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?
Steps/Progress
Background Research
- Find promoters that we can express constitutively
- 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.
- Kevin Xu Cheng 15:28, 23 July 2008 (UTC): there appears to be an assay for thiol groups using Ellman's reagent, which we can use to quantify AIP. This is described in this paper: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2074992 which also gives information on purifying AIP from cells (although they have the added complexity of distinguishing iAIP from normal AIP, which we don't need. I've found a more straight forward protocol): http://www.sciencemag.org/cgi/content/full/276/5321/2027.) It also mentions using a protease inhibitor cocktail, which we can look into as an alternative to protease deficient strains.
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
- Protocol from BCSG catalog
- used in Cornell bacteriology course, probably should start here
Resources
- Check out this huge database on B. subtilis genes, promoters and transcription factors. Use it in conjunction with the Bacillus Stock Center or to check and find papers and references on genes/promoters etc we are considering of using.
Notable Papers/Pages
Turing Patterns/Reaction Diffusion Patterns
Short introduction to reaction/diffusion systems and pattern formation
Bacillus subtilis
B. Subtilis Transformation Protcol - Electroporation
- 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...
- AIP is cleaved frm the propeptide on export, it looks like no other machinery is needed
- Question: does system need another intracellular processing enzyme to export AIP?
- Question: how leaky is P2 promoter?
- Question: where to get AIP? (check Norvick group)
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