Team:Edinburgh/Results

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Project Results

The following sections summarise the main results we achieved during the summer of working on iGEM, and on the various strands of our Communicating through BRIDGEs project. We would like to emphasise, both here and elsewhere, that although we were unable to accomplish absolutely everything that we aimed for, we made great progress in gaining preliminary results and in establishing the foundation of extremely useful (and promotable!) technologies. This section of our wiki will list what we have achieved so far; elsewhere in the individual 'Future' pages for each section, we will also summarise our progress on what we'd like to do next and any issues that remain, including listing possible reasons for the problems we currently face and the experiments that are currently undergoing to test these.

Of course, we are continuing experiments and hope to have more data to present in the talk... the results presented here are simply a snapshot in time, and it is highly likely that we'll have more to present at the Jamboree!

The results we have achieved...

• ... for the development of the BRIDGE protocol for markerless BioBrick insertion (genomic BRIDGEs).
• ... for the establishment of the FORTH framework for light-based communication (bacterial BRIDGEs).
• ... for the modelling aspects of the above BRIDGE and FORTH components.
• ... for the human practices component of our project (human BRIDGEs).

The future we foresee for...

• ... the applications for and usage of the BRIDGE markerless insertion protocol.
• ... the applications for and usage of FORTH light-based communication.
• ... the continued improvement of our models and the applications of the Kappa BioBrick modelling framework.
• ... more future applications for light-based communication, as discussed in a human BRIDGEs context.

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 the characterisation here). 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:

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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

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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

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Results: In conclusion

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