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 (derivative of strain 168)

• 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?

Plans

Test of Plasmid & Strains

• Grow B. subtilis (Done 22/7)
• Mini-prep plasmids
• Digest with enzymes to test size of plasmids

Test for Tranforming

• Make GFP inserts [AmpR - Promoter - GFP] [Can be done parallel with Plasmid testing]
• Input GFP insert into plasmid using E. coli
• Transform ligated plasmid into B. subtilis from E. coli

Quantifying promoters

• Make GFP inserts with different promoters [AmpR - Promoter - GFP] [Can be done parallel with Plasmid testing]
• Quantify GFP expression

Test AgR system

• Build [AgrC - AgrA] and [AgrB - AgrD] [PCR'ing out from 2007 tubes]
• Input into shuttle vectors
• Transform them into B. subtilis

Biobricking AgR system

• add in promoters
• place in cut sites

Steps/Progress

Background Research

• Find promoters that we can express constitutively
  • Promoters Page
• Look through Bacillus subtilis Stock Center for strains/promoters

• Do we need to be concerned about extracellular proteases in B. subtilis?
  • use mutant with these knocked out (WB600,WB700,WB800) -- see papers on 2007 subtilis page

Lab Work

Grow up shuttle vectors in E coli

• Daniel Goodman 18:11, 22 July 2008 (UTC): currently testing shuttle vectors & strains for correct resistances
• Daniel Goodman 18:11, 22 July 2008 (UTC): next, check to see if shuttle vectors run correctly on the gel

Extract biobricks, pull out the genes from the plasmid

• A BBa_I746210 Signalling AIP Sender 3353
• A BBa_I746211 Signalling AIP Sender 3353
• A BBa_I746220 Signalling AIP Detector 5504

Make shuttle vectors biobrick compatible

• remove unfriendly restriction sites

Standardize Bacillus transformation protocol

• use protocols listed below, as well as other resources, and vary parameters to figure out EXACTLY what conditions give the most competent cells

Test individual AIP sender-receiver parts:

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

Modeling/Computation

look over James' MATLAB code

• think about how to implement the diffusion model
• look at cellular automata tools in matlab

Protocols

Electroporation Competence protocol

Plasmid extraction protocol from Qiagen

• Chris French's lab page - protocols & documentation on the biobricking process.

• Chris French's protocol on transforming B. subtilis
• 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

links to 2007 wiki

B. subtilis group Peptide signaling group B. subtilis group: more info