IGEM:Cambridge/2008/Turing Pattern Formation

This project seeks to generate Turing Patterns by creating a Reaction-Diffusion system in the gram-positive bacteria Bacillus subtilis. We need to integrate two signalling systems into this bacterium and use an autofeedback mechanism to generate self-organizing patterns from random noise. We plan to incorporate the agr peptide signalling system from S. aureus and the lux AHL system from V. fisheri.

Introduction

The reaction-diffusion system depends on an activator and inhibitory signal that spread throughout the medium. The "grasshopper" example is quite intuitive: Imagine it is hot and there is a field of dry grass with grasshoppers. Suddenly, a fire starts burning at some point and spreads (the activator signal) so that the grasshoppers move away from that point to avoid the fire. However, the grasshoppers also generate moisture (the inhibitory signal) thus preventing the areas of dry grass the grasshoppers move to of catching fire. The result will be the initial patch of the field that has burnt down surrounded by moisture preventing the fire from spreading. Imagine now that at the beginning, not a single place but numerous randomly distributed places (resembling noise) of dry grass caught fire. The resulting patterning of charred grass and grasshoppers is called a Turing Pattern. It is important to note is that the inhibitory signal (grasshoppers) must travel faster than the activation signal (fire) as to prevent the whole field from burning down.

More to come. Pictures and such.

Materials

Bacillus strain 1A1

- deficient in tryptophan, have to add to media
- keep at room temp, aren't freezable

2 shuttle vectors:

• ppL82 (ampicillin) in DH5a
• pNZ8901 (SURE plasmid, chloramphenicol) in MC1061
• Daniel Goodman 10:00, 22 July 2008 (UTC): See paper below on SURE expression system
• Daniel Goodman 10:00, 22 July 2008 (UTC): Can we get/do we have sequences of these?

Steps/Progress

Lab Work

Test individual AIP sender-receiver parts:

• reciever: using supernatant or purified AIP
• sender: using some method to detect AIP

Protocols

Electroporation Competence protocol [www1.qiagen.com/literature/protocols/pdf/QP02.pdf Plasmid extraction protocol from Quiagen]

• We have Chris French's Bacillus protocol for growing/transformation, need to copy to wiki
• We have last year's protocol; not sure if it's trustworthy, need to compare with online resources

Notable Papers/Pages

Turing Patterns/Reaction Diffusion Patterns

Short introduction to reaction/diffusion systems and pattern formation

Bacillus subtilis

B. Subtilis Transformation Protcol - Electroporation

Bongers et al. … of a Subtilin-Regulated Expression System in Bacillus subtilis: Strict Control of Gene Expression …. Applied and Environmental Microbiology (2005)

• SURE: handy expression system for B. subtilis
• What about feedback/regulation effects of subtilin on the cell

Expression and characterization of aiiA gene from Bacillus subtilis BS-1.

• (Some strains of) Bacillus subtilis produces a gene called aiiA that degrades AHL molecules from gram-negative bacteria
• this is biobricked

agr system

mutual exclusion between agr systems from different S. aureus strains

• could we model this? could lead to interesting behavior...

Identification of the Putative Staphylococcal AgrB Catalytic Residues Involving the Proteolytic Cleavage of AgrD to Generate Autoinducing Peptide

• AIP is cleaved frm the propeptide on export, it looks like no other machinery is needed

lux system

cyclic dipeptides bind competitively and antagonize AHLs in luxR-based signalling..can we exploit?

Quorum Sensing/Cell-Cell Communication

great general overview of QS in both gram-neg and gram-pos bacteria