Imperial College 2006

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[[Image:800px-LogoRed1.PNG|400px|center]]
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<center>[[Image:Banner.png|800px]]</center>
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==Project Summary==
 
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The major goal of Imperial College’s 2006 entry into the iGEM competition was to create a stable biological oscillator, improving on past designs such as Elowitz’s repressilator.  The team investigated into natural biological oscillators and sought to mimic Lotka-Volterra predator-prey interactions with a molecular system.  The model was adapted to molecular interactions between prey (n-acyl homoserine lactone (AHL)) and predator (AiiA (AHL-lactonase) + LuxR).  The design strategy of the project was an engineering based cycle of specification, design, modeling, testing, and implementation. Parts were constructed and individually tested before the final construct was assembled.  Our ongoing parts testing shows correlation to our mathematical models, suggesting that the design could be successful.
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<center><font size="4">'''[http://openwetware.org/wiki/IGEM:IMPERIAL/2006 Full Documentation of the project on OpenWetWare]'''</font></center>
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<center><font size="3">'''[[Media:IGEM_Imperial_2006_presentation.pdf |Presentation ]]'''</font></center>
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<center><font size="3">'''[[Media:IGEM_Imperial_2006_Poster.pdf |Poster]]'''</font></center>
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==Introduction==
 
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Predator-prey interactions form stable oscillations with frequencies which can be calculated precisely given certain assumptions.  The team decided to model the biological oscillator on the basis of predator-prey interactions hoping that this novel approach would lead to success.  Since one of the key assumptions to the model is the self dependent growth of prey, a positive feedback loop for the production of AHL was implemented in E. coli strain DH5a.  Furthermore, a separate cell was constructed to sense the AHL molecules in the surrounding environment and respond by producing AiiA.  This will result in the AHL being degraded, or in terms of the model, being “eaten by the predator”.  Since AiiA production is dependent upon the concentration of AHL molecules, the action of AiiA itself, leading to a decrease in AHL concentration, would result in less AiiA being produced at a later time.  The advantage of a two cell construct instead of previous one cell construct designs is that Paremeters can be changed easily by altering cell ratios.  AHL is a suitable molecule to mimic the prey behavior. since it can be detected by a transcriptional regulator (LuxR) and therefore promote it's own growth. Also it easily diffuses outside of the cell as well having a complementary predator molecule AiiA.  The team attempted to use as many parts from the Registry of Standard Biological Parts, which are outlined in the biological parts section.  Once a stable oscillator has been created, several future applications into biological computing and synchronization become accessible.
 
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[[Image:800px-System overview.JPG|thumb|800px|center|Figure 1.  Full system overview showing interacting populations of cells. The Prey cell produces a constant amount of LuxR. The lux R expression is controlled by the tet promoter. LuxR binds AHL and this complex activates transcription at the Lux promoter (pLux). This leads to productin of the Enzyme LuxI. LuxI will produce AHL. The AHL will be bound by more LuxR and create a positive feedback loop. AHL can freely diffuse out of the prey cell and into the predator cell. Here it is bound by luxR. The AHL-LuxR complex will bind the pLux Promoter leading to more LuxR being made. The gene Aiia is also transcribed leading to the production of AHL-Lactonase which degrades AHL so the Predator cell senses and kills AHL. This system is based on Lotka Volterra. However there are important diferences. So the system has been re-modeled and is discussed below ]]
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{| border="0" width="100%" style="padding: 5px; background-color: #ffffff; border: 2px solid #2171B8;"
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|-
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|width="20%" style="background:#2171B8"|[[Image:LogoFinal2.png|210px|center]]  
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|width="40%" style="background:#2171B8"| <font face="arial" font style="color: #FFFFFF" >
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<center><big><u>'''Project Summary'''</u></big></center>
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==Biological Parts==
 
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===Final Constructs===
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*We have used the traditional engineering approach to build a stable and flexible molecular oscillator
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*Our original design relies on population dynamics and was inspired by the Lotka-Volterra predation model
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*Every step of the development cycle (Specifications, Design, Modelling, Implementation, Testing/Validation) has been fully documented on our OWW site</font>
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|width="40%" style="background:#2171B8" | <font face="arial" font style="color: #FFFFFF" >
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*<bbpart>J37015</bbpart> – Final Prey Cell
 
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**F2620 (AHL Receiver)
 
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**C0261 (LuxI)
 
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**I13504 (GFP Reporter)
 
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[[Image:J37015.png]]
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<center><big><u>'''Achievements'''</u></big></center>
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<br>
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*Derivation of the complete dynamical model, describing the main biochemical reactions driving our oscillator
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*Full theoretical analysis and detailed computer simulations, validating our design with regard to our specifications
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*Successful building and characterization of functional parts, providing the building blocks for the final oscillator
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<br>
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<br>
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<br>
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</font>
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|}
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*<bbpart>J37036</bbpart> – Final Predator Cell – Polycistronic Design
 
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**R0062 (Lux pR, AHL induced promoter)
 
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**B0034 (RBS)
 
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**C0062 (LuxR)
 
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**B0034 (RBS)
 
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**J37023 (AiiA w/ LVA & FLAG)
 
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**B0015 (terminator)
 
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[[Image:J37036.png]]
 
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*<bbpart>J37035</bbpart> – Final Predator Cell – Monocistronic Design
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==Main Project==
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**R0062 (Lux pR, AHL induced promoter)
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{| border="0" width="100%" align="center" style="padding: 5px; background-color: #ffffff; border: 2px solid #2171B8;"
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**B0034 (RBS)
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| width="20%" style="background:#2171B8"| <center>[[Image:Oscillator Icon.PNG|100px|center]] </center>
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**C0062 (LuxR)
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| style="background:#2171B8" colspan="3"| <font face="arial" font style="color: yellow"> <center><big>'''Engineering a Molecular Predation Oscillator'''</big><br>
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**I13033 (terminator + Lux pR)
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<font face="arial" font style="color: #ffffff">For building our biological oscillator, the engineering cycle below was followed.<br>
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**B0034 (RBS)
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'''<font style="color: orange">Click</font> on <font style="color: orange">each</font> of the stages of the <font style="color: orange">engineering cycle</font> below in order to find out about the different stages & aspects of the project. '''</font></center>
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**J37023 (AiiA w/ LVA & FLAG)
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|-
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**B0015 (terminator)
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| style="background:#2171B8" width="200pt" rowspan="4" |
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[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Full_System <font face="arial" font style="color: yellow"> '''1) Specifications: '''</font>]
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<font face="arial" font style="color: #ffffff">
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*Stable and robust oscillations
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*Controllable frequency/amplitude
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*Generic and modular</font>
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[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Full_System/Design <font face="arial" font style="color: yellow">'''2) Design: '''</font>]
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<font face="arial" font style="color: #ffffff">
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*Mimics predator-prey dynamics based on the Lotka-Volterra model
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</font>
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[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Full_System/Modelling <font face="arial" font style="color: yellow">'''3) Modelling: '''</font>]
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<font face="arial" font style="color: #ffffff">
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*[http://www.openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/Theoretical_Analyses <font face="arial" font style="color: #FFFFFF">Full theoretical analysis</font>]
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*Modelling of the full system
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</font>
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[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Full_System/Implementation <font face="arial" font style="color: yellow"> '''4) Implementation: '''</font>]
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<font face="arial" font style="color: #ffffff">
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*All parts designed and modelled
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*4 functional & 5 intermediate submitted to the Registry
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</font>
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[http://www.openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Full_System/TestingValidation <font face="arial" font style="color: yellow">'''5) Testing/ Validation: '''</font>]
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<font face="arial" font style="color: #ffffff">
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*Successful characterization of sub-components.
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</font>
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| align="right"|[[Image:ArrowCurvedRight.png|110px]]
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| align="center" |[http://www.openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Full_System http://openwetware.org/images/f/fd/IGEM_IMPERIAL_DevCycle_Specifications.png]<font style="color:red"><br>Click on picture for more info</font>
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| align="left"|[[Image:ArrowCurvedDown.PNG|120px]]
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|-
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| align="center"|[http://www.openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Full_System/TestingValidation http://openwetware.org/images/2/20/IGEM_IMPERIAL_DevCycle_Validation.png]<font style="color:red"><br>Click on picture for more info</font>
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| align="center" rowspan="2"|[[Image:IGEM_IMPERIAL_DevCycle_FullSystem.png |206px|center]]
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| align="center"|[http://www.openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Full_System/Design http://openwetware.org/images/c/ce/IGEM_IMPERIAL_DevCycle_Design.png]<font style="color:red"><br>Click on picture for more info</font>
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| align="center"|[[Image:ArrowUp.png|20px]]
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| align="center"|[[Image:ArrowDown.png|20px]]
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|-
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| align="center"|[http://www.openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Full_System/Implementation http://openwetware.org/images/0/04/IGEM_IMPERIAL_DevCycle_Implementation.png]<font style="color:red"><br>Click on picture for more info</font>
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| align="center"|[[Image:ArrowLeft.png|150px]]
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| align="center"|[http://www.openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Full_System/Modelling http://openwetware.org/images/5/5f/IGEM_IMPERIAL_DevCycle_Modelling.png]<font style="color:red"><br>Click on picture for more info</font>
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|}
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[[Image:J37035.png]]
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==Secondary Projects==
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{| border="0"  width="100%" style="padding: 5px; background-color: #ffffff; border: 2px solid #2171B8;"  width="100%"
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|-
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!  width="50%" | <big>'''[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/popsblocker <font face="arial" font style="color: #2171B8">PoPs Blocker</font>]  '''</big>!! width="50%"| <big>'''[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Bio_elec_interface <font face="arial" font style="color: #2171B8">Biological to Electrical Interface]</font>'''</big>
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|-
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| style="background:#2171B8" width="50%" |
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<font face="arial" font style="color: #FFFFFF"> As a method of controlling the activation of the positive-feedback loop in our predator-prey based oscillator, we successfully created this part, which can be used as a general Pops Blocker:
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*This part is placed downstream of a promoter and prevents any Pops from the promoter passing through this part
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*When an accompanying Cre Recombinase plasmid becomes activated, the enzyme produced will permanently cut a section of DNA from the plasmid containing this part
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*Only then, the polymerase can pass through this part and transcribe downstream genes.
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</font>
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| style="background:#2171B8" width="50%" |
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<font face="arial" font style="color: #FFFFFF">We also worked on a Biosensor for measuring AHL concentrations in order to establish a Biological to Electrical Interface this summer.
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*Using an enzyme to hydrolize the lactone AHL would result in a local change in pH
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*Measuring the change in pH gives a measurement of how much AHL is present.
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<br>
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<br>
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<br>
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<br>
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</font>
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|}
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===Test Constructs===
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==Our Contributions to the Registry==
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{|border="0" width="100%" style="padding: 5px; background-color: #ffffff; border: 2px solid #2171B8;"
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! style="background:#2171B8" | <font face="arial" font style="color: #FFFFFF">Part Logo !! style="background:#2171B8" |<font face="arial" font style="color: #FFFFFF">Description</font> !! style="background:#2171B8" |<font face="arial" font style="color: #FFFFFF">Link to registry</font> !! style="background:#2171B8" |<font face="arial" font style="color: #FFFFFF">Built</font> !! style="background:#2171B8" |<font face="arial" font style="color: #FFFFFF">Tested </font>!! style="background:#2171B8" |<font face="arial" font style="color: #FFFFFF">Characterized </font>!! style="background:#2171B8" |<font face="arial" font style="color: #FFFFFF">Sequenced</font> !! style="background:#2171B8" |<font face="arial" font style="color: #FFFFFF">Sent</font>
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|-
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!style="background:#008080" |<font face="arial" font style="color: #FFFFFF">Final Constructs</font>
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!style="background:#008080"|<font face="arial" font style="color: orange">(!) Favorite Part</font>
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!style="background:#008080"|
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!style="background:#008080"|
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!style="background:#008080"|
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!style="background:#008080"|
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|-
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![[Image:J37015.PNG|250px]]!! Final Prey Cell <font face="arial" font style="color: orange">(!) </font> !! <bbpart>J37015</bbpart> !! YES !! YES !! Pending !! YES !! YES
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|-
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! [[Image:J37027.png|120px]] !! Cre/Lox Prey Control <font face="arial" font style="color: orange">(!) </font>!! <bbpart>J37027</bbpart> !! YES !! YES !! YES !! YES !!YES
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|-
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!style="background:#008080" |<font face="arial" font style="color: #FFFFFF">Test Constructs</font>
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!style="background:#008080"|
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!style="background:#008080"|
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!style="background:#008080"|
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!style="background:#008080"|
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!style="background:#008080"|
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!style="background:#008080"|
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|-
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![[Image:J37016.PNG|150px]]!! Final Polycistronic Predator Cell Test Construct <font face="arial" font style="color: orange">(!) </font>!! <bbpart>J37016</bbpart> !! YES !! YES !! YES !! YES!! YES
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|-
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! [[Image:J37020.png|170px]] !! Predator Cell, pLux Transfer Function (two promoters)  !! <bbpart>J37020</bbpart> !! YES !! YES !! NO !! NO !! YES
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|-
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!style="background:#008080" |<font face="arial" font style="color: #FFFFFF">Intermediate Parts</font>
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!style="background:#008080"|
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!style="background:#008080"|
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!style="background:#008080"|
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!style="background:#008080"|
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!style="background:#008080"|
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|-
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! [[Image:J37019.PNG|80px]] !! AHL induced LuxR generator (for predator cell) !! <bbpart>J37019</bbpart> !! YES !! N/A !! N/A!! YES !!YES
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|-
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! [[Image:J37033.PNG|65px]] !! RBS + LuxR !! <bbpart>J37033</bbpart> !! YES !! N/A !! N/A!! YES !!YES
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|-
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! [[Image:J37034.PNG|90px]] !! Prey Cell Intermediate!! <bbpart>J37034</bbpart> !! YES !! N/A !! N/A!! YES !!YES
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|-
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! [[Image:J37032.png|90px]] !! LuxR + GFP !! <bbpart>J37032</bbpart> !! YES !! N/A !! N/A!! YES !!YES
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|}
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*<bbpart>J37016</bbpart> (Polycistronic Predator Cell Test Construct)
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==Our Open Documentation==
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**R0062 (Lux pR, AHL induced promoter)
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**B0034 (RBS)
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**C0062 (LuxR)
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**I13504 (GFP Reporter)
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[[Image:J37016.png]]
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*'''<font face="arial" font style="color: #2171B8">300+ Pages of documentation on OWW, 100's of Photographs, Extensive Modelling, Lab Notebook and much more!</font>'''
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__NOTOC__
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{| class="wikitable" border="0" cellpadding="10" cellspacing="1" style="padding: 5px; background-color: #ffffff; border: 2px solid #2171B8;text-align:center"
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!width="20%" align="left" valign="top" style="background:#2171B8; color:black"|
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*<bbpart>J37020</bbpart> (Monocistronic Predator Cell Test Construct)
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*[http://openwetware.org/wiki/IGEM:IMPERIAL/2006 <font face="arial" font style="color: #FFFFFF">iGEM@imperial</font>]
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**R0062 (Lux pR, AHL induced promoter)
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*[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser <font face="arial" font style="color: #FFFFFF">Oscillator project</font>]
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**B0034 (RBS)
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*[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/popsblocker <font face="arial" font style="color: #FFFFFF">PoPs Blocker project</font>]
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**C0062 (LuxR)
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*[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Bio_elec_interface <font face="arial" font style="color: #FFFFFF">AHL Biosensor project</font>]
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**I13033 (terminator + Lux pR)
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**I13504 (GFP Reporter)
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[[Image:J37020.png]]
 
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*<bbpart>J37022</bbpart> (AiiA Test Construct)
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!width="20%" align="left" valign="top" style="background:#2171B8; color:black"|
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**J04500 (LacI promoter + RBS)
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*[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/LabCalendar <font face="arial" font style="color: #FFFFFF">Lab Notebook</font>]
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**J37023 (AiiA w/ LVA & FLAG tag)
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*[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/Protocols <font face="arial" font style="color: #FFFFFF">Protocols</font>]
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**B0015 (terminator)
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*[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/Journal_Club<font face="arial" font style="color: #FFFFFF"> Journal Club</font>]<br\>
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*[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/Lecture_Notes <font face="arial" font style="color: #FFFFFF">Lecture Notes</font>]<br\>
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[[Image:J37022b.png]]
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!width="20%" align="left" valign="top" style="background:#2171B8; color:black"|
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*[http://openwetware.org/wiki/BioSysBio:abstracts/2007/iGEM2006_Imperial_College <font face="arial" font style="color: #FFFFFF">BioSysBio Conference, UK</font>]
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*[http://openwetware.org/wiki/IGEM:IMPERIAL/Methodology <font face="arial" font style="color: #FFFFFF">Description of our Methodology</font>]
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!width="20%" align="left" valign="top" style="background:#2171B8; color:black"|
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*[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/Calendar/2006-7-26 <font face="arial" font style="color: #FFFFFF">UK iGEM Teams Meeting</font>]
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*[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/Photos <font face="arial" font style="color: #FFFFFF">Photos</font>]
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*[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/Instructors_Feedbacks <font face="arial" font style="color: #FFFFFF">Feedbacks on our experience</font>]
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|}
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===New Individual Protein Coding Sequences===
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==The Team and Acknowledgements==
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*<bbpart>J37023</bbpart> (AiiA Protein Coding Sequence with LVA & FLAG immunotag)
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{| class="wikitable" border="0" width="100%" cellpadding="10" cellspacing="1" style="padding: 5px; background-color: #ffffff; border: 2px solid #2171B8;text-align:center"
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!width="20%" align="left" valign="top" style="background:#2171B8; color:black"|
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<center>
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==Experiments & Results==
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==<font face="arial" font style="color: #FFFFFF">Undergrads</font>==
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</center>
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*[http://openwetware.org/wiki/User:Cys <font face="arial" font style="color: #FFFFFF">Christin Sander</font>]
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*[http://openwetware.org/wiki/User:da104 <font face="arial" font style="color: #FFFFFF">Deepti Aswani</font>]
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*[http://openwetware.org/wiki/User:Farah <font face="arial" font style="color: #FFFFFF">Farah Vohra</font>]
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*[http://openwetware.org/wiki/User:Baijiongjun<font face="arial" font style="color: #FFFFFF"> Jiongjun Bai</font>]
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*[http://openwetware.org/wiki/User:Johnsy <font face="arial" font style="color: #FFFFFF">John Sy</font>]
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*[http://openwetware.org/wiki/User:JohnChattaway <font face="arial" font style="color: #FFFFFF">John Chattaway</font>]
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*[http://openwetware.org/wiki/User:Jonny<font face="arial" font style="color: #FFFFFF"> Jonathan Wells</font>]
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*[http://openwetware.org/wiki/User:Tom <font face="arial" font style="color: #FFFFFF">Tom Hinson</font>]
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Testing constructs above were made to characterise the parts necessary to build the biological oscillator using standard ligation procedures. The FLAG immunotag was added to the existing registry AiiA part with an LVA tag (<bbpart>C0060</bbpart>) using Polymerase Chain Reaction (PCR) methods. The resulting protein coding sequence was then ligated to the other BioBrick parts. 
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!width="25%" align="left" valign="top" color:black"|
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BioBricks composing the test constructs and the final oscillator were engineered and a number of important results were extracted from the modelling and testing stages: i) The test constructs/BioBricks representing the different parts of the oscillator were characterised from testing in E.Coli (DH5α) ([http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/parts testing page]). ii) The model of the test constructs, which included experimental data, predicted stable oscillations as output of the full system ([http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/Modelling modelling pages]). iii) The influence of specific system parameters on the frequency, amplitude and stability of the oscillations was investigated with the Lotka-Volterra model ([http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/Modelling/LV LV model]).
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[[Image:IGEM GroupPhotos0015.JPG|thumb|center|200px|The Imperial College iGEM team and advisors on the terrace of the Biochemistry building]]
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[[Image:300px-010906 J37016.png|thumb|300px|center|Figure 3.  Results of testing part <bbpart>J37016</bbpart>, AHL sensing construct to be implemented in the predator cell.]]
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!width="20%" align="left" valign="top" style="background:#2171B8; color:black"|
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<center>
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==Modelling==
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==<font face="arial" font style="color: #FFFFFF">Advisors</font>==
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</center>
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*[http://www3.imperial.ac.uk/people/r.kitney <font face="arial" font style="color: #FFFFFF">Prof. Richard Kitney</font>]
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*[http://www3.imperial.ac.uk/people/p.freemont <font face="arial" font style="color: #FFFFFF">Prof. Paul Freemont</font>]
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*[http://www3.imperial.ac.uk/people/d.mann <font face="arial" font style="color: #FFFFFF">Dr. David Mann</font>]
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*<font face="arial" font style="color: #FFFFFF">Kirsten Jensen</font>
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*[http://openwetware.org/wiki/User:Vincent <font face="arial" font style="color: #FFFFFF">Vincent Rouilly</font>]
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*[http://openwetware.org/wiki/User:chuehloo <font face="arial" font style="color: #FFFFFF">Chueh-Loo Poh</font>]
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*[http://openwetware.org/wiki/User:SxE00 <font face="arial" font style="color: #FFFFFF">Matthieu Bultelle</font>]
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===Assumptions for Modelling===
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!width="20%" align="left" valign="top" style="background:#2171B8; color:white"|
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*General assumptions on gene expression modelling:
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**Quasi-steady state hypothesis on mRNA expression.
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**Gene activation can be approximated by [http://en.wikipedia.org/wiki/Hill_equation Hill equations].
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*Assumptions linked to the quorum sensing:
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<center>
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**As a first approximation, we assume that luxR and AHL molecules form a heterodimer (even if it has been found that the complex formed is more complicated)
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**The concentration of the heterodimer is in equilibrium with the concentration of AHL
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**LuxR is constitutively produced and reaches steady state before AHL production begins. [LuxR] in the prey can be considered constant
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**The degradation rate of luxR and AHL-lactonase is due to the growth dilution which, in this case, is controlled by the chemostat
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**AHL is diffusing freely throughout the system
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===Differential Equations Derived===
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==<font face="arial" font style="color: #FFFFFF">Funding</font>==
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The following three differential equations completely describe the concentrations of AiiA, LuxR and AHL in our oscillator.
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</center>
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<br>  
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*European Commission
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[[Image:Eqn1.png]]
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*Imperial College Deputy Rector's Fund
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[[Image:Eqn2.png]]
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*Imperial College Faculty of Engineering
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[[Image:Eqn3.png]]
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*Imperial College Faculty of Natural Sciences
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|}
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==Applications==
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Once we are able to create a biological oscillator, we can then move to synchronise several biological computer paving the way for an internet-like system controlled by bacteria.  Further developments to electrical interfacing could mean that communication between electrical and biological devices would be seamless.  This can potentially integrate the existing infrastructure and novel biological approaches so the current technology would not be drastically displayed, but gradually replaced by better biological machines.  Moreover, the quest for self-reproducing machines has finally succeeded.  Wouldn’t it be great if our computers upgraded themselves?  Made themselves faster every 30 minutes?  Genetically engineered bacteria have this potential and are only limited by their lifespan and the biological reaction rates.  Unfortunately, biological reaction rates are relatively slow, so when compared to electrical signals, consider 100  years ago when we knew very little about electricity and how to harness the power of electricity.  Biological engineering is at that stage now, and we cannot expect to surpass in a few years the engineering foundations that have been perfected throughout the ages.
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==Future Extensions==
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Once a stable biological oscillator has been achieved, the next step is to connect two oscillators together and investigate into how two populations synchronize themselves.  This could lead to more information on the mechanism behind cellular communication and steps necessary to control it, especially in terms to controlling the amplitude and frequency of biological oscillations.  One major drawback to working with biological systems is their inherent noise and ability to mutate.  It is yet to be seen if any of these factors could affect the robustness of a biological oscillator. Furthermore, electrical to biological interfaces are the next step in bringing the power of biological systems to fruition.  This could lead to an internet communication using existing infrastructure between bacterial colonies, and the possibilities are limitless, just as they are with the internet today.
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==The Team==
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*Undergraduates
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**Christin Sander (Bioengineering)
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**Deepti Aswani (Biology)
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**Farah Vohra (Bioengineering)
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**Jiongjun Bai (Electrical Engineering)
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**John Sy (Bioengineering)
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**John Chattaway (Biology)
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**Jonathan Wells (Biology)
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**Tom Hinson (Biochemistry)
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*Advisors
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**[http://www3.imperial.ac.uk/people/r.kitney Prof. Richard Kitney]
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**[http://www3.imperial.ac.uk/people/p.freemont Prof. Paul Freemont]
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**[http://www3.imperial.ac.uk/people/d.mann Dr. David Mann]
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**Dr. Kirsten Jensen
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**Vincent Rouilly
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**Chueh-Loo Poh
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**Matthieu Bultelle
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*Contributors
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** Prof. Tony Cass
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** Dr. Anna Radomska
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** Dr. David Leak
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** Dr. Mauricio Barahona
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** Dr. Danny O'Hare
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** Ms. Susan E. Wryter
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** Mr. David Featherbe
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** Dr. Geoff Baldwin
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** Ciaran Mckeown
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** Dr. James Mansfield - Chemostat
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*Special thanks for their support to
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** The European Comission
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** Imperial College Deputy Rector's Fund
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** Imperial College Faculty of Engineering
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** Imperial College Faculty of Life Sciences
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Latest revision as of 16:42, 10 November 2006


Banner.png


[http://openwetware.org/wiki/IGEM:IMPERIAL/2006 Full Documentation of the project on OpenWetWare]
Presentation
Poster


LogoFinal2.png
Project Summary


  • We have used the traditional engineering approach to build a stable and flexible molecular oscillator
  • Our original design relies on population dynamics and was inspired by the Lotka-Volterra predation model
  • Every step of the development cycle (Specifications, Design, Modelling, Implementation, Testing/Validation) has been fully documented on our OWW site


Achievements


  • Derivation of the complete dynamical model, describing the main biochemical reactions driving our oscillator
  • Full theoretical analysis and detailed computer simulations, validating our design with regard to our specifications
  • Successful building and characterization of functional parts, providing the building blocks for the final oscillator





Main Project

Oscillator Icon.PNG
Engineering a Molecular Predation Oscillator

For building our biological oscillator, the engineering cycle below was followed.

Click on each of the stages of the engineering cycle below in order to find out about the different stages & aspects of the project.

[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Full_System 1) Specifications: ]

  • Stable and robust oscillations
  • Controllable frequency/amplitude
  • Generic and modular

[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Full_System/Design 2) Design: ]

  • Mimics predator-prey dynamics based on the Lotka-Volterra model

[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Full_System/Modelling 3) Modelling: ]

  • [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/Theoretical_Analyses Full theoretical analysis]
  • Modelling of the full system

[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Full_System/Implementation 4) Implementation: ]

  • All parts designed and modelled
  • 4 functional & 5 intermediate submitted to the Registry

[http://www.openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Full_System/TestingValidation 5) Testing/ Validation: ]

  • Successful characterization of sub-components.

ArrowCurvedRight.png [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Full_System http://openwetware.org/images/f/fd/IGEM_IMPERIAL_DevCycle_Specifications.png]
Click on picture for more info 		ArrowCurvedDown.PNG
[http://www.openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Full_System/TestingValidation http://openwetware.org/images/2/20/IGEM_IMPERIAL_DevCycle_Validation.png]
Click on picture for more info
IGEM IMPERIAL DevCycle FullSystem.png
[http://www.openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Full_System/Design http://openwetware.org/images/c/ce/IGEM_IMPERIAL_DevCycle_Design.png]
Click on picture for more info
ArrowUp.png ArrowDown.png
[http://www.openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Full_System/Implementation http://openwetware.org/images/0/04/IGEM_IMPERIAL_DevCycle_Implementation.png]
Click on picture for more info 		ArrowLeft.png [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser/Full_System/Modelling http://openwetware.org/images/5/5f/IGEM_IMPERIAL_DevCycle_Modelling.png]
Click on picture for more info

Secondary Projects

[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/popsblocker PoPs Blocker] [http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Bio_elec_interface Biological to Electrical Interface]

As a method of controlling the activation of the positive-feedback loop in our predator-prey based oscillator, we successfully created this part, which can be used as a general Pops Blocker:

  • This part is placed downstream of a promoter and prevents any Pops from the promoter passing through this part
  • When an accompanying Cre Recombinase plasmid becomes activated, the enzyme produced will permanently cut a section of DNA from the plasmid containing this part
  • Only then, the polymerase can pass through this part and transcribe downstream genes.

We also worked on a Biosensor for measuring AHL concentrations in order to establish a Biological to Electrical Interface this summer.

  • Using an enzyme to hydrolize the lactone AHL would result in a local change in pH
  • Measuring the change in pH gives a measurement of how much AHL is present.





Our Contributions to the Registry

Part Logo Description Link to registry Built Tested Characterized Sequenced Sent
Final Constructs (!) Favorite Part
J37015.PNG Final Prey Cell (!) J37015 YES YES Pending YES YES
J37027.png Cre/Lox Prey Control (!) J37027 YES YES YES YES YES
Test Constructs
J37016.PNG Final Polycistronic Predator Cell Test Construct (!) J37016 YES YES YES YES YES
J37020.png Predator Cell, pLux Transfer Function (two promoters) J37020 YES YES NO NO YES
Intermediate Parts
J37019.PNG AHL induced LuxR generator (for predator cell) J37019 YES N/A N/A YES YES
J37033.PNG RBS + LuxR J37033 YES N/A N/A YES YES
J37034.PNG Prey Cell Intermediate J37034 YES N/A N/A YES YES
J37032.png LuxR + GFP J37032 YES N/A N/A YES YES

Our Open Documentation

  • 300+ Pages of documentation on OWW, 100's of Photographs, Extensive Modelling, Lab Notebook and much more!
  • [http://openwetware.org/wiki/IGEM:IMPERIAL/2006 iGEM@imperial]
  • [http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Oscillator/project_browser Oscillator project]
  • [http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/popsblocker PoPs Blocker project]
  • [http://openwetware.org/wiki/IGEM:IMPERIAL/2006/project/Bio_elec_interface AHL Biosensor project]


  • [http://openwetware.org/wiki/IGEM:IMPERIAL/2006/LabCalendar Lab Notebook]
  • [http://openwetware.org/wiki/IGEM:IMPERIAL/2006/Protocols Protocols]
  • [http://openwetware.org/wiki/IGEM:IMPERIAL/2006/Journal_Club Journal Club]
  • [http://openwetware.org/wiki/IGEM:IMPERIAL/2006/Lecture_Notes Lecture Notes]
  • [http://openwetware.org/wiki/BioSysBio:abstracts/2007/iGEM2006_Imperial_College BioSysBio Conference, UK]
  • [http://openwetware.org/wiki/IGEM:IMPERIAL/Methodology Description of our Methodology]
  • [http://openwetware.org/wiki/IGEM:IMPERIAL/2006/Calendar/2006-7-26 UK iGEM Teams Meeting]
  • [http://openwetware.org/wiki/IGEM:IMPERIAL/2006/Photos Photos]
  • [http://openwetware.org/wiki/IGEM:IMPERIAL/2006/Instructors_Feedbacks Feedbacks on our experience]

The Team and Acknowledgements

Undergrads

  • [http://openwetware.org/wiki/User:Cys Christin Sander]
  • [http://openwetware.org/wiki/User:da104 Deepti Aswani]
  • [http://openwetware.org/wiki/User:Farah Farah Vohra]
  • [http://openwetware.org/wiki/User:Baijiongjun Jiongjun Bai]
  • [http://openwetware.org/wiki/User:Johnsy John Sy]
  • [http://openwetware.org/wiki/User:JohnChattaway John Chattaway]
  • [http://openwetware.org/wiki/User:Jonny Jonathan Wells]
  • [http://openwetware.org/wiki/User:Tom Tom Hinson]
The Imperial College iGEM team and advisors on the terrace of the Biochemistry building

Advisors

  • [http://www3.imperial.ac.uk/people/r.kitney Prof. Richard Kitney]
  • [http://www3.imperial.ac.uk/people/p.freemont Prof. Paul Freemont]
  • [http://www3.imperial.ac.uk/people/d.mann Dr. David Mann]
  • Kirsten Jensen
  • [http://openwetware.org/wiki/User:Vincent Vincent Rouilly]
  • [http://openwetware.org/wiki/User:chuehloo Chueh-Loo Poh]
  • [http://openwetware.org/wiki/User:SxE00 Matthieu Bultelle]

Funding

  • European Commission
  • Imperial College Deputy Rector's Fund
  • Imperial College Faculty of Engineering
  • Imperial College Faculty of Natural Sciences

Retrieved from "http://2006.igem.org/wiki/index.php/Imperial_College_2006"
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