to engage within the world of synthetic biology. Before the date of the event, the team increased awareness via a range of techniques, including posters, leaflets and newspaper and internet adverts, including the Forums website. This resulted with a large turnout at the event, including the local press. <br />
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At the Future of Science event, the team presented a selection of informative posters, revealed their new film on a large screen, as well as showing examples of work carried out by the students during iGEM. For example, the public could view ''E.coli'' which had been transformed with GFP within a UV light box, as well as carrying out a counting colonies exercise. The NRPUEA team also created a range of synthetic biology educational material for the event, to inform the public of the concepts and terminology of synthetic biology. The different activities were designed to interest a selection of different ages. The educational resources include a range of quizzes and worksheets such as crosswords and matching exercises.<br />
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[[File:Child_making_plasmid.jpg |300px | left]]<br />
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For younger children we designed a colourful jigsaw plasmid (as seen in image). The idea of this activity was for the participants to choose jigsaw pieces that represented the genes that code for certain characteristics, such as green or red hair, and piece them together to form a complete plasmid. Once they had designed their plasmid they made a biscuit creature that corresponded to the plasmid they had created. The learning outcome of this activity was to give the children an understanding that a different selection of genes, results in different characteristics, and that editing the DNA can result in a change in the characteristics. The activity was very popular and most of the participants seemed to understand this concept, as well as having great fun.<br />
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The event attracted a range of ages, as well as mixed levels of previous interest into synthetic biology. It also became clear that many members of the public knew very little about synthetic biology, and therefore were very interested in finding out more about the new branch of biology and the iGEM competition. The NRPUEA iGEM team also took the opportunity to talk to the public and listen to their opinions about the work the iGEM teams are carrying out, which demonstrated how mixed the view of the public were. The day was a huge success.<br />
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'''Synthetic Biology activity sheets'''<br />
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[[File:Word_Search_of_Synthetic_Biology.pdf]],<br />
[[File:Decode_Disney.pdf]],<br />
[[File:Questions_comprehensive.pdf]],[[File:Decode_Music_Artists_and_Films.pdf]],[[File:Word_Search_of_Synthetic_Biology.pdf]]<br />
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=== Creation of fictious futurist company, Quanticare ===<br />
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[[File:Tattoo1.png |300px]][[File:QuanticareLogo_(2).png |300px]][[File:Tattoo2.png |300px]]<br />
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The team created Quanticare, a fictitious company set in the future that had formed as a result of the UEA NRP iGEM 2012 team. The fictitious company's latest development is to introduce a visual bio sensors in to the human body,in the form of a tattoo based health moniture, Cura. Not only did the development of the fictitious company allow the team to explore potential future applications of synthetic biology and bio sensors, but also via the creation of a film , fuel a range of ethical questions.The team used novel marketing techniques, such as sending a range of transferable tattoos, representing the product Cure, to the other UK teams. They did this aiming to raise interest in the NRP UEA iGEM project and through curiosity of finding out what the different tattoos symbolized attracting fellow iGEMers to look at our wiki and therefore develop an interest in our presentation and poster at the European jamboree.<br />
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=== The team collaborated with Artist Amy to create this fictitious Film ===<br />
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The team worked closely with their artist Amy in order to produce a futurist film, demonstrating one of the many possible future potentials of our project, as well as synthetic biology as a whole.Together Amy and the team explored a range of potential ways to grab the public's attention, as well as fuel ethical thoughts. They decided that inventing a fictitious future business (Quanticare) which had used the developments of the teams project as a stepping stone towards a futuristic personal health monitoring tattoo would be a very visual and appealing method of communication. The film was released at the teams public event held at the forum in Norwich, attracting lots of attention due to both the plot of the film, and its artistic touches. Watch the film for yourself.<br />
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==Social Media==<br />
The NRPUEA iGEM team has been VERY active on a range of social media over the summer.Any one is welcome to follow the team on Facebook or twitter. The team keep everyone updated on what is going on, both in the lab and in our spare time. This has been a great method of connecting with the public, companies,researchers, as well as getting in touch with other iGEM teams. The NRPUEA iGEM team believe human out reach via social media is important since it allows the public to follow the teams achievements and lab results, as well as get to know the personality of the team members. please follow us on twitter @NRPiGEM or UEA iGEM 2012 page on Facebook.<br />
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===Twitter===<br />
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[[File:Twitter stats NRP iGEM.png]]<br />
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[[File:TweetReach_nrpigem.pdf| '''Download a PDF of our twitter statistics''']]<br />
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===Facebook===<br />
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Creating social media pages also allowed us to access information about who was checking our page out, showing interesting information, showing that social media techniques such as facebook attracted mostly 18-24 years olds interest, and that it attracted no attention from those above 65. Therefore the team though it was important to promote our project, not only through the internet, but also other techniques such as newspaper articles and radio shows. <br />
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The figure bellow hows the age ranges and gender of people who liked any posts from the NRP UEA iGEM team<br />
[[File:Likes.png |600px| centre]]<br />
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Below is a list of the location that people liked the teams statuses from. This highlighted that the majority of followers were located in Norwich, however also many from a range of places over the world, therefore highlighting the importance of using such social media to reach these followers. <br />
[[File:Location_of_likes.png |600px| centre]]<br />
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As mentioned the NRP UEA iGEM team made facebook statuses and tweets very regularly and as the graph below shows, there was consistently a increase in action and interest for the NRPUEA iGEM team after. <br />
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[[File:Posts_ratings.png |600px| centre]]<br />
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===Google+===<br />
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We championed the use of Google+, a new social network from Google. We encouraged the UK iGEM teams to utilise Google+ to help make the 2012 UK team meetup as open and accessible as possible.<br />
<br />
==Media appearances==<br />
<br />
===STAR Radio===<br />
<br />
[http://www.star107.co.uk/farming-show.php STAR 107.9 FM's Farming Show]<br />
<br />
[[Image:NRPSTARRadio.jpg | 300px | right]]<br />
<br />
[http://www.star107.co.uk/podcasts/the-farming-show/show-23.mp3 Listen To Our Radio Show Appearence!]<br />
<br />
In early July the NRP UEA team travelled to Cambridge to appear on STAR Radio's "The Business Hub" and "The Farming Show". They were interviewed by Mark, one of the presenters, about synthetic biology, iGEM, and our project. The interview went really well as Pascoe and Khadija got across all the information that was needed in a clear and concise way, and Mark was extremely helpful in making sure all the salient points of the project were brought out. <br />
<br />
The team were able to give a brief description of what synthetic biology is before talking about the future applications of synthetic biology for medicine, agriculture and business. We were also able to mention our event at the Norwich Forum in late August in order to help promote it!<br />
<br />
A big thanks goes out to all at STAR Radio 107.9 for their help with the project in setting up the interview and allowing us air time to discuss iGEM and synthetic biology. Please visit the link above to hear the radio show.<br />
<br />
<br />
<br />
[[File:Paper.jpg|100px|left]]<br />
<br />
===Newspapers===<br />
<br />
The team featured on the university website a few times through the summer, as well as a brief article in the Eastern daily press newspaper.This was great, as allowed the locals of Norwich to become aware not only of the team and iGEM, but also introduce many to the scientific branch of synthetic biology.<br />
<br />
==Engagement with the wider scientific community==<br />
<br />
=== The team presented at the John Innes Centre Synthetic Biology conference ===<br />
<br />
The john innes center (JIC) is a world leading center of plant research, the home of UEAs 2011 team, and is part of the Norwich research part. Researchers at the JIC recognized that synthetic biology was an important field and therefore hosted a synthetic biology confrere, with the aim of the day to enhance their reserches knowledge of what synthetic biology is, and projects in which synthetic biology is being used. when the NRP-UEA was invited to give a talk to the JIC researchers we were extremely excited, giving them an over view of what iGEM was, our project and how it developed over the summer, as well informing them about aspects of human practice the NRP- UEA team had carried out over the summer. the team took also showed them a clip of our futuristic applications film. The team enjoyed presenting their work and results, as well as using it as an opportunity to improve their presentation skills before the European jamboree. There seemed to be a real interest by their researchers in to our project, giving encouraging advice, with many asking further questions after the presentation. The researchers also seemed very interested to hear more about our human practices elements after the presentation, wanting to know what the public's opinion of the research carried out at Norwich research park was, as well as synthetic biology as a whole.<br />
[[File:JIC_Synthetic_Biology_Workshop.pdf]]<br />
<br />
<br />
===SGM microbiology synthetic biology (September 3rd -5th)===<br />
<br />
The society of general microbiology held a 3 day synthetic biology conference at the University of Warwick. Three members of the UEA NRP iGEM team were lucky enough to go and enjoy talks on streptococcus, molecular motors, and the dynamics of the genome and designer microbes. The event also gave the team member the opportunity to meet other synthetic biology scientists as well as informing them of the current advances in the field and inspiring them with the future possibilities. The event also allowed the team members to present a poster and therefore engage with the scientific community, both informing them more about the iGEM competition and our project . The team believed it was important for the scientists in the synthetic biology world to have the opportunity to meet undergraduate iGEM members, allowing them to express the skills it has allowed them to develop and to encourage further interest in to lab groups forming teams, and funding iGEM projects.</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/FutureTeam:NRP-UEA-Norwich/Future2012-09-26T13:28:27Z<p>Joyehicks: </p>
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<div>{{UEANRP}}<br />
Nitric oxide (NO) is produced in different environments, be that within humans or by bacteria in soil. It is a nitrogenous species that is involved in the nitrogen cycle. With further research, development and integration into various systems, the future of medicine and agriculture could involve NO sensors.<br />
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<br />
=='''Agriculture'''==<br />
<br />
NO can be produced by nitrifying bacteria during oxidation of ammonium (Lipschultz, F. et al. 1981). These gases can be released into the atmosphere. NO is also converted back to atmospheric nitrogen in a reductive process called denitrification. The differences in the bacterial species that compose soil flora produce and reduce nitric oxide lead to different levels of NO in soil, which can affect soil fertility and thus agricultural yield. Besides soil flora, soil composition, temperature, water levels (Davidson, E. 2012), tilling and fertiliser quantities (Civerolo, K.L. et al. 1998) can all affect the levels of NO. This is particularly significant now as the world population is ever increasing; the higher the agricultural yield the more people than can be supported. An accurate NO sensor can allow a farmer to be able to better utilise their resources and to accurately apply fertiliser, water and needed farming techniques to maximise the crop yield.<br />
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=='''Medicine'''==<br />
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[[File:JAMES_CAVALLINI_SCIENCE_PHOTO_LIBRARY.jpg| 200px | right | thumbnail | <html><u>This photograph is the property of James Cavallini/ Science Photo Library.</u></html>]]<br />
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<br />
Nitric oxide is an important physiological signalling molecule within the human body. This highly reactive molecule is produced through the action of enzymes called nitic oxide synthases (NOS). There are three main types of NOS in the body, which each produce NO for a different physiological role: inducible (iNOS), endothelial (eNOS) and neuronal (nNOS) (Xu, W. et al. 2002). NO in the blood, causes vasodilation through increase of cGMP, a second messenger which activates many receptors and processes (Ferreira, L.F. et al 2010). As NO signalling is so widespread within the body there are many applications NO sensors can have in medicine.<br />
<br><br>Whilst NO triggers several defence mechanisms within the innate immune response, it has been found that some bacteria, such as the food-borne pathogen ''Listeria monocytogenes'', are able to use increased NO levels (produced by nitric oxide synthase-2) to promote their colonisation of host cells; culminating in a condition known as listeriosis. ''L. monoctogenes'' is absorbed from donor to recipient cell within vacuoles, and as NO decreases the rate at which the immune response destroys these vacuoles the bacteria is increasingly successful (Cole, C. et al. 2012). A system which is able to both detect and decrease levels of nitric oxide would thus be highly advantageous in therapeutic strategies to treat ''L. monocytogenes'' infection.<br />
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<Br><Br><br />
<br />
=='''Cancer Research'''==<br />
<br />
[[File:Warrior_cell.png| 200px| left | thumbnail |This image is a logo which represents a future application of how our biobricks could be used to produce a bacteria capable of acting as a cancer thereapeutic.]]<br />
All the isoforms of NO synthases aforementioned are involved in the promotion or inhibition of the tumour cells (Xu, W. et al. 2002). Research has shown that high levels of NO can be cytotoxic /or cytostatic to tumour cells; however, at the low levels produced by tumour cells, NO signals angiogenic factors such as VEGF leading to angiogenesis and enlargement of the tumour (Xu, W. et al. 2002). Macrophages, as part of the innate immune system, produce NO to kill off the tumour. Research by Xu et al., shows that NO has greater activity when the tumour is less<br />
differentiated and hence a higher grade.<br />
<br />
Using NO as a chemoattractant, the low and high levels of NO produced by cancer cells and macrophages, respectively can be perceived by the sensor which can target drugs directly to the tumour. An alternative method is to use oxygen as a chemorepellent so modified cells can specifically target hypoxic cells. Specificity can be increased through the use of targeting using specific peptide sequences which home onto receptors or glycoproteins which are specific to tumour cells such as the NGR peptide (Pasqualini, R. et al. 2000).<br />
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<br><br><br><br><br><br><br><br><br><br />
<br />
=='''Diagnostics'''==<br />
<br />
NO is produced throughout the body. An abnormal level of NO is an indication of diseases and disorders such as hypertension, impotence and obesity. A NO sensor can be augmented into two ways to aid diagnosis and treatment. For example, measuring NO level of externally exhaled air can be an important, and non-invasive, diagnostic for airways inflammation. This is due to endothelial cells producing NO synthase (eNOS), both constitutive and inducible (iNOS). The iNOS is induced by pro inflammatory cytokines, as well as increase of the enzymes expression after expose to oxidants. Therefore measuring the NO level produced by eNOS, has led to finding that there are higher levels of epithelial nitric oxide when asthma is untreated, and that there is a fall after anti-inflammatory treatment (Narang ,et al., 2002) .<br />
<br />
Alternatively, Building upon the Cambridge 2009 iGEM E.Chromi project, the NO sensor can be incorporated to perceive abnormal levels of NO in the gut flora or damage to the gut, which can visually be seen in the form of colours in faecal matter (Cambridge iGEM team , 2009). For example NO levels increase during damage of the gut, potentially due to its role in functional repair ( Miller et al, 1993). Therefore,an NO sensor would produce particular colours that could correspond to different levels of NO expected for certain diseases and disorders.<br />
<br />
Another use is through the cells being stimulated to create a protein coat for resistance and persistence within the human body. These cells could be mass produced and contain vaccines which can be taken at birth. These cells can be used to target pathogens or other abnormalities that arise in the body.<br />
<br />
=='''Understanding Pathogens'''==<br />
<br />
Pathogens have many different mechanisms and strategies of invasion and attack. For example some pathogens produce biofilms. To create biofilms, bacteria signal to other bacteria to assess the population size. When numbers are sufficient, they switch on gene expression which leads to aggregation and adhesion. This is an example of quorum sensing. A greater understanding of NO can be applied to research into pathogenic diseases which could lead to advances in the creation of cures and vaccines.<br />
<br />
<br />
=='''NO sensing'''==<br />
<br />
Many NO sensors exist but none that can accurately and specifically measure the levels of NO. To do this a comparator circuit system can be put into place. The circuit could involve multiple sensors which sense different reactive oxygen and nitrogenous species. Within this system, short interfering RNA (siRNA) could be implemented to inhibit the expression certain enzymes and proteins. This would produce an overall output that corresponds only to NO levels. To give a visual quantitative output, the use of bioluminescence can be augmented. The brightness would relate to the amount of NO in cells environment.<br />
<br />
Furthermore, as mentioned previously the E.chromi project can be incorporated. In addition to its application in disease communication relative to NO levels, different sensors can be incorporated into the system which accurately measures all nitrogenous compounds. The levels of these can be qualitatively measured through the resulting colour product.<br />
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[[File:Future applications.png|950px]]<br />
<br />
===References===<br />
<br />
<br />
Cambridge iGEM team (2009) Available at: https://2009.igem.org/Team:Cambridge [Accessed on: 21/09.2012] <br />
<br />
<br />
Civerolo, K.L. and Dickerson, R.R. (1998) ‘Nitric oxide soil emissions from tilled and untilled corn®elds’, Agricultural and Forest Meteorology, 90; 307-311. <br />
<br><br><br />
<br />
Cole, C., Thomas, S., Filak, H., Henson, P., Lenz, L. (2012) ‘Nitric Oxide Increases Susceptibility of Toll-like Receptor-Activated Macrophages to Spreading Listeria monocytogenes’, Immunity, 36; 885.<br />
<br><br><br />
Davidson, E. (2012) Sources of Nitric Oxide and Nitrous Oxide following Wetting of Dry Soil, Soil Sci. Soc. Am. J. 56; 95–102<br />
<br><br><br />
Ferreira, L.F. and Behnke, B.J. (2010) ‘A toast to health and performance! Beetroot juice lowers blood pressure and the O2 cost of exercise’, Journal of Applied Physiology, 110; 585-586.<br />
<br><br><br />
Lipschultz, F., Zafiriou, O.C. Wofsy, S.C., Elroy, M.B., Valois, F.W. and Watson, S.W. (1981) ‘Production of NO and N2O by soil nitrifying bacteria’, Macmillan Journals, 294; 641-643.<br />
<br><br><br />
Pasqualini, R., Koivunen, E., Kain, R., Lahdenranta, J., Sakamoto, M., Stryhn, A., Ashmun, R.A., Shapiro, L.H., Arap, W. And Ruoslahti, E. (2000) ‘Aminopeptidase N is a receptor for tumour-homing peptides and a target for inhibiting angiogenesis’, The Journal of Cancer Research, 60; 722-727.<br />
<br><br><br />
Xu, W., Liu, L.Z., Loizidou, M., Ahmed, M. And Charles, I.G. (2002) ‘The role of nitric oxide in cancer’, Cell Research, 12; 311-320</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/SafetyTeam:NRP-UEA-Norwich/Safety2012-09-26T13:27:51Z<p>Joyehicks: </p>
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<div>{{UEANRP}}<br />
<br />
<br />
<br />
Use this page to answer the questions on the [[Safety | safety page]].<br />
<br />
<br />
=Potential safety issues our project could raise:=<br />
<br />
When designing the experiments the NRP-UEA-Norwich team would undertake they had to consider not only the safety of the researchers and others within the lab, but also the public and the environment. They had to consider carefully the organisms and chemicals they would be using within lab, how they would handle these safely and then dispose of them after to minimize their effect on the environment and the public.<br />
<br />
==Researcher safety==<br />
<br />
[[File:Joy_gloves.jpg |200px|left]]The University of East Anglia already has safety standards that have to be met, which included having basic safety training before starting wet lab work, as well as reading, understanding and signing relevant COSH forms for each potential lab procedure. Therefore, when designing experiments the NRP-UEA-Norwich team considered the risks associated and checked whether the COSH forms signed covered the procedures undertaken. The team also received training from advisors over the first few days of their time in the lab. They learnt what to do in emergencies, where to access the safety manual which is always within the lab, as well as being shown where to dispose of the different chemicals and used equipment. It is also vital that researchers within the university wear personal protective equipment (PPE), including lab coat, gloves and covered shoes. The members also wore protective face shields when using artificial UV light in the darkroom. In accordance with university regulation, the team was prohibited from eating, drinking or smoking in or near the lab. The team enjoyed their lunch well away from the lab, as well as being careful to wash their hands before leaving.[[File:Protection_of_ears.jpg |300px|right]]<br />
<br />
The lab facilities were designed to be safe for the team members, providing plenty of space and clean surfaces to work on. The lab was supplied with plastic handheld pipettes, and permanent pens; removing the need for use researchers to ever use their mouths during experimentation e.g. mouth pipettes and labels that require saliva to aid adhesion.<br />
<br />
The team had originally thought it would be interesting to look at NO levels within salmonella, but decided later that this would result in a lot more safety concerns and complications. Therefore, the team decided to only use strains of "E.coli" (which they had all used within their university studies before and were allowed to handle within the lab) within our projects. NEB 5-alpha ''E.coli'' was use to characterize existing bio bricks, as well as BL21 pLysS ''E. coli'' cells and Alpha select gold ''E.coli''. ''E.coli'' is a well-studied species of bacteria, with fairly predictable behavior, as well as being non-pathogenic (a bio safety level 1 bacteria). Therefore, all the students were able to use the ''E.coli'' within the university’s second year (category one and two) teaching labs.<br />
<br />
The hybrid promoter was further characterized by being placed into the mammalian cell. This was carried out by an experienced member of the team. This caused no concern as the mammalian cells were used and kept under standard laboratory conditions. However, if these cells somehow did make it in to the body they would be recognized as non self cells and consequently destroyed by the immune system. <br />
<br />
Since the team’s project involves Nitric Oxide (NO) they had to consider the safety of the chemicals involved. It is important to note that all chemicals within the lab are supplied with a safety advice sheet that has been generated by the company it was manufactured by. NO has been classed as a dangerous gas which can be directly toxic to blood, lungs, pancreas and nervous system. Therefore, caution was taken when using NO within the lab. Its clear appearance makes indicating the presence of the gas difficult, increasing the likelihood of the gas being unintentionally inhaled. However, the gas has an odor allowing researchers to detect its presence and act accordingly. To minimize this risk, NO was handled within fume cupboards and chambers, reducing researcher exposure to any released NO. <br />
<br />
In reality, although the team aimed to test the hybrid promoter with NO (as required for therapeutic effects) due to time restrictions this was inappropriate. If the team had more time they envisaged testing the comparator circuit using NO after its analysis when attached to promoters that respond to safer and simpler inducer molecules, such as arabinose. This would have been an ideal method of characterizing the comparator circuit, as well as a method of gaining experience in the relevant procedures before using more toxic chemicals.<br />
<br />
Another safety concern was the use of Ethidium Bromide (EtBr) during the agrose gel staining. Ethidium Bromide is a known carcinogen, therefore the team took care to follow the university's specific guidelines on working with carcinogens ([[File:UEA_Rules_for_working_with_carcinogens_in_laboratories.pdf]]), which include the use of protective equipment, gloves and the appropriate disposal of waste in order to minimize researcher exposure.<br />
<br />
In the preliminary discussions in which the experiments the team would carry out were discussed, careful consideration was taken to choose appropriate equipment. They considered the equipment’s safety to the researcher, as well as the practicality and quality of results gained. The team members used most of the discussed equipment and procedures already used during their first two years of their undergraduate degree. However, there were a few procedures such as NanoDrop spectrophotometery and fluorometery that had not been used by the team before. Therefore the team members received training from experienced members of staff in order to avoid accidents that could damage the researchers or the machine itself. Finally, there were always more experienced scientists present within the lab to advise the team further on how to use equipment safely.<br />
<br />
==Public safety==<br />
<br />
The team always removed their lab coats and gloves, as well as washed their hands before leaving the lab. This reduced the chance of bringing harmful bacteria or substances outside the laboratory environment. The team also took care to keep the laboratory windows shut, in order to reduce exposure to the outside world.<br />
<br />
All contaminated waste was autoclaved on site before being collected and disposed of under controlled conditions for cleaning for reuse.<br />
<br />
The team felt that it was important that the public were given the opportunity to understand and ask about procedures used in the laboratory, so they could form informed opinions about the safety and ethical implications of the teams work. The team took the opportunity at their public engagement project to explain the procedures they used in full detail.<br />
<br />
At this public engagement event the team took transformed ''E.coli'' samples to the venues and public safety was a priority. Therefore, the plates were maintained in a UV box behind a sheet of plastic, limiting public physical interaction with the laboratory specimens. These plates were also sealed extensively using parafilm and were kept well away form any part of the venue holding or serving food products.<br />
<br />
==Environmental safety==<br />
<br />
Data safety sheets that come with chemicals explain how to dispose of the chemicals correctly. These guidelines were adhered to rigidly since the local water providing company, Anglia water, carry out routine random samples on what is poured down the sink drains, and if a certain volume (volume is unknown to university, which encourages them not to put any waste down the sink) of listed chemicals are found, then the university is fined.<br />
<br />
There is a very low risk of damage to the environment as all bacteria will be stored safely within the lab. However, if the bacteria was to unexpectedly be released, there is very little threat on the environment, since the bacteria are standard autotrophic laboratory stains and therefore unlikely to survive. The ''E.coli'' stains used are also unlikely to replicate and therefore spread. Finally, due to using plasmids with a narrow host range horizontal gene transfer between bacteria within the environment is also very unlikely, as plasmids would not replicate if transferred to other organisms.<br />
<br />
=Safety issues our BioBrick parts could raise:=<br />
<br />
The first bio bricks made by the NRPUEA iGEM team are hybrid promoters consisting of a bacterial promoter (B) fused to a mammalian promoter (M); this has been carried out in two orientations, B upstream from M (B-M), and M upstream from B (M-B). As this is a new concept the biobricks are not well characterised, therefore raising safety concerns due to the lack of knowledge surrounding how the promoters will interact with one another. The team has not submitted any issues to the registry thus far as no specific issues have arisen, there are no specific concerns regarding the DNA sequence and both promoters are individually well characterised; however the team remains vigilant and understands that with unknown biobricks such as B-M and M-B there is always the chance for something unexpected to occur. <br />
<br />
The team has also synthesised a second new construct known as the comparator circuit, which again is not well characterised. Through its intended use as a gene regulation component a risk arises in that the comparator circuit could possibly join to unintended, functional genes and affect their regulation. As no specific issues have arisen yet no issues have been added to the registry, however upon characterisation of the biobricks any issues that do arise will immediately be included with the part.<br />
<br />
Throughout the project the team handled all safety issues by following and complying with both government safety standards as well as university safety standards. The team has also inserted the biobricks into narrow host-range plasmids (as produced by iGEM), thus reducing the chances of horizontal gene transfer. Biobricks of B-M, M-B, and the comparator circuit, were all transformed into standard BSL1 laboratory autotrophic strains of ''E. coli'' and are therefore unlikely to survive outside of the laboratory.<br />
<br />
Future iGEM teams could increase their safety by connecting the B-M and M-B biobricks to a suicide gene. This would mean that in the unlikely event of the transformed cells being released into the environment and unintended bacteria taking up the biobrick-containing plasmid, the unintended bacteria would be destroyed and further spread of the hybrid promoter biobricks would be prevented.<br />
<br />
=The University of East Anglia's biosafety review board=<br />
<br />
UEA takes both staff and student safety very seriously and has its own health and safety polices and biosaftey rules that must be followed:[[File:UEA_GMO_rules.pdf|UEA Genetically modified organisms guidelines]],[[File:UEA_statment_of_health_and_saftey_policy.pdf|UEA health and saftey policy]]and[[File:UEA_Microbiological_saftey_rules.pdf|UEA Microbiological saftey rules and guidelines]]. These have been created to follow the guidelines and biosafty rules that have to be considered in the United Kingdom as seen here http://www.hse.gov.uk/biosafety/gmo/law.htm, http://www.hse.gov.uk/biosafety/gmo/acgm/acgmcomp. All our projects are in compliance with both university requirements and national regulations.<br />
<br />
The university also has its own biosafety committee in which the responsibilities of different aspects of safety are split between departments and staff. Dr Andrew Hemmings is the BIO safety officer at UEA and spoke to members of the team about the project and discussed whether our COSH forms covered the processes carried out. The team then discussed their project further with other members of staff including Dr Mark Coleman, university GMO safety officer, and Dr Gabriella Kelemen, university microbial safety officer. <br />
<br />
When speaking to members of the UEA biosafty team there was no concern with the processes being carried out and the bacteria being used. When the team spoke to GMO safety officer, Dr Mark Coleman, about the safety of the biobricks they intended to create he said "there is a highly improbable chance this project poses a significant risk to either human health or the environment".<br />
<br />
=Making future iGEM competitions safer through biosafety engineering=<br />
<br />
Although the team started looking at NO, the dangers of unintentional exposure to NO soon became clear. Therefore, the team would hope that through using systems similar to our comparator sensor, NO sensors will be produced that are in the form of a sheet of paper and can be placed on the wall of labs, and buildings ect, to indicate excess levels of NO visually. <br />
<br />
The team feel that future iGEM teams could aim to reduce the chance of horizontal gene transfer further by reducing the adherence between the transformed cells and others, or via the Prevention of transformations without inducing competency. This would be a procedure to prevent some cases of antibiotic genes being passed through between bacterial species. However, a variety of other safeguards could also be deployed to prevent this.</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/NOSensingTeam:NRP-UEA-Norwich/NOSensing2012-09-26T13:25:15Z<p>Joyehicks: /* Generation of B-M and M-B with eCFP and RFP */</p>
<hr />
<div>{{UEANRP}}<br />
<br />
{{UEANRPProjects}}<br />
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[[File:NRPNOLogo.png | centre]]<br />
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<html><center><b><font size=4pt>Our hybrid promoter hopes to add to the systems already in the registry by creating a hybrid promoter that combines the bacterial promoter PyeaR and the mammalian CArG element , both of which respond to exogenous nitrogenous species. Combining the two would allow a more modular nitric oxide sensor that can be used in mammalian and bacterial cells interchangeably.</font> <br />
<br><br><br />
<font size=2pt>Six new biobricks produced and submitted to the registry with characterisation from fluorescence-based experiments.</font></b></center></html><br />
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Parts produced from this project:<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K774000 Bacterial-Mammalian/B-M (PyeaR-CArG) Hybrid Promoter] -- [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774001 Mammalian-Bacterial/M-B (CArG-PyeaR) Hybrid Promoter] -- [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774004 B-M + eCFP] -- [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774005 B-M + RFP] -- [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774006 M-B + eCFP] -- [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774004 M-B + RFP]<br />
<br />
Our main project has resulted in the production of a hybrid bacterial and mammalian promoter optimised for induction by nitric oxide, nitrates and nitrites. We have ligated PyeaR, a known bacterial promoter and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005 Part BBa_K216005] (Cambridge 2009) in the parts registry, with its mammalian counterpart, CArG. The resulting hybrid promoter has been synthesised in two orientations; PyeaR (bacterial, B) upstream of CArG (mammalian, M), nicknamed (B-M); and CArG upstream of PyeaR (M-B). These orientations were submitted to the parts registry as our first two biobricks.<br />
<br />
Each orientation of the promoter was ligated to enhanced cyan fluorescent protein (eCFP) and red fluorescent protein (RFP) to produce four new biobricks which have been submitted to the parts registry. These promoter + fluorescent protein biobricks have been characterised following transformation into ''Escherichia coli'' and induction by potassium nitrate using methods such as flow cytometry, fluorescence-activated cell sorting (FACS) and scanning with a fluorometer. The data from these experiments has proved that our promoter works as we expected it to. We have also transfected M-B + eCFP into a human breast cancer cell line, MCF7, and have proved the system is flexible and can be used in both eukaryotes and prokaryotes.<br />
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We believe the promoters we have produced have relevant uses in cancer therapeutics, soil fertilisation and detection of emissions from industries such as construction.<br />
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<br />
==Introduction==<br />
<br />
[[File:NRPPyeaR.png | 300px | thumbnail | '''''Figure 1.''''' ''A graphical representation of PyeaR. In the higher image PyeaR's activity is being repressed by both Nar and NsrR preventing transcription and the ultimate expression of a reporter. In the lower image nitrate/nitrite molecules have inhibited the activity of Nar, and nitric oxide has inhibited activity of NsrR, allowing for transcription to occur and subsequent expression of a reporter.'']]<br />
<br />
The University of East Anglia hosts many research teams whose work focuses on studying nitrogenous species and the way in which bacteria use and modify those species. One major problem that is faced by these teams involves working with nitric oxide (NO), as NO is highly reactive with a low half life, therefore making it difficult to trace and quantitatively measure accurately. Many of the methods currently used to measure NO levels are unable to distinguish between homogenous species, such as nitrates (NO3) and nitrites (NO2), therefore the figure given for NO levels is often inaccurate as other nitrogenous species are taken into account.<br />
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<br />
The ability to be able to accurately detect NO levels is one with a great deal of potential for the future. Nitric oxide has been noted as a possible cancer therapy due to its physiological use as an apoptosis inducer by macrophages, however NO is also known to be used by cancerous cells to establish a baseline and use it to induce apoptosis and promote proliferation of a tumour; being able to accurately sense nitric oxide and go on to act on that information could be very useful to prevent the NO baseline being established by cancerous cells, but to also use NO for its apoptosis-inducing abilities. There are also other potential applications in the construction business, in 2008 '''NEED TO FIND REFERENCE OF PEOPLE''' released legislation encouraging construction companies to monitor their NO output as it is inversely proportional to carbon monoxide (a toxic substance which needs to be regulated); the ability to accurately detail levels of NO being released in these circumstances would be highly useful.<br />
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<br />
Nitric oxide is an extremely physiologically relevant molecule found within both eukaryotes and prokaryotes, where it is utilised by different enzymes and systems for various roles. The aim of the experiment was to devise a hybrid promoter that could be applied to eukaryotes and prokaryotes in order to begin to more accurately sense NO and report on its specific levels.<br />
<br />
<br />
[[File:NRPBMandMB.png | 300px | thumbnail | '''''Figure 2.''''' ''A graphical representation of the hybrid promoter in its two orientations; the top image shows B-M with the bottom image showing M-B. Also included are the restriction sites and their location within the system, with the elements being read from left to right.'']]<br />
<br />
The hybrid promoter was designed to take on both bacterial and mammalian elements in order to be compatible with both bacterial and mammalian cells. After research around the subject and searching the parts registry a promoter known as PyeaR was decided upon as the bacterial element of the hybrid promoter; PyeaR is found in the ''yeaR/yoaG'' operon of ''Escherichia coli'' and is associated with induction by nitric oxide, nitrates and nitrites (Lin ''et al.'', 2007). PyeaR is repressed by two main repressors; Nar, which is regulated by nitrates and nitrites; and NsrR, which is regulated by nitric oxide (Figure 1.). One of the key elements of PyeaR is that it is not repressed in aerobic conditions, allowing for easier carrying out of experiments. The PyeaR aspect of the hybrid promoter has been known throughout the project as the bacterial promoter, or simply B.<br />
<br />
<br />
The mammalian element of the hybrid promoter was produced by nine CArG elements (repeated elements of CC(A/T)(6)GG), a promoter previously used synthetically for nitric oxide synthase as a cancer therapy (Worthington ''et al.'', 2005) and developed from the EGR1 gene for early growth response protein 1 (Scott ''et al.'', 2002). The CArG aspect of the hybrid promoter has been known throughout the project as the mammalian promoter, or simply M.<br />
<br />
<br />
Following identification of the two elements of the hybrid promoters the B (PyeaR) and M (CArG) aspects were ligated to one another in two orientations; B upstream of M (B-M) and M upstream of M (M-B) (Figure 2.). The hybrid promoters were synthesised in a pUC57 backbone with the standard iGEM restriction sites of EcoR1/Xba1 upstream of the promoter, and Spe1/Pst1 downstream of the promoter. A BamH1 restriction site was included in between the B and M sequences in order to allow for the B and M elements to be separated, as well as for easy verification of the promoter having been ligated into the iGEM backbone in future experiments (as BamH1 does not already exist in the pSB1C3 backbone).<br />
<br />
==Characterisation of Existing Biobrick: BBa_K381001 (PyeaR + GFP biobrick)==<br />
<br />
In order to begin to develop experiments to characterise the hybrid promoters + fluorescent proteins experiments were also carried out on a biobrick containing PyeaR + GFP (Part [http://partsregistry.org/Part:BBa_K381001 BBa_K381001], Bristol 2010). In these experiments transformed ''E. coli'' was inoculated into liquid culture, which in turn had varying potassium nitrate concentrations added to it. They were then left to grow before being spun down and viewed under a UV box in order to observe. The different concentrations of potassium nitrate that the transformed ''E. coli'' was grown in were: 0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 8 mM, 10 mM.<br />
<br />
[[File:GFP 4.JPG | 600 px | center | thumbnail | '''''Figure 3.''''' ''A photograph of spun-down media containing potassium nitrate (to induce the promoter) and E. coli transformed by PyeaR + GFP (art BBa_K381001). Each sample was grown with a different concentration of potassium nitrate, from left to right: 0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 8 mM, 10 mM.]]<br />
<br />
The figure suggests that fluorescent proteins have been expressed by the bacteria grown in media containing potassium nitrate due to the fluorescence shown under the UV box. It also suggests that different concentrations of potassium nitrate correlate with different intensities of expression due to the observable differences in fluorescence as the tubes are viewed from left to right, going up the gradient. The negative control of 0 mM potassium nitrate appears to show no fluorescence, suggesting it is indeed the potassium nitrate that is inducing the promoter.<br />
<br />
<br />
==Creating Novel Hybrid Promoters: B-M and M-B into Biobricks==<br />
<br />
The DNA for the synthesised genes of B-M and M-B had been supplied in the pUC57 backbone, therefore it was necessary for B-M and M-B to be digested from the pUC57 backbone and ligated into the pSB1C3 backbone. The synthesised gene was transformed into competent ’’E. coli’’ cells, which in turn were grown on agar plates containing 100 µg/ml ampicillin (due to pUC57 containing ampicillin resistance); colonies that had grown were then inoculated into liquid culture, and the liquid culture was subsequently mini-prepped using either the Bioline ISOLATE Plasmid DNA Mini Kit or the Promega Wizard® Plus SV Minipreps DNA Purification System. The DNA that had been extracted through mini-preps and the pSB1C3 backbone, as provided by the iGEM registry, were then digested using EcoR1 and Pst1 and a ligation was carried out using standard assembly protocol. The product of ligation was then transformed into competent ''E. coli'' cells, which were grown on agar plates containing 2.5 µg/ml chloramphenicol (due to pSB1C3 containing chloramphenicol resistance); this was done to eliminate any bacteria that had been transformed with undesirable ligation products. <br />
<br />
The colonies that had grown were then grown in liquid culture and mini-prepped in order to extract the DNA; the extracted DNA was then sent for sequencing, and the returned sequenced matched the expected sequence. The DNA was then sent to the parts registry as the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774000 Bacterial-Mammalian Hybrid Promoter] and the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774001 Mammalian-Bacterial Hybrid Promoter]. The biobricks for B-M and M-B were then used for further experiments, including ligation with a fluorescent protein reporter and growth studies.<br />
<br />
From weeks one through to five the team worked on producing the first biobricks of the hybrid promoter. Despite this proving difficult due to various reasons such as low amounts of DNA being produced from early mini-preps and ligation strategies not working, ''E. coli'' transformed by the biobrick DNA was successfully grown and proven to have the relevant antibiotic resistance by the beginning of [https://2012.igem.org/Team:NRP-UEA-Norwich/Week6 week six].<br />
<br />
===Studies into the effect of the hybrid promoter on growth of ''E. coli'' competent cells===<br />
<br />
[[File:NRPCalibration.png | 300 px | thumbnail | '''''Figure 4.''''' ''A calibration curve produced using E. coli alpha cells grown over time. The inoculations were run through a spectrophotometer at OD 600 nm every hour and then a sample was plated in order to calculate CFU/ml over time.]]<br />
<br />
As part of the characterisation of the hybrid promoters, as well as PyeaR alone ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005 Part BBa_K216005], studies were conducted to see if the growth of the ''E. coli'' was affected by transformation of the promoters. <br />
<br />
<br />
To being untransformed ''E. coli'' were inoculated into liquid culture and left to grow overnight at 37 ºC before being diluted down. The diluted liquid cultures were run through a spectrophotometer at OD 600 nm and their absorbance noted, before each culture was plated and once again left overnight at 37 ºC; the next morning the colonies on the plates were counted, allowing a calibration curve of absorbance at OD 600 nm against colony-forming units per ml (CFU/ml) to be set up (Figure 3.)<br />
<br />
<br />
Following establishment of the calibration curve liquid cultures were made up of ''E. coli'' that was:<br />
<br />
. Untransformed (otherwise referred to as alpha cells)<br />
<br />
. Transformed by B-M<br />
<br />
. Transformed by M-B<br />
<br />
. Transformed by PyeaR<br />
<br />
<br />
These cultures were then diluted down to the same starting level (an OD 600 nm absorbance of 0.2 +/- 0.1) and cuvettes filled with LB media were inoculated. The cuvettes were then placed in a spectrophotometer every hour and the absorbance at 600 nm was established; in between readings the cuvettes were placed into a 37 ºC incubator in order to encourage bacterial growth. This was repeated for 12 hours and the absorbance readings compared to the calibration curve in order to give data on the level of growth of ''E. coli'' transformed with the different promoters/untransformed over time.<br />
<br />
==Generation of B-M and M-B with eCFP and RFP==<br />
<br />
In order to test the activity of the hybrid promoters a reporter needed to be ligated. As the hybrid promoter did not already contain a ribosome binding site (RBS) both the RBS and the reporter were needed to be ligated to the promoter; in order to help improve experimental efficiency the parts registry was searched for relevant reporters that also contained an RBS. In [https://2012.igem.org/Team:NRP-UEA-Norwich/Week3 week three] two reporters were identified as [http://partsregistry.org/Part:BBa_E0420 BBa_E0420], a biobrick for enhanced CFP (eCFP) + RBS + terminators, and [http://partsregistry.org/Part:BBa_K081014 BBa_K081014], a biobrick for RFP + RBS + terminators.<br />
<br />
Once the B-M and M-B biobricks had been created in week six work began in earnest on the fluorescent proteins and ligating the promoters to them in order to begin characterisation. Due to many set-backs with low levels of DNA and having to order more biobricks from the registry, a successful ligation of the promoter to a fluorescent protein reporter was finally achieved in [https://2012.igem.org/Team:NRP-UEA-Norwich/Week10 week ten]. In order to carry out the ligation the promoter was first digested using Spe1 and Pst1 in order to linearise the backbone downstream of the promoter; the fluorescent proteins were digested using Xba1 and Pst1 in order to remove the insert. A ligation was then carried out using standard assembly protocol and the ligation products were transformed into ''E. coli'' competent cells, which in turn were grown on agar plates 2.5 µg/ml chloramphenicol (due to pSB1C3 containing chloramphenicol resistance). <br />
<br />
In order to quickly identify colonies of bacteria containing the promoter, RBS and reporter in a likely correct sequence a range of colonies were inoculated into media also containing potassium nitrate (KNO3) solution; this was done in order to inhibit the Nar repressor in PyeaR and result in activation of the promoter/induction of transcription/expression of the fluorescent protein reporter. Samples of the inoculated media containing KNO3 were then added to an eppendorf and spun down to form a pellet, which was viewed under a UV box and observed for fluorescence. After a week of various ligation and transformation experiments both promoters were successfully ligated to both fluorescent proteins and fluorescence of eCFP and RFP was observed under a UV box.<br />
<br />
From these experiments four more biobricks were produced and submitted to the registry: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774004 B-M + eCFP], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774005 B-M + RFP], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774006 M-B + eCFP], and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774004 M-B + RFP].<br />
<br />
In order to characterise the hybrid promoters ligated to fluorescent proteins a number of experiments were carried out to measure the level of fluorescent output at different concentrations of KNO3 (used to induce the promoter's activity). All of these experiments were carried out in [https://2012.igem.org/Team:NRP-UEA-Norwich/Week11 week eleven]<br />
<br />
===Qualitative Results===<br />
<br />
[[File:NRPCFPWorks.jpg | 600 px | center | thumbnail | '''''Figure 5.''''' ''A photograph of spun-down media containing potassium nitrate (to induce the hybrid promoters) and E. coli transformed by M-B + eCFP. The concentrations of potassium nitrate added to the media were, from left to right: 100 mM, 50 mM, 10 mM, 0 mM.]]<br />
<br />
This figure appears to show fluorescence from the spun-down pellets of each hybrid promoter + fluorescent protein that had been grown in media contain potassium nitrate; it can be inferred from this that the potassium nitrate has induced the hybrid promoter, resulting in expression of the fluorescent protein reporter. The negative control of 0 mM potassium nitrate appears to show no fluorescence, suggesting it is indeed the potassium nitrate that is inducing the promoter.<br />
<br />
[[File:NRPFluorescence.jpeg | 600 px | center | thumbnail | '''''Figure 6.''''' ''A photograph of spun-down media containing potassium nitrate (to induce the hybrid promoters) and E. coli transformed by the four biobricks containg the promoters and fluorescent proteins; the photograph has been taken from a UV box. From left to right: B-M + eCFP, M-B + eCFP, B-M + RFP, M-B + RFP.]]<br />
<br />
The figure appears to show fluorescence from the spun-down pellets of each hybrid promoter + fluorescent protein that had been grown in media contain potassium nitrate; it can be inferred from this that the potassium nitrate has induced the hybrid promoter, resulting in expression of the fluorescent protein reporter.<br />
<br />
===Fluorometer Experiments===<br />
<br />
The main characterisation of the biobricks was carried out using a fluorometer. Five tubes of media containing 200 µL transformed bacteria and potassium nitrate were grown for each biobrick in concentrations as follows:<br />
<br />
. B-M + RFP with 0 mM, 5 mM, 10 mM, 15 mM and 20 mM potassium nitrate<br />
<br />
. B-M + eCFP with 0 mM, 5 mM, 10 mM, 15 mM and 20 mM potassium nitrate<br />
<br />
. M-B + RFP with 0 mM, 5 mM, 10 mM, 15 mM and 20 mM potassium nitrate<br />
<br />
. M-B + eCFP with 0 mM, 5 mM, 10 mM, 15 mM and 20 mM potassium nitrate<br />
<br />
<br />
The samples were then spun down in a centrifuge and resuspended in Tris buffer before being subjected to sonication in order to lyse the cells. The resulting solution was then spun down in a centrifuge again before the supernatant was extracted (in order to separate the proteins from the rest of the cell). The supernatant was then run through a fluorometer in order to measure the intensity of fluorescence as a method to gauge the activity of the hybrid promoters. RFP-containing samples were excited with a wavelength of 560 nm and the emission measured from 600 – 650 nm; eCFP-containing samples were excited with a wavelength of 410 nm and emission measured from 440-490 nm.<br />
<br />
<br />
These characterisation experiments gave rise to the characterisation which is now available on the main page for all hybrid promoter + fluorescent protein parts on the registry, and the experience section of the hybrid promoters alone on the registry.<br />
<br />
<br><br><br />
<br />
[[Image:BM-CFP_Graph.png]]<br />
<br><br>The graph above shows the flourescence measured from the expression of eCFP due to the response of the bacterial-mammalian promoter to different concentrations of potassium nitrate. The wavelength reading which corresponds to eCFP is between 440-500nm. The graph clearly demonstrates that between 0mN and 15mM there is a proportional relationship between fluorescence intensity and potassium nitrate concentration. There appears to be a sharp increase in fluorescence intensity between 5mM and 10mM, and the rate at which intensity increase gradually decreases so that there is only a small increase between 15mM and 20mM.<br />
[[Image:MB-CFP_Graph.png]]<br />
<br><br><br />
The graph above shows the flourescence measured from the expression of eCFP due to the response of the mammalian-bacterial promoter to different concentrations of potassium nitrate. The wavelength reading which corresponds to eCFP is between 440-500nm. The graph clearly demonstrates that between 0mN and 15mM there is a proportional relationship between fluorescence intensity and potassium nitrate concentration. It can be noted that at a 20mM concentration the intensity of fluorescence sharply decreases back down to the level of 5mM potassium nitate concentration. This may be due to the cell overexpressing eCFP up to the point at which the excess protein begins to form inclusion bodies which can no longer fluoresce; alternatively, this could be due the potassium nitrate concentration reaching the critical concentration at which it becomes toxic to the cell. This data differs to the readings taken from the bacterial-mammalian promoter ligated to eCFP, as well as the hybrid promoters to RFP, which may suggest there is a difference in the molecular mechanisms that these promoters function by; however at this point the change in intensity at 20mM is inconclusive and is an area which we would like to look into further. <br />
<br />
<br><br><br />
<br />
[[File:CFP_Comparison_Graph.png]]<br />
<br><br><br />
We were initially unsure of the effect that the orientation of the bacterial (pYEAR) and the mammalian (CaRG) genes would have in gene expression, therefore we synthesised two hybrid promoters in the orientation bacterial-mammalian and mammalian-bacterial. The graph above compares the intensity of fluorescence of the two hybrid promoters (BBa_K774004 and BBa_K774006) ligated to eCFP. There is a distinct difference between the intensity of fluorescence produced by the bacterial-mammalian promoter and the mammalian-promoter which is something that we would like to look into further. It is particularly interesting that at an intensity of 109a.u. the mammalian-bacterial promoter returns to the same level of intensity as the apparent maxiumum of the bacterial-mammalian promoter at 40a.u.<br />
<br />
<br><br><br />
We also ligated both of our hybrid promoters to Red Fluorescent Protein (RFP), and the results can be seen below.<br />
<br />
<br><br><br />
[[File:BM-RFP_Graph.png]]<br />
<br><br><br />
The graph above shows the flourescence measured from the expression of RFP due to the response of the bacterial-mammalian promoter to different concentrations of potassium nitrate. The wavelength reading which corresponds to RFP is between 600-650nm. The graph clearly demonstrates that between 0mN and 15mM there is a proportional relationship between fluorescence intensity and potassium nitrate concentration. A similar pattern can be seen here as for the mammalian- bacterial promoter with eCFP as at a 20mM concentration the intensity of fluorescence sharply decreases, however the intensity here decreases down to a level between 10mM and 15mM potassium nitate concentration. There is also only a small difference between 5mM and 10mM potassium nitrate, which differs to the pattern seen with the bacterial-mammalian promoter ligated to eCFP. As previously stated, this may be due to the cell overexpressing eCFP up to the point at which the excess protein begins to form inclusion bodies which can no longer fluoresce; alternatively, this could be due the potassium nitrate concentration reaching the critical concentration at which it becomes toxic to the cell. This data differs to the readings taken from the bacterial-mammalian ligated to eCFP, as well as the hybrid promoters to RFP, which may suggest there is a difference in the molecular mechanisms that these promoters function by; however at this point the change in intensity at 20mM is inconclusive and is an area which we would like to look into further.<br />
<br><br><br />
<br><br><br />
[[File:MB-RFP_Graph.png]]<br />
<br><br><br />
The graph above shows the flourescence measured from the expression of RFP due to the response of the mammalian-bacterial promoter to different concentrations of potassium nitrate. The wavelength reading which corresponds to RFP is between 600-650nm. The graph clearly demonstrates that between 0mN and 15mM there is a proportional relationship between fluorescence intensity and potassium nitrate concentration. It has been found that for all biobricks apart from the mammalian-bacterial promoter ligated to eCFP at a 20mM concentration the intensity of fluorescence sharply decreases. <br />
<br><br><br />
[[File:RFP_Comparison_Graph.png]]<br />
<br><br><br />
As previously stated, we were initially unsure of the effect that the orientation of the bacterial (pYEAR) and the mammalian (CaRG) genes would have in gene expression, therefore we synthesised two hybrid promoters in the orientation bacterial-mammalian and mammalian-bacterial. The graph above compares the intensity of fluorescence of the two hybrid promoters (BBa_K774007 and BBa_K774005) ligated to RFP. There appears to be no pattern if the difference between the intensities of these two promoters; however both promoters do show a decrease in intensity at 20mM potassium nitrate and decrease from a maximum intensity of 82a.u. (bacterial-mammalian) and 66a.u. to approximately 36a.u.<br />
<br />
===Flow Cytometry===<br />
<br />
Three tubes of media were inoculated with E. coli transformed by the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774005 B-M + RFP] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774006 M-B + eCFP] biobrick. Each tube then had potassium nitrate added to it at different concentrations; 0 mM, 1 mM and 10 mM respectively. The E. coli were grown over night and then spun down, fixed in 4% PFA and re-suspened in 500ul PBS. The samples were then analysed in an Acuri C6 or BD Aria II flow cytometer.<br />
<br />
[https://2012.igem.org/Team:NRP-UEA-Norwich/Protocol Full Protocol]<br />
<br />
''Note: This was the first time the flow cytometers at the University of East Anglia had been used with E. coli''<br />
<br />
====B-M + RFP Flow Cytometry Data====<br />
<br />
[[File:BM-RFP_18-9-12.png | 300 px | center | thumbnail | '''''Figure 7.''''' ''Flow cytometry data for B-M RFP transformed E. coli that were grown in either 0 mM, 1 mM or 10 mM potassium nitrate. Top row: Scatter plot of raw data and gating strategy utilised. Middle row: RFP Fluorescence profiles of samples. Lower left: Fluroescence profiles of the three samples overlai on the same plot.]]<br />
<br />
[[File:BM-RFP.jpg | 300 px | center | thumbnail | '''''Figure 8.''''' ''Flow cytometry fluorescence data: B-M RFP transformed E. coli that were grown in either 0 mM, 1 mM or 10 mM potassium nitrate.]]<br />
<br />
<br><br />
====M-B + eCFP Flow Cytometry Data====<br />
<br />
[[File:MB-CFP_data.jpg | 300 px | center | thumbnail | '''''Figure 9.''''' ''Flow cytometry data for M-B eCFP transformed E. coli that were grown in either 0 mM, 1 mM or 10 mM potassium nitrate. Top row: Scatter plot of raw data and gating strategy utilised. Middle row: RFP Fluorescence profiles of samples.]]<br />
<br />
[[File:MB-CFP.png | 300 px | center | thumbnail | '''''Figure 10.''''' ''Flow cytometry fluorescence data: M-B eCFP transformed E. coli that were grown in either 0 mM, 1 mM or 10 mM potassium nitrate.]]<br />
<br><br />
<br />
==Transfection of M-B + eCFP into the MCF7 human breast cancer cell line==<br />
<br />
In order to ascertain the flexibility of the hybrid promoter and help prove our original aim of producing a promoter that could be used in both eukaryotes and prokaryotes correct, the DNA for M-B + eCFP was transfected into the MCF7 human breast cancer cell line. An experiment was set up using S-Nitroso-N-acetyl-DL-penicillamine (SNAP), a nitric oxide donor, in order to induce expression of the fluorescent protein. A six-well transfection slide was produced containing:<br />
<br />
[[File:slide wells labelled.png | 500px | center]]<br />
<br />
The cells were then left for a day before they were imaged with a fluorescence microscope in order to observe expression of eCFP.<br />
<br />
[[File: Transfection.png | thumb | 500px |center | '''''Figure 11.''''' '' Transfection of MCF7 cells with images taken via a Zeiss CCD2 inverted microscope to detect CFP expression. Images in the left two columns are controls and have not been transfected, images in the right two columns have been transfected with M-B + CFP DNA; SNAP is a nitric oxide donor, therefore addition of SNAP was used to try and induce promoter activity.'']]<br />
<br />
The figure appears to show fluorescence in the mammalian cells that had been transfected with M-B + CFP compared with the cells that had not been transfected. The figure also appears to show stronger fluorescence in the cells that had been transfected with M-B + CFP and had been grown with the nitric oxide donor SNAP compared to the cells that had been transfected with M-B + CFP and grown without SNAP.<br />
<br />
[[File: NRPMBCFP.JPG | thumb | 500px |center | '''''Figure 12.''''' '' Transfection of MCF7 cells with images taken via a Zeiss CCD2 inverted microscope to detect CFP expression. Image shows mammalian cells transfected with M-B + CFP DNA however no SNAP (a nitric oxide donor) has been administered.'']]<br />
<br />
[[File:MCF7 blue.png | 300px | center ]]<br />
<br />
Close up image of what appears to be eCFP fluorescence from a transfected MCF7 cell.<br />
<br />
The figure appears to show low levels of fluorescence in the cells as there are small blue circles appearing in the cells indicative of CFP expression. MCF7 cells do endogenously express endothelial cell nitric-oxide synthase (ecNOS), which can produce low levels of NO. Perhaps it is endogenous NO production which is inducing the M-B promoter to express low levels of eCFP.<br />
<br><br />
<br />
[[File: NRPMBCFPSNAP.JPG | thumb | 500px |center | '''''Figure 13''''' '' Transfection of MCF7 cells with images taken via a Zeiss CCD2 inverted microscope to detect CFP expression. Image shows mammalian cells transfected with M-B + CFP DNA where SNAP (a nitric oxide donor) has been administered.'']]<br />
<br />
<br />
The figure suggests that expression of CFP has occurred due to the large blue shape appearing in the centre of the image following the use of a filter to view fluorescent cyan more easily.<br />
<br />
Future experiments are needed to confirm these results and also to provide more information.<br />
<br />
==Discussion==<br />
<br />
BM/MB better or the same, RFP/CFP better or the same etc.<br />
<br />
==Future Experiments==<br />
<br />
. Full quantitative analysis to see where the values lie; combine with tuners for different sensitivity levels <br />
<br />
. With different substrates (e.g. nitrite salt, NO donor)<br />
<br />
. With different reporter/effector enzyme<br />
<br />
. Repeat MB-eCFP transfection into MCF7 cells. Utilise more experimental controls (e.g. M-B only transfection).<br />
<br />
. Construct an MB-eCFP plasmid which is optimised for mammalian systems.<br />
<br />
. Optimise use of SNAP NO donor and use alternatives to enhance the induction of Nitric Oxide production.<br />
<br />
. Generate a new biobrick: M-B + Nitric Oxide Synthase + eCFP to test the concept of generating a synthetic gene network which can act as a cancer theraeutic.<br />
<br />
==References==<br />
<br />
Civerolo, K.L. and Dickerson, R.R. (1998) ''Nitric oxide soil emissions from tilled and untilled cornfields'', Agricultural and Forest Meteorology, '''90; 307-311''' <br />
<br />
<br />
Davidson, E., (2012), ''Sources of Nitric Oxide and Nitrous Oxide following Wetting of Dry Soil'', Soil Sci. Soc. Am. J. '''56; 95–102'''<br />
<br />
<br />
Lin H.Y., Bledsoe P.J., Stewart V., (2007), ''Activation of yeaR-yoaG Operon Transcription by the Nitrate-Responsive Regulator NarL Is Independent of Oxygen- Responsive Regulator Fnr in Escherichia coli K-12▿'', Journal of Bacteriology, '''189: 7539 - 7548'''<br />
<br />
<br />
Lipschultz, F., Zafiriou, O.C. Wofsy, S.C., Elroy, M.B., Valois, F.W. and Watson, S.W. (1981) ''Production of NO and N2O by soil nitrifying bacteria'', Macmillan Journals, '''294; 641-643''' <br />
<br />
<br />
Pasqualini, R., Koivunen, E., Kain, R., Lahdenranta, J., Sakamoto, M., Stryhn, A., Ashmun, R.A., Shapiro, L.H., Arap, W. And Ruoslahti, E. (2000) ''Aminopeptidase N is a receptor for tumour-homing peptides and a target for inhibiting angiogenesis'', The Journal of Cancer Research, '''60; 722-727''' <br />
<br />
<br />
Scott S.D., Joiner M.C., Marples B., (2002), ''Optimizing radiation-responsive gene promoters for radiogenetic cancer therapy.'', Gene Therapy, '''9: 1396-1402'''<br />
<br />
<br />
Worthington J., Robson T., Scott S., Hirst, D., (2005), ''Evaluation of a synthetic CArG promoter for nitric oxide synthase gene therapy of cancer'', Gene Therapy, '''12: 1417–1423'''<br />
<br />
<br />
Xu, W., Liu, L.Z., Loizidou, M., Ahmed, M. And Charles, I.G. (2002) ''The role of nitric oxide in cancer'', Cell Research, '''12; 311-320'''</div>Joyehickshttp://2012.igem.org/File:RFP_Comparison_Graph.pngFile:RFP Comparison Graph.png2012-09-26T13:24:21Z<p>Joyehicks: </p>
<hr />
<div></div>Joyehickshttp://2012.igem.org/File:MB-RFP_Graph.pngFile:MB-RFP Graph.png2012-09-26T13:24:08Z<p>Joyehicks: </p>
<hr />
<div></div>Joyehickshttp://2012.igem.org/File:BM-RFP_Graph.pngFile:BM-RFP Graph.png2012-09-26T13:23:53Z<p>Joyehicks: </p>
<hr />
<div></div>Joyehickshttp://2012.igem.org/File:CFP_Comparison_Graph.pngFile:CFP Comparison Graph.png2012-09-26T13:20:59Z<p>Joyehicks: </p>
<hr />
<div></div>Joyehickshttp://2012.igem.org/File:MB-CFP_Graph.pngFile:MB-CFP Graph.png2012-09-26T13:20:44Z<p>Joyehicks: </p>
<hr />
<div></div>Joyehickshttp://2012.igem.org/File:BM-CFP_Graph.pngFile:BM-CFP Graph.png2012-09-26T13:20:26Z<p>Joyehicks: </p>
<hr />
<div></div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/ProjectTeam:NRP-UEA-Norwich/Project2012-09-26T13:07:31Z<p>Joyehicks: </p>
<hr />
<div>{{UEANRP}}<br />
{{UEANRPProjects}}<br />
<br />
'''RUSSELL, KHADIJA AND PASCOE TO ORGANISE SUMMARIES OF THE THREE PAGES'''<br />
<br />
Sensory BioBrick systems have been a large constituent of previous iGEM projects in which teams have combined impressive amounts of logic with limitless creativity in order to produce synthetically engineered organisms with the ability to detect the presence of specific substrates; this was achieved by combining various promoters and reporters to produce novel gene systems of great breadth and depth.<br />
<br />
<br />
We too have taken a sensory approach to our project and have produced systems involved in the sensation of nitric oxide (NO). Originally we set out to develop a bacterial and mammalian hybrid NO-sensing promoter (which we have achieved); we then looked into ways of quantifying the levels of highly reactive and difficult to measure NO within a system, leading to us producing a novel gene regulation system known as the comparator circuit. Throughout the project we went on to look at theoretical alternative approaches to the gene systems we have produced.<br />
<br />
<br />
Overall in our project we have produced 6 sensory BioBricks, 2 BioBricks involved in gene regulation, and have further characterised 4 more BioBricks. All 8 of our BioBricks have been submitted to the registry and the 6 sensory BioBricks have been characterised.<br />
<br />
<br />
----<br />
<br />
[[File:NRPNOLogo.png | centre | link=Team:NRP-UEA-Norwich/NOSensing]]<br />
<br />
<br />
<br />
[[File:MB.png | 200px | right | thumbnail | '' '''Figure 1.''' A graphical representation of the hybrid promoter in its Mammalian-Bacterial orientation, also showing restriction sites.'' ]]<br />
<br />
[[File:BM.png | 200px | right| thumbnail | '' '''Figure 2.''' A graphical representation of the hybrid promoter in its Bacterial-Mammalian orientation, also showing restriction sites.'']]<br />
<br />
Our own hybrid promoter hopes to add to the systems already in the registry by creating a hybrid promoter that combines the bacterial promoter PyeaR and the mammalian CArG element , both of which respond to exogenous nitrogenous species. Combining the two would allow a more modular NO sensor that can be used in mammalian and bacterial cells interchangeably.<br />
<br />
<br />
The University of East Anglia has a great deal of research groups studying nitrogenous species, therefore we decided to look into using synthetic biology to try and help solve any problems they had experienced; one of the main issues was with the use of nitric oxide, as it is difficult to work with due to its highly reactive nature. We decided to take on this challenge and investigate how we could use synthetic biology to quantitatively measure NO levels in an environment.<br />
<br />
<br />
We began by synthesising a dual promoter taking elements of bacterial NO-sensing promoters and mammalian NO-sensing promoters; this has led to our hybrid promoter, a combination of the PyeaR promoter (bacterial, given as 'B' in our project) and the CArG promoter (mammalian, given as 'M' in our project). These two elements have been synthesised in two orientations; mammalian upstream of bacterial (M-B) (Figure 1) and bacterial upstream of mammalian (B-M) (Figure 2); this allows our promoter to be flexible and have use in both prokarayotes and eukarayotes.<br />
<br />
<br />
Over the course of our project we have produced six individual BioBricks using these hybrid promoters; M-B alone, M-B with RFP and M-B with CFP; and B-M alone, B-M with RFP and B-M with CFP. The BioBricks containing fluorescent proteins were transformed into an ''Escherichia Coli'' chassis and through induction by potassium nitrate (using the nitrate as a homogenous substitute for nitric oxide) we have observed expression of RFP and CFP. We have also conducted experiments to measure the level of fluorescence (and thus transcription) under different concentrations of potassium nitrate; to do this we carried out flow cytometry, fluorescence-activated cell sorting, and scanned samples using a fluorometer. Our data thus far has suggested that in the case of CFP, M-B has a higher transcription rate than B-M; however, in the case of RFP both M-B and B-M appear to have similar rates of transcription. In the majority of fluorometer experiments it appeared that the maximum concentration of potassium nitrate that the cell could cope with was between 15 and 20 mM, as 15 mM gave the highest fluorescence intensity, while 20 mM showed much lower fluorescence intensity; we hypothesise that this is due to 20 mM potassium nitrate being toxic, or due to over-expression of the fluorescent proteins resulting in aggregation and the formation of inclusion bodies.<br />
<br />
<br />
We have also transfected mammalian cells with M-B attached to CFP and added SNAP (a nitricoxide-producing agent) in order to induce transcription. Results appeared to show low levels of CFP expression in cells without SNAP (pertaining to normal physiological levels of nitric oxide) and higher levels of CFP expression once SNAP had been added.<br />
<br />
<br />
The flexibility of the hybrid mammalian and bacterial dual promoter allows for many advances in sensation and control of nitric oxide. For instance NO has been recognised for its implications as a potential cancer therapeutic (due to macrophage usage of NO as an immune response) therefore this promoters allows for the development of systems in easier to use, faster-growing bacteria that can then be applied to mammalian cells for testing. We believe this to be a novel approach made possible through our BioBricks.<br />
<br />
<br />
----<br />
<br />
<br />
[[File:NRPCompLogo.png | centre | link=Team:NRP-UEA-Norwich/ComparatorCircuit]]<br />
<br />
[[File:Comparator circuit.png | 200px |right | thumbnail | '' '''Figure 3.''' The electrical circuitary of the comparator circuit.'']]<br />
<br />
The lack of specificity of the bacterial promoter, pYEAR, used in the hybrid promoter was a pitfall that was always a concern. From this potential problem spawned a potential solution; the Comparator Circuit.This pair of BioBricks is designed to specifically bind to each other while ligated to two different promoters of overlapping specificity to result in an integrating of the conflicting outputs of the two opposing gene systems.<br />
<br />
<br />
Our system relies on two constructs that interact via complimentary base pair sequences both before and after the ribosome binding site of the reporter protein. The idea being that, when both transcripts are present in the chassis, they would bind together, inhibiting the translation of the reporter proteins.<br />
<br />
<br />
Any imbalance of transcription due to the presence of the substrate of interest results in free mRNA of the gene system that detects that substrate. Crucially, if both promoters detect the same substrates but differ with one extra substrate being detected by one of the promoters, it is this substrate and this substrate only that our system will be able to detect in a simple and quantitative way. <br />
<br />
<br />
Our team have constructed a countercurrent comparator circuit in which the reporter proteins are at the same end of the complimentary region, although a contracurrent system has been theorised. Both systems share a crucial subtractive nature comparable to an analogue computer. We envisage that, should the system be fine-tuned and expanded on, a variety of different business sectors from agriculture to spinoff pharmaceutical companies (such as the fictious QuantaCare) could capitalise on this novel genetic technology.<br />
<br />
<br />
What we have produced is a biobrick pair that work in harmony, when ligated to promoters of interest and genes of interest, to sequester translation when both mRNA transcripts are present in the cell. The use of quantative tuners with these biobricks is encouraged to ensure that the transcription rate both gene constructs are equal when both promoters are transcribing at their optimal rate. Although the parts have been submitted to the registry and theoretically characterised, time constraints have meant that further lab-based characterisation could not occur.<br />
<br />
<br />
However, we hope to utilise any free time in our timetables sduring the next semester to characterise the biobricks further (please see our project proposal), and hope that we will be given a chance to present our further findings at MIT! <br />
<br />
<br />
To conclude, what we have created is a pair of antagonistic BioBricks that turned the pair of mRNAs in which they reside into translational repressor molecules when both are transcribed in tandum within a specific chassis of interest, a new application for mRNA complimentary base pairing within the registry and a project we feel could go very far indeed.<br />
<br />
<br />
----<br />
<br />
<br />
[[File:NRPTheoreticalLogo.png | centre | link=Team:NRP-UEA-Norwich/TheoreticalProjects]]<br />
<br />
<br />
'''Division circuit'''<br />
<br />
The comparator circuit that we have created integrates two different transcription levels in a negative manner (subtraction) to perform a range of functions a range of different integrations (calculations) are necessary to this end we have also designed a system that would allow one signal (transcription rate) to be divided by the transcription level of another promoter. To achieve this we have used the processes of attenuation and the three loop system (in the tryp leader). the system has also been mathematically modelled. to find more please click the image above. <br />
<br />
'''Multisensor'''<br />
<br />
There are many groups of chemical species for which there are no current biological techniques for distinguishing between each of these species and quantitatively analysing its concentration. Here we outline a possible approach for solving this problem using non-specific transcription factors and promoters. We use nitrates nitrites and nitric oxide as our example group. To find more please click the image above.<br />
<br />
in addition to the two main projects, we have also worked to elaborate on some of the teams earlier ideas during the project.</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/ProjectTeam:NRP-UEA-Norwich/Project2012-09-26T13:02:52Z<p>Joyehicks: </p>
<hr />
<div>{{UEANRP}}<br />
{{UEANRPProjects}}<br />
<br />
'''RUSSELL, KHADIJA AND PASCOE TO ORGANISE SUMMARIES OF THE THREE PAGES'''<br />
<br />
Sensory BioBrick systems have been a large constituent of previous iGEM projects in which teams have combined impressive amounts of logic with limitless creativity in order to produce synthetically engineered organisms with the ability to detect the presence of specific substrates; this was achieved by combining various promoters and reporters to produce novel gene systems of great breadth and depth.<br />
<br />
<br />
We too have taken a sensory approach to our project and have produced systems involved in the sensation of nitric oxide (NO). Originally we set out to develop a bacterial and mammalian hybrid NO-sensing promoter (which we have achieved); we then looked into ways of quantifying the levels of highly reactive and difficult to measure NO within a system, leading to us producing a novel gene regulation system known as the comparator circuit. Throughout the project we went on to look at theoretical alternative approaches to the gene systems we have produced.<br />
<br />
<br />
Overall in our project we have produced 6 sensory BioBricks, 2 BioBricks involved in gene regulation, and have further characterised 4 more BioBricks. All 8 of our BioBricks have been submitted to the registry and the 6 sensory BioBricks have been characterised.<br />
<br />
<br />
----<br />
<br />
[[File:NRPNOLogo.png | centre | link=Team:NRP-UEA-Norwich/NOSensing]]<br />
<br />
<br />
<br />
[[File:MB.png | 200px | right | thumbnail | '' '''Figure 1.''' A graphical representation of the hybrid promoter in its Mammalian-Bacterial orientation, also showing restriction sites.'' ]]<br />
<br />
[[File:BM.png | 200px | right| thumbnail | '' '''Figure 2.''' A graphical representation of the hybrid promoter in its Bacterial-Mammalian orientation, also showing restriction sites.'']]<br />
<br />
Our own hybrid promoter hopes to add to the systems already in the registry by creating a hybrid promoter that combines the bacterial promoter PyeaR and the mammalian CArG element , both of which respond to exogenous nitrogenous species. Combining the two would allow a more modular NO sensor that can be used in mammalian and bacterial cells interchangeably.<br />
<br />
<br />
The University of East Anglia has a great deal of research groups studying nitrogenous species, therefore we decided to look into using synthetic biology to try and help solve any problems they had experienced; one of the main issues was with the use of nitric oxide, as it is difficult to work with due to its highly reactive nature. We decided to take on this challenge and investigate how we could use synthetic biology to quantitatively measure NO levels in an environment.<br />
<br />
<br />
We began by synthesising a dual promoter taking elements of bacterial NO-sensing promoters and mammalian NO-sensing promoters; this has led to our hybrid promoter, a combination of the PyeaR promoter (bacterial, given as 'B' in our project) and the CArG promoter (mammalian, given as 'M' in our project). These two elements have been synthesised in two orientations; mammalian upstream of bacterial (M-B) (Figure 1) and bacterial upstream of mammalian (B-M) (Figure 2); this allows our promoter to be flexible and have use in both prokarayotes and eukarayotes.<br />
<br />
<br />
Over the course of our project we have produced six individual BioBricks using these hybrid promoters; M-B alone, M-B with RFP and M-B with CFP; and B-M alone, B-M with RFP and B-M with CFP. The BioBricks containing fluorescent proteins were transformed into an ''Escherichia Coli'' chassis and through induction by potassium nitrate (using the nitrate as a homogenous substitute for nitric oxide) we have observed expression of RFP and CFP in the relevant systems. We have also conducted experiments to measure the level of fluorescence (and thus transcription) under different concentrations of potassium nitrate; to do this we carried out flow cytometry, fluorescence-activated cell sorting, and scanned samples using a fluorometer. Our data thus far has suggested that in the case of CFP, M-B has a higher transcription rate than B-M; however, in the case of RFP both M-B and B-M appear to have similar rates of transcription. In the majority of fluorometer experiments it appeared that the maximum concentration of potassium nitrate that the cell could cope with was between 15 and 20 mM, as 15 mM gave the highest fluorescence intensity, while 20 mM showed much lower fluorescence intensity; we hypothesise that this is due to 20 mM potassium nitrate being toxic, or due to over-expression of the fluorescent proteins resulting in aggregation and the formation of inclusion bodies.<br />
<br />
<br />
We have also transfected mammalian cells with M-B attached to CFP and added SNAP (a nitricoxide-producing agent) in order to induce transcription. Results appeared to show low levels of CFP expression in cells without SNAP (pertaining to normal physiological levels of nitric oxide) and higher levels of CFP expression once SNAP had been added.<br />
<br />
<br />
The flexibility of the hybrid mammalian and bacterial dual promoter allows for many advances in sensation and control of nitric oxide. For instance NO has been recognised for its implications as a potential cancer therapeutic (due to macrophage usage of NO as an immune response) therefore this promoters allows for the development of systems in easier to use, faster-growing bacteria that can then be applied to mammalian cells for testing. We believe this to be a novel approach made possible through our BioBricks.<br />
<br />
<br />
----<br />
<br />
<br />
[[File:NRPCompLogo.png | centre | link=Team:NRP-UEA-Norwich/ComparatorCircuit]]<br />
<br />
[[File:Comparator circuit.png | 200px |right | thumbnail | '' '''Figure 3.''' The electrical circuitary of the comparator circuit.'']]<br />
<br />
The lack of specificity of the bacterial promoter, pYEAR, used in the hybrid promoter was a pitfall that was always a concern. From this potential problem spawned a potential solution; the Comparator Circuit.This pair of BioBricks is designed to specifically bind to each other while ligated to two different promoters of overlapping specificity to result in an integrating of the conflicting outputs of the two opposing gene systems.<br />
<br />
<br />
Our system relies on two constructs that interact via complimentary base pair sequences both before and after the ribosome binding site of the reporter protein. The idea being that, when both transcripts are present in the chassis, they would bind together, inhibiting the translation of the reporter proteins.<br />
<br />
<br />
Any imbalance of transcription due to the presence of the substrate of interest results in free mRNA of the gene system that detects that substrate. Crucially, if both promoters detect the same substrates but differ with one extra substrate being detected by one of the promoters, it is this substrate and this substrate only that our system will be able to detect in a simple and quantitative way. <br />
<br />
<br />
Our team have constructed a countercurrent comparator circuit in which the reporter proteins are at the same end of the complimentary region, although a contracurrent system has been theorised. Both systems share a crucial subtractive nature comparable to an analogue computer. We envisage that, should the system be fine-tuned and expanded on, a variety of different business sectors from agriculture to spinoff pharmaceutical companies (such as the fictious QuantaCare) could capitalise on this novel genetic technology.<br />
<br />
<br />
What we have produced is a biobrick pair that work in harmony, when ligated to promoters of interest and genes of interest, to sequester translation when both mRNA transcripts are present in the cell. The use of quantative tuners with these biobricks is encouraged to ensure that the transcription rate both gene constructs are equal when both promoters are transcribing at their optimal rate. Although the parts have been submitted to the registry and theoretically characterised, time constraints have meant that further lab-based characterisation could not occur.<br />
<br />
<br />
However, we hope to utilise any free time in our timetables sduring the next semester to characterise the biobricks further (please see our project proposal), and hope that we will be given a chance to present our further findings at MIT! <br />
<br />
<br />
To conclude, what we have created is a pair of antagonistic BioBricks that turned the pair of mRNAs in which they reside into translational repressor molecules when both are transcribed in tandum within a specific chassis of interest, a new application for mRNA complimentary base pairing within the registry and a project we feel could go very far indeed.<br />
<br />
<br />
----<br />
<br />
<br />
[[File:NRPTheoreticalLogo.png | centre | link=Team:NRP-UEA-Norwich/TheoreticalProjects]]<br />
<br />
<br />
'''Division circuit'''<br />
<br />
The comparator circuit that we have created integrates two different transcription levels in a negative manner (subtraction) to perform a range of functions a range of different integrations (calculations) are necessary to this end we have also designed a system that would allow one signal (transcription rate) to be divided by the transcription level of another promoter. To achieve this we have used the processes of attenuation and the three loop system (in the tryp leader). the system has also been mathematically modelled. to find more please click the image above. <br />
<br />
'''Multisensor'''<br />
<br />
There are many groups of chemical species for which there are no current biological techniques for distinguishing between each of these species and quantitatively analysing its concentration. Here we outline a possible approach for solving this problem using non-specific transcription factors and promoters. We use nitrates nitrites and nitric oxide as our example group. To find more please click the image above.<br />
<br />
in addition to the two main projects, we have also worked to elaborate on some of the teams earlier ideas during the project.</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/ProjectTeam:NRP-UEA-Norwich/Project2012-09-26T13:00:36Z<p>Joyehicks: </p>
<hr />
<div>{{UEANRP}}<br />
{{UEANRPProjects}}<br />
<br />
'''RUSSELL, KHADIJA AND PASCOE TO ORGANISE SUMMARIES OF THE THREE PAGES'''<br />
<br />
Sensory BioBrick systems have been a large constituent of previous iGEM projects in which teams have combined impressive amounts of logic with limitless creativity in order to produce synthetically engineered organisms with the ability to detect the presence of specific substrates; this was achieved by combining various promoters and reporters to produce novel gene systems of great breadth and depth.<br />
<br />
<br />
We too have taken a sensory approach to our project and have produced systems involved in the sensation of nitric oxide (NO). Originally we set out to develop a bacterial and mammalian hybrid NO-sensing promoter (which we have achieved); we then looked into ways of quantifying the levels of highly reactive and difficult to measure NO within a system, leading to us producing a novel gene regulation system known as the comparator circuit. Throughout the project we went on to look at theoretical alternative approaches to the gene systems we have produced.<br />
<br />
<br />
Overall in our project we have produced 6 sensory BioBricks, 2 BioBricks involved in gene regulation, and have further characterised 4 more BioBricks. All 8 of our BioBricks have been submitted to the registry and the 6 sensory BioBricks have been characterised.<br />
<br />
<br />
----<br />
<br />
[[File:NRPNOLogo.png | centre | link=Team:NRP-UEA-Norwich/NOSensing]]<br />
<br />
<br />
<br />
[[File:MB.png | 200px | right | thumbnail | '' '''Figure 1.''' A graphical representation of the hybrid promoter in its Mammalian-Bacterial orientation, also showing restriction sites.'' ]]<br />
<br />
[[File:BM.png | 200px | right| thumbnail | '' '''Figure 2.''' A graphical representation of the hybrid promoter in its Bacterial-Mammalian orientation, also showing restriction sites.'']]<br />
<br />
Our own hybrid promoter hopes to add to the systems already in the registry by creating a hybrid promoter that combines the bacterial promoter PyeaR and the mammalian CArG element , both of which respond to exogenous nitrogenous species. Combining the two would allow a more modular NO sensor that can be used in mammalian and bacterial cells interchangeably.<br />
<br />
<br />
The University of East Anglia has a great deal of research groups studying nitrogenous species, therefore we decided to look into using synthetic biology to try and help solve any problems they had experienced; one of the main issues was with the use of nitric oxide, as it is difficult to work with due to its highly reactive nature. We decided to take on this challenge and investigate how we could use synthetic biology to quantitatively measure NO levels in an environment.<br />
<br />
<br />
We began by synthesising a dual promoter taking elements of bacterial NO-sensing promoters and mammalian NO-sensing promoters; this has led to our hybrid promoter, a combination of the PyeaR promoter (bacterial, given as 'B' in our project) and the CArG promoter (mammalian, given as 'M' in our project). These two elements have been synthesised in two orientations; mammalian upstream of bacterial (M-B) (Figure 1) and bacterial upstream of mammalian (B-M) (Figure 2); this allows our promoter to be flexible and have use in both prokarayotes and eukarayotes.<br />
<br />
<br />
Following the ten weeks of our project we have produced six individual BioBricks using these hybrid promoters; M-B alone, M-B with RFP and M-B with CFP; and B-M alone, B-M with RFP and B-M with CFP. The BioBricks containing fluorescent proteins were transformed into an ''Escherichia Coli'' chassis and through induction by potassium nitrate (using the nitrate as a homogenous substitute for nitric oxide) we have observed expression of RFP and CFP in the relevant systems. We have also conducted experiments to measure the level of fluorescence (and thus transcription) under different concentrations of potassium nitrate; to do this we carried out flow cytometry, fluorescence-activated cell sorting, and scanned samples using a fluorometer. Our data thus far has suggested that in the case of CFP, M-B has a higher transcription rate than B-M; however, in the case of RFP both M-B and B-M appear to have similar rates of transcription. In the majority of fluorometer experiments it appeared that the maximum concentration of potassium nitrate that the cell could cope with was between 15 and 20 mM, as 15 mM gave the highest fluorescence intensity, while 20 mM showed much lower fluorescence intensity; we hypothesise that this is due to 20 mM potassium nitrate being toxic, or due to over-expression of the fluorescent proteins resulting in aggregation and the formation of inclusion bodies.<br />
<br />
<br />
We have also transfected mammalian cells with M-B attached to CFP and added SNAP (a nitricoxide-producing agent) in order to induce transcription. Results appeared to show low levels of CFP expression in cells without SNAP (pertaining to normal physiological levels of nitric oxide) and higher levels of CFP expression once SNAP had been added.<br />
<br />
<br />
The flexibility of the hybrid mammalian and bacterial dual promoter allows for many advances in sensation and control of nitric oxide. For instance NO has been recognised for its implications as a potential cancer therapeutic (due to macrophage usage of NO as an immune response) therefore this promoters allows for the development of systems in easier to use, faster-growing bacteria that can then be applied to mammalian cells for testing. We believe this to be a novel approach made possible through our BioBricks.<br />
<br />
<br />
----<br />
<br />
<br />
[[File:NRPCompLogo.png | centre | link=Team:NRP-UEA-Norwich/ComparatorCircuit]]<br />
<br />
[[File:Comparator circuit.png | 200px |right | thumbnail | '' '''Figure 3.''' The electrical circuitary of the comparator circuit.'']]<br />
<br />
The lack of specificity of the bacterial promoter, pYEAR, used in the hybrid promoter was a pitfall that was always a concern. From this potential problem spawned a potential solution; the Comparator Circuit.This pair of BioBricks is designed to specifically bind to each other while ligated to two different promoters of overlapping specificity to result in an integrating of the conflicting outputs of the two opposing gene systems.<br />
<br />
<br />
Our system relies on two constructs that interact via complimentary base pair sequences both before and after the ribosome binding site of the reporter protein. The idea being that, when both transcripts are present in the chassis, they would bind together, inhibiting the translation of the reporter proteins.<br />
<br />
<br />
Any imbalance of transcription due to the presence of the substrate of interest results in free mRNA of the gene system that detects that substrate. Crucially, if both promoters detect the same substrates but differ with one extra substrate being detected by one of the promoters, it is this substrate and this substrate only that our system will be able to detect in a simple and quantitative way. <br />
<br />
<br />
Our team have constructed a countercurrent comparator circuit in which the reporter proteins are at the same end of the complimentary region, although a contracurrent system has been theorised. Both systems share a crucial subtractive nature comparable to an analogue computer. We envisage that, should the system be fine-tuned and expanded on, a variety of different business sectors from agriculture to spinoff pharmaceutical companies (such as the fictious QuantaCare) could capitalise on this novel genetic technology.<br />
<br />
<br />
What we have produced is a biobrick pair that work in harmony, when ligated to promoters of interest and genes of interest, to sequester translation when both mRNA transcripts are present in the cell. The use of quantative tuners with these biobricks is encouraged to ensure that the transcription rate both gene constructs are equal when both promoters are transcribing at their optimal rate. Although the parts have been submitted to the registry and theoretically characterised, time constraints have meant that further lab-based characterisation could not occur.<br />
<br />
<br />
However, we hope to utilise any free time in our timetables sduring the next semester to characterise the biobricks further (please see our project proposal), and hope that we will be given a chance to present our further findings at MIT! <br />
<br />
<br />
To conclude, what we have created is a pair of antagonistic BioBricks that turned the pair of mRNAs in which they reside into translational repressor molecules when both are transcribed in tandum within a specific chassis of interest, a new application for mRNA complimentary base pairing within the registry and a project we feel could go very far indeed.<br />
<br />
<br />
----<br />
<br />
<br />
[[File:NRPTheoreticalLogo.png | centre | link=Team:NRP-UEA-Norwich/TheoreticalProjects]]<br />
<br />
<br />
'''Division circuit'''<br />
<br />
The comparator circuit that we have created integrates two different transcription levels in a negative manner (subtraction) to perform a range of functions a range of different integrations (calculations) are necessary to this end we have also designed a system that would allow one signal (transcription rate) to be divided by the transcription level of another promoter. To achieve this we have used the processes of attenuation and the three loop system (in the tryp leader). the system has also been mathematically modelled. to find more please click the image above. <br />
<br />
'''Multisensor'''<br />
<br />
There are many groups of chemical species for which there are no current biological techniques for distinguishing between each of these species and quantitatively analysing its concentration. Here we outline a possible approach for solving this problem using non-specific transcription factors and promoters. We use nitrates nitrites and nitric oxide as our example group. To find more please click the image above.<br />
<br />
in addition to the two main projects, we have also worked to elaborate on some of the teams earlier ideas during the project.</div>Joyehickshttp://2012.igem.org/File:Thankyou.pngFile:Thankyou.png2012-09-26T12:52:10Z<p>Joyehicks: uploaded a new version of &quot;File:Thankyou.png&quot;</p>
<hr />
<div></div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/AttributionsTeam:NRP-UEA-Norwich/Attributions2012-09-26T12:51:17Z<p>Joyehicks: </p>
<hr />
<div>{{UEANRP}}<br />
<br />
'''RUSSELL TO DO'''<br />
<br />
'''[http://www.amycongdon.com/#!mainPage Amy Congdon]''' has acted as our team artist and has designed our logos as well as helping with the design and production of our video and various pieces of equipment for our human outreach work.<br />
<br />
<br />
'''[http://www.biochemistry.org The Biochemical Society]''' were kind enough to offer funding to go towards videos that have proven to be central to the human outreach aspect of our project.<br />
<br />
<br />
'''[http://www.bioline.com/h_uk.asp Bioline]''' and their representative, Josh Wright, have been extremely supportive in offering us a discount from their catalogued products as well as donating plasmid isolation mini kits, PCR gel clean-up kits and enzymes.<br />
<br />
<br />
'''[http://www.promega.com Promega]''' have been extremely supportive in offering us a discount from their catalogued products as well as donating PCR gel clean-up kits and DNA purification mini-preps.<br />
<br />
<br />
'''Sebastian Runkel''' and '''Anke Arkenberg''' are both PhD students at the University of East Anglia whose research involve nitric oxide. Both gave in-depth presentations on nitric oxide and how to work with it. Their experience and advice has proven invaluable to our own project.<br />
<br />
<br />
'''[http://www.star107.co.uk/ STAR Radio 107.9/1 FM]''' allowed us interview time on their radio show to discuss the iGEM competition, synthetic biology, and the future applications of this branch of science. They gave us a lengthy slot on the show and helped us promote the competition and synthetic biology as a whole.<br />
<br />
<br />
'''[http://www.wellcome.ac.uk/ The Wellcome Trust]''' kindly provided us with both stipends that have allowed the team to work on the project for the ten week period allotted. They also provided us with an Annual fund, without which the lab work performed could not have been performed.<br />
<br />
<br />
'''[http://www.uea.ac.uk/bio/eande/eandeclub The UEA Enterprise and Engagement Club]''' were able to provide vital funding towards travel expenses, lab consumables and other necessities to the project.<br />
<br><br><br />
[[File:Thankyou.png|centre]]</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/AttributionsTeam:NRP-UEA-Norwich/Attributions2012-09-26T12:50:38Z<p>Joyehicks: </p>
<hr />
<div>{{UEANRP}}<br />
<br />
'''RUSSELL TO DO'''<br />
<br />
'''[http://www.amycongdon.com/#!mainPage Amy Congdon]''' has acted as our team artist and has designed our logos as well as helping with the design and production of our video and various pieces of equipment for our human outreach work.<br />
<br />
<br />
'''[http://www.biochemistry.org The Biochemical Society]''' were kind enough to offer funding to go towards videos that have proven to be central to the human outreach aspect of our project.<br />
<br />
<br />
'''[http://www.bioline.com/h_uk.asp Bioline]''' and their representative, Josh Wright, have been extremely supportive in offering us a discount from their catalogued products as well as donating plasmid isolation mini kits, PCR gel clean-up kits and enzymes.<br />
<br />
<br />
'''[http://www.promega.com Promega]''' have been extremely supportive in offering us a discount from their catalogued products as well as donating PCR gel clean-up kits and DNA purification mini-preps.<br />
<br />
<br />
'''Sebastian Runkel''' and '''Anke Arkenberg''' are both PhD students at the University of East Anglia whose research involve nitric oxide. Both gave in-depth presentations on nitric oxide and how to work with it. Their experience and advice has proven invaluable to our own project.<br />
<br />
<br />
'''[http://www.star107.co.uk/ STAR Radio 107.9/1 FM]''' allowed us interview time on their radio show to discuss the iGEM competition, synthetic biology, and the future applications of this branch of science. They gave us a lengthy slot on the show and helped us promote the competition and synthetic biology as a whole.<br />
<br />
<br />
'''[http://www.wellcome.ac.uk/ The Wellcome Trust]''' kindly provided us with both stipends that have allowed the team to work on the project for the ten week period allotted. They also provided us with an Annual fund, without which the lab work performed could not have been performed.<br />
<br />
<br />
'''[http://www.uea.ac.uk/bio/eande/eandeclub The UEA Enterprise and Engagement Club]''' were able to provide vital funding towards travel expenses, lab consumables and other necessities to the project.<br />
[[File:Thankyou.png]]</div>Joyehickshttp://2012.igem.org/File:Thankyou.pngFile:Thankyou.png2012-09-26T12:49:57Z<p>Joyehicks: uploaded a new version of &quot;File:Thankyou.png&quot;</p>
<hr />
<div></div>Joyehickshttp://2012.igem.org/File:Thankyou.pngFile:Thankyou.png2012-09-26T12:47:39Z<p>Joyehicks: </p>
<hr />
<div></div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/ComparatorCircuitTeam:NRP-UEA-Norwich/ComparatorCircuit2012-09-26T10:25:21Z<p>Joyehicks: /* Future Applications */</p>
<hr />
<div>{{UEANRP}}<br />
<br />
{{UEANRPProjects}}<br />
<br />
KHADIJA TO DO<br />
<br />
[[File:NRPCompLogo.png | centre]]<br />
<br />
Biological systems function on a great variety of different integrative mechanisms which include post-transcriptional attenuation. We believe that Synthetic Biology is at its most effective when these natural mechanisms are taken and applied in novel situations. This ethos we have sought to emulate by creating our own mechanism of post-transcriptional attentuation; the Comparator Circuit.<br />
<br />
<br />
==Introduction==<br />
<br />
In a previous project, the apparent lack of specificity of the promoter BioBrick we looked to improve upon proved to be a minor difficulty that we felt had not been addressed previously in the Registry. Thus we decided to tackle the issue by devising a way of quantitatively measuring the output of NO with the non-specific promoter we were using through a novel gene regulation system.<br />
<br />
Using pairs of BioBricks that result in the complimentary binding of a pair of otherwise standard promoter and reporter constructs, a subtractive effect can be gained to result in altered translation relative to the availability of various substrates within the chassis environment.<br />
<br />
The video below describes the project in further detail:<br />
<br />
<br />
<html><align=center><br />
<table align=center width=100% cellpadding=0 cellspacing=0><br />
<tr><br />
<td valign=absmiddle align=center><br />
<iframe width="420" height="315" src="http://www.youtube.com/embed/LDpXYcmpZPc?rel=0" frameborder="0" allowfullscreen></iframe><br />
</td><br />
</tr><br />
</table><br />
</align><br />
</html><br />
<br />
==Planning==<br />
<br />
[[File:CombinedMRNA.png | thumb | Figure 1 - Both BioBricks of the Comparator Circuit bound together.]]<br />
<br />
Assembling the various gene constructs was not without its challenges. Due to the fact that complimentary ‘zips’ within the sequence were designed to surround the ribosome binding site it was often the case that the DNA sequence would form unwanted secondary structures that could serve to inhibit the translation of the mRNA in its uncoupled state. <br />
<br />
Therefore, when designing the DNA, care was taken to avoid these structures obstructing sequences required for translation of the mRNA. Simultaneously, only common codons for the chassis of interest, ''E. coli'', could be used and these codon had to code for amino acids that were unlikely to change the function of the protein product. This is required since the zip sequences extend past the translational start codon, thus our construct will add a small N-terminal tag to any reporter protein it was attached to.<br />
<br />
Using IDT's very helpful online tool, Oligo Analyser, we were able to produce sequences in both constructs required in the Comparator Circuit system that, theoretically, bound together when transcribed at around the same within the chassis, yet left the ribosome bind site open and unfolded when either strand of mRNA lacks its counterpart to complimentarily bind to. The software produced figures demonstrating the likely secondary structure of the constructs made, and their Gibbs Free Energy value at specific temperatures. From this, trial and error eventually resulted in the construction of two BioBricks that have free ribosome binding sites when in isolation, but bind to sequester translation of both mRNAs when bound together (Figure 1).<br />
<br />
Due to the stop codon present in the scars of Assembly Standard 10 BioBricks, we decided that our constructs would have to be an Assembly Standard 23 BioBrick. Although the use of Bioscaffolds produced by previous iGEM teams was considered, time constraints meant that changing the Assembly Standard we put our BioBricks in would be the most convenient solution.<br />
<br />
==Experiments==<br />
<br />
Due to the limited time we had available, it was decided fairly early on that the comparator circuit BioBricks could only be characterised theoretically. However, our confidence in the system meant that we felt obliged to synthesise these BioBricks and submit them to the registry. It is our hope that we will be granted lab space after the regional jamboree to characterise these BioBricks and submit our findings onto their registry experience page in due course. In preparation for this, the team have devised a provisional experimentation design for these characterisation studies.<br />
<br />
==Future Experiments==<br />
<br />
Further cloning experiments hope to be carried out, ligating each comparator circuit biobrick with a different reporter and promoter. Initially, for proof of concept, the promoters used would be simple promoters that have overlapping specificity, but one substance will only act as an inducer for one of these promoters (e.g. the pYEAR and SoxR promoters, which both sense small nitrogen species, but SoxR also senses superoxides). <br />
<br />
Reporter biobricks would also be ligated, with these reporters being as far apart in wavelength of the florescence produced as possible. Both DNA sequences, each containing one half of the comparator circuit system, with be transformed into an E. coli chassis and the amount of either fluorophore (as seen via florometer studies) would indicate the translation of either sequences. It is hoped that in simulated environments containing higher levels of, using the example above, superoxides, the corresponding fluorescent protein will be translated over its counterpart.<br />
<br />
Ultimately it is hoped that we would be able to ligate our comparator circuit BioBricks to NO-sensing promoters and effecter enzymes to control NO levels/reporters to detect NO levels quantitatively - which is where our fictitious brand Quanticare comes in. Should we have time, we'd like to investigate if the transfection of mammalian cells with these constructs, as seen in our science-fiction self-diagnostic tattoos, is plausible via the transfection of cancer cell lines with the DNA constructs.<br />
<br />
==Theoretical Characterisation==<br />
The idea of the comparator circuit is to provide a modular method of signal integration that can produce a sensor which can specifically and quantitatively measure different chemical species within the cell using non specific promoters. Through mathematical modelling, an equation has been assembled which can predict the expression of each of the reporter proteins such as RFP and CFP. <br />
<br />
[[File:Equation_7.png| 300px | center]] <br />
Figure: Theoretical equation to predict the degree of expression of Construct 1 and 2. <br />
The full equation has been laid out in a way that is relevant only to Construct 1, however, the numbers can be reversed to be relevant to Construct 2. For ease of explanation, everything described will be relevant to Construct 1.<br />
<br />
<br />
E = Proportion of expression rate of Construct 1 when both constructs are transcribed (i.e. there is knockdown of one construct) relative to the non-knocked down expression of Construct 1 when only Construct 1 is expressed. <br />
<br />
A = The rate of transcription of Construct 1 as a proportion of the maximum transcription rate. As a proportion this is measured on a scale of 0 - 1. As an example if the rate of transcription is half of the maximum rate, rate would be 0.5 (arbituary units). It can be assumed the rate of transcription of construct 1 and 2 due to cellular components (e.g. RNA polymerase) is the same, however, the rate of transcription initiation will dictate the transcription rate. The initiation is reliant on the chemical species interacting with the transcription factor which binds to the promoter (i.e. nitric oxide,nitrates,nitrites to PyeaR). The '1' and '2' refer to the Construct 1 or 2 and hence the promoter and the measured fluorescent protein attached (e.g GFP, RFP, CFP, etc).<br />
<br />
L = The length of the Construct 1 in the DNA form that is transcribed (i.e the leader and protein coding region).<br />
<br />
Note: Leader refers to the section of RNA at the start of the mRNA that is not translated but has an effect on translation rate.<br />
<br />
C = The rate of transcription. Assuming the rate of transcription of Construct 1 and 2 are the same because the same ribosomes and RBS are involved.<br />
<br />
T = Half life of Construct 1 when only Construct 1 is present; the natural half life of Construct 1.<br />
<br />
K = A constant of the biological system. This can only be measured through observation. <br />
<br />
<br />
The full equation is modelled on the basic equation of:<br />
<br />
[[File:Equation_2.png| 400px | center]]<br />
<br />
where E is the rate of expression and E(A1) is the same as that explained above.<br />
<br />
<br />
The additional complexity factors in less assumptions, and mimics a biological system, more closely. Below is a breakdown of the full equation.<br />
<br />
[[File:Equation_3.png| 50px | left]]<br />
<br />
This refers to the number of Construct 1 RNA transcripts undergoing transcription at any one time. The length of DNA is particularly important when the chassis is bacterial. In bacteria, as there is no true nucleus, translation occurs simultaneously with transcription. Transcription affects the probability of interaction between construct 1 and 2 and therefore, they are less likely to be translated. As the measurement of fluorescence is the output directly related to the rate of translation, the overall equation measures translation, however, translation rate iis dependent on rate of transcription and degree of knockdown, and hence transcription is factored in here. L/C is the period of time taken for transcription to take place. It is the time in which translation can be initiated but it is unlikely that the two leaders will bind to one another <br />
<br />
[[File:Equation_5.png| 150px | left]]<br />
<br />
This part of the equation is the deduction of the knockdown of Construct 1 when there is Construct 2 expression and interaction. The biological constant, k, factors in that not all of construct 2 that is expressed will interact with construct 1 and vice versa. Hence, both exist despite construct 2 existing in small quantities. We believe that depending upon the assembly of the orientation of the two constructs within the plasmid, the interaction and hence the binding efficiency can be altered vastly. If the genes have opposite orientations, so that the termination sites are very close then the reduction of distance will increase the chances of interaction and hence make the sensory system more accurate.<br />
[[File:Equation_6.png| 120px | left]]<br />
<br />
This part of the equation encompasses the natural half life of Construct 1 when it alone is expressed (i.e. no expression of or interaction with Construct 2). As described before in the modelling from the basic equation, this is the lower part of the equation and puts it in perspective of Construct 1 and gives expression as a porportion of the maximum transcription. The half life is also Construct 1's half life.<br />
<br />
So to bring it all together; the top half of the equation indicates the degree of translation of the RNA transcribed by the first promoter under any particular transcription rate of the two promoters in arbitrary units. To make this into a meaningful output it is divided by the maximum translation rate at that rate of transcription to equal E(A1); this indicates the degree of attenuation of one RNA from the other.<br />
<br />
==Future Applications==<br />
<br />
The Comparator Circuit has the potential to have real world applications, particularly, as QuantiCare shows, in medicine. To give an example, by monitoring blood sugar levels quantitatively via specific promoters, the sequence following the comparator circuit could encode for insulin. This could be transfected into human cells and could be used to alleviate the symptoms associated with type I diabetes. <br />
<br />
Moving back to the specific nitrogen sensor, attaching these promoters to the comparator circuit biobricks and a gene for the synthesis of nitrogen reductase could result in a positive feedback loop to result in the tumour reducing in size. Macrophages naturally sense the presence of tumours in the body via their emission of nitric oxide. This could be taken one step further by adding nitrogen reductase to this system, where an excess of nitric oxide in the tumour environment. NO was seen to have cytotoxic properties in large amounts and, thus, a positive feedback loop could result in tumour apoptosis.</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/ComparatorCircuitTeam:NRP-UEA-Norwich/ComparatorCircuit2012-09-26T10:24:14Z<p>Joyehicks: /* Future Applications */</p>
<hr />
<div>{{UEANRP}}<br />
<br />
{{UEANRPProjects}}<br />
<br />
KHADIJA TO DO<br />
<br />
[[File:NRPCompLogo.png | centre]]<br />
<br />
Biological systems function on a great variety of different integrative mechanisms which include post-transcriptional attenuation. We believe that Synthetic Biology is at its most effective when these natural mechanisms are taken and applied in novel situations. This ethos we have sought to emulate by creating our own mechanism of post-transcriptional attentuation; the Comparator Circuit.<br />
<br />
<br />
==Introduction==<br />
<br />
In a previous project, the apparent lack of specificity of the promoter BioBrick we looked to improve upon proved to be a minor difficulty that we felt had not been addressed previously in the Registry. Thus we decided to tackle the issue by devising a way of quantitatively measuring the output of NO with the non-specific promoter we were using through a novel gene regulation system.<br />
<br />
Using pairs of BioBricks that result in the complimentary binding of a pair of otherwise standard promoter and reporter constructs, a subtractive effect can be gained to result in altered translation relative to the availability of various substrates within the chassis environment.<br />
<br />
The video below describes the project in further detail:<br />
<br />
<br />
<html><align=center><br />
<table align=center width=100% cellpadding=0 cellspacing=0><br />
<tr><br />
<td valign=absmiddle align=center><br />
<iframe width="420" height="315" src="http://www.youtube.com/embed/LDpXYcmpZPc?rel=0" frameborder="0" allowfullscreen></iframe><br />
</td><br />
</tr><br />
</table><br />
</align><br />
</html><br />
<br />
==Planning==<br />
<br />
[[File:CombinedMRNA.png | thumb | Figure 1 - Both BioBricks of the Comparator Circuit bound together.]]<br />
<br />
Assembling the various gene constructs was not without its challenges. Due to the fact that complimentary ‘zips’ within the sequence were designed to surround the ribosome binding site it was often the case that the DNA sequence would form unwanted secondary structures that could serve to inhibit the translation of the mRNA in its uncoupled state. <br />
<br />
Therefore, when designing the DNA, care was taken to avoid these structures obstructing sequences required for translation of the mRNA. Simultaneously, only common codons for the chassis of interest, ''E. coli'', could be used and these codon had to code for amino acids that were unlikely to change the function of the protein product. This is required since the zip sequences extend past the translational start codon, thus our construct will add a small N-terminal tag to any reporter protein it was attached to.<br />
<br />
Using IDT's very helpful online tool, Oligo Analyser, we were able to produce sequences in both constructs required in the Comparator Circuit system that, theoretically, bound together when transcribed at around the same within the chassis, yet left the ribosome bind site open and unfolded when either strand of mRNA lacks its counterpart to complimentarily bind to. The software produced figures demonstrating the likely secondary structure of the constructs made, and their Gibbs Free Energy value at specific temperatures. From this, trial and error eventually resulted in the construction of two BioBricks that have free ribosome binding sites when in isolation, but bind to sequester translation of both mRNAs when bound together (Figure 1).<br />
<br />
Due to the stop codon present in the scars of Assembly Standard 10 BioBricks, we decided that our constructs would have to be an Assembly Standard 23 BioBrick. Although the use of Bioscaffolds produced by previous iGEM teams was considered, time constraints meant that changing the Assembly Standard we put our BioBricks in would be the most convenient solution.<br />
<br />
==Experiments==<br />
<br />
Due to the limited time we had available, it was decided fairly early on that the comparator circuit BioBricks could only be characterised theoretically. However, our confidence in the system meant that we felt obliged to synthesise these BioBricks and submit them to the registry. It is our hope that we will be granted lab space after the regional jamboree to characterise these BioBricks and submit our findings onto their registry experience page in due course. In preparation for this, the team have devised a provisional experimentation design for these characterisation studies.<br />
<br />
==Future Experiments==<br />
<br />
Further cloning experiments hope to be carried out, ligating each comparator circuit biobrick with a different reporter and promoter. Initially, for proof of concept, the promoters used would be simple promoters that have overlapping specificity, but one substance will only act as an inducer for one of these promoters (e.g. the pYEAR and SoxR promoters, which both sense small nitrogen species, but SoxR also senses superoxides). <br />
<br />
Reporter biobricks would also be ligated, with these reporters being as far apart in wavelength of the florescence produced as possible. Both DNA sequences, each containing one half of the comparator circuit system, with be transformed into an E. coli chassis and the amount of either fluorophore (as seen via florometer studies) would indicate the translation of either sequences. It is hoped that in simulated environments containing higher levels of, using the example above, superoxides, the corresponding fluorescent protein will be translated over its counterpart.<br />
<br />
Ultimately it is hoped that we would be able to ligate our comparator circuit BioBricks to NO-sensing promoters and effecter enzymes to control NO levels/reporters to detect NO levels quantitatively - which is where our fictitious brand Quanticare comes in. Should we have time, we'd like to investigate if the transfection of mammalian cells with these constructs, as seen in our science-fiction self-diagnostic tattoos, is plausible via the transfection of cancer cell lines with the DNA constructs.<br />
<br />
==Theoretical Characterisation==<br />
The idea of the comparator circuit is to provide a modular method of signal integration that can produce a sensor which can specifically and quantitatively measure different chemical species within the cell using non specific promoters. Through mathematical modelling, an equation has been assembled which can predict the expression of each of the reporter proteins such as RFP and CFP. <br />
<br />
[[File:Equation_7.png| 300px | center]] <br />
Figure: Theoretical equation to predict the degree of expression of Construct 1 and 2. <br />
The full equation has been laid out in a way that is relevant only to Construct 1, however, the numbers can be reversed to be relevant to Construct 2. For ease of explanation, everything described will be relevant to Construct 1.<br />
<br />
<br />
E = Proportion of expression rate of Construct 1 when both constructs are transcribed (i.e. there is knockdown of one construct) relative to the non-knocked down expression of Construct 1 when only Construct 1 is expressed. <br />
<br />
A = The rate of transcription of Construct 1 as a proportion of the maximum transcription rate. As a proportion this is measured on a scale of 0 - 1. As an example if the rate of transcription is half of the maximum rate, rate would be 0.5 (arbituary units). It can be assumed the rate of transcription of construct 1 and 2 due to cellular components (e.g. RNA polymerase) is the same, however, the rate of transcription initiation will dictate the transcription rate. The initiation is reliant on the chemical species interacting with the transcription factor which binds to the promoter (i.e. nitric oxide,nitrates,nitrites to PyeaR). The '1' and '2' refer to the Construct 1 or 2 and hence the promoter and the measured fluorescent protein attached (e.g GFP, RFP, CFP, etc).<br />
<br />
L = The length of the Construct 1 in the DNA form that is transcribed (i.e the leader and protein coding region).<br />
<br />
Note: Leader refers to the section of RNA at the start of the mRNA that is not translated but has an effect on translation rate.<br />
<br />
C = The rate of transcription. Assuming the rate of transcription of Construct 1 and 2 are the same because the same ribosomes and RBS are involved.<br />
<br />
T = Half life of Construct 1 when only Construct 1 is present; the natural half life of Construct 1.<br />
<br />
K = A constant of the biological system. This can only be measured through observation. <br />
<br />
<br />
The full equation is modelled on the basic equation of:<br />
<br />
[[File:Equation_2.png| 400px | center]]<br />
<br />
where E is the rate of expression and E(A1) is the same as that explained above.<br />
<br />
<br />
The additional complexity factors in less assumptions, and mimics a biological system, more closely. Below is a breakdown of the full equation.<br />
<br />
[[File:Equation_3.png| 50px | left]]<br />
<br />
This refers to the number of Construct 1 RNA transcripts undergoing transcription at any one time. The length of DNA is particularly important when the chassis is bacterial. In bacteria, as there is no true nucleus, translation occurs simultaneously with transcription. Transcription affects the probability of interaction between construct 1 and 2 and therefore, they are less likely to be translated. As the measurement of fluorescence is the output directly related to the rate of translation, the overall equation measures translation, however, translation rate iis dependent on rate of transcription and degree of knockdown, and hence transcription is factored in here. L/C is the period of time taken for transcription to take place. It is the time in which translation can be initiated but it is unlikely that the two leaders will bind to one another <br />
<br />
[[File:Equation_5.png| 150px | left]]<br />
<br />
This part of the equation is the deduction of the knockdown of Construct 1 when there is Construct 2 expression and interaction. The biological constant, k, factors in that not all of construct 2 that is expressed will interact with construct 1 and vice versa. Hence, both exist despite construct 2 existing in small quantities. We believe that depending upon the assembly of the orientation of the two constructs within the plasmid, the interaction and hence the binding efficiency can be altered vastly. If the genes have opposite orientations, so that the termination sites are very close then the reduction of distance will increase the chances of interaction and hence make the sensory system more accurate.<br />
[[File:Equation_6.png| 120px | left]]<br />
<br />
This part of the equation encompasses the natural half life of Construct 1 when it alone is expressed (i.e. no expression of or interaction with Construct 2). As described before in the modelling from the basic equation, this is the lower part of the equation and puts it in perspective of Construct 1 and gives expression as a porportion of the maximum transcription. The half life is also Construct 1's half life.<br />
<br />
So to bring it all together; the top half of the equation indicates the degree of translation of the RNA transcribed by the first promoter under any particular transcription rate of the two promoters in arbitrary units. To make this into a meaningful output it is divided by the maximum translation rate at that rate of transcription to equal E(A1); this indicates the degree of attenuation of one RNA from the other.<br />
<br />
==Future Applications==<br />
<br />
The Comparator Circuit has the potential to have real world applications, particularly, as QuantiCare shows, in medicine. To give an example, by monitoring blood sugar levels quantitatively via specific promoters, the sequence following the comparator circuit could encode for insulin. This could be transfected into human cells and could be used to alleviate the symptoms associated with type I diabetes. <br />
<br />
Moving back to the specific nitrogen sensor, attaching these promoters to the comparator circuit biobricks and a gene for the synthesis of nitrogen reductase could result in a positive feedback loop to result in the tumour reducing in size. Macrophages naturally sense the presence of tumours in the body via their emission of nitric oxide. This could be taken one step further by adding nitrogen reductase to this system, where an excess of nitric oxide in the tumour environment. NO was seen to have cytotoxic properties in large amounts and, thus, a positive feedback loop could result in tumour necrosis.</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/ComparatorCircuitTeam:NRP-UEA-Norwich/ComparatorCircuit2012-09-26T10:23:33Z<p>Joyehicks: /* Future Applications */</p>
<hr />
<div>{{UEANRP}}<br />
<br />
{{UEANRPProjects}}<br />
<br />
KHADIJA TO DO<br />
<br />
[[File:NRPCompLogo.png | centre]]<br />
<br />
Biological systems function on a great variety of different integrative mechanisms which include post-transcriptional attenuation. We believe that Synthetic Biology is at its most effective when these natural mechanisms are taken and applied in novel situations. This ethos we have sought to emulate by creating our own mechanism of post-transcriptional attentuation; the Comparator Circuit.<br />
<br />
<br />
==Introduction==<br />
<br />
In a previous project, the apparent lack of specificity of the promoter BioBrick we looked to improve upon proved to be a minor difficulty that we felt had not been addressed previously in the Registry. Thus we decided to tackle the issue by devising a way of quantitatively measuring the output of NO with the non-specific promoter we were using through a novel gene regulation system.<br />
<br />
Using pairs of BioBricks that result in the complimentary binding of a pair of otherwise standard promoter and reporter constructs, a subtractive effect can be gained to result in altered translation relative to the availability of various substrates within the chassis environment.<br />
<br />
The video below describes the project in further detail:<br />
<br />
<br />
<html><align=center><br />
<table align=center width=100% cellpadding=0 cellspacing=0><br />
<tr><br />
<td valign=absmiddle align=center><br />
<iframe width="420" height="315" src="http://www.youtube.com/embed/LDpXYcmpZPc?rel=0" frameborder="0" allowfullscreen></iframe><br />
</td><br />
</tr><br />
</table><br />
</align><br />
</html><br />
<br />
==Planning==<br />
<br />
[[File:CombinedMRNA.png | thumb | Figure 1 - Both BioBricks of the Comparator Circuit bound together.]]<br />
<br />
Assembling the various gene constructs was not without its challenges. Due to the fact that complimentary ‘zips’ within the sequence were designed to surround the ribosome binding site it was often the case that the DNA sequence would form unwanted secondary structures that could serve to inhibit the translation of the mRNA in its uncoupled state. <br />
<br />
Therefore, when designing the DNA, care was taken to avoid these structures obstructing sequences required for translation of the mRNA. Simultaneously, only common codons for the chassis of interest, ''E. coli'', could be used and these codon had to code for amino acids that were unlikely to change the function of the protein product. This is required since the zip sequences extend past the translational start codon, thus our construct will add a small N-terminal tag to any reporter protein it was attached to.<br />
<br />
Using IDT's very helpful online tool, Oligo Analyser, we were able to produce sequences in both constructs required in the Comparator Circuit system that, theoretically, bound together when transcribed at around the same within the chassis, yet left the ribosome bind site open and unfolded when either strand of mRNA lacks its counterpart to complimentarily bind to. The software produced figures demonstrating the likely secondary structure of the constructs made, and their Gibbs Free Energy value at specific temperatures. From this, trial and error eventually resulted in the construction of two BioBricks that have free ribosome binding sites when in isolation, but bind to sequester translation of both mRNAs when bound together (Figure 1).<br />
<br />
Due to the stop codon present in the scars of Assembly Standard 10 BioBricks, we decided that our constructs would have to be an Assembly Standard 23 BioBrick. Although the use of Bioscaffolds produced by previous iGEM teams was considered, time constraints meant that changing the Assembly Standard we put our BioBricks in would be the most convenient solution.<br />
<br />
==Experiments==<br />
<br />
Due to the limited time we had available, it was decided fairly early on that the comparator circuit BioBricks could only be characterised theoretically. However, our confidence in the system meant that we felt obliged to synthesise these BioBricks and submit them to the registry. It is our hope that we will be granted lab space after the regional jamboree to characterise these BioBricks and submit our findings onto their registry experience page in due course. In preparation for this, the team have devised a provisional experimentation design for these characterisation studies.<br />
<br />
==Future Experiments==<br />
<br />
Further cloning experiments hope to be carried out, ligating each comparator circuit biobrick with a different reporter and promoter. Initially, for proof of concept, the promoters used would be simple promoters that have overlapping specificity, but one substance will only act as an inducer for one of these promoters (e.g. the pYEAR and SoxR promoters, which both sense small nitrogen species, but SoxR also senses superoxides). <br />
<br />
Reporter biobricks would also be ligated, with these reporters being as far apart in wavelength of the florescence produced as possible. Both DNA sequences, each containing one half of the comparator circuit system, with be transformed into an E. coli chassis and the amount of either fluorophore (as seen via florometer studies) would indicate the translation of either sequences. It is hoped that in simulated environments containing higher levels of, using the example above, superoxides, the corresponding fluorescent protein will be translated over its counterpart.<br />
<br />
Ultimately it is hoped that we would be able to ligate our comparator circuit BioBricks to NO-sensing promoters and effecter enzymes to control NO levels/reporters to detect NO levels quantitatively - which is where our fictitious brand Quanticare comes in. Should we have time, we'd like to investigate if the transfection of mammalian cells with these constructs, as seen in our science-fiction self-diagnostic tattoos, is plausible via the transfection of cancer cell lines with the DNA constructs.<br />
<br />
==Theoretical Characterisation==<br />
The idea of the comparator circuit is to provide a modular method of signal integration that can produce a sensor which can specifically and quantitatively measure different chemical species within the cell using non specific promoters. Through mathematical modelling, an equation has been assembled which can predict the expression of each of the reporter proteins such as RFP and CFP. <br />
<br />
[[File:Equation_7.png| 300px | center]] <br />
Figure: Theoretical equation to predict the degree of expression of Construct 1 and 2. <br />
The full equation has been laid out in a way that is relevant only to Construct 1, however, the numbers can be reversed to be relevant to Construct 2. For ease of explanation, everything described will be relevant to Construct 1.<br />
<br />
<br />
E = Proportion of expression rate of Construct 1 when both constructs are transcribed (i.e. there is knockdown of one construct) relative to the non-knocked down expression of Construct 1 when only Construct 1 is expressed. <br />
<br />
A = The rate of transcription of Construct 1 as a proportion of the maximum transcription rate. As a proportion this is measured on a scale of 0 - 1. As an example if the rate of transcription is half of the maximum rate, rate would be 0.5 (arbituary units). It can be assumed the rate of transcription of construct 1 and 2 due to cellular components (e.g. RNA polymerase) is the same, however, the rate of transcription initiation will dictate the transcription rate. The initiation is reliant on the chemical species interacting with the transcription factor which binds to the promoter (i.e. nitric oxide,nitrates,nitrites to PyeaR). The '1' and '2' refer to the Construct 1 or 2 and hence the promoter and the measured fluorescent protein attached (e.g GFP, RFP, CFP, etc).<br />
<br />
L = The length of the Construct 1 in the DNA form that is transcribed (i.e the leader and protein coding region).<br />
<br />
Note: Leader refers to the section of RNA at the start of the mRNA that is not translated but has an effect on translation rate.<br />
<br />
C = The rate of transcription. Assuming the rate of transcription of Construct 1 and 2 are the same because the same ribosomes and RBS are involved.<br />
<br />
T = Half life of Construct 1 when only Construct 1 is present; the natural half life of Construct 1.<br />
<br />
K = A constant of the biological system. This can only be measured through observation. <br />
<br />
<br />
The full equation is modelled on the basic equation of:<br />
<br />
[[File:Equation_2.png| 400px | center]]<br />
<br />
where E is the rate of expression and E(A1) is the same as that explained above.<br />
<br />
<br />
The additional complexity factors in less assumptions, and mimics a biological system, more closely. Below is a breakdown of the full equation.<br />
<br />
[[File:Equation_3.png| 50px | left]]<br />
<br />
This refers to the number of Construct 1 RNA transcripts undergoing transcription at any one time. The length of DNA is particularly important when the chassis is bacterial. In bacteria, as there is no true nucleus, translation occurs simultaneously with transcription. Transcription affects the probability of interaction between construct 1 and 2 and therefore, they are less likely to be translated. As the measurement of fluorescence is the output directly related to the rate of translation, the overall equation measures translation, however, translation rate iis dependent on rate of transcription and degree of knockdown, and hence transcription is factored in here. L/C is the period of time taken for transcription to take place. It is the time in which translation can be initiated but it is unlikely that the two leaders will bind to one another <br />
<br />
[[File:Equation_5.png| 150px | left]]<br />
<br />
This part of the equation is the deduction of the knockdown of Construct 1 when there is Construct 2 expression and interaction. The biological constant, k, factors in that not all of construct 2 that is expressed will interact with construct 1 and vice versa. Hence, both exist despite construct 2 existing in small quantities. We believe that depending upon the assembly of the orientation of the two constructs within the plasmid, the interaction and hence the binding efficiency can be altered vastly. If the genes have opposite orientations, so that the termination sites are very close then the reduction of distance will increase the chances of interaction and hence make the sensory system more accurate.<br />
[[File:Equation_6.png| 120px | left]]<br />
<br />
This part of the equation encompasses the natural half life of Construct 1 when it alone is expressed (i.e. no expression of or interaction with Construct 2). As described before in the modelling from the basic equation, this is the lower part of the equation and puts it in perspective of Construct 1 and gives expression as a porportion of the maximum transcription. The half life is also Construct 1's half life.<br />
<br />
So to bring it all together; the top half of the equation indicates the degree of translation of the RNA transcribed by the first promoter under any particular transcription rate of the two promoters in arbitrary units. To make this into a meaningful output it is divided by the maximum translation rate at that rate of transcription to equal E(A1); this indicates the degree of attenuation of one RNA from the other.<br />
<br />
==Future Applications==<br />
<br />
The Comparator Circuit has the potential to have real world applications, particularly, as QuantiCare shows, in medicine. To give an example, by monitoring blood sugar levels quantitatively via specific promoters, the sequence following the comparator circuit could encode for insulin. This could be transfected into human cells and could be used to eleviate the symptoms associated with type I diabetes. <br />
<br />
Moving back to the specific nitrogen sensor, attaching these promoters to the comparator circuit biobricks and a gene for the synthesis of nitrogen reductase could result in a positive feedback loop to result in the tumour reducing in size. Macrophages naturally sense the presence of tumours in the body via their emission of nitric oxide. This could be taken one step further by adding nitrogen reductase to this system, where an excess of nitric oxide in the tumour environment. NO was seen to have cytotoxic properties in large amounts and, thus, a positive feedback loop could result in tumour necrosis.</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/NOSensingTeam:NRP-UEA-Norwich/NOSensing2012-09-26T09:46:50Z<p>Joyehicks: /* B-M + RFP Flow Cytometry Data */</p>
<hr />
<div>{{UEANRP}}<br />
<br />
{{UEANRPProjects}}<br />
<br />
[[File:NRPNOLogo.png | centre]]<br />
<br />
<html><center><b><font size=4pt>Our hybrid promoter hopes to add to the systems already in the registry by creating a hybrid promoter that combines the bacterial promoter PyeaR and the mammalian CArG element , both of which respond to exogenous nitrogenous species. Combining the two would allow a more modular nitric oxide sensor that can be used in mammalian and bacterial cells interchangeably.</font> <br />
<br><br><br />
<font size=2pt>Six new biobricks produced and submitted to the registry with characterisation from fluorescence-based experiments.</font></b></center></html><br />
<br />
Parts produced from this project:<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K774000 Bacterial-Mammalian/B-M (PyeaR-CArG) Hybrid Promoter] -- [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774001 Mammalian-Bacterial/M-B (CArG-PyeaR) Hybrid Promoter] -- [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774004 B-M + eCFP] -- [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774005 B-M + RFP] -- [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774006 M-B + eCFP] -- [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774004 M-B + RFP]<br />
<br />
Our main project has resulted in the production of a hybrid bacterial and mammalian promoter optimised for induction by nitric oxide, nitrates and nitrites. We have ligated PyeaR, a known bacterial promoter and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005 Part BBa_K216005] (Cambridge 2009) in the parts registry, with its mammalian counterpart, CArG. The resulting hybrid promoter has been synthesised in two orientations; PyeaR (bacterial, B) upstream of CArG (mammalian, M), nicknamed (B-M); and CArG upstream of PyeaR (M-B). These orientations were submitted to the parts registry as our first two biobricks.<br />
<br />
Each orientation of the promoter was ligated to enhanced cyan fluorescent protein (eCFP) and red fluorescent protein (RFP) to produce four new biobricks which have been submitted to the parts registry. These promoter + fluorescent protein biobricks have been characterised following transformation into ''Escherichia coli'' and induction by potassium nitrate using methods such as flow cytometry, fluorescence-activated cell sorting (FACS) and scanning with a fluorometer. The data from these experiments has proved that our promoter works as we expected it to. We have also transfected M-B + eCFP into a human breast cancer cell line, MCF7, and have proved the system is flexible and can be used in both eukaryotes and prokaryotes.<br />
<br />
We believe the promoters we have produced have relevant uses in cancer therapeutics, soil fertilisation and detection of emissions from industries such as construction.<br />
<br />
<br />
<br />
==Introduction==<br />
<br />
[[File:NRPPyeaR.png | 300px | thumbnail | '''''Figure 1.''''' ''A graphical representation of PyeaR. In the higher image PyeaR's activity is being repressed by both Nar and NsrR preventing transcription and the ultimate expression of a reporter. In the lower image nitrate/nitrite molecules have inhibited the activity of Nar, and nitric oxide has inhibited activity of NsrR, allowing for transcription to occur and subsequent expression of a reporter.'']]<br />
<br />
The University of East Anglia hosts many research teams whose work focuses on studying nitrogenous species and the way in which bacteria use and modify those species. One major problem that is faced by these teams involves working with nitric oxide (NO), as NO is highly reactive with a low half life, therefore making it difficult to trace and quantitatively measure accurately. Many of the methods currently used to measure NO levels are unable to distinguish between homogenous species, such as nitrates (NO3) and nitrites (NO2), therefore the figure given for NO levels is often inaccurate as other nitrogenous species are taken into account.<br />
<br />
<br />
The ability to be able to accurately detect NO levels is one with a great deal of potential for the future. Nitric oxide has been noted as a possible cancer therapy due to its physiological use as an apoptosis inducer by macrophages, however NO is also known to be used by cancerous cells to establish a baseline and use it to induce apoptosis and promote proliferation of a tumour; being able to accurately sense nitric oxide and go on to act on that information could be very useful to prevent the NO baseline being established by cancerous cells, but to also use NO for its apoptosis-inducing abilities. There are also other potential applications in the construction business, in 2008 '''NEED TO FIND REFERENCE OF PEOPLE''' released legislation encouraging construction companies to monitor their NO output as it is inversely proportional to carbon monoxide (a toxic substance which needs to be regulated); the ability to accurately detail levels of NO being released in these circumstances would be highly useful.<br />
<br />
<br />
Nitric oxide is an extremely physiologically relevant molecule found within both eukaryotes and prokaryotes, where it is utilised by different enzymes and systems for various roles. The aim of the experiment was to devise a hybrid promoter that could be applied to eukaryotes and prokaryotes in order to begin to more accurately sense NO and report on its specific levels.<br />
<br />
<br />
[[File:NRPBMandMB.png | 300px | thumbnail | '''''Figure 2.''''' ''A graphical representation of the hybrid promoter in its two orientations; the top image shows B-M with the bottom image showing M-B. Also included are the restriction sites and their location within the system, with the elements being read from left to right.'']]<br />
<br />
The hybrid promoter was designed to take on both bacterial and mammalian elements in order to be compatible with both bacterial and mammalian cells. After research around the subject and searching the parts registry a promoter known as PyeaR was decided upon as the bacterial element of the hybrid promoter; PyeaR is found in the ''yeaR/yoaG'' operon of ''Escherichia coli'' and is associated with induction by nitric oxide, nitrates and nitrites (Lin ''et al.'', 2007). PyeaR is repressed by two main repressors; Nar, which is regulated by nitrates and nitrites; and NsrR, which is regulated by nitric oxide (Figure 1.). One of the key elements of PyeaR is that it is not repressed in aerobic conditions, allowing for easier carrying out of experiments. The PyeaR aspect of the hybrid promoter has been known throughout the project as the bacterial promoter, or simply B.<br />
<br />
<br />
The mammalian element of the hybrid promoter was produced by nine CArG elements (repeated elements of CC(A/T)(6)GG), a promoter previously used synthetically for nitric oxide synthase as a cancer therapy (Worthington ''et al.'', 2005) and developed from the EGR1 gene for early growth response protein 1 (Scott ''et al.'', 2002). The CArG aspect of the hybrid promoter has been known throughout the project as the mammalian promoter, or simply M.<br />
<br />
<br />
Following identification of the two elements of the hybrid promoters the B (PyeaR) and M (CArG) aspects were ligated to one another in two orientations; B upstream of M (B-M) and M upstream of M (M-B) (Figure 2.). The hybrid promoters were synthesised in a pUC57 backbone with the standard iGEM restriction sites of EcoR1/Xba1 upstream of the promoter, and Spe1/Pst1 downstream of the promoter. A BamH1 restriction site was included in between the B and M sequences in order to allow for the B and M elements to be separated, as well as for easy verification of the promoter having been ligated into the iGEM backbone in future experiments (as BamH1 does not already exist in the pSB1C3 backbone).<br />
<br />
==Characterisation of Existing Biobrick: BBa_K381001 (PyeaR + GFP biobrick)==<br />
<br />
In order to begin to develop experiments to characterise the hybrid promoters + fluorescent proteins experiments were also carried out on a biobrick containing PyeaR + GFP (Part [http://partsregistry.org/Part:BBa_K381001 BBa_K381001], Bristol 2010). In these experiments transformed ''E. coli'' was inoculated into liquid culture, which in turn had varying potassium nitrate concentrations added to it. They were then left to grow before being spun down and viewed under a UV box in order to observe. The different concentrations of potassium nitrate that the transformed ''E. coli'' was grown in were: 0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 8 mM, 10 mM.<br />
<br />
[[File:GFP 4.JPG | 600 px | center | thumbnail | '''''Figure 3.''''' ''A photograph of spun-down media containing potassium nitrate (to induce the promoter) and E. coli transformed by PyeaR + GFP (art BBa_K381001). Each sample was grown with a different concentration of potassium nitrate, from left to right: 0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 8 mM, 10 mM.]]<br />
<br />
The figure suggests that fluorescent proteins have been expressed by the bacteria grown in media containing potassium nitrate due to the fluorescence shown under the UV box. It also suggests that different concentrations of potassium nitrate correlate with different intensities of expression due to the observable differences in fluorescence as the tubes are viewed from left to right, going up the gradient. The negative control of 0 mM potassium nitrate appears to show no fluorescence, suggesting it is indeed the potassium nitrate that is inducing the promoter.<br />
<br />
<br />
==Creating Novel Hybrid Promoters: B-M and M-B into Biobricks==<br />
<br />
The DNA for the synthesised genes of B-M and M-B had been supplied in the pUC57 backbone, therefore it was necessary for B-M and M-B to be digested from the pUC57 backbone and ligated into the pSB1C3 backbone. The synthesised gene was transformed into competent ’’E. coli’’ cells, which in turn were grown on agar plates containing 100 µg/ml ampicillin (due to pUC57 containing ampicillin resistance); colonies that had grown were then inoculated into liquid culture, and the liquid culture was subsequently mini-prepped using either the Bioline ISOLATE Plasmid DNA Mini Kit or the Promega Wizard® Plus SV Minipreps DNA Purification System. The DNA that had been extracted through mini-preps and the pSB1C3 backbone, as provided by the iGEM registry, were then digested using EcoR1 and Pst1 and a ligation was carried out using standard assembly protocol. The product of ligation was then transformed into competent ''E. coli'' cells, which were grown on agar plates containing 2.5 µg/ml chloramphenicol (due to pSB1C3 containing chloramphenicol resistance); this was done to eliminate any bacteria that had been transformed with undesirable ligation products. <br />
<br />
The colonies that had grown were then grown in liquid culture and mini-prepped in order to extract the DNA; the extracted DNA was then sent for sequencing, and the returned sequenced matched the expected sequence. The DNA was then sent to the parts registry as the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774000 Bacterial-Mammalian Hybrid Promoter] and the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774001 Mammalian-Bacterial Hybrid Promoter]. The biobricks for B-M and M-B were then used for further experiments, including ligation with a fluorescent protein reporter and growth studies.<br />
<br />
From weeks one through to five the team worked on producing the first biobricks of the hybrid promoter. Despite this proving difficult due to various reasons such as low amounts of DNA being produced from early mini-preps and ligation strategies not working, ''E. coli'' transformed by the biobrick DNA was successfully grown and proven to have the relevant antibiotic resistance by the beginning of [https://2012.igem.org/Team:NRP-UEA-Norwich/Week6 week six].<br />
<br />
===Studies into the effect of the hybrid promoter on growth of ''E. coli'' competent cells===<br />
<br />
[[File:NRPCalibration.png | 300 px | thumbnail | '''''Figure 4.''''' ''A calibration curve produced using E. coli alpha cells grown over time. The inoculations were run through a spectrophotometer at OD 600 nm every hour and then a sample was plated in order to calculate CFU/ml over time.]]<br />
<br />
As part of the characterisation of the hybrid promoters, as well as PyeaR alone ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005 Part BBa_K216005], studies were conducted to see if the growth of the ''E. coli'' was affected by transformation of the promoters. <br />
<br />
<br />
To being untransformed ''E. coli'' were inoculated into liquid culture and left to grow overnight at 37 ºC before being diluted down. The diluted liquid cultures were run through a spectrophotometer at OD 600 nm and their absorbance noted, before each culture was plated and once again left overnight at 37 ºC; the next morning the colonies on the plates were counted, allowing a calibration curve of absorbance at OD 600 nm against colony-forming units per ml (CFU/ml) to be set up (Figure 3.)<br />
<br />
<br />
Following establishment of the calibration curve liquid cultures were made up of ''E. coli'' that was:<br />
<br />
. Untransformed (otherwise referred to as alpha cells)<br />
<br />
. Transformed by B-M<br />
<br />
. Transformed by M-B<br />
<br />
. Transformed by PyeaR<br />
<br />
<br />
These cultures were then diluted down to the same starting level (an OD 600 nm absorbance of 0.2 +/- 0.1) and cuvettes filled with LB media were inoculated. The cuvettes were then placed in a spectrophotometer every hour and the absorbance at 600 nm was established; in between readings the cuvettes were placed into a 37 ºC incubator in order to encourage bacterial growth. This was repeated for 12 hours and the absorbance readings compared to the calibration curve in order to give data on the level of growth of ''E. coli'' transformed with the different promoters/untransformed over time.<br />
<br />
==Generation of B-M and M-B with eCFP and RFP==<br />
<br />
In order to test the activity of the hybrid promoters a reporter needed to be ligated. As the hybrid promoter did not already contain a ribosome binding site (RBS) both the RBS and the reporter were needed to be ligated to the promoter; in order to help improve experimental efficiency the parts registry was searched for relevant reporters that also contained an RBS. In [https://2012.igem.org/Team:NRP-UEA-Norwich/Week3 week three] two reporters were identified as [http://partsregistry.org/Part:BBa_E0420 BBa_E0420], a biobrick for enhanced CFP (eCFP) + RBS + terminators, and [http://partsregistry.org/Part:BBa_K081014 BBa_K081014], a biobrick for RFP + RBS + terminators.<br />
<br />
Once the B-M and M-B biobricks had been created in week six work began in earnest on the fluorescent proteins and ligating the promoters to them in order to begin characterisation. Due to many set-backs with low levels of DNA and having to order more biobricks from the registry, a successful ligation of the promoter to a fluorescent protein reporter was finally achieved in [https://2012.igem.org/Team:NRP-UEA-Norwich/Week10 week ten]. In order to carry out the ligation the promoter was first digested using Spe1 and Pst1 in order to linearise the backbone downstream of the promoter; the fluorescent proteins were digested using Xba1 and Pst1 in order to remove the insert. A ligation was then carried out using standard assembly protocol and the ligation products were transformed into ''E. coli'' competent cells, which in turn were grown on agar plates 2.5 µg/ml chloramphenicol (due to pSB1C3 containing chloramphenicol resistance). <br />
<br />
In order to quickly identify colonies of bacteria containing the promoter, RBS and reporter in a likely correct sequence a range of colonies were inoculated into media also containing potassium nitrate (KNO3) solution; this was done in order to inhibit the Nar repressor in PyeaR and result in activation of the promoter/induction of transcription/expression of the fluorescent protein reporter. Samples of the inoculated media containing KNO3 were then added to an eppendorf and spun down to form a pellet, which was viewed under a UV box and observed for fluorescence. After a week of various ligation and transformation experiments both promoters were successfully ligated to both fluorescent proteins and fluorescence of eCFP and RFP was observed under a UV box.<br />
<br />
From these experiments four more biobricks were produced and submitted to the registry: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774004 B-M + eCFP], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774005 B-M + RFP], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774006 M-B + eCFP], and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774004 M-B + RFP].<br />
<br />
In order to characterise the hybrid promoters ligated to fluorescent proteins a number of experiments were carried out to measure the level of fluorescent output at different concentrations of KNO3 (used to induce the promoter's activity). All of these experiments were carried out in [https://2012.igem.org/Team:NRP-UEA-Norwich/Week11 week eleven]<br />
<br />
===Qualitative Results===<br />
<br />
[[File:NRPCFPWorks.jpg | 600 px | center | thumbnail | '''''Figure 5.''''' ''A photograph of spun-down media containing potassium nitrate (to induce the hybrid promoters) and E. coli transformed by M-B + eCFP. The concentrations of potassium nitrate added to the media were, from left to right: 100 mM, 50 mM, 10 mM, 0 mM.]]<br />
<br />
This figure appears to show fluorescence from the spun-down pellets of each hybrid promoter + fluorescent protein that had been grown in media contain potassium nitrate; it can be inferred from this that the potassium nitrate has induced the hybrid promoter, resulting in expression of the fluorescent protein reporter. The negative control of 0 mM potassium nitrate appears to show no fluorescence, suggesting it is indeed the potassium nitrate that is inducing the promoter.<br />
<br />
[[File:NRPFluorescence.jpeg | 600 px | center | thumbnail | '''''Figure 6.''''' ''A photograph of spun-down media containing potassium nitrate (to induce the hybrid promoters) and E. coli transformed by the four biobricks containg the promoters and fluorescent proteins; the photograph has been taken from a UV box. From left to right: B-M + eCFP, M-B + eCFP, B-M + RFP, M-B + RFP.]]<br />
<br />
The figure appears to show fluorescence from the spun-down pellets of each hybrid promoter + fluorescent protein that had been grown in media contain potassium nitrate; it can be inferred from this that the potassium nitrate has induced the hybrid promoter, resulting in expression of the fluorescent protein reporter.<br />
<br />
===Fluorometer Experiments===<br />
<br />
The main characterisation of the biobricks was carried out using a fluorometer. Five tubes of media containing 200 µL transformed bacteria and potassium nitrate were grown for each biobrick in concentrations as follows:<br />
<br />
. B-M + RFP with 0 mM, 5 mM, 10 mM, 15 mM and 20 mM potassium nitrate<br />
<br />
. B-M + eCFP with 0 mM, 5 mM, 10 mM, 15 mM and 20 mM potassium nitrate<br />
<br />
. M-B + RFP with 0 mM, 5 mM, 10 mM, 15 mM and 20 mM potassium nitrate<br />
<br />
. M-B + eCFP with 0 mM, 5 mM, 10 mM, 15 mM and 20 mM potassium nitrate<br />
<br />
<br />
The samples were then spun down in a centrifuge and resuspended in Tris buffer before being subjected to sonication in order to lyse the cells. The resulting solution was then spun down in a centrifuge again before the supernatant was extracted (in order to separate the proteins from the rest of the cell). The supernatant was then run through a fluorometer in order to measure the intensity of fluorescence as a method to gauge the activity of the hybrid promoters. RFP-containing samples were excited with a wavelength of 560 nm and the emission measured from 600 – 650 nm; eCFP-containing samples were excited with a wavelength of 410 nm and emission measured from 440-490 nm.<br />
<br />
<br />
These characterisation experiments gave rise to the characterisation which is now available on the main page for all hybrid promoter + fluorescent protein parts on the registry, and the experience section of the hybrid promoters alone on the registry.<br />
<br />
===Flow Cytometry===<br />
<br />
Three tubes of media were inoculated with E. coli transformed by the B-M + RFP biobrick. Each tube then had potassium nitrate added to it at different concentrations; 0 mM, 1 mM and 10 mM respectively. The E. coli were grown over night and then spun down, fixed in 4% PFA and re-suspened in 500ul PBS. The samples were then analysed in an Acuri C6 or BD Aria II flow cytometer.<br />
<br />
[https://2012.igem.org/Team:NRP-UEA-Norwich/Protocol Full Protocol]<br />
<br />
''Note: This was the first time the flow cytometers at the University of East Anglia had been used with E. coli''<br />
<br />
====B-M + RFP Flow Cytometry Data====<br />
<br />
[[File:BM-RFP_18-9-12.png | 300 px | center | thumbnail | '''''Figure 7.''''' ''Flow cytometry data for B-M RFP transformed E. coli that were grown in either 0 mM, 1 mM or 10 mM potassium nitrate. Top row: Scatter plot of raw data and gating strategy utilised. Middle row: RFP Fluorescence profiles of samples. Lower left: Fluroescence profiles of the three samples overlai on the same plot.]]<br />
<br />
[[File:BM-RFP.jpg | 300 px | center | thumbnail | '''''Figure 7.''''' ''Flow cytometry fluorescence data: B-M RFP transformed E. coli that were grown in either 0 mM, 1 mM or 10 mM potassium nitrate.]]<br />
<br />
<br />
[[File:MB-CFP_data.jpg | 300 px | center | thumbnail | '''''Figure 7.''''' ''Flow cytometry data for M-B RFP transformed E. coli that were grown in either 0 mM, 1 mM or 10 mM potassium nitrate. Top row: Scatter plot of raw data and gating strategy utilised. Middle row: RFP Fluorescence profiles of samples.]]<br />
<br />
[[File:MB-CFP.png | 300 px | center | thumbnail | '''''Figure 7.''''' ''Flow cytometry fluorescence data: B-M RFP transformed E. coli that were grown in either 0 mM, 1 mM or 10 mM potassium nitrate.]]<br />
<br />
==Transfection of M-B + eCFP into the MCF7 human breast cancer cell line==<br />
<br />
In order to ascertain the flexibility of the hybrid promoter and help prove our original aim of producing a promoter that could be used in both eukaryotes and prokaryotes correct, the DNA for M-B + eCFP was transfected into the MCF7 human breast cancer cell line. An experiment was set up using S-Nitroso-N-acetyl-DL-penicillamine (SNAP), a nitric oxide donor, in order to induce expression of the fluorescent protein. A six-well transfection slide was produced containing:<br />
<br />
[[File:slide wells labelled.png | 500px | center]]<br />
<br />
The cells were then left for a day before they were imaged with a fluorescence microscope in order to observe expression of eCFP.<br />
<br />
[[File: Transfection.png | thumb | 500px |center | '''''Figure X.''''' '' Transfection of MCF7 cells with images taken via a Zeiss CCD2 inverted microscope to detect CFP expression. Images in the left two columns are controls and have not been transfected, images in the right two columns have been transfected with M-B + CFP DNA; SNAP is a nitric oxide donor, therefore addition of SNAP was used to try and induce promoter activity.'']]<br />
<br />
[[File:MCF7 blue.png | 400px | center| '''''Figure X.''''' '' Close up image of what appears to be eCFP fluorescence from a transfected MCF7 cell]]<br />
<br />
<br />
The figure appears to show fluorescence in the mammalian cells that had been transfected with M-B + CFP compared with the cells that had not been transfected. The figure also appears to show stronger fluorescence in the cells that had been transfected with M-B + CFP and had been grown with the nitric oxide donor SNAP compared to the cells that had been transfected with M-B + CFP and grown without SNAP.<br />
<br />
<br />
[[File: NRPMBCFP.JPG | thumb | 500px |center | '''''Figure x.''''' '' Transfection of MCF7 cells with images taken via a Zeiss CCD2 inverted microscope to detect CFP expression. Image shows mammalian cells transfected with M-B + CFP DNA however no SNAP (a nitric oxide donor) has been administered.'']]<br />
<br />
<br />
The figure appears to show low levels of fluorescence in the cells as there are small blue circles appearing in the cells indicative of CFP expression.<br />
<br />
<br />
[[File: NRPMBCFPSNAP.JPG | thumb | 500px |center | '''''Figure x.''''' '' Transfection of MCF7 cells with images taken via a Zeiss CCD2 inverted microscope to detect CFP expression. Image shows mammalian cells transfected with M-B + CFP DNA where SNAP (a nitric oxide donor) has been administered.'']]<br />
<br />
<br />
The figure suggests that expression of CFP has occurred due to the large blue shape appearing in the centre of the image following the use of a filter to view fluorescent cyan more easily.<br />
<br />
==Discussion==<br />
<br />
BM/MB better or the same, RFP/CFP better or the same etc.<br />
<br />
==Future Experiments==<br />
<br />
. Full quantitative analysis to see where the values lie; combine with tuners for different sensitivity levels <br />
<br />
. With different substrates (e.g. nitrite salt, NO donor)<br />
<br />
. With different reporter/effector enzyme<br />
<br />
==References==<br />
<br />
Civerolo, K.L. and Dickerson, R.R. (1998) ''Nitric oxide soil emissions from tilled and untilled cornfields'', Agricultural and Forest Meteorology, '''90; 307-311''' <br />
<br />
<br />
Davidson, E., (2012), ''Sources of Nitric Oxide and Nitrous Oxide following Wetting of Dry Soil'', Soil Sci. Soc. Am. J. '''56; 95–102'''<br />
<br />
<br />
Lin H.Y., Bledsoe P.J., Stewart V., (2007), ''Activation of yeaR-yoaG Operon Transcription by the Nitrate-Responsive Regulator NarL Is Independent of Oxygen- Responsive Regulator Fnr in Escherichia coli K-12▿'', Journal of Bacteriology, '''189: 7539 - 7548'''<br />
<br />
<br />
Lipschultz, F., Zafiriou, O.C. Wofsy, S.C., Elroy, M.B., Valois, F.W. and Watson, S.W. (1981) ''Production of NO and N2O by soil nitrifying bacteria'', Macmillan Journals, '''294; 641-643''' <br />
<br />
<br />
Pasqualini, R., Koivunen, E., Kain, R., Lahdenranta, J., Sakamoto, M., Stryhn, A., Ashmun, R.A., Shapiro, L.H., Arap, W. And Ruoslahti, E. (2000) ''Aminopeptidase N is a receptor for tumour-homing peptides and a target for inhibiting angiogenesis'', The Journal of Cancer Research, '''60; 722-727''' <br />
<br />
<br />
Scott S.D., Joiner M.C., Marples B., (2002), ''Optimizing radiation-responsive gene promoters for radiogenetic cancer therapy.'', Gene Therapy, '''9: 1396-1402'''<br />
<br />
<br />
Worthington J., Robson T., Scott S., Hirst, D., (2005), ''Evaluation of a synthetic CArG promoter for nitric oxide synthase gene therapy of cancer'', Gene Therapy, '''12: 1417–1423'''<br />
<br />
<br />
Xu, W., Liu, L.Z., Loizidou, M., Ahmed, M. And Charles, I.G. (2002) ''The role of nitric oxide in cancer'', Cell Research, '''12; 311-320'''</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/NOSensingTeam:NRP-UEA-Norwich/NOSensing2012-09-26T09:45:34Z<p>Joyehicks: /* M-B + eCFP Flow Cytometry Data */</p>
<hr />
<div>{{UEANRP}}<br />
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{{UEANRPProjects}}<br />
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[[File:NRPNOLogo.png | centre]]<br />
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<html><center><b><font size=4pt>Our hybrid promoter hopes to add to the systems already in the registry by creating a hybrid promoter that combines the bacterial promoter PyeaR and the mammalian CArG element , both of which respond to exogenous nitrogenous species. Combining the two would allow a more modular nitric oxide sensor that can be used in mammalian and bacterial cells interchangeably.</font> <br />
<br><br><br />
<font size=2pt>Six new biobricks produced and submitted to the registry with characterisation from fluorescence-based experiments.</font></b></center></html><br />
<br />
Parts produced from this project:<br />
<br />
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K774000 Bacterial-Mammalian/B-M (PyeaR-CArG) Hybrid Promoter] -- [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774001 Mammalian-Bacterial/M-B (CArG-PyeaR) Hybrid Promoter] -- [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774004 B-M + eCFP] -- [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774005 B-M + RFP] -- [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774006 M-B + eCFP] -- [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774004 M-B + RFP]<br />
<br />
Our main project has resulted in the production of a hybrid bacterial and mammalian promoter optimised for induction by nitric oxide, nitrates and nitrites. We have ligated PyeaR, a known bacterial promoter and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005 Part BBa_K216005] (Cambridge 2009) in the parts registry, with its mammalian counterpart, CArG. The resulting hybrid promoter has been synthesised in two orientations; PyeaR (bacterial, B) upstream of CArG (mammalian, M), nicknamed (B-M); and CArG upstream of PyeaR (M-B). These orientations were submitted to the parts registry as our first two biobricks.<br />
<br />
Each orientation of the promoter was ligated to enhanced cyan fluorescent protein (eCFP) and red fluorescent protein (RFP) to produce four new biobricks which have been submitted to the parts registry. These promoter + fluorescent protein biobricks have been characterised following transformation into ''Escherichia coli'' and induction by potassium nitrate using methods such as flow cytometry, fluorescence-activated cell sorting (FACS) and scanning with a fluorometer. The data from these experiments has proved that our promoter works as we expected it to. We have also transfected M-B + eCFP into a human breast cancer cell line, MCF7, and have proved the system is flexible and can be used in both eukaryotes and prokaryotes.<br />
<br />
We believe the promoters we have produced have relevant uses in cancer therapeutics, soil fertilisation and detection of emissions from industries such as construction.<br />
<br />
<br />
<br />
==Introduction==<br />
<br />
[[File:NRPPyeaR.png | 300px | thumbnail | '''''Figure 1.''''' ''A graphical representation of PyeaR. In the higher image PyeaR's activity is being repressed by both Nar and NsrR preventing transcription and the ultimate expression of a reporter. In the lower image nitrate/nitrite molecules have inhibited the activity of Nar, and nitric oxide has inhibited activity of NsrR, allowing for transcription to occur and subsequent expression of a reporter.'']]<br />
<br />
The University of East Anglia hosts many research teams whose work focuses on studying nitrogenous species and the way in which bacteria use and modify those species. One major problem that is faced by these teams involves working with nitric oxide (NO), as NO is highly reactive with a low half life, therefore making it difficult to trace and quantitatively measure accurately. Many of the methods currently used to measure NO levels are unable to distinguish between homogenous species, such as nitrates (NO3) and nitrites (NO2), therefore the figure given for NO levels is often inaccurate as other nitrogenous species are taken into account.<br />
<br />
<br />
The ability to be able to accurately detect NO levels is one with a great deal of potential for the future. Nitric oxide has been noted as a possible cancer therapy due to its physiological use as an apoptosis inducer by macrophages, however NO is also known to be used by cancerous cells to establish a baseline and use it to induce apoptosis and promote proliferation of a tumour; being able to accurately sense nitric oxide and go on to act on that information could be very useful to prevent the NO baseline being established by cancerous cells, but to also use NO for its apoptosis-inducing abilities. There are also other potential applications in the construction business, in 2008 '''NEED TO FIND REFERENCE OF PEOPLE''' released legislation encouraging construction companies to monitor their NO output as it is inversely proportional to carbon monoxide (a toxic substance which needs to be regulated); the ability to accurately detail levels of NO being released in these circumstances would be highly useful.<br />
<br />
<br />
Nitric oxide is an extremely physiologically relevant molecule found within both eukaryotes and prokaryotes, where it is utilised by different enzymes and systems for various roles. The aim of the experiment was to devise a hybrid promoter that could be applied to eukaryotes and prokaryotes in order to begin to more accurately sense NO and report on its specific levels.<br />
<br />
<br />
[[File:NRPBMandMB.png | 300px | thumbnail | '''''Figure 2.''''' ''A graphical representation of the hybrid promoter in its two orientations; the top image shows B-M with the bottom image showing M-B. Also included are the restriction sites and their location within the system, with the elements being read from left to right.'']]<br />
<br />
The hybrid promoter was designed to take on both bacterial and mammalian elements in order to be compatible with both bacterial and mammalian cells. After research around the subject and searching the parts registry a promoter known as PyeaR was decided upon as the bacterial element of the hybrid promoter; PyeaR is found in the ''yeaR/yoaG'' operon of ''Escherichia coli'' and is associated with induction by nitric oxide, nitrates and nitrites (Lin ''et al.'', 2007). PyeaR is repressed by two main repressors; Nar, which is regulated by nitrates and nitrites; and NsrR, which is regulated by nitric oxide (Figure 1.). One of the key elements of PyeaR is that it is not repressed in aerobic conditions, allowing for easier carrying out of experiments. The PyeaR aspect of the hybrid promoter has been known throughout the project as the bacterial promoter, or simply B.<br />
<br />
<br />
The mammalian element of the hybrid promoter was produced by nine CArG elements (repeated elements of CC(A/T)(6)GG), a promoter previously used synthetically for nitric oxide synthase as a cancer therapy (Worthington ''et al.'', 2005) and developed from the EGR1 gene for early growth response protein 1 (Scott ''et al.'', 2002). The CArG aspect of the hybrid promoter has been known throughout the project as the mammalian promoter, or simply M.<br />
<br />
<br />
Following identification of the two elements of the hybrid promoters the B (PyeaR) and M (CArG) aspects were ligated to one another in two orientations; B upstream of M (B-M) and M upstream of M (M-B) (Figure 2.). The hybrid promoters were synthesised in a pUC57 backbone with the standard iGEM restriction sites of EcoR1/Xba1 upstream of the promoter, and Spe1/Pst1 downstream of the promoter. A BamH1 restriction site was included in between the B and M sequences in order to allow for the B and M elements to be separated, as well as for easy verification of the promoter having been ligated into the iGEM backbone in future experiments (as BamH1 does not already exist in the pSB1C3 backbone).<br />
<br />
==Characterisation of Existing Biobrick: BBa_K381001 (PyeaR + GFP biobrick)==<br />
<br />
In order to begin to develop experiments to characterise the hybrid promoters + fluorescent proteins experiments were also carried out on a biobrick containing PyeaR + GFP (Part [http://partsregistry.org/Part:BBa_K381001 BBa_K381001], Bristol 2010). In these experiments transformed ''E. coli'' was inoculated into liquid culture, which in turn had varying potassium nitrate concentrations added to it. They were then left to grow before being spun down and viewed under a UV box in order to observe. The different concentrations of potassium nitrate that the transformed ''E. coli'' was grown in were: 0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 8 mM, 10 mM.<br />
<br />
[[File:GFP 4.JPG | 600 px | center | thumbnail | '''''Figure 3.''''' ''A photograph of spun-down media containing potassium nitrate (to induce the promoter) and E. coli transformed by PyeaR + GFP (art BBa_K381001). Each sample was grown with a different concentration of potassium nitrate, from left to right: 0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 8 mM, 10 mM.]]<br />
<br />
The figure suggests that fluorescent proteins have been expressed by the bacteria grown in media containing potassium nitrate due to the fluorescence shown under the UV box. It also suggests that different concentrations of potassium nitrate correlate with different intensities of expression due to the observable differences in fluorescence as the tubes are viewed from left to right, going up the gradient. The negative control of 0 mM potassium nitrate appears to show no fluorescence, suggesting it is indeed the potassium nitrate that is inducing the promoter.<br />
<br />
<br />
==Creating Novel Hybrid Promoters: B-M and M-B into Biobricks==<br />
<br />
The DNA for the synthesised genes of B-M and M-B had been supplied in the pUC57 backbone, therefore it was necessary for B-M and M-B to be digested from the pUC57 backbone and ligated into the pSB1C3 backbone. The synthesised gene was transformed into competent ’’E. coli’’ cells, which in turn were grown on agar plates containing 100 µg/ml ampicillin (due to pUC57 containing ampicillin resistance); colonies that had grown were then inoculated into liquid culture, and the liquid culture was subsequently mini-prepped using either the Bioline ISOLATE Plasmid DNA Mini Kit or the Promega Wizard® Plus SV Minipreps DNA Purification System. The DNA that had been extracted through mini-preps and the pSB1C3 backbone, as provided by the iGEM registry, were then digested using EcoR1 and Pst1 and a ligation was carried out using standard assembly protocol. The product of ligation was then transformed into competent ''E. coli'' cells, which were grown on agar plates containing 2.5 µg/ml chloramphenicol (due to pSB1C3 containing chloramphenicol resistance); this was done to eliminate any bacteria that had been transformed with undesirable ligation products. <br />
<br />
The colonies that had grown were then grown in liquid culture and mini-prepped in order to extract the DNA; the extracted DNA was then sent for sequencing, and the returned sequenced matched the expected sequence. The DNA was then sent to the parts registry as the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774000 Bacterial-Mammalian Hybrid Promoter] and the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774001 Mammalian-Bacterial Hybrid Promoter]. The biobricks for B-M and M-B were then used for further experiments, including ligation with a fluorescent protein reporter and growth studies.<br />
<br />
From weeks one through to five the team worked on producing the first biobricks of the hybrid promoter. Despite this proving difficult due to various reasons such as low amounts of DNA being produced from early mini-preps and ligation strategies not working, ''E. coli'' transformed by the biobrick DNA was successfully grown and proven to have the relevant antibiotic resistance by the beginning of [https://2012.igem.org/Team:NRP-UEA-Norwich/Week6 week six].<br />
<br />
===Studies into the effect of the hybrid promoter on growth of ''E. coli'' competent cells===<br />
<br />
[[File:NRPCalibration.png | 300 px | thumbnail | '''''Figure 4.''''' ''A calibration curve produced using E. coli alpha cells grown over time. The inoculations were run through a spectrophotometer at OD 600 nm every hour and then a sample was plated in order to calculate CFU/ml over time.]]<br />
<br />
As part of the characterisation of the hybrid promoters, as well as PyeaR alone ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K216005 Part BBa_K216005], studies were conducted to see if the growth of the ''E. coli'' was affected by transformation of the promoters. <br />
<br />
<br />
To being untransformed ''E. coli'' were inoculated into liquid culture and left to grow overnight at 37 ºC before being diluted down. The diluted liquid cultures were run through a spectrophotometer at OD 600 nm and their absorbance noted, before each culture was plated and once again left overnight at 37 ºC; the next morning the colonies on the plates were counted, allowing a calibration curve of absorbance at OD 600 nm against colony-forming units per ml (CFU/ml) to be set up (Figure 3.)<br />
<br />
<br />
Following establishment of the calibration curve liquid cultures were made up of ''E. coli'' that was:<br />
<br />
. Untransformed (otherwise referred to as alpha cells)<br />
<br />
. Transformed by B-M<br />
<br />
. Transformed by M-B<br />
<br />
. Transformed by PyeaR<br />
<br />
<br />
These cultures were then diluted down to the same starting level (an OD 600 nm absorbance of 0.2 +/- 0.1) and cuvettes filled with LB media were inoculated. The cuvettes were then placed in a spectrophotometer every hour and the absorbance at 600 nm was established; in between readings the cuvettes were placed into a 37 ºC incubator in order to encourage bacterial growth. This was repeated for 12 hours and the absorbance readings compared to the calibration curve in order to give data on the level of growth of ''E. coli'' transformed with the different promoters/untransformed over time.<br />
<br />
==Generation of B-M and M-B with eCFP and RFP==<br />
<br />
In order to test the activity of the hybrid promoters a reporter needed to be ligated. As the hybrid promoter did not already contain a ribosome binding site (RBS) both the RBS and the reporter were needed to be ligated to the promoter; in order to help improve experimental efficiency the parts registry was searched for relevant reporters that also contained an RBS. In [https://2012.igem.org/Team:NRP-UEA-Norwich/Week3 week three] two reporters were identified as [http://partsregistry.org/Part:BBa_E0420 BBa_E0420], a biobrick for enhanced CFP (eCFP) + RBS + terminators, and [http://partsregistry.org/Part:BBa_K081014 BBa_K081014], a biobrick for RFP + RBS + terminators.<br />
<br />
Once the B-M and M-B biobricks had been created in week six work began in earnest on the fluorescent proteins and ligating the promoters to them in order to begin characterisation. Due to many set-backs with low levels of DNA and having to order more biobricks from the registry, a successful ligation of the promoter to a fluorescent protein reporter was finally achieved in [https://2012.igem.org/Team:NRP-UEA-Norwich/Week10 week ten]. In order to carry out the ligation the promoter was first digested using Spe1 and Pst1 in order to linearise the backbone downstream of the promoter; the fluorescent proteins were digested using Xba1 and Pst1 in order to remove the insert. A ligation was then carried out using standard assembly protocol and the ligation products were transformed into ''E. coli'' competent cells, which in turn were grown on agar plates 2.5 µg/ml chloramphenicol (due to pSB1C3 containing chloramphenicol resistance). <br />
<br />
In order to quickly identify colonies of bacteria containing the promoter, RBS and reporter in a likely correct sequence a range of colonies were inoculated into media also containing potassium nitrate (KNO3) solution; this was done in order to inhibit the Nar repressor in PyeaR and result in activation of the promoter/induction of transcription/expression of the fluorescent protein reporter. Samples of the inoculated media containing KNO3 were then added to an eppendorf and spun down to form a pellet, which was viewed under a UV box and observed for fluorescence. After a week of various ligation and transformation experiments both promoters were successfully ligated to both fluorescent proteins and fluorescence of eCFP and RFP was observed under a UV box.<br />
<br />
From these experiments four more biobricks were produced and submitted to the registry: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774004 B-M + eCFP], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774005 B-M + RFP], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774006 M-B + eCFP], and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K774004 M-B + RFP].<br />
<br />
In order to characterise the hybrid promoters ligated to fluorescent proteins a number of experiments were carried out to measure the level of fluorescent output at different concentrations of KNO3 (used to induce the promoter's activity). All of these experiments were carried out in [https://2012.igem.org/Team:NRP-UEA-Norwich/Week11 week eleven]<br />
<br />
===Qualitative Results===<br />
<br />
[[File:NRPCFPWorks.jpg | 600 px | center | thumbnail | '''''Figure 5.''''' ''A photograph of spun-down media containing potassium nitrate (to induce the hybrid promoters) and E. coli transformed by M-B + eCFP. The concentrations of potassium nitrate added to the media were, from left to right: 100 mM, 50 mM, 10 mM, 0 mM.]]<br />
<br />
This figure appears to show fluorescence from the spun-down pellets of each hybrid promoter + fluorescent protein that had been grown in media contain potassium nitrate; it can be inferred from this that the potassium nitrate has induced the hybrid promoter, resulting in expression of the fluorescent protein reporter. The negative control of 0 mM potassium nitrate appears to show no fluorescence, suggesting it is indeed the potassium nitrate that is inducing the promoter.<br />
<br />
[[File:NRPFluorescence.jpeg | 600 px | center | thumbnail | '''''Figure 6.''''' ''A photograph of spun-down media containing potassium nitrate (to induce the hybrid promoters) and E. coli transformed by the four biobricks containg the promoters and fluorescent proteins; the photograph has been taken from a UV box. From left to right: B-M + eCFP, M-B + eCFP, B-M + RFP, M-B + RFP.]]<br />
<br />
The figure appears to show fluorescence from the spun-down pellets of each hybrid promoter + fluorescent protein that had been grown in media contain potassium nitrate; it can be inferred from this that the potassium nitrate has induced the hybrid promoter, resulting in expression of the fluorescent protein reporter.<br />
<br />
===Fluorometer Experiments===<br />
<br />
The main characterisation of the biobricks was carried out using a fluorometer. Five tubes of media containing 200 µL transformed bacteria and potassium nitrate were grown for each biobrick in concentrations as follows:<br />
<br />
. B-M + RFP with 0 mM, 5 mM, 10 mM, 15 mM and 20 mM potassium nitrate<br />
<br />
. B-M + eCFP with 0 mM, 5 mM, 10 mM, 15 mM and 20 mM potassium nitrate<br />
<br />
. M-B + RFP with 0 mM, 5 mM, 10 mM, 15 mM and 20 mM potassium nitrate<br />
<br />
. M-B + eCFP with 0 mM, 5 mM, 10 mM, 15 mM and 20 mM potassium nitrate<br />
<br />
<br />
The samples were then spun down in a centrifuge and resuspended in Tris buffer before being subjected to sonication in order to lyse the cells. The resulting solution was then spun down in a centrifuge again before the supernatant was extracted (in order to separate the proteins from the rest of the cell). The supernatant was then run through a fluorometer in order to measure the intensity of fluorescence as a method to gauge the activity of the hybrid promoters. RFP-containing samples were excited with a wavelength of 560 nm and the emission measured from 600 – 650 nm; eCFP-containing samples were excited with a wavelength of 410 nm and emission measured from 440-490 nm.<br />
<br />
<br />
These characterisation experiments gave rise to the characterisation which is now available on the main page for all hybrid promoter + fluorescent protein parts on the registry, and the experience section of the hybrid promoters alone on the registry.<br />
<br />
===Flow Cytometry===<br />
<br />
Three tubes of media were inoculated with E. coli transformed by the B-M + RFP biobrick. Each tube then had potassium nitrate added to it at different concentrations; 0 mM, 1 mM and 10 mM respectively. The E. coli were grown over night and then spun down, fixed in 4% PFA and re-suspened in 500ul PBS. The samples were then analysed in an Acuri C6 or BD Aria II flow cytometer.<br />
<br />
[https://2012.igem.org/Team:NRP-UEA-Norwich/Protocol Full Protocol]<br />
<br />
''Note: This was the first time the flow cytometers at the University of East Anglia had been used with E. coli''<br />
<br />
====B-M + RFP Flow Cytometry Data====<br />
<br />
[[File:BM-RFP_18-9-12.png | 300 px | center | thumbnail | '''''Figure 7.''''' ''Flow cytometry data for B-M RFP transformed E. coli that were grown in either 0 mM, 1 mM or 10 mM potassium nitrate. Top row: Scatter plot of raw data and gating strategy utilised. Middle row: RFP Fluorescence profiles of samples. Lower left: Fluroescence profiles of the three samples overlai on the same plot.]]<br />
<br />
[[File:BM-RFP.jpg | 300 px | center | thumbnail | '''''Figure 7.''''' ''Flow cytometry fluorescence data: B-M RFP transformed E. coli that were grown in either 0 mM, 1 mM or 10 mM potassium nitrate.]]<br />
<br />
<br />
[[File:MB-CFP_data.jpg | 300 px | center | thumbnail | '''''Figure 7.''''' ''Flow cytometry data for M-B RFP transformed E. coli that were grown in either 0 mM, 1 mM or 10 mM potassium nitrate. Top row: Scatter plot of raw data and gating strategy utilised. Middle row: RFP Fluorescence profiles of samples.]]<br />
<br />
[[File:MB-CFP.png | 300 px | center | thumbnail | '''''Figure 7.''''' ''Flow cytometry fluorescence data: M-B RFP transformed E. coli that were grown in either 0 mM, 1 mM or 10 mM potassium nitrate.]]<br />
<br />
==Transfection of M-B + eCFP into the MCF7 human breast cancer cell line==<br />
<br />
In order to ascertain the flexibility of the hybrid promoter and help prove our original aim of producing a promoter that could be used in both eukaryotes and prokaryotes correct, the DNA for M-B + eCFP was transfected into the MCF7 human breast cancer cell line. An experiment was set up using S-Nitroso-N-acetyl-DL-penicillamine (SNAP), a nitric oxide donor, in order to induce expression of the fluorescent protein. A six-well transfection slide was produced containing:<br />
<br />
[[File:slide wells labelled.png | 500px | center]]<br />
<br />
The cells were then left for a day before they were imaged with a fluorescence microscope in order to observe expression of eCFP.<br />
<br />
[[File: Transfection.png | thumb | 500px |center | '''''Figure X.''''' '' Transfection of MCF7 cells with images taken via a Zeiss CCD2 inverted microscope to detect CFP expression. Images in the left two columns are controls and have not been transfected, images in the right two columns have been transfected with M-B + CFP DNA; SNAP is a nitric oxide donor, therefore addition of SNAP was used to try and induce promoter activity.'']]<br />
<br />
[[File:MCF7 blue.png | 400px | center| '''''Figure X.''''' '' Close up image of what appears to be eCFP fluorescence from a transfected MCF7 cell]]<br />
<br />
<br />
The figure appears to show fluorescence in the mammalian cells that had been transfected with M-B + CFP compared with the cells that had not been transfected. The figure also appears to show stronger fluorescence in the cells that had been transfected with M-B + CFP and had been grown with the nitric oxide donor SNAP compared to the cells that had been transfected with M-B + CFP and grown without SNAP.<br />
<br />
<br />
[[File: NRPMBCFP.JPG | thumb | 500px |center | '''''Figure x.''''' '' Transfection of MCF7 cells with images taken via a Zeiss CCD2 inverted microscope to detect CFP expression. Image shows mammalian cells transfected with M-B + CFP DNA however no SNAP (a nitric oxide donor) has been administered.'']]<br />
<br />
<br />
The figure appears to show low levels of fluorescence in the cells as there are small blue circles appearing in the cells indicative of CFP expression.<br />
<br />
<br />
[[File: NRPMBCFPSNAP.JPG | thumb | 500px |center | '''''Figure x.''''' '' Transfection of MCF7 cells with images taken via a Zeiss CCD2 inverted microscope to detect CFP expression. Image shows mammalian cells transfected with M-B + CFP DNA where SNAP (a nitric oxide donor) has been administered.'']]<br />
<br />
<br />
The figure suggests that expression of CFP has occurred due to the large blue shape appearing in the centre of the image following the use of a filter to view fluorescent cyan more easily.<br />
<br />
==Discussion==<br />
<br />
BM/MB better or the same, RFP/CFP better or the same etc.<br />
<br />
==Future Experiments==<br />
<br />
. Full quantitative analysis to see where the values lie; combine with tuners for different sensitivity levels <br />
<br />
. With different substrates (e.g. nitrite salt, NO donor)<br />
<br />
. With different reporter/effector enzyme<br />
<br />
==References==<br />
<br />
Civerolo, K.L. and Dickerson, R.R. (1998) ''Nitric oxide soil emissions from tilled and untilled cornfields'', Agricultural and Forest Meteorology, '''90; 307-311''' <br />
<br />
<br />
Davidson, E., (2012), ''Sources of Nitric Oxide and Nitrous Oxide following Wetting of Dry Soil'', Soil Sci. Soc. Am. J. '''56; 95–102'''<br />
<br />
<br />
Lin H.Y., Bledsoe P.J., Stewart V., (2007), ''Activation of yeaR-yoaG Operon Transcription by the Nitrate-Responsive Regulator NarL Is Independent of Oxygen- Responsive Regulator Fnr in Escherichia coli K-12▿'', Journal of Bacteriology, '''189: 7539 - 7548'''<br />
<br />
<br />
Lipschultz, F., Zafiriou, O.C. Wofsy, S.C., Elroy, M.B., Valois, F.W. and Watson, S.W. (1981) ''Production of NO and N2O by soil nitrifying bacteria'', Macmillan Journals, '''294; 641-643''' <br />
<br />
<br />
Pasqualini, R., Koivunen, E., Kain, R., Lahdenranta, J., Sakamoto, M., Stryhn, A., Ashmun, R.A., Shapiro, L.H., Arap, W. And Ruoslahti, E. (2000) ''Aminopeptidase N is a receptor for tumour-homing peptides and a target for inhibiting angiogenesis'', The Journal of Cancer Research, '''60; 722-727''' <br />
<br />
<br />
Scott S.D., Joiner M.C., Marples B., (2002), ''Optimizing radiation-responsive gene promoters for radiogenetic cancer therapy.'', Gene Therapy, '''9: 1396-1402'''<br />
<br />
<br />
Worthington J., Robson T., Scott S., Hirst, D., (2005), ''Evaluation of a synthetic CArG promoter for nitric oxide synthase gene therapy of cancer'', Gene Therapy, '''12: 1417–1423'''<br />
<br />
<br />
Xu, W., Liu, L.Z., Loizidou, M., Ahmed, M. And Charles, I.G. (2002) ''The role of nitric oxide in cancer'', Cell Research, '''12; 311-320'''</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-NorwichTeam:NRP-UEA-Norwich2012-09-26T09:40:41Z<p>Joyehicks: </p>
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<img src="https://static.igem.org/mediawiki/2012/5/5f/NRP1.gif"><a href=https://2012.igem.org/Team:NRP-UEA-Norwich/Project><img src="https://static.igem.org/mediawiki/2012/7/75/NRP2.gif"></a><img src="https://static.igem.org/mediawiki/2012/0/00/NRP3.gif"><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Parts"><img src=https://static.igem.org/mediawiki/2012/d/df/NRP4.gif></a><img src="https://static.igem.org/mediawiki/2012/9/96/NRP5.gif"><br />
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<img src="https://static.igem.org/mediawiki/2012/8/8b/NRP6.gif"><img src="https://static.igem.org/mediawiki/2012/d/da/NRP7.gif"><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Project"><img src="https://static.igem.org/mediawiki/2012/7/79/NRP8.gif"></a><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Project"><img src="https://static.igem.org/mediawiki/2012/0/09/NRP9.gif"></a><img src="https://static.igem.org/mediawiki/2012/9/94/NRP10.gif"><br />
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<a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Future"><img src="https://static.igem.org/mediawiki/2012/e/e5/NRP11.gif"></a><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Future"><img src="https://static.igem.org/mediawiki/2012/8/86/NRP12.gif"></a><img src="https://static.igem.org/mediawiki/2012/f/fe/NRP13.gif"><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Notebook"><img src="https://static.igem.org/mediawiki/2012/3/3b/NRP14.gif"></a><img src="https://static.igem.org/mediawiki/2012/d/d4/NRP15.gif"><br />
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<img src="https://static.igem.org/mediawiki/2012/b/bc/NRP16.gif"><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Future"><img src="https://static.igem.org/mediawiki/2012/0/01/NRP17.gif"></a><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Quanticare"><img src="https://static.igem.org/mediawiki/2012/2/2d/NRP18.gif"></a><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Notebook"><img src="https://static.igem.org/mediawiki/2012/e/ee/NRP19.gif"></a><img src="https://static.igem.org/mediawiki/2012/0/0a/NRP20.gif"><br />
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</html></div>Joyehickshttp://2012.igem.org/Template:UEANRPTemplate:UEANRP2012-09-26T09:38:04Z<p>Joyehicks: </p>
<hr />
<div>[[Image:Header1NewGreen.png|top]]<br />
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<br />
<div id="colortab" class="ddcolortabs"><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich" title="Home"><span>Home</span></a></li><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Team" rel="dropmenu1_a"><span>Team</span></a></li><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Project" title="Home"><span>Project</span></a></li><br />
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<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Notebook" rel="dropmenu1_a"><span>Lab Book</span></a></li><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Parts" rel="dropmenu1_a"><span>Parts</span></a></li><br />
<br />
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<div class="ddcolortabsline">&nbsp;</div><br />
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<a href="https://2008.igem.org/Team:Edinburgh/Team">Overview</a><br />
<a href="https://2008.igem.org/Team:Edinburgh/Team">Step1</a><br />
<a href="https://2008.igem.org/Team:Edinburgh/Team">Step2</a><br />
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</html></div>Joyehickshttp://2012.igem.org/Template:UEANRPQuanticareTemplate:UEANRPQuanticare2012-09-26T09:37:40Z<p>Joyehicks: </p>
<hr />
<div>[[Image:Header1NewGreen.png|top]]<br />
<br />
<html><style><br />
#content a[href ^="https://"].external<br />
{<br />
background: center right no-repeat; padding-right: 18px;<br />
}</style></html><br />
<br />
<html><style type="text/css"><br />
#edi-twitter-widget .plainlinks{<br />
border: 1px solid #ccc;<br />
}<br />
#welcome a,<br />
#welcome a:hover,<br />
#welcome a:visited{<br />
color: #009443;<br />
}<br />
</style></html><br />
<br />
<html><br />
<br />
<style><br />
body{<br />
background-image:url("https://static.igem.org/mediawiki/2012/b/b2/WikiBGNewGreen.png");<br />
background-position:center top;<br />
background-repeat:no-repeat;<br />
background-color:#009443;<br />
<br />
}<br />
<br />
/*Browser History*/<br />
a:link {ccolor:#20548f;}<br />
a:visited {color:#20548f;}<br />
a:hover {color:#20548f;}<br />
a:active {color:#20548f;}<br />
<br />
#menubar.top-menu {<br />
position: absolute;<br />
top:0px;<br />
left:0px;<br />
}<br />
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#menubar.slide-menu {<br />
position: absolute;<br />
top:0px;<br />
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#menubar a:active,#menubar a:hover,#menubar a:link,#menubar a:visited {<br />
color: #1D1D1B;<br />
}<br />
<br />
<br />
/*Code to remove/overlay standard iGEM Header for blank canvas*/<br />
#top-section{<br />
background-color: #009443;<br />
height: 100px;<br />
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width: 975px;<br />
height:20px<br />
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.left-menu:hover {<br />
background-color: transparent;<br />
}<br />
<br />
.right-menu li a, .right-menu li a:hover {<br />
color: #dddddd;<br />
background-color: transparent;<br />
}<br />
<br />
<br />
<br />
#menubar {<br />
width: auto;<br />
}<br />
<br />
#p-logo{<br />
position: absolute;<br />
display: none;<br />
}<br />
<br />
#search-controls{<br />
display: none;<br />
}<br />
<br />
.firstHeading{<br />
display: none;<br />
}<br />
<br />
a{<br />
outline:none;<br />
}<br />
<br />
/*Page Controls(how wide the content section is within the window - background colour)*/ <br />
#content {<br />
width: 950x;<br />
border: none;<br />
font-family: helvetica, arial, sans-serif;<br />
color: #555555;<br />
}<br />
<br />
#footer-box {<br />
width:1075px;<br />
}<br />
</style><br />
<br />
<head><br />
<title>NRP UEA iGEM 2012</title><br />
<script type="text/javascript"><br />
<br />
var tabdropdown={<br />
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//No need to edit beyond here////////////////////////<br />
dropmenuobj: null, ie: document.all, firefox: document.getElementById&&!document.all, previousmenuitem:null,<br />
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this.dropmenuobj.onmouseover=function(){tabdropdown.clearhidemenu()}<br />
this.dropmenuobj.onmouseout=function(e){tabdropdown.dynamichide(e, obj)}<br />
this.dropmenuobj.onclick=function(){tabdropdown.delayhidemenu(obj)}<br />
this.showhide(this.dropmenuobj.style, e, obj)<br />
this.dropmenuobj.x=this.getposOffset(obj, "center")<br />
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this.dropmenuobj.style.left=this.dropmenuobj.x-this.clearbrowseredge(obj, "rightedge")+"px"<br />
this.dropmenuobj.style.top=this.dropmenuobj.y-this.clearbrowseredge(obj, "bottomedge")+obj.offsetHeight+1+"px"<br />
this.previousmenuitem=obj //remember main menu item mouse moved out from (and into current menu item)<br />
this.positionshim() //call iframe shim function<br />
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},<br />
<br />
contains_firefox:function(a, b) {<br />
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if ((b = b.parentNode) == a)<br />
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return false;<br />
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<br />
dynamichide:function(e, obj2){ //obj2 refers to tab menu item mouse is currently over<br />
var evtobj=window.event? window.event : e<br />
if (this.ie&&!this.dropmenuobj.contains(evtobj.toElement))<br />
this.delayhidemenu(obj2)<br />
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hideshim:function(){<br />
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isSelected:function(menuurl){<br />
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},<br />
<br />
init:function(menuid, dselected){<br />
this.standardbody=(document.compatMode=="CSS1Compat")? document.documentElement : document.body //create reference to common "body" across doctypes<br />
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<style><br />
.ddcolortabs{<br />
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<br />
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background: transparent url(media/color_tabs_right.gif) no-repeat right top;<br />
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<br />
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<br />
.ddcolortabs a:hover{<br />
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<br />
.ddcolortabs a:hover span{<br />
background-color: #00632d;<br />
}<br />
<br />
.ddcolortabs .selected a, #ddcolortabs .selected a span{ /*currently selected tab*/<br />
background-color: #00632d;<br />
}<br />
<br />
.ddcolortabsline{<br />
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padding: 0;<br />
width: 100%;<br />
height: 8px;<br />
line-height: 8px;<br />
background: #009443;<br />
border-top: 1px solid #fff; /*Remove this to remove border between bar and tabs*/<br />
}<br />
<br />
/* ######### Style for Drop Down Menu ######### */<br />
<br />
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position:absolute;<br />
top: 0;<br />
border: 1px solid black; /*THEME CHANGE HERE*/<br />
border-top-width: 8px; /*Top border width. Should match height of .ddcolortabsline above*/<br />
border-bottom-width: 0;<br />
font:normal 12px Arial;<br />
line-height:18px;<br />
z-index:100;<br />
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<br />
<br />
.dropmenudiv_a a{<br />
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text-indent: 5px;<br />
border-top: 0 solid #678b3f;<br />
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color: black;<br />
}<br />
<br />
* html .dropmenudiv_a a{ /*IE only hack*/<br />
width: 100%;<br />
}<br />
<br />
.dropmenudiv_a a:hover{ /*THEME CHANGE HERE*/<br />
background-color: #8a3c3d;<br />
color: white;<br />
}<br />
</style><br />
</head><br />
<!-- CSS for Drop Down Tabs Menu #1 --><br />
<link rel="stylesheet" type="text/css" href="ddcolortabs.css" /><br />
<br />
<div id="colortab" class="ddcolortabs"><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich" title="Home"><span>Home</span></a></li><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Team" rel="dropmenu1_a"><span>Team</span></a></li><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Project" title="Home"><span>Project</span></a></li><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Safety" rel="dropmenu1_a"><span>Safety</span></a></li><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Notebook" rel="dropmenu1_a"><span>Lab Book</span></a></li><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Parts" rel="dropmenu1_a"><span>Parts</span></a></li><br />
<br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Future" rel="dropmenu1_a"><span>The Future</span></a></li><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Quanticare" rel="dropmenu1_a"><span>Quanticare</span></a></li><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Human Outreach" rel="dropmenu1_a"><span>Human Practices</span></a></li><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Attributions" rel="dropmenu1_a"><span>Attributions</span></a></li><br />
</ul><br />
</div><br />
<div class="ddcolortabsline">&nbsp;</div><br />
<div id="dropmenu1_a" class="dropmenudiv_a"><br />
<a href="https://2008.igem.org/Team:Edinburgh/Team">Overview</a><br />
<a href="https://2008.igem.org/Team:Edinburgh/Team">Step1</a><br />
<a href="https://2008.igem.org/Team:Edinburgh/Team">Step2</a><br />
</div><br />
<script type="text/javascript"><br />
//SYNTAX: tabdropdown.init("menu_id", [integer OR "auto"])<br />
tabdropdown.init("colortab", 3)<br />
</script><br />
</body><br />
</html></div>Joyehickshttp://2012.igem.org/Template:UEANRPQuanticareTemplate:UEANRPQuanticare2012-09-26T09:37:15Z<p>Joyehicks: </p>
<hr />
<div>[[Image:Header1NewGreen.png|top]]<br />
<br />
<html><style><br />
#content a[href ^="https://"].external<br />
{<br />
background: center right no-repeat; padding-right: 18px;<br />
}</style></html><br />
<br />
<html><style type="text/css"><br />
#edi-twitter-widget .plainlinks{<br />
border: 1px solid #ccc;<br />
}<br />
#welcome a,<br />
#welcome a:hover,<br />
#welcome a:visited{<br />
color: #009443;<br />
}<br />
</style></html><br />
<br />
<html><br />
<br />
<style><br />
body{<br />
background-image:url("https://static.igem.org/mediawiki/2012/b/b2/WikiBGNewGreen.png");<br />
background-position:center top;<br />
background-repeat:no-repeat;<br />
background-color:#009443;<br />
<br />
}<br />
<br />
/*Browser History*/<br />
a:link {ccolor:#20548f;}<br />
a:visited {color:#20548f;}<br />
a:hover {color:#20548f;}<br />
a:active {color:#20548f;}<br />
<br />
#menubar.top-menu {<br />
position: absolute;<br />
top:0px;<br />
left:0px;<br />
}<br />
<br />
#menubar.slide-menu {<br />
position: absolute;<br />
top:0px;<br />
right:0px;<br />
}<br />
/*Top Control Bar*/ <br />
#menubar a:active,#menubar a:hover,#menubar a:link,#menubar a:visited {<br />
color: #1D1D1B;<br />
}<br />
<br />
<br />
/*Code to remove/overlay standard iGEM Header for blank canvas*/<br />
#top-section{<br />
background-color: #009443;<br />
height: 100px;<br />
border: none;<br />
width: 975px;<br />
height:20px<br />
}<br />
<br />
.left-menu:hover {<br />
background-color: transparent;<br />
}<br />
<br />
.right-menu li a, .right-menu li a:hover {<br />
color: #dddddd;<br />
background-color: transparent;<br />
}<br />
<br />
<br />
<br />
#menubar {<br />
width: auto;<br />
}<br />
<br />
#p-logo{<br />
position: absolute;<br />
display: none;<br />
}<br />
<br />
#search-controls{<br />
display: none;<br />
}<br />
<br />
.firstHeading{<br />
display: none;<br />
}<br />
<br />
a{<br />
outline:none;<br />
}<br />
<br />
/*Page Controls(how wide the content section is within the window - background colour)*/ <br />
#content {<br />
width: 950x;<br />
border: none;<br />
font-family: helvetica, arial, sans-serif;<br />
color: #555555;<br />
}<br />
<br />
#footer-box {<br />
width:1075px;<br />
}<br />
</style><br />
<br />
<head><br />
<title>NRP UEA iGEM 2012</title><br />
<script type="text/javascript"><br />
<br />
var tabdropdown={<br />
disappeardelay: 200, //set delay in miliseconds before menu disappears onmouseout<br />
disablemenuclick: false, //when user clicks on a menu item with a drop down menu, disable menu item's link?<br />
enableiframeshim: 1, //1 or 0, for true or false<br />
<br />
//No need to edit beyond here////////////////////////<br />
dropmenuobj: null, ie: document.all, firefox: document.getElementById&&!document.all, previousmenuitem:null,<br />
currentpageurl: window.location.href.replace("http://"+window.location.hostname, "").replace(/^\//, ""), //get current page url (minus hostname, ie: http://www.dynamicdrive.com/)<br />
<br />
getposOffset:function(what, offsettype){<br />
var totaloffset=(offsettype=="center")? what.offsetcenter : what.offsetTop;<br />
var parentEl=what.offsetParent;<br />
while (parentEl!=null){<br />
totaloffset=(offsettype=="center")? totaloffset+parentEl.offsetcenter : totaloffset+parentEl.offsetTop;<br />
parentEl=parentEl.offsetParent;<br />
}<br />
return totaloffset;<br />
},<br />
<br />
showhide:function(obj, e, obj2){ //obj refers to drop down menu, obj2 refers to tab menu item mouse is currently over<br />
if (this.ie || this.firefox)<br />
this.dropmenuobj.style.center=this.dropmenuobj.style.top="-500px"<br />
if (e.type=="click" && obj.visibility==hidden || e.type=="mouseover"){<br />
if (obj2.parentNode.className.indexOf("default")==-1) //if tab isn't a default selected one<br />
obj2.parentNode.className="selected"<br />
obj.visibility="visible"<br />
}<br />
else if (e.type=="click")<br />
obj.visibility="hidden"<br />
},<br />
<br />
iecompattest:function(){<br />
return (document.compatMode && document.compatMode!="BackCompat")? document.documentElement : document.body<br />
},<br />
<br />
clearbrowseredge:function(obj, whichedge){<br />
var edgeoffset=0<br />
if (whichedge=="rightedge"){<br />
var windowedge=this.ie && !window.opera? this.standardbody.scrollLeft+this.standardbody.clientWidth-15 : window.pageXOffset+window.innerWidth-15<br />
this.dropmenuobj.contentmeasure=this.dropmenuobj.offsetWidth<br />
if (windowedge-this.dropmenuobj.x < this.dropmenuobj.contentmeasure) //move menu to the left?<br />
edgeoffset=this.dropmenuobj.contentmeasure-obj.offsetWidth<br />
}<br />
else{<br />
var topedge=this.ie && !window.opera? this.standardbody.scrollTop : window.pageYOffset<br />
var windowedge=this.ie && !window.opera? this.standardbody.scrollTop+this.standardbody.clientHeight-15 : window.pageYOffset+window.innerHeight-18<br />
this.dropmenuobj.contentmeasure=this.dropmenuobj.offsetHeight<br />
if (windowedge-this.dropmenuobj.y < this.dropmenuobj.contentmeasure){ //move up?<br />
edgeoffset=this.dropmenuobj.contentmeasure+obj.offsetHeight<br />
if ((this.dropmenuobj.y-topedge)<this.dropmenuobj.contentmeasure) //up no good either?<br />
edgeoffset=this.dropmenuobj.y+obj.offsetHeight-topedge<br />
}<br />
this.dropmenuobj.firstlink.style.borderTopWidth=(edgeoffset==0)? 0 : "1px" //Add 1px top border to menu if dropping up<br />
}<br />
return edgeoffset<br />
},<br />
<br />
dropit:function(obj, e, dropmenuID){<br />
if (this.dropmenuobj!=null){ //hide previous menu<br />
this.dropmenuobj.style.visibility="hidden" //hide menu<br />
if (this.previousmenuitem!=null && this.previousmenuitem!=obj){<br />
if (this.previousmenuitem.parentNode.className.indexOf("default")==-1) //If the tab isn't a default selected one<br />
this.previousmenuitem.parentNode.className=""<br />
}<br />
}<br />
this.clearhidemenu()<br />
if (this.ie||this.firefox){<br />
obj.onmouseout=function(){tabdropdown.delayhidemenu(obj)}<br />
obj.onclick=function(){return !tabdropdown.disablemenuclick} //disable main menu item link onclick?<br />
this.dropmenuobj=document.getElementById(dropmenuID)<br />
this.dropmenuobj.onmouseover=function(){tabdropdown.clearhidemenu()}<br />
this.dropmenuobj.onmouseout=function(e){tabdropdown.dynamichide(e, obj)}<br />
this.dropmenuobj.onclick=function(){tabdropdown.delayhidemenu(obj)}<br />
this.showhide(this.dropmenuobj.style, e, obj)<br />
this.dropmenuobj.x=this.getposOffset(obj, "center")<br />
this.dropmenuobj.y=this.getposOffset(obj, "top")<br />
this.dropmenuobj.style.left=this.dropmenuobj.x-this.clearbrowseredge(obj, "rightedge")+"px"<br />
this.dropmenuobj.style.top=this.dropmenuobj.y-this.clearbrowseredge(obj, "bottomedge")+obj.offsetHeight+1+"px"<br />
this.previousmenuitem=obj //remember main menu item mouse moved out from (and into current menu item)<br />
this.positionshim() //call iframe shim function<br />
}<br />
},<br />
<br />
contains_firefox:function(a, b) {<br />
while (b.parentNode)<br />
if ((b = b.parentNode) == a)<br />
return true;<br />
return false;<br />
},<br />
<br />
dynamichide:function(e, obj2){ //obj2 refers to tab menu item mouse is currently over<br />
var evtobj=window.event? window.event : e<br />
if (this.ie&&!this.dropmenuobj.contains(evtobj.toElement))<br />
this.delayhidemenu(obj2)<br />
else if (this.firefox&&e.currentTarget!= evtobj.relatedTarget&& !this.contains_firefox(evtobj.currentTarget, evtobj.relatedTarget))<br />
this.delayhidemenu(obj2)<br />
},<br />
<br />
delayhidemenu:function(obj2){<br />
this.delayhide=setTimeout(function(){tabdropdown.dropmenuobj.style.visibility='hidden'; if (obj2.parentNode.className.indexOf('default')==-1) obj2.parentNode.className=''},this.disappeardelay) //hide menu<br />
},<br />
<br />
clearhidemenu:function(){<br />
if (this.delayhide!="undefined")<br />
clearTimeout(this.delayhide)<br />
},<br />
<br />
positionshim:function(){ //display iframe shim function<br />
if (this.enableiframeshim && typeof this.shimobject!="undefined"){<br />
if (this.dropmenuobj.style.visibility=="visible"){<br />
this.shimobject.style.width=this.dropmenuobj.offsetWidth+"px"<br />
this.shimobject.style.height=this.dropmenuobj.offsetHeight+"px"<br />
this.shimobject.style.center=this.dropmenuobj.style.center<br />
this.shimobject.style.top=this.dropmenuobj.style.top<br />
}<br />
this.shimobject.style.display=(this.dropmenuobj.style.visibility=="visible")? "block" : "none"<br />
}<br />
},<br />
<br />
hideshim:function(){<br />
if (this.enableiframeshim && typeof this.shimobject!="undefined")<br />
this.shimobject.style.display='none'<br />
},<br />
<br />
isSelected:function(menuurl){<br />
var menuurl=menuurl.replace("http://"+menuurl.hostname, "").replace(/^\//, "")<br />
return (tabdropdown.currentpageurl==menuurl)<br />
},<br />
<br />
init:function(menuid, dselected){<br />
this.standardbody=(document.compatMode=="CSS1Compat")? document.documentElement : document.body //create reference to common "body" across doctypes<br />
var menuitems=document.getElementById(menuid).getElementsByTagName("a")<br />
for (var i=0; i<menuitems.length; i++){<br />
if (menuitems[i].getAttribute("rel")){<br />
var relvalue=menuitems[i].getAttribute("rel")<br />
document.getElementById(relvalue).firstlink=document.getElementById(relvalue).getElementsByTagName("a")[0]<br />
menuitems[i].onmouseover=function(e){<br />
var event=typeof e!="undefined"? e : window.event<br />
tabdropdown.dropit(this, event, this.getAttribute("rel"))<br />
}<br />
}<br />
if (dselected=="auto" && typeof setalready=="undefined" && this.isSelected(menuitems[i].href)){<br />
menuitems[i].parentNode.className+=" selected default"<br />
var setalready=true<br />
}<br />
else if (parseInt(dselected)==i)<br />
menuitems[i].parentNode.className+=" selected default"<br />
}<br />
}<br />
<br />
}<br />
</script><br />
<style><br />
.ddcolortabs{<br />
padding: 0;<br />
width: 100%;<br />
background: transparent;<br />
voice-family: "\"}\"";<br />
voice-family: inherit;<br />
}<br />
<br />
.ddcolortabs ul{<br />
font: normal 16px Calibri, sans-serif;<br />
margin:0;<br />
padding:0;<br />
list-style:none;<br />
}<br />
<br />
.ddcolortabs li{<br />
display:inline;<br />
margin:0 2px 0 0;<br />
padding:0;<br />
text-transform:uppercase;<br />
}<br />
<br />
<br />
.ddcolortabs a{<br />
float: left;<br />
color: white;<br />
background: #009443 url(media/color_tabs_left.gif) no-repeat left top;<br />
margin:0 2px 0 0;<br />
padding:0 0 1px 2px;<br />
text-decoration:none;<br />
letter-spacing: 1px;<br />
}<br />
<br />
.ddcolortabs a span{<br />
float:left;<br />
display:block;<br />
background: transparent url(media/color_tabs_right.gif) no-repeat right top;<br />
padding: 4px 8px 2px 7px;<br />
}<br />
<br />
.ddcolortabs a span{<br />
float:none;<br />
}<br />
<br />
.ddcolortabs a:hover{<br />
background-color: #00632d;<br />
}<br />
<br />
.ddcolortabs a:hover span{<br />
background-color: #00632d;<br />
}<br />
<br />
.ddcolortabs .selected a, #ddcolortabs .selected a span{ /*currently selected tab*/<br />
background-color: #00632d;<br />
}<br />
<br />
.ddcolortabsline{<br />
clear: both;<br />
padding: 0;<br />
width: 100%;<br />
height: 8px;<br />
line-height: 8px;<br />
background: #009443;<br />
border-top: 1px solid #fff; /*Remove this to remove border between bar and tabs*/<br />
}<br />
<br />
/* ######### Style for Drop Down Menu ######### */<br />
<br />
.dropmenudiv_a{<br />
position:absolute;<br />
top: 0;<br />
border: 1px solid black; /*THEME CHANGE HERE*/<br />
border-top-width: 8px; /*Top border width. Should match height of .ddcolortabsline above*/<br />
border-bottom-width: 0;<br />
font:normal 12px Arial;<br />
line-height:18px;<br />
z-index:100;<br />
background-color: white;<br />
width: 200px;<br />
visibility: hidden;<br />
}<br />
<br />
<br />
.dropmenudiv_a a{<br />
width: auto;<br />
display: block;<br />
text-indent: 5px;<br />
border-top: 0 solid #678b3f;<br />
border-bottom: 1px solid #678b3f; /*THEME CHANGE HERE*/<br />
padding: 2px 0;<br />
text-decoration: none;<br />
color: black;<br />
}<br />
<br />
* html .dropmenudiv_a a{ /*IE only hack*/<br />
width: 100%;<br />
}<br />
<br />
.dropmenudiv_a a:hover{ /*THEME CHANGE HERE*/<br />
background-color: #8a3c3d;<br />
color: white;<br />
}<br />
</style><br />
</head><br />
<!-- CSS for Drop Down Tabs Menu #1 --><br />
<link rel="stylesheet" type="text/css" href="ddcolortabs.css" /><br />
<br />
<div id="colortab" class="ddcolortabs"><br />
<ul><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich" title="Home"><span>Home</span></a></li><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Team" rel="dropmenu1_a"><span>Team</span></a></li><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Project" title="Home"><span>Project</span></a></li><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Safety" rel="dropmenu1_a"><span>Safety</span></a></li><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Notebook" rel="dropmenu1_a"><span>Lab Book</span></a></li><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Parts" rel="dropmenu1_a"><span>Parts</span></a></li><br />
<br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Future" rel="dropmenu1_a"><span>Future Apps</span></a></li><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Quanticare" rel="dropmenu1_a"><span>Quanticare</span></a></li><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Human Outreach" rel="dropmenu1_a"><span>Human Practices</span></a></li><br />
<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich/Attributions" rel="dropmenu1_a"><span>Attributions</span></a></li><br />
</ul><br />
</div><br />
<div class="ddcolortabsline">&nbsp;</div><br />
<div id="dropmenu1_a" class="dropmenudiv_a"><br />
<a href="https://2008.igem.org/Team:Edinburgh/Team">Overview</a><br />
<a href="https://2008.igem.org/Team:Edinburgh/Team">Step1</a><br />
<a href="https://2008.igem.org/Team:Edinburgh/Team">Step2</a><br />
</div><br />
<script type="text/javascript"><br />
//SYNTAX: tabdropdown.init("menu_id", [integer OR "auto"])<br />
tabdropdown.init("colortab", 3)<br />
</script><br />
</body><br />
</html></div>Joyehickshttp://2012.igem.org/Template:UEANRPQuanticareTemplate:UEANRPQuanticare2012-09-26T09:36:47Z<p>Joyehicks: Created page with "<html> <style> body{ background-image:url("https://static.igem.org/mediawiki/2012/f/fc/NRPMainPageBG.png"); background-position:center top; background-repeat:no-repeat; background-col..."</p>
<hr />
<div><html><br />
<br />
<style><br />
body{<br />
background-image:url("https://static.igem.org/mediawiki/2012/f/fc/NRPMainPageBG.png");<br />
background-position:center top;<br />
background-repeat:no-repeat;<br />
background-color:#009443;<br />
<br />
}<br />
<br />
/*Browser History*/<br />
a:link {ccolor:#20548f;}<br />
a:visited {color:#20548f;}<br />
a:hover {color:#20548f;}<br />
a:active {color:#20548f;}<br />
<br />
#menubar.top-menu {<br />
position: absolute;<br />
top:0px;<br />
left:0px;<br />
}<br />
<br />
#menubar.slide-menu {<br />
position: absolute;<br />
top:0px;<br />
right:0px;<br />
}<br />
/*Top Control Bar*/ <br />
#menubar a:active,#menubar a:hover,#menubar a:link,#menubar a:visited {<br />
color: #ffffff;<br />
}<br />
<br />
<br />
/*Code to remove/overlay standard iGEM Header for blank canvas*/<br />
#top-section{<br />
background-color: 009443;<br />
height: 0px;<br />
border: none;<br />
width: 975px;<br />
height: 20px<br />
}<br />
<br />
.left-menu:hover {<br />
background-color: transparent;<br />
}<br />
<br />
.right-menu li a, .right-menu li a:hover {<br />
color: #dddddd;<br />
background-color: transparent;<br />
}<br />
<br />
<br />
<br />
#menubar {<br />
width: auto;<br />
}<br />
<br />
#p-logo{<br />
position: absolute;<br />
display: none;<br />
}<br />
<br />
#search-controls{<br />
display: none;<br />
}<br />
<br />
.firstHeading{<br />
display: none;<br />
}<br />
<br />
a{<br />
outline:none;<br />
}<br />
<br />
/*Page Controls(how wide the content section is within the window - background colour)*/ <br />
#content {<br />
width: 950x;<br />
border: none;<br />
font-family: helvetica, arial, sans-serif;<br />
color: #009443;<br />
}<br />
<br />
#footer-box {<br />
width:1075px;<br />
}<br />
</style><br />
</body><br />
</html></div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/QuanticareTeam:NRP-UEA-Norwich/Quanticare2012-09-26T09:35:23Z<p>Joyehicks: Created page with "{{UEANRPQuanticare}}"</p>
<hr />
<div>{{UEANRPQuanticare}}</div>Joyehickshttp://2012.igem.org/Template:UEANRPTemplate:UEANRP2012-09-26T09:35:05Z<p>Joyehicks: </p>
<hr />
<div>[[Image:Header1NewGreen.png|top]]<br />
<br />
<html><style><br />
#content a[href ^="https://"].external<br />
{<br />
background: center right no-repeat; padding-right: 18px;<br />
}</style></html><br />
<br />
<html><style type="text/css"><br />
#edi-twitter-widget .plainlinks{<br />
border: 1px solid #ccc;<br />
}<br />
#welcome a,<br />
#welcome a:hover,<br />
#welcome a:visited{<br />
color: #009443;<br />
}<br />
</style></html><br />
<br />
<html><br />
<br />
<style><br />
body{<br />
background-image:url("https://static.igem.org/mediawiki/2012/b/b2/WikiBGNewGreen.png");<br />
background-position:center top;<br />
background-repeat:no-repeat;<br />
background-color:#009443;<br />
<br />
}<br />
<br />
/*Browser History*/<br />
a:link {ccolor:#20548f;}<br />
a:visited {color:#20548f;}<br />
a:hover {color:#20548f;}<br />
a:active {color:#20548f;}<br />
<br />
#menubar.top-menu {<br />
position: absolute;<br />
top:0px;<br />
left:0px;<br />
}<br />
<br />
#menubar.slide-menu {<br />
position: absolute;<br />
top:0px;<br />
right:0px;<br />
}<br />
/*Top Control Bar*/ <br />
#menubar a:active,#menubar a:hover,#menubar a:link,#menubar a:visited {<br />
color: #1D1D1B;<br />
}<br />
<br />
<br />
/*Code to remove/overlay standard iGEM Header for blank canvas*/<br />
#top-section{<br />
background-color: #009443;<br />
height: 100px;<br />
border: none;<br />
width: 975px;<br />
height:20px<br />
}<br />
<br />
.left-menu:hover {<br />
background-color: transparent;<br />
}<br />
<br />
.right-menu li a, .right-menu li a:hover {<br />
color: #dddddd;<br />
background-color: transparent;<br />
}<br />
<br />
<br />
<br />
#menubar {<br />
width: auto;<br />
}<br />
<br />
#p-logo{<br />
position: absolute;<br />
display: none;<br />
}<br />
<br />
#search-controls{<br />
display: none;<br />
}<br />
<br />
.firstHeading{<br />
display: none;<br />
}<br />
<br />
a{<br />
outline:none;<br />
}<br />
<br />
/*Page Controls(how wide the content section is within the window - background colour)*/ <br />
#content {<br />
width: 950x;<br />
border: none;<br />
font-family: helvetica, arial, sans-serif;<br />
color: #555555;<br />
}<br />
<br />
#footer-box {<br />
width:1075px;<br />
}<br />
</style><br />
<br />
<head><br />
<title>NRP UEA iGEM 2012</title><br />
<script type="text/javascript"><br />
<br />
var tabdropdown={<br />
disappeardelay: 200, //set delay in miliseconds before menu disappears onmouseout<br />
disablemenuclick: false, //when user clicks on a menu item with a drop down menu, disable menu item's link?<br />
enableiframeshim: 1, //1 or 0, for true or false<br />
<br />
//No need to edit beyond here////////////////////////<br />
dropmenuobj: null, ie: document.all, firefox: document.getElementById&&!document.all, previousmenuitem:null,<br />
currentpageurl: window.location.href.replace("http://"+window.location.hostname, "").replace(/^\//, ""), //get current page url (minus hostname, ie: http://www.dynamicdrive.com/)<br />
<br />
getposOffset:function(what, offsettype){<br />
var totaloffset=(offsettype=="center")? what.offsetcenter : what.offsetTop;<br />
var parentEl=what.offsetParent;<br />
while (parentEl!=null){<br />
totaloffset=(offsettype=="center")? totaloffset+parentEl.offsetcenter : totaloffset+parentEl.offsetTop;<br />
parentEl=parentEl.offsetParent;<br />
}<br />
return totaloffset;<br />
},<br />
<br />
showhide:function(obj, e, obj2){ //obj refers to drop down menu, obj2 refers to tab menu item mouse is currently over<br />
if (this.ie || this.firefox)<br />
this.dropmenuobj.style.center=this.dropmenuobj.style.top="-500px"<br />
if (e.type=="click" && obj.visibility==hidden || e.type=="mouseover"){<br />
if (obj2.parentNode.className.indexOf("default")==-1) //if tab isn't a default selected one<br />
obj2.parentNode.className="selected"<br />
obj.visibility="visible"<br />
}<br />
else if (e.type=="click")<br />
obj.visibility="hidden"<br />
},<br />
<br />
iecompattest:function(){<br />
return (document.compatMode && document.compatMode!="BackCompat")? document.documentElement : document.body<br />
},<br />
<br />
clearbrowseredge:function(obj, whichedge){<br />
var edgeoffset=0<br />
if (whichedge=="rightedge"){<br />
var windowedge=this.ie && !window.opera? this.standardbody.scrollLeft+this.standardbody.clientWidth-15 : window.pageXOffset+window.innerWidth-15<br />
this.dropmenuobj.contentmeasure=this.dropmenuobj.offsetWidth<br />
if (windowedge-this.dropmenuobj.x < this.dropmenuobj.contentmeasure) //move menu to the left?<br />
edgeoffset=this.dropmenuobj.contentmeasure-obj.offsetWidth<br />
}<br />
else{<br />
var topedge=this.ie && !window.opera? this.standardbody.scrollTop : window.pageYOffset<br />
var windowedge=this.ie && !window.opera? this.standardbody.scrollTop+this.standardbody.clientHeight-15 : window.pageYOffset+window.innerHeight-18<br />
this.dropmenuobj.contentmeasure=this.dropmenuobj.offsetHeight<br />
if (windowedge-this.dropmenuobj.y < this.dropmenuobj.contentmeasure){ //move up?<br />
edgeoffset=this.dropmenuobj.contentmeasure+obj.offsetHeight<br />
if ((this.dropmenuobj.y-topedge)<this.dropmenuobj.contentmeasure) //up no good either?<br />
edgeoffset=this.dropmenuobj.y+obj.offsetHeight-topedge<br />
}<br />
this.dropmenuobj.firstlink.style.borderTopWidth=(edgeoffset==0)? 0 : "1px" //Add 1px top border to menu if dropping up<br />
}<br />
return edgeoffset<br />
},<br />
<br />
dropit:function(obj, e, dropmenuID){<br />
if (this.dropmenuobj!=null){ //hide previous menu<br />
this.dropmenuobj.style.visibility="hidden" //hide menu<br />
if (this.previousmenuitem!=null && this.previousmenuitem!=obj){<br />
if (this.previousmenuitem.parentNode.className.indexOf("default")==-1) //If the tab isn't a default selected one<br />
this.previousmenuitem.parentNode.className=""<br />
}<br />
}<br />
this.clearhidemenu()<br />
if (this.ie||this.firefox){<br />
obj.onmouseout=function(){tabdropdown.delayhidemenu(obj)}<br />
obj.onclick=function(){return !tabdropdown.disablemenuclick} //disable main menu item link onclick?<br />
this.dropmenuobj=document.getElementById(dropmenuID)<br />
this.dropmenuobj.onmouseover=function(){tabdropdown.clearhidemenu()}<br />
this.dropmenuobj.onmouseout=function(e){tabdropdown.dynamichide(e, obj)}<br />
this.dropmenuobj.onclick=function(){tabdropdown.delayhidemenu(obj)}<br />
this.showhide(this.dropmenuobj.style, e, obj)<br />
this.dropmenuobj.x=this.getposOffset(obj, "center")<br />
this.dropmenuobj.y=this.getposOffset(obj, "top")<br />
this.dropmenuobj.style.left=this.dropmenuobj.x-this.clearbrowseredge(obj, "rightedge")+"px"<br />
this.dropmenuobj.style.top=this.dropmenuobj.y-this.clearbrowseredge(obj, "bottomedge")+obj.offsetHeight+1+"px"<br />
this.previousmenuitem=obj //remember main menu item mouse moved out from (and into current menu item)<br />
this.positionshim() //call iframe shim function<br />
}<br />
},<br />
<br />
contains_firefox:function(a, b) {<br />
while (b.parentNode)<br />
if ((b = b.parentNode) == a)<br />
return true;<br />
return false;<br />
},<br />
<br />
dynamichide:function(e, obj2){ //obj2 refers to tab menu item mouse is currently over<br />
var evtobj=window.event? window.event : e<br />
if (this.ie&&!this.dropmenuobj.contains(evtobj.toElement))<br />
this.delayhidemenu(obj2)<br />
else if (this.firefox&&e.currentTarget!= evtobj.relatedTarget&& !this.contains_firefox(evtobj.currentTarget, evtobj.relatedTarget))<br />
this.delayhidemenu(obj2)<br />
},<br />
<br />
delayhidemenu:function(obj2){<br />
this.delayhide=setTimeout(function(){tabdropdown.dropmenuobj.style.visibility='hidden'; if (obj2.parentNode.className.indexOf('default')==-1) obj2.parentNode.className=''},this.disappeardelay) //hide menu<br />
},<br />
<br />
clearhidemenu:function(){<br />
if (this.delayhide!="undefined")<br />
clearTimeout(this.delayhide)<br />
},<br />
<br />
positionshim:function(){ //display iframe shim function<br />
if (this.enableiframeshim && typeof this.shimobject!="undefined"){<br />
if (this.dropmenuobj.style.visibility=="visible"){<br />
this.shimobject.style.width=this.dropmenuobj.offsetWidth+"px"<br />
this.shimobject.style.height=this.dropmenuobj.offsetHeight+"px"<br />
this.shimobject.style.center=this.dropmenuobj.style.center<br />
this.shimobject.style.top=this.dropmenuobj.style.top<br />
}<br />
this.shimobject.style.display=(this.dropmenuobj.style.visibility=="visible")? "block" : "none"<br />
}<br />
},<br />
<br />
hideshim:function(){<br />
if (this.enableiframeshim && typeof this.shimobject!="undefined")<br />
this.shimobject.style.display='none'<br />
},<br />
<br />
isSelected:function(menuurl){<br />
var menuurl=menuurl.replace("http://"+menuurl.hostname, "").replace(/^\//, "")<br />
return (tabdropdown.currentpageurl==menuurl)<br />
},<br />
<br />
init:function(menuid, dselected){<br />
this.standardbody=(document.compatMode=="CSS1Compat")? document.documentElement : document.body //create reference to common "body" across doctypes<br />
var menuitems=document.getElementById(menuid).getElementsByTagName("a")<br />
for (var i=0; i<menuitems.length; i++){<br />
if (menuitems[i].getAttribute("rel")){<br />
var relvalue=menuitems[i].getAttribute("rel")<br />
document.getElementById(relvalue).firstlink=document.getElementById(relvalue).getElementsByTagName("a")[0]<br />
menuitems[i].onmouseover=function(e){<br />
var event=typeof e!="undefined"? e : window.event<br />
tabdropdown.dropit(this, event, this.getAttribute("rel"))<br />
}<br />
}<br />
if (dselected=="auto" && typeof setalready=="undefined" && this.isSelected(menuitems[i].href)){<br />
menuitems[i].parentNode.className+=" selected default"<br />
var setalready=true<br />
}<br />
else if (parseInt(dselected)==i)<br />
menuitems[i].parentNode.className+=" selected default"<br />
}<br />
}<br />
<br />
}<br />
</script><br />
<style><br />
.ddcolortabs{<br />
padding: 0;<br />
width: 100%;<br />
background: transparent;<br />
voice-family: "\"}\"";<br />
voice-family: inherit;<br />
}<br />
<br />
.ddcolortabs ul{<br />
font: normal 16px Calibri, sans-serif;<br />
margin:0;<br />
padding:0;<br />
list-style:none;<br />
}<br />
<br />
.ddcolortabs li{<br />
display:inline;<br />
margin:0 2px 0 0;<br />
padding:0;<br />
text-transform:uppercase;<br />
}<br />
<br />
<br />
.ddcolortabs a{<br />
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<li><a href="https://2012.igem.org/Team:NRP-UEA-Norwich" title="Home"><span>Home</span></a></li><br />
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<a href="https://2008.igem.org/Team:Edinburgh/Team">Overview</a><br />
<a href="https://2008.igem.org/Team:Edinburgh/Team">Step1</a><br />
<a href="https://2008.igem.org/Team:Edinburgh/Team">Step2</a><br />
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</html></div>Joyehickshttp://2012.igem.org/Template:UEANRPTemplate:UEANRP2012-09-26T09:29:42Z<p>Joyehicks: </p>
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<div class="ddcolortabsline">&nbsp;</div><br />
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<a href="https://2008.igem.org/Team:Edinburgh/Team">Overview</a><br />
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<a href="https://2008.igem.org/Team:Edinburgh/Team">Step2</a><br />
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</html></div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/Human_OutreachTeam:NRP-UEA-Norwich/Human Outreach2012-09-25T22:53:21Z<p>Joyehicks: /* Newspapers */</p>
<hr />
<div>{{UEANRP}}<br />
<br />
'''RACHEL TO FINISH FILM STUFF, RUSSELL TO PUT IN SOCIAL MEDIA'''<br />
<br />
<br />
=Human Practices=<br />
<br />
<br />
The UEA NRP team really went up and beyond this year, caring out a wide selection of activity's to inform the public of both synthetic biology and iGEM, aiming to raise awareness of this branch of science. We used many methods of communication in order to cater for a range of audiences, including a radio show, newspaper articles, pubic events and talks as well as collaborating with all the other UK teams, in order to organize the UK team meet up and steam it live on the internet. <br />
<br />
<br />
=== Creation of fictious futurist company, Quanticare===<br />
<br />
<br />
[[File:Tattoo1.png |300px]][[File:QuanticareLogo_(2).png |300px]][[File:Tattoo2.png |300px]]<br />
<br />
<br />
The team created Quanticare, a fictitious company set in the future that had formed as a result of the UEA NRP iGEM 2012 team. The fictitious company's latest development is to introduce a visual bio sensors in to the human body,in the form of a tattoo based health moniture, Cura. Not only did the development of the fictitious company allow the team to explore potential future applications of synthetic biology and bio sensors, but also via the creation of a film , fuel a range of ethical questions.The team used novel marketing techniques, such as sending a range of transferable tattoos, representing the product Cure, to the other UK teams. They did this aiming to raise interest in the NRP UEA iGEM project and through curiosity of finding out what the different tattoos symbolized attracting fellow iGEMers to look at our wiki and therefore develop an interest in our presentation and poster at the European jamboree.<br />
<br />
=== The team collaborated with Artist Amy to create this fictitious Film ===<br />
<br />
The team worked closely with their artist Amy in order to produce a futurist film, demonstrating one of the many possible future potentials of our project, as well as synthetic biology as a whole.Together Amy and the team explored a range of potential ways to grab the public's attention, as well as fuel ethical thoughts. They decided that inventing a fictitious future business (Quanticare) which had used the developments of the teams project as a stepping stone towards a futuristic personal health monitoring tattoo would be a very visual and appealing method of communication. The film was released at the teams public event held at the forum in Norwich, attracting lots of attention due to both the plot of the film, and its artistic touches. Watch the film for yourself at this link (insert link).<br />
<br />
<br />
<br />
[[File:Mish mash.png | 300px| centre]]<br />
<br />
<br />
<br />
===The iGEM UK team meet up, hosted by the NRP-UEA team at Google campus London (Friday 17th August)===<br />
<br />
<br />
[[File:UKiGEMteams.jpg | 300px | right]]<br />
<br />
<br />
The NRPUEA iGEM team organised and hosted the UK team meet up at Google campus in London. The NRPUEA iGEM team greeted the UK teams, provided refreshments and a lovely buffet lunch, as well as chairing the speeches and making sure the day ran smoothly. There was a great atmosphere at the meet up, and the event was a huge success.<br />
<br />
<br />
The day consisted of a number of guest speakers, including advice and tips for iGEM success from Tom Ellis (advisor of the 2009 winning iGEM project E.chromi) and an interactive talk on synthetic biology and science communication from the BBC science presenter, Nature editor and Guardian writer,Adam Rutherford. The UK iGEM teams presented a poster and a 15 minute presentation about their project. This gave the members of each team the chance to practice their presentation skills before the European team meet up, as well as a chance for others to ask questions and see what other project routes the other teams have taken. <br />
<br />
<br />
The event was also available live on the internet via Google hangout, to allow the public and those that were unable to attend to still watch the presentations. Despite there being a few technical difficulties with the quality of the image, figures have shown that over 600 people watched the event.<br />
<br />
<br />
[https://dl.dropbox.com/u/9957127/UK%20iGEM%20Team%20Hangout%2017th%20Aug%202012%20Programme.pdf '''Click here for programme PDF''']<br />
<br />
<br />
<html><table align=center cellspacing=0 cellpadding=0 width=100%><tr><td valign=absmiddle><br />
<iframe width="560" height="315" src="http://www.youtube.com/embed/Kv_Z14OIrKc" frameborder="0" allowfullscreen></iframe></td></tr></table></html><br />
<br />
<br />
<br />
===The Future of Science event held at the forum, Norwich (Sunday 19th August)===<br />
<br />
<br />
<br />
[[File:Forum_team.JPG | 300px| right]]<br />
<br />
<br />
The Forum is located at the heart of the city of Norwich, and hosts a wide range of free events and exhibits to be enjoyed by the whole community. The venue is packed with restaurants, cafes and shops, as well as being home to Norwich library and BBC East Anglia. <br />
<br />
The NRPUEA iGEM team were fortunate enough to hold an interactive day at the Forum, giving the public a chance to engage within the world of synthetic biology. Before the date of the event, the team increased awareness via a range of techniques, including posters, leaflets and newspaper and internet adverts, including the Forums website. This resulted with a large turnout at the event, including the local press. <br />
<br />
At the Future of Science event, the team presented a selection of informative posters, revealed their new film on a large screen, as well as showing examples of work carried out by the students during iGEM. For example, the public could view ''E.coli'' which had been transformed with GFP within a UV light box, as well as carrying out a counting colonies exercise. The NRPUEA team also created a range of synthetic biology educational material for the event, to inform the public of the concepts and terminology of synthetic biology. The different activities were designed to interest a selection of different ages. The educational resources include a range of quizzes and worksheets such as crosswords and matching exercises.<br />
<br />
[[File:Child_making_plasmid.jpg |300px | left]]<br />
<br />
For younger children we designed a colourful jigsaw plasmid (as seen in image). The idea of this activity was for the participants to choose jigsaw pieces that represented the genes that code for certain characteristics, such as green or red hair, and piece them together to form a complete plasmid. Once they had designed their plasmid they made a biscuit creature that corresponded to the plasmid they had created. The learning outcome of this activity was to give the children an understanding that a different selection of genes, results in different characteristics, and that editing the DNA can result in a change in the characteristics. The activity was very popular and most of the participants seemed to understand this concept, as well as having great fun.<br />
<br />
The event attracted a range of ages, as well as mixed levels of previous interest into synthetic biology. It also became clear that many members of the public knew very little about synthetic biology, and therefore were very interested in finding out more about the new branch of biology and the iGEM competition. The NRPUEA iGEM team also took the opportunity to talk to the public and listen to their opinions about the work the iGEM teams are carrying out, which demonstrated how mixed the view of the public were. This was again another great day, and a huge success.<br />
<br />
'''Synthetic Biology activity sheets'''<br />
<br />
[[File:Word_Search_of_Synthetic_Biology.pdf]],<br />
[[File:Decode_Disney.pdf]],<br />
[[File:Questions_comprehensive.pdf]],[[File:Decode_Music_Artists_and_Films.pdf]],[[File:Word_Search_of_Synthetic_Biology.pdf]]<br />
<br />
===STAR Radio===<br />
<br />
[http://www.star107.co.uk/farming-show.php STAR 107.9 FM's Farming Show]<br />
<br />
[[Image:NRPSTARRadio.jpg | 300px | right]]<br />
<br />
[http://www.star107.co.uk/podcasts/the-farming-show/show-23.mp3 Listen To Our Radio Show Appearence!]<br />
<br />
In early July the NRP UEA team travelled to Cambridge to appear on STAR Radio's "The Business Hub" and "The Farming Show". They were interviewed by Mark, one of the presenters, about synthetic biology, iGEM, and our project. The interview went really well as Pascoe and Khadija got across all the information that was needed in a clear and concise way, and Mark was extremely helpful in making sure all the salient points of the project were brought out. <br />
<br />
The team were able to give a brief description of what synthetic biology is before talking about the future applications of synthetic biology for medicine, agriculture and business. We were also able to mention our event at the Norwich Forum in late August in order to help promote it!<br />
<br />
A big thanks goes out to all at STAR Radio 107.9 for their help with the project in setting up the interview and allowing us air time to discuss iGEM and synthetic biology. Please visit the link above to hear the radio show.<br />
<br />
=== The team presented at the John Innes Centre Synthetic Biology conference ===<br />
<br />
The john innes center (JIC) is a world leading center of plant research, the home of UEAs 2011 team, and is part of the Norwich research part. Researchers at the JIC recognized that synthetic biology was an important field and therefore hosted a synthetic biology confrere, with the aim of the day to enhance their reserches knowledge of what synthetic biology is, and projects in which synthetic biology is being used. when the NRP-UEA was invited to give a talk to the JIC researchers we were extremely excited, giving them an over view of what iGEM was, our project and how it developed over the summer, as well informing them about aspects of human practice the NRP- UEA team had carried out over the summer. the team took also showed them a clip of our futuristic applications film. The team enjoyed presenting their work and results, as well as using it as an opportunity to improve their presentation skills before the European jamboree. There seemed to be a real interest by their researchers in to our project, giving encouraging advice, with many asking further questions after the presentation. The researchers also seemed very interested to hear more about our human practices elements after the presentation, wanting to know what the public's opinion of the research carried out at Norwich research park was, as well as synthetic biology as a whole.<br />
<br />
===Social Media===<br />
The NRPUEA iGEM team has been VERY active on a range of social media over the summer.Any one is welcome to follow the team on Facebook or twitter. The team keep everyone updated on what is going on, both in the lab and in our spare time. This has been a great method of connecting with the public, companies,researchers, as well as getting in touch with other iGEM teams. The NRPUEA iGEM team believe human out reach via social media is important since it allows the public to follow the teams achievements and lab results, as well as get to know the personality of the team members. please follow us on twitter @NRPiGEM or UEA iGEM 2012 page on Facebook.<br />
<br />
creating social media pages also allowed us to access information about who was checking our page out, showing interesting information, showing that social media techniques such as facebook attracted mostly 18-24 years olds interest, and that it attracted no attention from those above 65. Therefore the team though it was important to promote our project, not only through the internet, but also other techniques such as newspaper articles and radio shows. <br />
<br />
The figure bellow hows the age ranges and gender of people who liked any posts from the NRP UEA iGEM team<br />
[[File:Likes.png |600px| centre]]<br />
<br />
Below is a list of the location that people liked the teams statuses from. This highlighted that the majority of followers were located in Norwich, however also many from a range of places over the world, therefore highlighting the importance of using such social media to reach these followers. <br />
[[File:Location_of_likes.png |600px| centre]]<br />
<br />
As mentioned the NRP UEA iGEM team made facebook statuses and tweets very regularly and as the graph below shows, there was consistently a increase in action and interest for the NRPUEA iGEM team after. <br />
<br />
[[File:Posts_ratings.png |600px| centre]]<br />
<br />
===SGM microbiology synthetic biology (September 3rd -5th)===<br />
<br />
The society of general microbiology held a 3 day synthetic biology conference at the University of Warwick. Three members of the UEA NRP iGEM team were lucky enough to go and enjoy talks on streptococcus, molecular motors, and the dynamics of the genome and designer microbes. The event also gave the team member the opportunity to meet other synthetic biology scientists as well as informing them of the current advances in the field and inspiring them with the future possibilities. The event also allowed the team members to present a poster and therefore engage with the scientific community, both informing them more about the iGEM competition and our project . The team believed it was important for the scientists in the synthetic biology world to have the opportunity to meet undergraduate iGEM members, allowing them to express the skills it has allowed them to develop and to encourage further interest in to lab groups forming teams, and funding iGEM projects.<br />
<br />
===Newspapers===<br />
[[File:Paper.jpg|200px|left]]<br />
The team featured on the university website a few times through the summer, as well as a brief article in the Eastern daily press newspaper.This was great, as allowed the locals of Norwich to become aware not only of the team and iGEM, but also introduce many to the scientific branch of synthetic biology.</div>Joyehickshttp://2012.igem.org/File:Paper.jpgFile:Paper.jpg2012-09-25T22:52:14Z<p>Joyehicks: </p>
<hr />
<div></div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/ExperimentsTeam:NRP-UEA-Norwich/Experiments2012-09-25T20:42:53Z<p>Joyehicks: /* Flexibility of the hybrid promoters: BM and MB */</p>
<hr />
<div>{{UEANRP}}<br />
<br />
{{UEANRPLabs}}<br />
<br />
=Characterisation Experiments=<br />
<br />
The following experiments as the title suggests are the experiments carried out to characterise both our BioBricks and others. In total we characterised PyeaR, BM and MB as well as gained experience in use of CFP and RFP. The experiments written below are not in chronological order as we received the BioBricks at different points of our project. Given more time, there are further characterisation studies we would like to carry out. These are also described below.<br />
<br />
==Functionality Studies==<br />
<br />
===Flexibility of the hybrid promoters: BM and MB===<br />
<br />
The hybrid promoters, BM and MB were created to increase the flexibility of chassis a promoter can be used in. To fully characterise the functionality of BM and MB, it was therefore, important that it can in fact work within mammalian and bacterial chassis. Through transformations and successful growth in potassium nitrate, it has been shown that BM and MB both function in bacterial cells. <br />
<br />
To incorporate BM and MB into a mammalian system, transformations were not possible, instead a transfection was carried out. Unlike the growth studies, where growing colonies were sufficient to prove that the DNA within plasmids were incorporated, reporter proteins: CFP and RFP were attached to allow visual characterisation. <br />
<br />
To transfect the cells we used lipid based transfection. To do this, media, transfection reagent, nitric oxide donor, DNA and cells were required. Below is the full list of reagents used:<br />
<br />
. DMEM (Dulbecco’s Modified Eagle Medium) without serum to transfect and later treated with DMEM with serum and 10% Fetal Calf Serum. Cells are transfected without serum as serum interferes with the process but cells can die without serum so later is treated with serum.<br />
<br />
. M-B +CFP was the DNA used. More than one sample was used. From nanodropping the concentrations of DNA were found to be both 500nm/µL. To transfect, 6.5 µL of DNA was used. The exact DNA we used was labelled MB2-C11a and MB2-C12a. The DNA was obtained from different colonies from the same plate.<br />
<br />
. SNAP (S-Nitroso-N-Acetylpenicillamine) at a final concentration of 500µM. SNAP is the nitric oxide donor. Unlike bacterial cells where potassium nitrate could be used as a direct source. SNAP is metabolised by cells to produce NO which then induces the BioSensor.<br />
<br />
. LipoD293 which is the transfection agent. This creates a membrane around the DNA which then binds to the mammalian cell and allows entrance of DNA into cell. This is much like endocytosis. <br />
<br />
. The chassis used was MCF7 which is a human breast cancer cell line. The cells (30 µL) were seeded into a 6 channel slide at a concentration of 3 x 105 cells/ml.<br />
<br />
In the process of transfection, LipoD293 was mixed with DNA at left for 15mins. Meanwhile, the media was removed from the cells and washed with serum free DMEM (100 µL). The transfection mixture was then added to the relevant channels on the slide. Below is how our channels were labelled.<br />
<br />
<br />
[[File:slide wells labelled.png | 500px | center]]<br />
<br />
For the full transfection protocol please click [https://2012.igem.org/Team:NRP-UEA-Norwich/Experiments '''here''']<br />
<br />
After the transfection, the media was changed to media containing serum and also SNAP was added. The cells were then viewed and the following images were obtained.<br />
<br />
[[File: Transfection.png | thumb | 500px |center | '' Transfection of MCF7 cells. Images in the left two columns are controls and have not been transfected, images in the right two columns have been transfected with MB2-C11a DNA'']]<br />
<br />
The images show what looks like exclusion bodies which have a greater concentration of fluorescent proteins than within cells. In the control without SNAP, there are none of these exclusion bodies found. In the control photo with SNAP and also the transfected cells without SNAP added, there are a few exclusion bodies. However, in the photos that showed transfected cells with SNAP added, there are a large number of these.<br />
<br />
We do not know for certain what these may be, but a possibility is that the transfection was successful and the MB promoter does work. It may be that due to MB attachment to fluorescent proteins, the cells are producing exclusion bodies to rid the cells of these. In the control with SNAP, the cancer cells react to NO in the human body. These may be exclusion bodies formed from cells in general in reaction of NO. As to the transfected cell with exclusion bodies, there are very few of these. MB may be very sensitive to NO. As NO is naturally produced by cancer cells to induce angiogenesis, these may be for that reason.<br />
<br />
Another possibility is that NO has induced the cells to apoptose and this has lead to vesicles forming containing the fluorescent proteins (Yu, ''et al''., 1999). In non transfected cells, there is less fluorescent proteins compared to the transfected cells and hence there are more fluorescing vesicles.<br />
<br />
Following transfection, to test the cytotoxicity of NO, the number of cells after addition of SNAP was calculated. The MCF7 cells were seeded into 6 well plates again at a concentration of 2.5 x 105 cells/ml. SNAP was then added at 500µM 2 days after plating. The cells were counted 24 hours after the addition of SNAP. For the full cell count protocol please click [https://2012.igem.org/Team:NRP-UEA-Norwich/Protocol#Cell_Counting '''here'''].<br />
<br />
From previous studies such as that by Lala and Chakraborty, 2001 have shown that NO can lead to cytostasis and apoptosis. Our assay further confirmed this.<br />
<br />
<br />
References:<br />
<br />
Lala, P.K. and Chakraborty, C. (2001) 'Role of nitricoxide in carcinogenesis and tumour progression', ''The Lancelet'', 2;149–156.<br />
<br />
Yu, W., Simmons-Menchaca, M., Gapor, A., Sanders, B.G. and Kline, K. (1999) 'Induction of Apoptosis in Human Breast<br />
Cancer Cells by Tocopherols and Tocotrienols', ''Nutrition and Cancer'', 33;26-32.<br />
<br />
===Cell Identification===<br />
<br />
===Study of BM and MB in different concentrations of potassium nitrate===<br />
<br />
Following the experiment measuring the affect on the intensity of GFP fluorescence of PyeaR-GFP at different concentrations of potassium nitrate, a study on how different levels of potassium nitrate affect fluorescence intensity of BM and MB attached to fluorescent proteins was tested. Where previously the study was carried out in a qualitative fashion, this was carried out to collect data quantitatively using a fluorometer. <br />
Overnight cultures of BM and MB attached to RFPs and CFPs containing different levels of potassium nitrate prepared. Using 5ml of the cultures, cells were peletted. The overnight cultures contained different concentrations of potassium nitrate (0mM, 5mM, 10mM, 15mM and 20mM). These cells were then resuspended and lysed (full protocol on protocol page). These cultures were then diluted using Tris buffer (0.5ml sample to 1.5ml buffer). Readings from the fluorometer wavescans were recorded. <br />
<br />
Comparing BM and MB with CFP fluorescence showed that BM-CFP intensity is relatively low when compared to MB. MB seems more sensitive to nitrate levels than BM. However, for both, it seems that once a threshold of nitrate levels is reached, there is not additional expression. Beyond this concentration, the intensity drops. This may be due to a number of factors. The cells may die due to overload of proteins which may have a toxic affect. For BM this threshold seems to be just above 20mM and for MB this appears to be around 15mM. This is seen in the figure below.<br />
<br />
[[File:CFPgraph.png | 500px | center]]<br />
<br />
Comparing BM and MB with RFP fluorescence showed a similar result to the CFP study. However, both BM and MB have similar sensitivities to potassium nitrate levels, BM seems to be more sensitive to potassium nitrate concentration this time. From this study it is clear that beyond a certain concentration of nitrates, the fluorescence intensity stops increasing and decreases instead. Again, this may be due to the capacity or tolerance of the cell to hold the volume of fluorescence. protein. <br />
<br />
[[File:RFPgraph.png | 500px | center]]<br />
<br />
<br />
==Growth Studies==<br />
<br />
===A study of growth of PyeaR in different concentrations of potassium nitrate===<br />
<br />
===A comparison between the growth of E.coli cells, before and after transformation with PyeaR + GFP (BBa_K381001) and B-M and M-B (in pSB1C3)===<br />
<br />
The study involved testing the affects of transforming E.coli with different promoters on its growth over time. The promoters E.coli had been transformed with were PyeaR, M-B and B-M. These are promoters which all react to nitrogenous species. By running these together, we can obtain a direct comparison between all three of these promoters on the growth of E.coli. To see if there are any significant changes, the study was run alongside E.coli cells which had not been transformed with anything. For the rest of this brief report, untransformed cells will be referred to as Alpha cells and the other E.coli cells with transformations will be referred to as the promoter with which they were transformed with. <br />
<br />
The E.coli cells used in the study and for the transformation are the same type of cells (Alpha select gold standard cells from Bioline). A colony was inoculated into 5ml of LB media overnight and the cells spun down the following morning and diluted with fresh LB until an OD reading at 600nm of 0.2 ± 0.01 was obtained. Three repeats were made of each sample. <br />
<br />
The study lasted for 12 hours. An OD reading at 600nm was taken once an hour. Between the hour, the cuvettes were put into a 37ᵒC incubator to encourage growth and for standardising measurements with other growth studies. <br />
To calculate the number of cells in the samples, a calibration curve was set up. This involved using cultures of the E.coli cells without transformations. The E.coli cells were diluted with different volumes of LB and OD readings were taken as well as plating on Agar plates. After a day of growth, the numbers on these plates were counted and recorded. The CFU/ml was calculated. When the OD readings (x axis) and the CFU/ml (y axis) readings are plotted, the equation of the line of best fit, gives a conversion for the absorbance readings. This allowed us to measure the growth. This is demonstrated in figure 1. <br />
<br />
[[File:Calibration curve.png | 500px | center]]<br />
Figure 1: Calibration curve to calculate the conversion factor between OD reading at 600nm and the number of colony forming units growing per ml (CFU/ml)<br />
<br />
We found that there was a significant difference between Alpha cells and PyeaR cells. Initially, Alpha cells had a greater growth rate, but after the third hour into the study, the growth rate of PyeaR was faster than that of Alpha cells. The overall growth rate of PyeaR cells was significantly faster that Alpha cells (Levenes Test, F = 1.009 p = 0.372; T Test, t = 4.196, df = 4, p = 0.014).<br />
<br />
<br />
[[File:A + P.png | 500px | center]]<br />
<br />
<br />
Figure 2: Growth of PyeaR transformed E.coli cells relative to Alpha cell (untransformed cells. Error bars show the standard deviation between the three repeats. For clarity reasons, lines of best fit are not shown<br />
The growth pattern and rate of E.coli cells with or without transformation with B-M and M-B show little difference. Any differences in growth rate were not significant. There was lots of overlap. As previously described, there was a significant difference between the growth rate of PyeaR and Alpha cells. There was also a significant difference between MB/BM and PyeaR cells. The statistical results can be seen in Table 1<br />
<br />
[[File:A+M+B.png | 500px | center]]<br />
<br />
Figure 3: Growth over 12 hours of Alpha, M-B and B-M. Error bars and lines of best fit are not shown for clarity reasons.<br />
<br />
<br />
Table 1: ANOVA readings of statistical differences between Alpha (1) PyeaR (2), MB (3) and BM (4).<br />
[[File:ANOVA.png | 500px | center]]<br />
<br />
From all the above graphs, it can be seen that with the starting concentration of cells as high as they are, the cultures are in exponential stage and do not undergo lag phase. A further growth study will be carried out on purely the lag phase with lower starting concentrations. As the starting absorbances here are approximately 0.2 at a wavelength of 600nm, the lag phase study will involve starting absorbances of 0.04 and lower.<br />
<br />
===A comparison between the growth of E.coli cells, before and after transformation with PyeaR + GFP (BBa_K381001) and B-M and M-B (in pSB1C3)- Lag Phase Study===<br />
<br />
Following the above study, we found that a lag phase only study needed to be carried out to see if there was a significant difference in the lag phase. Again the study protocol was the same except that the starting concentration absorbances at 600nm was lowered to <0.04. It was extremely difficult to keep the absorbances ranges within 0.005 so the range is actually 0.3±0.1.<br />
The below graph shows the mean average of all the data; using the data from the calibration curve, the absorbances were converted to colony forming units per ml (CFU/ml). The trend lines of alpha cells, BM/MB and PyeaR transformed cells are shown within this order from highest to lowest trendlines. One single trendline was used to represent BM and MB because the actual trendlines were extremely similar. Using the initial concentrations of 0.3±0.1 showed that there is little difference between the growth rates. Using statistical analysis, it was found that there was no significant difference between any of the transformed cells relative to Alpha cells or to each other (Anova, p > 0.05). <br />
<br />
[[File:PyeaR, BM, MB, alpha lag phase.png| 500px | center]]<br />
<br />
From this study we have found that changes in growth occur during exponential growth phase and not the lag growth phase. <br />
<br />
<br />
<br />
==Future Experiments==<br />
<br />
=== Test of the effect transforming E.coli with construct 1 and construct 2 has on the rate of growth===<br />
<br />
The team plan to carry out a growth study similar to that above for the BM MB, as when working with construct transformed e.coli team members have noticed quick growth . This will allow analysis of any differences in the rate of growth between E.coli transformed with either constructs from the alpha gold control.</div>Joyehickshttp://2012.igem.org/Team:NRP-UEA-Norwich/PartsTeam:NRP-UEA-Norwich/Parts2012-09-24T17:55:43Z<p>Joyehicks: /* Characterisation of our parts */</p>
<hr />
<div>{{UEANRP}}<br />
<br />
==Our Parts==<br />
<br />
<groupparts>iGEM012 NRP-UEA-Norwich</groupparts><br />
<br />
<br><br><br />
<br />
== Characterisation of other parts ==<br />
<br />
1) Part:BBa_K216005:Please see experience page<br />
[http://partsregistry.org/Part:BBa_K216005:Experience] (Group: iGEM09_Edinburgh (2009-09-25))<br />
[[File:PYEAR_page.png|800px]] <br />
<br />
<br><br><br />
2) Part:BBa_K381001:Please see experience page [http://partsregistry.org/Part:BBa_K381001:Experience] (Group: iGEM10_BCCS-Bristol (2010-10-05))<br />
[[File:K381001.png|800px]]<br />
<br><br><br />
<br />
== Characterisation of our parts ==<br />
1)Bacterial-Mammalian Promoter, Part:BBa_K774000[http://partsregistry.org/wiki/index.php?title=Part:BBa_K774000]<br />
[[File:4000.png|800px]]<br />
<br><br><br />
2)Mammalian-Bacterial Promoter, Part:BBa_K774001[http://partsregistry.org/wiki/index.php?title=Part:BBa_K774001]<br />
[[File:001.png|800px]]<br />
<br><br><br />
3)Bacterial-Mammalian promoter with eCFP reporter, Part:BBa_K774004[http://partsregistry.org/wiki/index.php?title=Part:BBa_K774004]<br />
[[File:004.png|800px]]<br />
<br><br><br />
4)Bacterial-Mammalian promoter with RFP reporter,Part:BBa_K774005[http://partsregistry.org/wiki/index.php?title=Part:BBa_K774005]<br />
[[File:005.png|800px]]<br />
<br><br><br />
5)Mammalian-Bacterial promoter with eCFP reporter, Part:BBa_K774006[http://partsregistry.org/wiki/index.php?title=Part:BBa_K774006]<br />
[[File:006.png|800px]]<br />
<br><br><br />
6)Mammalian-Bacterial promoter with RFP reporter, Part:BBa_K774007[http://partsregistry.org/wiki/index.php?title=Part:BBa_K774007]<br />
[[File:007.png|800px]]<br />
<br />
== Parts to characterise in the future ==<br />
1)Comparator Circuit Part 1, Part:BBa_K774002<br />
<br><br><br />
2)Comparator Circuit Part 2, Part:BBa_K774003<br />
<br><br><br />
This system relies on two interacting mRNA transcripts, both of which would ordinary be translated into a reporter (a fluorescent protein in our case) in the presence of a particular substrate. The idea being that these transcripts will only be made in the presence of certain substrates due to differing promoter activity. Two promoters with overlapping specificity would be used and, crucially, if both promoters detect the same substrates but differ in that one extra substrate is detected by one of the promoters, it is this substrate and this substrate only that our system will be able to detect in a simple and quantitative way.<br />
Our system relies on two constructs that interact via complimentary base pair sequences both before and after the ribosome binding site of the reporter protein. The idea being that, when both transcripts are present in the chassis, they would bind together, inhibiting the translation of the reporter proteins. Any imbalance of transcription due to the presence of the substrate of interest results in free mRNA of the gene system that detects that substrate.<br />
Our team have constructed a countercurrent comparator circuit in which the reporter proteins are at the same end of the complimentary region, although a contracurrent system has been theorised. Both systems share a crucial subtractive nature comparable to an analogue computer. We envisage that, should the system be fine-tuned and expanded on, a variety of different business sectors from agriculture to spinoff pharmaceutical companies (such as the fictious QuantiCare) could capitalise on this novel genetic technology.</div>Joyehickshttp://2012.igem.org/File:007.pngFile:007.png2012-09-24T17:55:09Z<p>Joyehicks: </p>
<hr />
<div></div>Joyehicks