Team:Edinburgh/Results

From 2010.igem.org

(Difference between revisions)
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<br>
<br>
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<p>
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<h3>Characterisation of <i>sacB</i>:</h3><br>
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<b>Characterisation of <i>sacB</i>:</b><br><br>
+
 
-
We demonstrated that cell lines containing <i>sacB</i> are unable to grow in sucrose at a concentration of 10% compared to control cells which grew normally (see BRIDGE: The Protocol). This confirms it's usefulness as a negative selection marker in the BRIDGE protocol and provided us with conditions for selection at the end of the second step of the protocol.<br><br>
+
<p>We demonstrated that cell lines containing <i>sacB</i> are unable to grow in sucrose at a concentration of 10% compared to control cells which grew normally (see <a href="http://2010.igem.org/Team:Edinburgh/Project/Protocol">BRIDGE: The Protocol</a>). This confirms its usefulness as a negative selection marker in the BRIDGE protocol and provided us with conditions for selection at the end of the second step of the protocol.</p><br><br>
-
<b>Characterisation of <i>cat</i>:</b><br><br>
+
 
-
<br>
+
<h3>Characterisation of <i>cat</i>:</h3><br>
-
<b>Protocol:</b><br><br>
+
 
-
The majority of ground gained in our time working on the actual protocol for BRIDGE was in the adjustment of experimental procedure and the design of control experiments.<br><br>
+
<p>***</p><br><br>
-
We managed to fine tune the protocol to our resources and particular strains without causing damage to the cells. For example, we confirmed that ddH20 is not necessary for the washing step. Despite having access to ddH20 we used cold sterile water, which is just as effective at removing LB and antibiotic from cell cultures. These tweaks make the procedure slightly more cost and time effective, especially for labs with limited resources.<br><br>
+
 
-
We narrowed down the source of entry of a contaminant which was repeatedly infecting our selection plates by sampling the cells at every stage after exposure to unsterile air, a solution or instrument. Through use of negative controls and stringent aseptic technique we managed to eliminate contamination entirely. For example, we sampled the sterile water we had been using and moved our washing step the clean hood, away from sterile air. These controls enabled us to edit the protocol to advise others on how to avoid contamination and how to find the source of a contaminant should it occur. <br><br>
+
<h3>Protocol:</h3><br>
-
We developed a simple titre of antibiotic resistance by growing our transformants in increasing concentrations of chloramphenicol along with negative controls. This can be used to determine a) the level of success of the procedure and b) the level of resistance of an antibiotic resistance gene (making good characterisation data for the registry.<br><br>
+
 
-
Unfortunately we never achieved our goal of replacing <i>tnaA</i> with the <i>cat/sacB</i> construct via recombineering, but our lab will continue to work with this protocol, possibly replacing the <i>cat</i> gene with a kanamycin resistance gene.<br>
+
<p>The majority of ground gained in our time working on the actual protocol for BRIDGE was in the adjustment of experimental procedure and the design of control experiments.</p>
-
For anyone wishing to use this protocol we recommend focusing on the recombinase induction step, as this seems to be the problem area which results in the lack of recombinants. <br><br>
+
 
-
</p>
+
<p>We managed to fine tune the protocol to our resources and particular strains without causing damage to the cells. For example, we confirmed that ddH20 is not necessary for the washing step. Despite having access to ddH20 we used cold sterile water, which is just as effective at removing LB and antibiotic from cell cultures. These tweaks make the procedure slightly more cost and time effective, especially for labs with limited resources.</p>
 +
 
 +
<p>We narrowed down the source of entry of a contaminant which was repeatedly infecting our selection plates by sampling the cells at every stage after exposure to unsterile air, a solution, or an instrument. Through use of negative controls and stringent aseptic technique we managed to eliminate contamination entirely. For example, we sampled the sterile water we had been using and moved our washing step the clean hood, away from sterile air. These controls enabled us to edit the protocol to advise others on how to avoid contamination and how to find the source of a contaminant should it occur.</p>
 +
 
 +
<p>We developed a simple titre of antibiotic resistance by growing our transformants in increasing concentrations of chloramphenicol along with negative controls. This can be used to determine a) the level of success of the procedure and b) the level of resistance of an antibiotic resistance gene (making good characterisation data for the registry.</p>
 +
 
 +
<p>Unfortunately we never achieved our goal of replacing <i>tnaA</i> with the <i>cat/sacB</i> construct via recombineering, but our lab will continue to work with this protocol, possibly replacing the <i>cat</i> gene with a kanamycin resistance gene.</p>
 +
 
 +
<p>For anyone wishing to use this protocol we recommend focusing on the recombinase induction step, as this seems to be the problem area which results in the lack of recombinants.</p>
<br>
<br>
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<a name="Modelling" id="Modelling"></a><h2>Results: Modelling BRIDGEs</h2>
<a name="Modelling" id="Modelling"></a><h2>Results: Modelling BRIDGEs</h2>
<br>
<br>
 +
 +
<h3>The completed models:</h3><br>
<p>The main <b>result</b> achieved by the modelling component of our project was the theoretical <b>conclusion</b> that, given the <b>information</b> available and the <b>assumptions</b> made, the biological systems <b>proposed</b> throughout our project should work. The <a href="http://2010.igem.org/Team:Edinburgh/Modelling/Genomic">genomic BRIDGEs model</a> provided <b>verification</b> of the time course of the <a href="http://2010.igem.org/Team:Edinburgh/Project/Protocol">BRIDGE protocol</a>. The <a href="http://2010.igem.org/Team:Edinburgh/Modelling/Bacterial">intracellular bacterial BRIDGEs model</a> acted to verify the idea of a <a href="http://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">light-based repressilating system</a>, and to confirm that the responses of the <a href="http://2010.igem.org/Team:Edinburgh/Modelling/Bacterial">various light pathways</a> were as expected. Finally, the <a href="http://2010.igem.org/Team:Edinburgh/Modelling/Signalling">intercellular bacterial BRIDGEs model</a> established the concept of light-based communication within colonies of cells, in all its <b>complexity</b>.</p>
<p>The main <b>result</b> achieved by the modelling component of our project was the theoretical <b>conclusion</b> that, given the <b>information</b> available and the <b>assumptions</b> made, the biological systems <b>proposed</b> throughout our project should work. The <a href="http://2010.igem.org/Team:Edinburgh/Modelling/Genomic">genomic BRIDGEs model</a> provided <b>verification</b> of the time course of the <a href="http://2010.igem.org/Team:Edinburgh/Project/Protocol">BRIDGE protocol</a>. The <a href="http://2010.igem.org/Team:Edinburgh/Modelling/Bacterial">intracellular bacterial BRIDGEs model</a> acted to verify the idea of a <a href="http://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">light-based repressilating system</a>, and to confirm that the responses of the <a href="http://2010.igem.org/Team:Edinburgh/Modelling/Bacterial">various light pathways</a> were as expected. Finally, the <a href="http://2010.igem.org/Team:Edinburgh/Modelling/Signalling">intercellular bacterial BRIDGEs model</a> established the concept of light-based communication within colonies of cells, in all its <b>complexity</b>.</p>
-
<p>Each of the above models was extensively <b>analysed</b> via a variety of <b>methods</b> available, in an attempt to <b>push the boundaries</b> of <b>understanding</b> regarding the biological processes <b>embodied</b> within. Some interesting <b>results</b> were revealed, but overall this extensive analysis did much to <b>reinforce</b> the conclusion made above - that theoretically, our systems should work!</p>
+
<p>Each of the above models was extensively <b>analysed</b> via a variety of <b>methods</b> available, in an attempt to <b>push the boundaries</b> of <b>understanding</b> regarding the biological processes <b>embodied</b> within. Some interesting <b>results</b> were revealed, but overall this extensive analysis did much to <b>reinforce</b> the conclusion made above - that theoretically, our systems should work!</p><br><br>
 +
 
 +
<h3>The Kappa BioBrick framework:</h3><br>
-
<p>Throughout the process, Ty Thomson's <b>framework</b> for modelling BioBricks in Kappa was found to be an <b>invaluable</b> aid in <b>organising</b> and <b>thoroughly describing</b> the biological parts involved. Whether in simply <b>ensuring</b> that the entirety of the BioBrick's actions were <b>described</b>, or in making <b>explicit</b> the <b>correlation</b> between various rate parameters and their <b>effects</b> upon the model, the usefulness of such a <b>structured</b> and <b>standardised</b> framework in developing biological models cannot be <b>understated</b>.</p>
+
<p>Throughout the process, Ty Thomson's <b>framework</b> for modelling BioBricks in Kappa was found to be an <b>invaluable</b> aid in <b>organising</b> and <b>thoroughly describing</b> the biological parts involved. Whether in simply <b>ensuring</b> that the entirety of the BioBrick's actions were <b>described</b>, or in making <b>explicit</b> the <b>correlation</b> between various rate parameters and their <b>effects</b> upon the model, the usefulness of such a <b>structured</b> and <b>standardised</b> framework in developing biological models cannot be <b>understated</b>. We hope in the near future to be able to <b>extend</b> this into the basis of a 'Virtual Registry' of modelled and characterised BioBrick parts and devices, with corresponding <b>tools</b> to aid design and simulation..</p>
<p>One final result that hopefully was achieved by our modelling is the <b>establishment</b> and <b>promotion</b> of <a href="http://2010.igem.org/Team:Edinburgh/Modelling/Kappa">the Kappa stochastic rule-based modelling language</a> as a <b>BioBrick-friendly alternative</b> to traditional methods of modelling such as differential equations. From its <b>introduction</b> to iGEM by last year's Edinburgh team, this year its use has <b>spread</b> to a handful of other teams as well... we are hopeful that this <b>trend</b> will continue in the near future, due to the numerous <b>advantages</b> that are inherent in the approach.</p>
<p>One final result that hopefully was achieved by our modelling is the <b>establishment</b> and <b>promotion</b> of <a href="http://2010.igem.org/Team:Edinburgh/Modelling/Kappa">the Kappa stochastic rule-based modelling language</a> as a <b>BioBrick-friendly alternative</b> to traditional methods of modelling such as differential equations. From its <b>introduction</b> to iGEM by last year's Edinburgh team, this year its use has <b>spread</b> to a handful of other teams as well... we are hopeful that this <b>trend</b> will continue in the near future, due to the numerous <b>advantages</b> that are inherent in the approach.</p>

Revision as of 09:30, 27 October 2010







Project Results


***



Results: Genomic BRIDGEs


Characterisation of sacB:


We demonstrated that cell lines containing sacB are unable to grow in sucrose at a concentration of 10% compared to control cells which grew normally (see BRIDGE: The Protocol). This confirms its usefulness as a negative selection marker in the BRIDGE protocol and provided us with conditions for selection at the end of the second step of the protocol.



Characterisation of cat:


***



Protocol:


The majority of ground gained in our time working on the actual protocol for BRIDGE was in the adjustment of experimental procedure and the design of control experiments.

We managed to fine tune the protocol to our resources and particular strains without causing damage to the cells. For example, we confirmed that ddH20 is not necessary for the washing step. Despite having access to ddH20 we used cold sterile water, which is just as effective at removing LB and antibiotic from cell cultures. These tweaks make the procedure slightly more cost and time effective, especially for labs with limited resources.

We narrowed down the source of entry of a contaminant which was repeatedly infecting our selection plates by sampling the cells at every stage after exposure to unsterile air, a solution, or an instrument. Through use of negative controls and stringent aseptic technique we managed to eliminate contamination entirely. For example, we sampled the sterile water we had been using and moved our washing step the clean hood, away from sterile air. These controls enabled us to edit the protocol to advise others on how to avoid contamination and how to find the source of a contaminant should it occur.

We developed a simple titre of antibiotic resistance by growing our transformants in increasing concentrations of chloramphenicol along with negative controls. This can be used to determine a) the level of success of the procedure and b) the level of resistance of an antibiotic resistance gene (making good characterisation data for the registry.

Unfortunately we never achieved our goal of replacing tnaA with the cat/sacB construct via recombineering, but our lab will continue to work with this protocol, possibly replacing the cat gene with a kanamycin resistance gene.

For anyone wishing to use this protocol we recommend focusing on the recombinase induction step, as this seems to be the problem area which results in the lack of recombinants.



Results: Bacterial BRIDGEs


***





Results: Modelling BRIDGEs


The completed models:


The main result achieved by the modelling component of our project was the theoretical conclusion that, given the information available and the assumptions made, the biological systems proposed throughout our project should work. The genomic BRIDGEs model provided verification of the time course of the BRIDGE protocol. The intracellular bacterial BRIDGEs model acted to verify the idea of a light-based repressilating system, and to confirm that the responses of the various light pathways were as expected. Finally, the intercellular bacterial BRIDGEs model established the concept of light-based communication within colonies of cells, in all its complexity.

Each of the above models was extensively analysed via a variety of methods available, in an attempt to push the boundaries of understanding regarding the biological processes embodied within. Some interesting results were revealed, but overall this extensive analysis did much to reinforce the conclusion made above - that theoretically, our systems should work!



The Kappa BioBrick framework:


Throughout the process, Ty Thomson's framework for modelling BioBricks in Kappa was found to be an invaluable aid in organising and thoroughly describing the biological parts involved. Whether in simply ensuring that the entirety of the BioBrick's actions were described, or in making explicit the correlation between various rate parameters and their effects upon the model, the usefulness of such a structured and standardised framework in developing biological models cannot be understated. We hope in the near future to be able to extend this into the basis of a 'Virtual Registry' of modelled and characterised BioBrick parts and devices, with corresponding tools to aid design and simulation..

One final result that hopefully was achieved by our modelling is the establishment and promotion of the Kappa stochastic rule-based modelling language as a BioBrick-friendly alternative to traditional methods of modelling such as differential equations. From its introduction to iGEM by last year's Edinburgh team, this year its use has spread to a handful of other teams as well... we are hopeful that this trend will continue in the near future, due to the numerous advantages that are inherent in the approach.



Results: Human BRIDGEs


***



Results: In conclusion


***




Throughout this wiki there are words in bold that indicate a relevance to human aspects. It will become obvious that human aspects are a part of almost everything in iGEM.