Project Overview

Planning

In the process of deciding on a project for iGEM 2013 we've considered a multitude of ideas, including bacterial consortia against desertification, genomic encryption, anti-algal bacterial paint, and bacterial nano wires. Each of them was found to have its own strengths and weaknesses. For example bacterial consortia was a wonderful big idea, with a fundamental background from recent research, however due to the competition format and time limit on iGEM it was considered unreliable, where the level of input would have had little effect on the output due to the novelty of the subject, limited time and resourced to build such a system. The genomic encryption and nano wires ware not taken any further from the discussion at a number of meetings due to the lack of comprehensive information on the matters. While researching modern synthetic biology techniques we have found that a cell wall, one of the standard components of the bacterial cell may often cause difficulties in transformation efficiency, secretion of recombinant proteins, adapting to the environment etc. That is how the idea of removing a cell wall crossed our minds. As a result of this we have found a project we believe to be destined to revolutionise the approach to synthetic biology by introducing a BioBrick which would act as a switch for production and loss of bacterial cell wall in the model Gram positive bacteria Bacillus subtilis on our demand, while still allowing it to grow and divide. B. subtilis without a cell wall is better in many different ways and should be used as a new chassis for use in Synthetic Biology.

Bacteria which have lost their cell wall which are still able to grow and propagate are called L-forms, or as we prefer to call them, naked bacteria. There are loads of things that you can do with naked bacteria, we explored a few of them:

• Genome Shuffling
• Introduction and Detection of Naked Bacteria in Plants
• Shape Shifting

[http://www.youtube.com/watch?v=b0Kk6bKKOQ0 Click here to view a short video which explains or project.]

This isn’t a finite list of what can be done with naked bacteria, there’s loads more! L-forms are currently used to discover novel antibiotics which don’t act on the cell wall. L-forms can teach us a great deal about how bacterial life has evolved, through acting as a model for a cell wall-less bacterial progenitor, and through being able to test the ease of induction of endosymbiosis in cell wall-less organisms (Mercier et al. 2013).

L-form bacteria can be used in any process which protoplasts are currently used for. Protoplasts are bacteria which have been chemically induced to lose their cell wall. They cannot however grow or divide (as L-forms can) and are not classified as being alive. L-forms can be used to transform bacteria which are recalcitrant to transformation (Chang and Cohen 1979).

And a very important bonus, which comes with our naked bacteria is that they are osmotically sensitive, meaning that they will lyse if they escape into the environment. Therefore they can be used in non-contained environments (e.g agriculture).

Analysis

List of requirements for our system (i.e desired qualities of the L-forms) This could be our mini themes descriptions.

Modelling

The next step on the diagram is modelling. This step is an essential part of a successful synthetic biology project. Although it requires a lot of time and effort, and therefore is often neglected. We believe that in the long run modelling can save a lot of time, effort and resources to those who take their time in the beginning, simulating all the possible outcomes of the system and refining it at an early stage, before any in vitro and in vivo experiments have been planned and conducted. Another positive side to modelling prior to the "wet lab" sessions is the fact that a model behaves according to the known facts and principles, and if in real life the outcome drastically differs from the simulation, there's a good chance of finding out what may be causing the difference through adjusting the model and repeating the experiments.

For every research theme we have constructed a model to help us understand the systems we engineered. Click on the links to view each model or visit our modelling page:

• L-form switch
• Cell Shape
• Cell Fusion
• Hbsu-xFP

Implementation

What we've done for each project (brief summary)

Testing

Blah blah blah

All you need to start using an L-form chassis is a culture of Bacillus subtilis, our L-form switch BioBrick and a set of instructions from us.

References

[http://www.ncbi.nlm.nih.gov/pubmed/11849491 Walker R, Ferguson CMJ, Booth NA and Allan EJ (2002) The symbiosis of Bacillus subtilis L-forms with Chinese cabbage seedlings inhibits conidial germination of ‘Botrytis cinerea. Letters in Applied Microbiology, 34, 42-45.]

[http://www.ncbi.nlm.nih.gov/pubmed/107388 Chang S and Cohen SN (1979)High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Molecular Genetics & Genomics, 168, 111–115.]

[http://www.cell.com/abstract/S0092-8674(13)00135-9 Mercier R, Kawai Y and Errington J. (2013) Excess Membrane Synthesis Drives a Primitive Mode of Cell Proliferation, Cell, 152, 997–1007.]