IGEM:Cambridge/2008/Turing Pattern Formation

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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

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_I746000 AIP Generator (agrB + agrD+ RBSes) 759bp
  • A BBa_I746001 AIP Generator (agrB + agrD+ RBSes) + B0015 terminator 896bp
  • A BBa_I746100 AIP Receiver (agrC + agrA + RBSes) 1920bp
  • A BBa_I746101 AIP Receiver (agrC + agrA + RBSes) + B0015 terminator 2057bp

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.


Modelling/Computation

Modelling reaction-diffusion using finite difference method

  • Implement simple reaction-diffusion systems numerically using finite difference methods. Determine parameter space.
  • Xiao-Hu Yan 10:29, 1 August 2008 (UTC):DONE

Modelling intra-/intercellular behaviour

  • Need to think about which approach is most appropriate to model agr/lux kinematics.

Beyond reaction-diffusion

  • Is it possible to arrive at Turing-like patterns using a different approach than the activator-inhibitor system?
  • In particular, can we generate patterns with a single signalling molecule?
  • A genetic toggle switch Would be great to run some models using this "memory" device. Possible use of leaky promoter?

Resources

Protocols

Electroporation Competence protocol

Plasmid extraction protocol from Qiagen

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

Turing Patterns/Reaction Diffusion Patterns

Short introduction to reaction/diffusion systems and pattern formation

An extensive chapter on pattern formation and mathematical models

Modeling of spatiotemporal patterns in bacterial colonies

  • paper on the range of B.subtilis patterns on different media. 2nd species is nutrient. Can we exploit? Also includes realistic models we should consider.

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
  • Question: does system need another intracellular processing enzyme to export AIP?
  • Question: how leaky is P2 promoter?
  • Question: where to get AIP? (check Novick 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 Cambridge 2007 Wiki


Notes

  • Team discussion 9.05 am Genetics Department


Here's a copy of the email I sent on the 20th, with some early thoughts:

Here's an interesting one: http://www.pnas.org/content/96/4/1218.abstract

Did you know that there are four different AIPs (and associated machinery) in different strains of S. aureus, and that they are mutually exclusive? In other words, if you use one strain's AIP in another's receptor histidine kinase, it inhibits phosphorylation and stops the cascade? This could have some interesting potential applications in our project, and would be cool to model.

All of that is discussed in the following paper, which I have yet to read through thoroughly: http://pubs.acs.org/cgi-bin/abstract.cgi/bichaw/2002/41/i31/abs/bi026049u.html

The following is also a great paper, and but think you probably both have seen it: http://arjournals.annualreviews.org/doi/full/10.1146/annurev.cellbio.21.012704.131001?cookieSet=1

It goes over quorum sensing in both gram-neg and gram-pos bacteria, and describes exhaustively all the well-known systems and examples. I think you probably know this already James, but there is a well-characterized exporter that pumps out LasI, thus mitigating its toxicity. My guess is you had trouble expressing/folding/localizing the exporter to the membrane in E. coli. It might work better in Bacillus?

I know I mentioned this before, but working with as many different signalling systems as possible will give us more options when it comes to putting the whole system together. I know it's rather ambitious, but once we get transformation working this week (and possibly next), and assuming we get more people to work in parallel on this, we could examine transplanting multiple different QS systems into B. subtilis so we can do different combinations if need be. Maybe I'm getting ahead of myself.

In any case, I'd like to know why we're going with the AIP/lux combo specifically - what do we know about these systems so that we're confident that they will yield the right parameters for pattern formation? The idea of using one peptide and one AHL seems intuitive, as one will diffuse faster than the other, but if lux is 100s of times faster, instead of just 10-15x faster, won't lateral inhibition totally quash any activation at all?

I'm also concerned that, if we use a subtilin-based expression system like SURE, we will get some weird effects, as subtilin is responsible for modulating sporulation/competence in B. subtilis. I think this expression system works great if you're just trying to overexpress something, but I'm worried we could get medium- to long-term feedback effects that we're not currently anticipating. I will try to read more into this.

I'll keep reading for now, let me know your thoughts.

Cheers, Daniel


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