Team:Imperial College/Summary

From 2008.igem.org

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{{Imperial/StartPage2}}
{{Imperial/StartPage2}}
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=== Summer Summary ===
 
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{{Imperial/Box2|Our Approach|[[Image:Cycle.PNG|center|500px]]<br>
 
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The Imperial College 2008 team chose to take the engineering approach to the biofabricator project. This framework has helped us organised the project and team.  |}}
 
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=== Project Summary ===
{{Imperial/Box1|Design|
{{Imperial/Box1|Design|
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In order to achieve our specifications of design previously described, we require the following devices;  
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In order to achieve our specifications of design, we require the following devices;  
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*'''Light sensing device''' - Converting a light input into a PoPS output,
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*'''Light sensing device''' - Converting a light input into a PoPS output
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*'''Biomaterial production device'''- Converting a PoPS input into an output of biomaterial production,
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*'''Biomaterial production device''' - Converting a PoPS input into an output of biomaterial production
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*'''Motility Control device''' - Converting a PoPS input into an output of motility arrest,
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*'''Motility Control device''' - Converting a PoPS input into an output of motility arrest
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*'''Integration device''' - To allow integration and selection of our genetic constructs and devices into ''B,subtilis'',
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*'''Integration device''' - To allow integration and selection of our genetic constructs and devices into ''B,subtilis''
<br>
<br>
Each of these constructs makes up the '''final device''' which is shown below:
Each of these constructs makes up the '''final device''' which is shown below:
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(AB is our antibiotic resistance cassette, ''ytvA'' is the gene controlling the light-sensing pathway, ''SB'' is the biomaterial, ''epsE'' the clutch and the 5' and 3' sections are integration sites. Light-inducible promoters are labelled with an 'L')
(AB is our antibiotic resistance cassette, ''ytvA'' is the gene controlling the light-sensing pathway, ''SB'' is the biomaterial, ''epsE'' the clutch and the 5' and 3' sections are integration sites. Light-inducible promoters are labelled with an 'L')
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{{Imperial/Box1|Modeling - Overview|
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{{Imperial/Box1|Modelling|
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Basically our dry lab team concentrated on characterising the chassis. In the dry lab section you'll find pages on the genetic circuit, growth curve and motility analysis from our project; this section will give a brief brief overview of each area (no pictures?). It will also include (in the motility analysis part) a movie (y/n?) of the motility response of ''B. subtilis'' with an inducible ''epsE'' gene BioBricked in.
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The Dry Lab used computational simulations to explore the different properties of the Biofabricator. Our activities are summarized on this page. To find out more please visit the [https://2008.igem.org/Team:Imperial_College/Dry_Lab '''Dry Lab Hub'''].
====== Growth Curve ======
====== Growth Curve ======
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The growth curve of ''B. subtilis'' was modelled by superposition of three more basic ODE models, which were constructed and simulated in MATLAB. The lag, exponential and stationary phases are modelled and combined to produce the curve on the right (Image here?).
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We have developed a simple model for the growth of B. subtilis where the rate of growth is related to the amount of nutrients available. To this purpose we have exploited the ideas put forward by last year's Imperial College  iGEM team for their modelling of F2620 in a cell-free system.
====== Genetic Circuit ======
====== Genetic Circuit ======
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We feel accurate modelling of the genetic circuit contributes greatly to the characterisation of synthetic systems. As part of the project, the behaviours of constitutive and inducible promoters were modelled for comparison with our experimental data.
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We have also built mathematical models for the time evolution of the basic genetic circuits that comprise our device. We have verified which model best describe the behaviour of the circuit better by using laboratory data.
====== Motility Analysis ======
====== Motility Analysis ======
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A major component of the system is the motility and shift between a motile and arrested state with the expression of EpsE.  
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Finally, we have carried out a detailed analysis of the swimming motility of B. subtilis, which led us, among other  things, to develop a simple mechanical model for the swimming motility of B. subtilis. Using manual tracking, we were able to extract x,y coordinate data from the cell trajectory. This has allowed us to fit experimental data with our model. The data suggest that flagellar force of ''B. subtilis'' is Exponentially distributed.
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All model simulations and motility data analysis were carried out with MATLAB. Cell tracking was done with ImageJ via the Manual Tracking Plugin. All our MATLAB files can be found in the Appendices section.
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[[Image:Motility_Summary.jpg|center|600px]]<br>
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{{Imperial/Box1|Implementation|
{{Imperial/Box1|Implementation|
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Think what we can refer to as implementation is the construction of the parts. Last week Kirsten had the idea that we could make a big gel with the digests from all our parts, I will look into this week to get an image. \We could use this then put some stuff about no. of parts etc.}}
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Following the design stage of our project we moved on to the implementation stage. This involved construction of a cloning strategy, construction of our biobricks and transformation and characterisation of these biobricks in ''B. subtilis''. For more information on this aspect of the project please see the [https://2008.igem.org/Team:Imperial_College/Wet_Lab '''Wet Lab Hub'''].
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[[Image:Implementation.PNG|center|600px]]
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|}}
{{Imperial/Box1|Testing|
{{Imperial/Box1|Testing|
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The wet lab team was responsible for designing the constructs and setting up the cloning strategy to get us from the starting parts to the finished system. We were also responsible for designing and BioBricking the starting parts, of course, and designing and implementing the integration brick technique. This section shows some of the major results that came from the wet lab over the summer. Motility results can go in the dry lab overview above.
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The testing and validation of our project can be split into three main areas;
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====== Transformation ======
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*'''Work with ''B. subtilis''''' - Including characterisation of growth curves and transformation,
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Transformation stuff goes here...
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*'''Extensive Characterisation ''' of new ''B.subtilis'' biobricks, Chloramphenicol resistance gene and motility
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====== Calibration Curve ======
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*'''Production of Biomaterials in ''B. subtilis'''''
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Calibration curve results go here...
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|Stuff}}
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If you'd like to see more information on the key results from the testing and validation, you can find it on the [https://2008.igem.org/Team:Imperial_College/Major_Results '''Results Page'''].
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=====Results=====
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|[[Image:Result.PNG|center|300px]]}}
{{Imperial/Box1|Achievements|
{{Imperial/Box1|Achievements|
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*Helped Bristol by sending them a mini-iGEM project: ''Chemotactic dot-to-dot'' with information on quorum sensing and directed movement
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Here is a summary of the achievements of the Imperial College 2008 team:
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*Helped Bristol by sending them a part (BBa_J37015) from our 2007 stock which was an empty vector in the Registry
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*Submitted 45 documented parts to the Registry
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*Developed integration bricks, to allow devices to be constructed that can then be excised and planted into ''B. subtilis''
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*<html><a target="_blank" href="https://2008.igem.org/Team:Imperial_College/CAT"><b>Characterized</b></a></html> and improved the existing part <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_J31005"  target="_blank">BBa J31005</a></html> (chloramphenicol acetyl transferase, CAT)
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*Layed the groundwork for future teams to work with ''B. subtilis'' by BioBricking promoters, RBSs, terminators and so on and characterising them
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*<html><a target="_blank" href="https://2008.igem.org/Team:Imperial_College/Biobricks"><b>Characterized</b></a></html> the new promoter and ribosome binding sites biobricks <html><a target="_blank" href="http://partsregistry.org/wiki/index.php/Part:BBa_K143079">BBa K143079</a></html> and <html><a target="_blank" href="http://partsregistry.org/wiki/index.php/Part:BBa_K143082">BBa K143082</a></html> that we submitted this year.
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*Developed integration sequences for Biobricks, to allow devices to be constructed that can then be excised and planted into ''B. subtilis''
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*Laid the groundwork for future teams to work with ''B. subtilis'' by BioBricking and characterising promoters, RBSs, integration sequences, coding sequences and complex devices
*Showed that expansion into other organisms is a definite possibility!
*Showed that expansion into other organisms is a definite possibility!
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|<html><align="right"><img width="100px" src="http://i59.photobucket.com/albums/g305/Timpski/Gold_Medal.png"></align></html>}}
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*Developed a method for tracking and analysing bacterial motility
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*Helped Bristol by sending them a mini-iGEM project: ''Chemotactic dot-to-dot'' with information on quorum sensing and directed movement
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*Helped Bristol by sending them part <html><a href="http://partsregistry.org/wiki/index.php/Part:BBa_J37015" target="_blank">BBa_J37015</a></html> (AHL generator + GFP) from our 2006 stock which was an empty vector in the Registry
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*Helped Cambridge by sending them a plate of ''Synechocystis'' PCC680 and a genome preparation
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|}}
<hr><br>
<hr><br>
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{{Imperial/Box2||Of course, that's a very simplified description of our project. We expanded upon our project by looking into possible areas for real-world application; for a case-study of such an implementation check out how our project fits in with [[Team:Imperial_College/Biocouture | '''>>> Biocouture >>>''']]}}
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{{Imperial/Box2||Of course, that is a very simplified description of our project. We expanded upon our project by looking into possible areas for real-world applications. For a case-study of such an implementation, check out how our project fits in with [[Team:Imperial_College/Cellulose | '''>>> Biocouture >>>''']]|}}
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{{Imperial/EndPage|Chassis_2|Biocouture}}
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{{Imperial/EndPage|Chassis_2|Cellulose}}

Latest revision as of 03:12, 30 October 2008


Project Summary

Design

In order to achieve our specifications of design, we require the following devices;

  • Light sensing device - Converting a light input into a PoPS output
  • Biomaterial production device - Converting a PoPS input into an output of biomaterial production
  • Motility Control device - Converting a PoPS input into an output of motility arrest
  • Integration device - To allow integration and selection of our genetic constructs and devices into B,subtilis


Each of these constructs makes up the final device which is shown below:

Genetic circuit.PNG

(AB is our antibiotic resistance cassette, ytvA is the gene controlling the light-sensing pathway, SB is the biomaterial, epsE the clutch and the 5' and 3' sections are integration sites. Light-inducible promoters are labelled with an 'L')


Modelling

The Dry Lab used computational simulations to explore the different properties of the Biofabricator. Our activities are summarized on this page. To find out more please visit the Dry Lab Hub.

Growth Curve

We have developed a simple model for the growth of B. subtilis where the rate of growth is related to the amount of nutrients available. To this purpose we have exploited the ideas put forward by last year's Imperial College iGEM team for their modelling of F2620 in a cell-free system.

Genetic Circuit

We have also built mathematical models for the time evolution of the basic genetic circuits that comprise our device. We have verified which model best describe the behaviour of the circuit better by using laboratory data.

Motility Analysis

Finally, we have carried out a detailed analysis of the swimming motility of B. subtilis, which led us, among other things, to develop a simple mechanical model for the swimming motility of B. subtilis. Using manual tracking, we were able to extract x,y coordinate data from the cell trajectory. This has allowed us to fit experimental data with our model. The data suggest that flagellar force of B. subtilis is Exponentially distributed.

All model simulations and motility data analysis were carried out with MATLAB. Cell tracking was done with ImageJ via the Manual Tracking Plugin. All our MATLAB files can be found in the Appendices section.

Motility Summary.jpg

Implementation

Following the design stage of our project we moved on to the implementation stage. This involved construction of a cloning strategy, construction of our biobricks and transformation and characterisation of these biobricks in B. subtilis. For more information on this aspect of the project please see the Wet Lab Hub.

Implementation.PNG

Testing

The testing and validation of our project can be split into three main areas;

  • Work with B. subtilis - Including characterisation of growth curves and transformation,
  • Extensive Characterisation of new B.subtilis biobricks, Chloramphenicol resistance gene and motility
  • Production of Biomaterials in B. subtilis

If you'd like to see more information on the key results from the testing and validation, you can find it on the Results Page.

Results
Result.PNG

Achievements

Here is a summary of the achievements of the Imperial College 2008 team:

  • Submitted 45 documented parts to the Registry
  • Characterized and improved the existing part BBa J31005 (chloramphenicol acetyl transferase, CAT)
  • Characterized the new promoter and ribosome binding sites biobricks BBa K143079 and BBa K143082 that we submitted this year.
  • Developed integration sequences for Biobricks, to allow devices to be constructed that can then be excised and planted into B. subtilis
  • Laid the groundwork for future teams to work with B. subtilis by BioBricking and characterising promoters, RBSs, integration sequences, coding sequences and complex devices
  • Showed that expansion into other organisms is a definite possibility!
  • Developed a method for tracking and analysing bacterial motility
  • Helped Bristol by sending them a mini-iGEM project: Chemotactic dot-to-dot with information on quorum sensing and directed movement
  • Helped Bristol by sending them part BBa_J37015 (AHL generator + GFP) from our 2006 stock which was an empty vector in the Registry
  • Helped Cambridge by sending them a plate of Synechocystis PCC680 and a genome preparation


Of course, that is a very simplified description of our project. We expanded upon our project by looking into possible areas for real-world applications. For a case-study of such an implementation, check out how our project fits in with >>> Biocouture >>>



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