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| − | <link rel="stylesheet" href="https://maxcdn.bootstrapcdn.com/bootstrap/4.0.0-beta/css/bootstrap.min.css" integrity="sha384-/Y6pD6FV/Vv2HJnA6t+vslU6fwYXjCFtcEpHbNJ0lyAFsXTsjBbfaDjzALeQsN6M" crossorigin="anonymous"> | + | <style> |
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| | a, abbr, acronym, address, big, cite, code, | | a, abbr, acronym, address, big, cite, code, |
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| − | <link href="https://fonts.googleapis.com/css?family=Roboto" rel="stylesheet"> | + | .dropdown-menu{ |
| − | <link href="https://fonts.googleapis.com/css?family=Rubik" rel="stylesheet"> | + | background-color: #e8e8e8; |
| | + | font-family: 'Roboto', sans-serif; |
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| − | font-family: Rubik | + | min-height: 80px; |
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| | + | <link href="https://fonts.googleapis.com/css?family=Roboto" rel="stylesheet"> |
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| − | </style> | + | </head> |
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| | <div class="container-fluid" id="main" style="width: 100%; padding-top: 0.5%"> | | <div class="container-fluid" id="main" style="width: 100%; padding-top: 0.5%"> |
| − | <div class="container-fluid text-center" style="max-width: 80%; font-size: 1em"> | + | <div class="container-fluid" style="max-width: 60%"> |
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| | + | <h1 class="text-center" style="font-family: Rubik; margin-bottom: 2%">Attributions</h1> |
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| − | <h1 style="font-weight:normal; font-family: Rubik">Our Experimental Results</h1>
| + | <img src="https://static.igem.org/mediawiki/2017/7/7b/T--newcastle--zw-team_pic_1.png" class="img-fluid border border-dark rounded float-left" style="margin-top: 1%; margin-bottom: 2%; margin-right: 1%; max-width: 48%;" alt=""> |
| − | <div style="height: 400px; border: 1px solid black">
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| − | <h1 class="display-1" style="font-weight:normal;">Image will go here</h1>
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| − | </div>
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| − | <nav class="nav nav-tabs" id="myTab" role="tablist"> | + | <img src="https://static.igem.org/mediawiki/2017/3/37/T--newcastle--zw-team_pic_2.png" class="img-fluid border border-dark rounded float-right" style="margin-top: 1%; margin-bottom: 2%; margin-left: 1%; max-width: 30%;" alt=""> |
| − | <a class="nav-item nav-link active" id="nav-adaptor-tab" data-toggle="tab" href="#nav-adaptor" role="tab" aria-controls="nav-adaptor" aria-selected="true" style="font-weight:normal; font-size: 0.8em">Adaptor Modules</a>
| + | </br></br></br></br></br></br></br></br></br></br></br> |
| − | <a class="nav-item nav-link" id="nav-detector-tab" data-toggle="tab" href="#nav-detector" role="tab" aria-controls="nav-detector" aria-selected="false" style="font-weight:normal; font-size: 0.8em">Detector Modules</a>
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| − | <a class="nav-item nav-link" id="nav-processor-tab" data-toggle="tab" href="#nav-processor" role="tab" aria-controls="nav-processor" aria-selected="false" style="font-weight:normal; font-size: 0.8em">Processor Modules</a>
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| − | <a class="nav-item nav-link" id="nav-reporter-tab" data-toggle="tab" href="#nav-reporter" role="tab" aria-controls="nav-reporter" aria-selected="false" style="font-weight:normal; font-size: 0.8em">Reporter Modules</a>
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| − | <a class="nav-item nav-link" id="nav-framework-tab" data-toggle="tab" href="#nav-framework" role="tab" aria-controls="nav-framework" aria-selected="false" style="font-weight:normal; font-size: 0.8em">Framework Testing (IPTG Sensor)</a>
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| − | <a class="nav-item nav-link" id="nav-cellfree-tab" data-toggle="tab" href="#nav-cellfree" role="tab" aria-controls="nav-cellfree" aria-selected="false" style="font-weight:normal; font-size: 0.8em">Cell Free Protein Synthesis System Optimisation</a>
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| | | | |
| − | </nav>
| + | <p style="clear: both; margin-bottom: 2%">This year our team consisted of 8 molecular biologists, a life scientist, a computer scientist, a forensic scientist, a chemical engineer and an English student. We bring different branches of expertise together and are a true example of an interdisciplinary team. We decided on our project idea during April, after months of intense and independent discussions. Unless otherwise stated, all the experiments, constructs, code, wiki and results documented on our Wiki were performed, written and collected by the Sensynova team.</p> |
| | + | </br> |
| | + | <p style="float: left; margin-bottom: 2%"><b>Team Leader</b>: Jessica Birt</p> |
| | + | <p style="float: right; margin-bottom: 2%"><b>Vice Team Leader</b>: Declan Kohl</p> |
| | + | </br> |
| | + | <nav class="nav nav-tabs justify-content-center" role="tablist" style="clear: both;"> |
| | + | <a class="nav-item nav-link active" id="nav-experimental-tab" data-toggle="tab" href="#nav-experimental" role="tab" aria-controls="nav-experimental" aria-selected="true">Team Members</a> |
| | + | <a class="nav-item nav-link" id="nav-hp-tab" data-toggle="tab" href="#nav-hp" role="tab" aria-controls="nav-hp" aria-selected="false">Supervisors and Advisers</a> |
| | + | <a class="nav-item nav-link" id="nav-model-tab" data-toggle="tab" href="#nav-model" role="tab" aria-controls="nav-model" aria-selected="false">Additional Acknowledgements</a> |
| | + | </nav> |
| | + | |
| | <div class="tab-content" id="nav-tabContent"> | | <div class="tab-content" id="nav-tabContent"> |
| − | <div class="tab-pane fade show active" id="nav-adaptor" role="tabpanel" aria-labelledby="nav-adaptor-tab"> | + | |
| − | | + | <div class="tab-pane fade show active" id="nav-experimental" role="tabpanel" aria-labelledby="nav-experimental-tab"> |
| − | <h1 style="font-family: Rubik"> Sarcosine Oxidase <span style="font-family:arial">(</span>Glyphosate to Formaldehyde<span style="font-family:arial">)</span><button class="btn btn-primary collapsed" type="button" data-toggle="collapse" data-target="#sox" aria-expanded="false" aria-controls="sox" style="margin-left: 1%"></button></h1> | + | <p class="text-left" style="font-family: Rubik; margin-top: 2%"> |
| − | <div id="sox" class="collapse"> | + | <b>Experimental Lead</b>: Valeria Verrone</br> |
| − | | + | <b>Sensynova Framework Proof of Concept</b>: Jessica Birt (design), Valeria Verrone, Declan Kohl (characterisation) </br> |
| − | <h2 style="font-size: 1em"> BioBricks used: BBa_0123456 (New), BBa_7890123 (Team_Name 20XX) </h2>
| + | <b>Arsenic Biosensor</b>: Valeria Verrone (characterisation), Declan Kohl (design) </br> |
| − | | + | <b>Sarcosine Oxidase</b>: Sophie Badger (characterisation), Jessica Birt (design) </br> |
| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Rationale and Aim </h2>
| + | <b>FimE Switch</b>: Marcia Pryce (characterisation), Bradley Brown (design) </br> |
| − | <p>Sarcosine Oxidase (SOX) is an enzyme that oxidatively demethylates sarcosine to form glycine, hydrogen peroxide and formaldehyde (Trickey et al. 1999). SOX was selected to be an example of a possible solution to one of the 5 problems in biosensor production that we identified - unconventional substrates. We defined an unconventional substrate as a substrate that we have little prior knowledge of but that can be adapted into something with an existing biosensor. SOX was specifically chosen to demonstrate that glyphosate, an unconventional substrate which there is not a lot information on, can be converted into formaldehyde which there are existing biosensors for (Ling and Heng 2010).
| + | <b> Cell Free Protein Synthesis Systems</b>: Bradley Brown </br> |
| − | </br></br>
| + | <b>deGFP</b>: Bradley Brown (design and characterisation), Sophie Badger (implementation) </br> |
| − | As part of our project, SOX was designed to be an ‘adapter’ that could link glyphosate into our framework via a formaldehyde detector module. This concept could then be applied to other molecules that have easily detectable substrates in their degradation pathways. The aim of this part of the project was to demonstrate that SOX can be expressed by E. coli cells and that when glyphosate is added SOX can convert it to formaldehyde to be detected via a biosensor.
| + | <b>Chromoproteins</b>: Lais Takiguchi (design, implementation, characterisation), Valeria Verrone (characterisation within framework) </br> |
| − | </p>
| + | <b>Processing Variants</b>: Jessica Birt (design), Lais Takiguchi (characterisation) </br> |
| − | | + | <b>Synthetic Promoter Library</b>: Lais Takiguchi </br> |
| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Background Information </h2>
| + | <b>Fluorescene Normalisation</b>: Michaela Chapman, Ansh Vyas </br> |
| − | <p>Text goes here
| + | <b>Assembly Methods</b>: Anna Walsh, Michaela Chapman, Evie Whittaker </br> |
| − | </p>
| + | <b> Microfluidics Software</b>: Jack Cooper </br> |
| − | | + | <b>Interlab Study</b>: Michaela Chapman (Head), Declan Kohl, Zoe Wilson, Marcia Pryce, Ansh Vyas</br> |
| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Design Stage </h2>
| + | </p> |
| − | <p>To ensure the codon usage of our SOX protein was not differing significantly from the average codon usage of E. coli, rare codons were removed from the sequence using the IDT codon optimisation toolto produce high protein expression.
| + | <p class="text-left" style="font-family: Rubik; margin-top: 2%"> |
| − | </br></br>
| + | <b>Human Practices Lead</b>: Declan Kohl</br> |
| − | E. coli BL21-DE3 cells have higher levels of protein expression than DH5α cells and so were a more practical choice. This led to the expression of SOX being placed under the control of a T7 promoter due to BL21-DE3 cells producing T7 polymerase after the addition of IPTG.
| + | <b>Integrated HP</b>: Declan Kohl, Jessica Birt </br> |
| − | </br></br>
| + | <b>Outreach and Education</b>: Zoe Wilson (lead), Declan Kohl, Sophie Badger, Marcia Pryce </br> |
| − | During the initial design stage of the protein, parts of the sequence were lost between optimisation and sending it to be synthesised into a gBlock. This was not discovered until expression of SOX was induced by IPTG in BL21-DE3 cells and a sample analysed by SDS-Page gel electrophoresis (picture). It was noticed that the band we were expecting was of a lower weight than what it should have been; ~35kDa instead of ~42kDa. It was realised that the sequence in the PSB1C3 plasmid was different to the sequence origin. Therefore a new gBlock was synthesised using the proper sequence and an SDS-Page gel used to confirm that the protein expressed was of the correct weight (picture).
| + | <b>Scientific Communication Analysis</b>: Zoe Wilson, Declan Kohl</br> |
| − | </p>
| + | </p> |
| − | | + | <p class="text-left" style="font-family: Rubik; margin-top: 2%"> |
| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Implementation </h2>
| + | <b>Modelling Lead</b>: Jessica Birt</br> |
| − | <p>Text goes here
| + | <b>Simbiotics</b>: Jessica Birt, Bradley Brown </br> |
| − | </p>
| + | <b>Design of Experiments for CFPS</b>: Bradley Brown </br> |
| − | | + | <b>Microfluidics Software</b>: Jack Cooper</br> |
| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Characterisation </h2>
| + | </p> |
| − | <p>Text goes here
| + | <p class="text-left" style="font-family: Rubik; margin-top: 2%"> |
| − | </p>
| + | <b>Wiki Coding and Design</b>: Jack Cooper, Bradley Brown</br> |
| − | | + | <b>Poster Development</b>: All Team Members</br> |
| − | <h2 style="text-align: left; clear: both"> Conclusions and Future Work </h2>
| + | <b>Presentation</b>: Declan Kohl, Sophie Badger and Zoe Wilson</br> |
| − | <p>Text goes here
| + | </p> |
| − | </p>
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| − | | + | |
| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> References </h2>
| + | |
| − | <p>Ling YP, Heng LY (2010). A Potentiometric Formaldehyde Biosensor Based on Immobilization of Alcohol Oxidase on Acryloxysuccinimide-modified Acrylic Microspheres. Sensors 10:9963-9981.
| + | |
| − | </br></br>
| + | |
| − | Trickey P, Wagner MA, Jorns MS, Mathews FS (1999). Monomeric sarcosine oxidase: structure of a covalently flavinylated amine oxidizing enzyme. Structure 7:331-345.
| + | |
| − | </p>
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| − | </div> | + | |
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| | </div> | | </div> |
| − | <div class="tab-pane fade" id="nav-detector" role="tabpanel" aria-labelledby="nav-detector-tab"> | + | |
| | + | <div class="tab-pane fade" id="nav-hp" role="tabpanel" aria-labelledby="nav-hp-tab"> |
| | + | <p class="text-left" style="font-family: Rubik; margin-top: 2%"> |
| | + | All supervisors and advisers gave advice to the team over a range of areas, including the initial project idea and help in the laboratory.</br></br> |
| | + | <b>Anil Wipat</b>: PI - general advice</br> |
| | + | <b>Thomas Howard</b>: Second PI - general advice</br> |
| | + | <b>Jon Marles-Wright</b>: Instructor - general advice</br> |
| | + | <b>Dana Ofiteru</b>: Instructor - general advice</br> |
| | + | <b>Angel Goñi-Moreno</b>: Instructor - modelling advice</br> |
| | + | <b>Wendy Smith</b>: Adviser - general and laboratory advice</br> |
| | + | <b>Michael White</b>: Adviser - laboratory advice</br> |
| | + | <b>Alice Banks</b>: Adviser - laboratory advice</br> |
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| − | <h1 style="font-family: Rubik"> Synthetic Promoter Library <button class="btn btn-primary collapsed" type="button" data-toggle="collapse" data-target="#syn-prom-lib" aria-expanded="false" aria-controls="syn-prom-lib" style="margin-left: 1%"></button></h1> | + | </p> |
| − | <div id="syn-prom-lib" class="collapse">
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| − | <h2 style="font-size: 1em"> BioBricks used: BBa_0123456 (New), BBa_7890123 (Team_Name 20XX) </h2>
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Rationale and Aim </h2>
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| − | <p>Text goes here.</p>
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Background Information </h2>
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Design Stage </h2>
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Implementation </h2>
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Characterisation </h2>
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| − | <h2 style="text-align: left; clear: both"> Conclusions and Future Work </h2>
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> References </h2>
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| − | </div>
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| − | <hr>
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| − | <h1 style="font-family: Rubik"> Arsenic Biosensor <button class="btn btn-primary collapsed" type="button" data-toggle="collapse" data-target="#arsenic" aria-expanded="false" aria-controls="arsenic" style="margin-left: 1%"></button></h1>
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| − | <div id="arsenic" class="collapse">
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| − | <h2 style="font-size: 1em"> BioBricks used: BBa_0123456 (New), BBa_7890123 (Team_Name 20XX) </h2>
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Rationale and Aim </h2>
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| − | <p>The Sensynova multicellular biosensor platform has been developed to overcome the limitations identified by our team [hyperlink to human practices] that hamper the success in biosensors development. One of these limits regards the lack of modularity and reusability of the various components. Our platform design, based on the expression of three main modules (Detector, Processor and Output) by three E.coli strains in co-culture, allows the switch of possible variances for each module and the production of multiple customised biosensors.
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| − | </br></br>
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| − | This section of the project is based on testing the modularity of the system by replacing the IPTG detector part of the Sensynova design with different detecting parts. In particular, an Arsenic sensing part will be used.</p>
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Background Information </h2>
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| − | <p></p>
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Design Stage </h2>
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Implementation </h2>
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Characterisation </h2>
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| − | <h2 style="text-align: left; clear: both"> Conclusions and Future Work </h2>
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> References </h2>
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| − | <p></p>
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| − | <hr>
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| − | <h1 style="font-family: Rubik">Psicose Biosensor <span style="font-family: Arial">(</span><a href=#>Evry Paris-Saclay Collaboration</a><span style="font-family: Arial">)</span> <button class="btn btn-primary collapsed" type="button" data-toggle="collapse" data-target="#psicose" aria-expanded="false" aria-controls="psicose" style="margin-left: 1%"></button></h1>
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| − | <div id="psicose" class="collapse">
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| − | <h2 style="font-size: 1em"> BioBricks used: BBa_K2205023 (New), BBa_??? (Evry Paris-Saclay 2017) </h2>
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Rationale and Aim </h2>
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| − | <p>The Sensynova multicellular biosensor platform has been developed to overcome the limitations identified by our team [hyperlink to human practices] that hamper the success in biosensors development. One of these limits regards the lack of modularity and reusability of the various components. Our platform design, based on the expression of three main modules (Detector, Processor and Output) by three E.coli strains in co-culture, allows the switch of possible variances for each module and the production of multiple customised biosensors.
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| − | </br></br>
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| − | This section of the project is based on testing the modularity of the system by implementing the biosensor created by the 2017 Evry Paris-Saclay iGEM team into the Sensynova platform as part of our collaboration requirement.</p>
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Background Information </h2>
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| − | <p>This biosensor was designed, made and submitted to the iGEM registry by the Evry Paris-Saclay 2017 team.
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| − | </br></br>
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| − | We chose to use this system as a variant to the IPTG detector module present in the Sensynova platform in order to fulfil the requirement of collaborating with another iGEM team.
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| − | </br></br>
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| − | The image below, provided to us by the Evry Paris-Saclay 2017 team, details the psicose biosensor design. It features the pLac derivative promoter pTAC (BBa_K180000), a RBS (BBa_B0034), the PsiR coding sequence, the terminator (BBa_B0015), the synthetic promoter pPsitac, a RBS (BBa_B0034), a mCherry coding sequence and finally the terminator (BBa_B0015) flanked by the iGEM prefix and suffix.</p>
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| − |
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| − | <img src="https://static.igem.org/mediawiki/2017/1/1b/T--Newcastle--Lais--Evry--Biosensor.png" class="img-fluid border border-dark rounded" style="margin: 2%">
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| − |
| |
| − | <p>The inducible system works as detailed in the diagram below. When pTAC is induced due to the presence of IPTG, PsiR is transcribed and binds to the pPsitac promoter repressing the transcription of the mCherry protein. When psicose is present, the sugar binds to PsiR, freeing up the promoter and subsequently the colour output</p>
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| − |
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| − | <img src="https://static.igem.org/mediawiki/2017/e/ef/T--Newcastle--Lais--Evry--System--SBOL.png" class="img-fluid border border-dark rounded" style="margin: 2%">
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| − |
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Design Stage </h2>
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| − | <p>In order to implement the psicose biosensor variant to the Sensynova platform, a design was created by replacing the IPTG sensing system in the original detector module with the construct detailed above, creating part K2205023.
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| − | </br></br>
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| − | We chose to replace the pTAC promoter with the constitutive promoter present within the platform in order to eliminate the need for induction with IPTG. In place of the colour output present in the Evry Paris-Saclay design, we have added our part K2205008, which produces our first connector in order to trigger a response from following modules of the Sensynova platform.</p>
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| − |
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| − | <img src="https://static.igem.org/mediawiki/2017/2/28/T--Newcastle--Lais--Evry--SBOL.png" class="img-fluid border border-dark rounded" style="margin: 2%">
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| − |
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| − | <p>Part K2205023 detailed above was designed using Benchling and ordered for synthesis through IDT. Using Benchling, virtual digestions and ligations were simulated resulting in the plasmid map detailed below.</p>
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| − |
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| − | <a target="_blank" href="https://static.igem.org/mediawiki/2017/4/49/T--Newcastle--Lais--Evry--Plasmid--Map.png">
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| − | <img src="https://static.igem.org/mediawiki/2017/4/49/T--Newcastle--Lais--Evry--Plasmid--Map.png" class="img-fluid border border-dark rounded" style="margin: 2%; max-width: 40%">
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| − | </a>
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| − |
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Implementation </h2>
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| − | <p>The Psicose detector construct obtained by gBlock synthesis has been designed to include required overhangs for Gibson assembly into the linearized plasmid pSB1C3.
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| − | </br></br>
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| − | The plasmid backbone was acquired by digestion [Protocol link] of the part K2205015 with XbaI and SpeI, cutting out the original sfGFP construct.
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| − | </br></br>
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| − | The Psicose detector construct was assembled into the plasmid backbone using the NEB Hi-Fi kit [Protocol link] and transformed into DH5α E. coli cells [Protocol link].
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| − | </br></br>
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| − | Colonies picked from streaked plates and cultures were prepared for miniprepping [Protocol link]. DNA samples were then sent off for sequencing [Website link] to ensure that the constructs were correct.</p>
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| − |
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Characterisation </h2>
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| − | <p>Text goes here.</p>
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| − |
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Conclusions and Future Work </h2>
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| − | <p>Due to time constraints resulted from synthesis delays, we lacked the time to co-culture this part with the Sensynova platform's multiple modules in order for the creation of variants. The part K2205023, the Evry Pasir-Sclay's psicose biosensor system as the detecting unit of the platform, has been submitted to the iGEM registry for future work and characterisation by future teams.</p>
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> References </h2>
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| − | <p>Text goes here.</p>
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| − | </div>
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| | </div> | | </div> |
| − | <div class="tab-pane fade" id="nav-processor" role="tabpanel" aria-labelledby="nav-processor-tab"> | + | |
| − | | + | <div class="tab-pane fade" id="nav-model" role="tabpanel" aria-labelledby="nav-model-tab"> |
| − | <h1 style="font-family: Rubik"> Fim Standby Switch<button class="btn btn-primary collapsed" type="button" data-toggle="collapse" data-target="#fim" aria-expanded="false" aria-controls="fim" style="margin-left: 1%"></button></h1> | + | <p class="text-left" style="font-family: Rubik; margin-top: 2%"> |
| − | <div id="fim" class="collapse">
| + | |
| − | <h2 style="font-size: 1em"> BioBricks used: BBa_0123456 (New), BBa_7890123 (Team_Name 20XX) </h2>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/a/a4/T--Newcastle--MP_FimON-OFF_diagram.jpeg" class="img-fluid border border-dark rounded" style="margin: 2%" style="max-wdith: 70%">
| + | |
| − | <p style="font-family: Rubik; margin-left: 12%; max-width: 70%">Diagrammatic Overview: Representation of the switching mechanism of the Fim Switch, in the native [OFF] state the eforRED reporter is expressed (shown in red) allowing direct visualisation of the cells. Following the inversion of the promoter region (K1632004), eforRED expression is halted and the RhlI gene is expressed (J64718), this is now the [ON] state.</p> | + | |
| − | | + | |
| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Rationale and Aim </h2>
| + | |
| − | <p>Sensynova multicellular biosensor platform has been developed to overcome the limitations that hamper the success in biosensors development. One of these limits regards the lack of modularity and reusability of the various components. Our platform design, based on the expression of three main modules (Detector, Processor and Output) by three E.coli strains in co-culture, allows the switch of possible variances for each module and the production of multiple customised biosensors.
| + | |
| − | </br></br>
| + | |
| − | This part can be used within the platform as a Processor unit. Real world applications of biosensors are limited by many factors, one of which is that with most biosensors there is not a readout signal showing if the biosensor is working when not in use, i.e that the cells are still alive and have not lost their biosensor phenotypes. This can make them difficult to use, as well as market, since their viability comes into question as well as leading to false negatives/positives. Biosensors which rely on expression of a reporter signal may also suffer from unobserved activation due to weak or inconstant induction.
| + | |
| − | </br></br>
| + | |
| − | For this section of the project, as an improvement on a part by the Tokyo Tech team, we aim to produce a biobrick compatible part which is able to constitutively express a reporter signal prior to activation (to show that it is functioning) and to amplify a weak or inconsistent induction signal by permanently switching from an [OFF] to [ON] state after induction.</p>
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| − | | + | |
| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Background Information </h2>
| + | |
| − | <p>Expression of the E. coli type 1 fimbriae gene is tightly regulated and phase dependent, i.e expression is either completely [ON] or [OFF] >>FimB FimE fimbrae control - Klemm 1986<<. This change in expression is controlled by the action of two proteins FimB and FimE which independently act upon a 300bp promoter region upstream of the fimbriae gene. The 300bp promoter region is inverted to either activate or suppress expression >> Roles of FimB FimE in site specific inversion - McClain 1991<<. Typical gene regulation mechanisms rely on up or down regulation of a promoter from a baseline expression, the fimbriae mechanism of ‘ALL’ or ‘NONE’ makes it a useful tool for synthetic biology applications. While the FimB protein inverts the promoter back and forth between [ON] and [OFF] states the FimE protein permanently inverts the promoter from [ON] to [OFF]. This inversion can be used to amplify weak or inconsistent induction signals.
| + | |
| − | </br></br>
| + | |
| − | The part we are producing is a combination of the 2015 Tokyo Tech part (BBa_K1632013, BBa_K1632007) and the Voight lab 2007 part (BBa_J64718) quorum sensing RhlI an autoinducer synthesis protein which produces C4-HSL. The part by Tokyo Tech used FimE in combination with GFP to switch from an [ON] to [OFF] state, we have produced a construct where in the [ON] state the non-fluorescense based reporter protein eforRed (Uppsala 2011 BBa_K592012) is expressed. In the [OFF] state the RhlI quorum sensing protein from P. aeruginosa is expressed. The eforRed chromoprotein was chosen as fluorescence based reporters often require complex laboratory equipment to measure expression.
| + | |
| − | </br></br>
| + | |
| − | Using a chromoprotein allows for the switch from [ON] to [OFF] to be verified by sight alone. The promoter flip also creates expression of the quorum sensing protein RhlI. This allows for a downstream activation of a reporter cell, meaning that the Fim Standby Switch has dual reporting properties.
| + | |
| − | </br></br>
| + | |
| − | The use of the FimE switch as opposed to FimB (which is reversible) means that even with a low or inconsistent induction signal, the permanent inversion of the 300bp fim promoter region leads to a signal which can be observed. This may have applications in agricultural biosensors where there can be plant uptake of the potential induction compound (reference ). FimE was also chosen because FimB can switch the promoter back and forth, so it is likely to result in about half the plasmids with the promoter in one direction and half in the other over time, resulting in no strong signal either direction.
| + | |
| − | </br></br>
| + | |
| − | The inactivation of the eforRed chromoprotein following an induction signal visibly displays the [ON] to [OFF] inversion. Expression of this chromoprotein also displays that the [ON] section of the fim switch is working correctly.</p>
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| − | | + | |
| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Design Stage </h2>
| + | |
| − | <p>To construct the Fim reporter switch 3 separate gBlocks were designed with overlapping adapter regions homologous to the iGEM prefix and suffix to allow for Gibson assembly into the pSB1C3 backbone whilst retaining biobrick compatibility. The individual genes and other components are shown in (Table 1). The 1st gBlock sequence starts with a RBS (B0034) upstream of the fimE ORF (K137007) with no promoter region, this is to allow for other promoters to be cloned in upstream of the part. Downstream of the fimE gene is a double terminator (B0015). All RBS and terminator sequences used are B0034 and B0015 respectively.</p>
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| − | | + | |
| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Implementation </h2>
| + | |
| − | <p> Text goes here.</p>
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| − | | + | |
| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Characterisation </h2>
| + | |
| − | <p>The fim Standby Switch has two main functions; a visual signal to show that the target compound has been detected and AHL production so that the part can be detected by a reporter cell. To characterise the part these functions are individually tested, in aim to further isolate issues if they occur.
| + | |
| − | To test the detecting function of the Fim Standby Switch it was assembled with the PBAD/AraC promotor. The PBAD/AraC promotor with Standby Switch parts were plated out onto four different LB plates containing chloramphenicol, two plates with different concentrations of glucose and chloramphenicol, and another containing arabinose. As colonies for the Fim Switch section were red and white when plated onto chloramphenicol plates due to leakiness, the plates with glucose in theory should suppress this switching and a greater percentage colonies on these plates should be red after transformation. The colonies on the arabinose plate should be white as translation of fimE leads to the flipping fimS, and expression of RHlI.
| + | |
| − | </br></br>
| + | |
| − | The number of colonies on the plate that are white and red confirm inversion, this will show the percentage of colonies in the [ON] and [OFF] states. DNA sequencing will show inversion of the switch. As fimE is unidirectional over time the colonies should all become white on the plate containing arabinose. The plate containing glucose should repress leakage and the medium is supplemented by some percentage glucose.
| + | |
| − | </br></br>
| + | |
| − | To ensure that AHL is produced a white colony is chosen, where the fimS section has flipped, and a red colony which has not yet inverted. These colonies are then co-cultured with a successfully independently tested reporter cell. This reporter cell detects AHL production and as a result GFP produced. This also shows that the reversed sequences for RHLI and B0034 are working as expected.</p>
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| − | | + | |
| − | <img src="https://static.igem.org/mediawiki/2017/b/bb/T--Newcastle--MP_Char1.jpeg" class="img-fluid border border-dark rounded rounded float-left" style="max-width: 40%" alt="">
| + | |
| − | | + | |
| − | <h2 style="text-align: left; clear: both"> Conclusions and Future Work </h2>
| + | |
| − | | + | |
| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> References </h2>
| + | |
| − | | + | |
| − | </div>
| + | |
| − |
| + | |
| − | <hr>
| + | |
| − | <!---------------------------------------------------------------->
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| − | | + | |
| − | <h1 style="font-family: Rubik"> Signal Tuners <button class="btn btn-primary collapsed" type="button" data-toggle="collapse" data-target="#signal" aria-expanded="false" aria-controls="signal" style="margin-left: 1%"></button></h1>
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| − | <div id="signal" class="collapse">
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| − |
| + | |
| − | <h2 style="font-size: 1em"> BioBricks used: BBa_K2205024 (New),BBa_K2205025 (New), BBa_K274371 (Cambridge 2009), BBa_K274381 (Cambridge 2009) </h2>
| + | |
| − | | + | |
| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Rationale and Aim </h2>
| + | |
| − | <p>The Sensynova multicellular biosensor platform has been developed to overcome the limitations identified by our team [hyperlink to human practices] that hamper the success in biosensors development. One of these limits regards the lack of modularity and reusability of the various components. Our platform design, based on the expression of three main modules (Detector, Processor and Output) by three E.coli strains in co-culture, allows the switch of possible variances for each module and the production of multiple customised biosensors.
| + | |
| − | </br></br>
| + | |
| − | This section of the project is based on testing the modularity of the system by inserting two different sensitivity tuner constructs between the processing units of the Sensynova platform; BBa_K274371 and BBa_K274381.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Background Information </h2>
| + | |
| − | <p>Both selected sensitivity tuner constructs were made and submitted to the iGEM registry by the Cambridge 2009 team.
| + | |
| − | </br></br>
| + | |
| − | They were chosen as variants to the empty processing module present in the Sensynova platform due to the fact that, although they have been included in the iGEM distribution kit since their submission in 2009, they have yet to be successfully implemented into a team’s system, as far as we are aware.
| + | |
| − | </br></br>
| + | |
| − | The 2007 Cambridge iGEM team built 15 different constructs that amplified the PoPS output of the promoter pBad/AraC detailed by image below taken from the Cambridge 2009 team's wiki.
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| − | </p>
| + | |
| − | <img class="img-fluid border border-dark rounded" style="margin: 2%" src="https://static.igem.org/mediawiki/2017/a/a4/T--Newcastle--Lais--ST--C1--SBOL.png"></img>
| + | |
| − |
| + | |
| − | <p>The 2009 Cambridge iGEM team then re-designed these constructs to be PoPS converters, as image below taken from their wiki details, and generated a set sensitivity tuners corresponding to Cambridge 2007’s amplifiers.</p>
| + | |
| − | <img class="img-fluid border border-dark rounded" style="margin: 2%" style="margin: 2%" src="https://static.igem.org/mediawiki/2017/b/b9/T--Newcastle--Lais--ST--C2--SBOL.png"></img>
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| − |
| + | |
| − | <h3> BBa_K274371 </h3>
| + | |
| − | <p>This part is made up of an RBS (BBa_B0034), an org activator coding sequence (BBa_I746350) from P2 phage, the double terminator BBa_B0015 (made up of BBa_B0010 and BBa_B0012) and the inducible promoter PO (BBa_I746361) from P2 phage.</p>
| + | |
| − | <img class="img-fluid border border-dark rounded" style="margin: 2%" src="https://static.igem.org/mediawiki/2017/6/6f/T--Newcastle--Lais--ST--C71.png"></img>
| + | |
| − |
| + | |
| − | <h3> BBa_ K274381 </h3>
| + | |
| − | <p>This part is made up of an RBS (BBa_B0034), a pag activator coding sequence (BBa_I746351) from PSP3 phage, the double terminator BBa_B0015 (made up of BBa_B0010 and BBa_B0012) and the inducible promoter PO (BBa_I746361) from P2 phage.</p>
| + | |
| − | <img class="img-fluid border border-dark rounded" style="margin: 2%" src="https://static.igem.org/mediawiki/2017/e/ef/T--Newcastle--Lais--ST--C81.png"></img>
| + | |
| − | | + | |
| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Design Stage </h2>
| + | |
| − | <p>In order to implement these two sensitivity tuner variants into the Sensynova platform, designs were made by inserting the above parts between the two constructs forming the empty processor module of our framework.</p>
| + | |
| − | <img class="img-fluid border border-dark rounded" style="margin: 2%" src="https://static.igem.org/mediawiki/2017/9/95/T--Newcastle--Lais--ST--SBOL.png"></img>
| + | |
| − |
| + | |
| − | <p>Using Benchling, virtual digestions of the two sensitivity tuners and ligations to the part K2205010, the connector 1 receiver module, were carried out. These two new constructs were then virtual digested and ligated to the part K2205011, the connector 2 reporter module, resulting in the two plasmid maps detailed below; parts K2205024 and K2205025.</p>
| + | |
| − | <a target="_blank" href="https://static.igem.org/mediawiki/2017/3/36/T--Newcastle--Lais--ST--C71--Map.png">
| + | |
| − | <img class="img-fluid border border-dark rounded" style="margin: 2%; max-width: 40%" src="https://static.igem.org/mediawiki/2017/3/36/T--Newcastle--Lais--ST--C71--Map.png"></img>
| + | |
| − | </a>
| + | |
| − | <a target="_blank" href="https://static.igem.org/mediawiki/2017/4/42/T--Newcastle--Lais--ST--C81--Map.png">
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| − | <img class="img-fluid border border-dark rounded" style="margin: 2%; max-width: 40%" src="https://static.igem.org/mediawiki/2017/4/42/T--Newcastle--Lais--ST--C81--Map.png"></img>
| + | |
| − | </a>
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| − |
| + | |
| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Implementation </h2>
| + | |
| − | <p>The sensitivity tuners parts BBa_K274371 and BBa_K274381 were requested from the iGEM parts registry. Upon arrival, parts were transformed in DH5α E. coli cells [Protocol link]. Colonies were picked and cultures were prepared for miniprepping [Protocol link]. Minipreps were digested [Protocol link] with XbaI and PstI for BioBrick assembly [Protocol link].
| + | |
| − | </br></br>
| + | |
| − | The part K2205010 contained in pSB1C3, was digested [Protocol link] using SpeI and PstI to allow for the insertion of the processing variants directly after the Las controlled promoter (pLas) that would trigger transcription of sensitivity tuners in the presence of connector 1 of the Sensynova platform.
| + | |
| − | </br></br>
| + | |
| − | Ligations were set up overnight [Protocol link] using NEB’s T4 ligase and transformed in DH5α E. coli cells [Protocol link]. Colony PCR [Protocol link] was performed to check ligations. Colonies picked for this protocol were streaked onto a LB-agar plate.
| + | |
| − | </br></br>
| + | |
| − | Colonies picked from streaked plates and cultures were prepared for miniprepping [Protocol link]. Minipreps were digested [Protocol link] with SpeI and PstI to allow for the insertion of the part K2205011 directly after the PO promoter.
| + | |
| − | </br></br>
| + | |
| − | The part K2205010 contained in pSB1C3, was digested [Protocol link] using XbaI and PstI for BioBrick assembly [Protocol link]. Ligations were set up overnight [Protocol link] using NEB’s T4 ligase and transformed in DH5α E. coli cells [Protocol link]. Colony PCR [Protocol link] was performed to check ligations. Colonies picked for this protocol were streaked onto a LB-agar plate.
| + | |
| − | </br></br>
| + | |
| − | Colonies picked from streaked plates and cultures were prepared for miniprepping [Protocol link]. DNA samples were then sent off for sequencing [Website link] to ensure that the constructs were correct.</p>
| + | |
| − | | + | |
| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Characterisation </h2>
| + | |
| − | <p>Text goes here
| + | |
| − | </p>
| + | |
| | | | |
| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Conclusions and Future Work </h2>
| + | <b>Jonny Naylor</b>: Help with the modelling aspect of the project, specifically with his Simbiotics software (which he customised to our needs!)</br> |
| − | <p>Due to time constraints, we lacked the time to characterise these parts into the Sensynova platform within the lab. The parts K2205024 and K2205025, the parts BBa_K274371 and BBa_K274381 respectively as processing units of the platform, were been submitted to the iGEM registry for future work and characterisation by future teams.
| + | <b>James McLaughlin</b>: Assisted us with the technical aspects of our wiki.</br> |
| − | </p>
| + | |
| | | | |
| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> References </h2>
| + | <b>Exeter iGEM 2017</b>: Analysed our deGFP construct for us using their fluorescence Activated Cell Sorting machine.</br> |
| − | <p>Text goes here
| + | <b>Evry Paris-Saclay 2017</b>: collaborated with us to test their novel synthetic promoter regulated by psicose in our Sensynova framework.</br> |
| − | </p>
| + | <b>Dr Chris French, Dr Martin Peacock, Dr Karen Polizzi</b> and <b>Dr Oliver Purcell</b> for their invaluable insight into biosensor development and their help in influencing our project.</br> |
| − | </div>
| + | <b>Ivy Wellman from DEFRA</b>: For her insight into the legislative procedure deliberate release of GMOs into the environment.</br> |
| | + | <b>Jasmine Bird</b>: Took time to help proof-read the wiki and suggest improvements.<br /> |
| | | | |
| | + | </p> |
| | </div> | | </div> |
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| | + | <div class="tab-pane fade" id="nav-wap" role="tabpanel" aria-labelledby="nav-wap-tab"> |
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| − | <div class="tab-pane fade" id="nav-reporter" role="tabpanel" aria-labelledby="nav-reporter-tab">
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| − | <h1 style="font-family: Rubik"> deGFP <button class="btn btn-primary collapsed" type="button" data-toggle="collapse" data-target="#degfp" aria-expanded="false" aria-controls="degfp" style="margin-left: 1%"></button></h1>
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| − | <h2 style="font-size: 1em"> BioBricks used: BBa_0123456 (New), BBa_7890123 (Team_Name 20XX) </h2>
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Rationale and Aim </h2>
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| − | <p>Text goes here.</p>
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Background Information </h2>
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| − | <p>Text goes here.</p>
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Design Stage </h2>
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| − | <p>Text goes here.</p>
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Implementation </h2>
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| − | <p>Text goes here.</p>
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Characterisation </h2>
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| − | <p>Text goes here.</p>
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| − | <h2 style="text-align: left; clear: both"> Conclusions and Future Work </h2>
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| − | <p>Text goes here.</p>
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| − | <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> References </h2>
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| − | <p>Text goes here.</p>
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| − | </div>
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| − | <hr>
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| − | <h1 style="font-family: Rubik"> Chromoproteins <button class="btn btn-primary collapsed" type="button" data-toggle="collapse" data-target="#chromoproteins" aria-expanded="false" aria-controls="chromoproteins" style="margin-left: 1%"></button></h1>
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| − | <div id="bioS_what" class="content-div">
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| − | <center><h2>Attributions</h2></center>
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| − | <table class="content-layout">
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| − | <tr>
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| − | <td>
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| − | <p style="text-align:left;">
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| − | <br />
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| − | Team Leader: Jessica Birt <br />
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| − | Second Team Leader: Declan Kohl
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| − | <br />
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| − | <br />
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| − | Experimental: Valeria Verrone (Head)
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| − | <br />
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| − | IPTG Sensor: Declan Kohl, Jessica Birt
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| − | <br />
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| − | Arsenic Biosensor: Valeria Verrone (characterisation), Declan Kohl (design)
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| − | <br />
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| − | Sarcosine Oxidase: Sophie Badger (characterisation), Jessica Birt (design)
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| − | <br />
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| − | FimE Switch: Marcia Pryce (characterisation), Bradley Brown (design)
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| − | <br />
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| − | Cell Free Protein Synthesis Systems: Bradley Brown
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| − | <br />
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| − | deGFP: Sophie Badger, Bradley Brown, Lais Takiguchi
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| − | <br />
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| − | Chromoproteins: Sophie Badger, Lais Takiguchi
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| − | <br />
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| − | Processing Variants: Jessica Birt, Lais Takiguchi
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| − | <br />
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| − | Synthetic Promoter Library: Lais Takiguchi
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| − | <br />
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| − | Fluorescene Normalisation: Michaela Chapman, Ansh Vyas
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| − | <br />
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| − | Assembly Methods: Anna Walsh, Michaela Chapman, Evie Whittaker
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| − | <br />
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| − | Microfluidics Software: Jack Cooper
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| − | <br />
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| − | Robotics: Jessica Birt, Declan Kohl
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| − | <br />
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| − | Interlab Study: Michaela Chapman (Head), Declan Kohl, Zoe Wilson, Marcia Pryce, Ansh Vyas
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| − | <br />
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| − | <br />
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| − | Human Practices: Declan Kohl (Head), Zoe Wilson, Jessica Birt
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| − | <br />
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| − | Integrated HP: Declan Kohl, Jessica Birt
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| − | <br />
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| − | Outreach and Education: Declan Kohl, Zoe Wilson, Sophie Badger, Marcia Pryce
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| − | <br />
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| − | Scientific Communication Analysis: Zoe Wilson, Declan Kohl<br />
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| − | <br />
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| − | Modelling: Jessica Birt (Head), Bradley Brown
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| − | <br />
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| − | Simbiotics: Jessica Birt
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| − | <br />
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| − | Design of Experiments: Bradley Brown
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| − | <br /><br />
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| − | Wiki: Declan Kohl, Bradley Brown, Jack Cooper, Marcia Pryce
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| − | <br /><br />
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| − | Poster Development: All Team Members
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| − | <br /><br />
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| − | Presentation Development: All Team Members
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| − | <br /><br />
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| − | Presenters: Declan Kohl, Sophie Badger and Zoe Wilson
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| − |
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| − | </p>
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