IGEM:Cambridge/2008/Turing Pattern Formation: Difference between revisions

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This project seeks to generate [[Wikipedia:Turing Patterns|Turing Patterns]] by creating a [[Wikipedia:Reaction-Diffusion System|Reaction-Diffusion system]] in the [[Wikipedia:gram-positive bacteria|gram-positive bacteria]] [[Wikipedia:Bacillus subtilis|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''.
<hr width="810px">
 
<div id="about">
 
{{Cambridge08a}}
== Introduction ==
</div>
 
<hr width="810px">
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. 
= Background Research =
 
More to come. Pictures and such.
 
== Whiteboard ==
 
[[image:igem-whiteboard.jpg|thumb|right|Daniel Goodman]]
 
== 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
*'''[[User:Daniel Goodman|Daniel Goodman]] 10:00, 22 July 2008 (UTC)''': See paper below on SURE expression system
*'''[[User:Daniel Goodman|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  
*Find promoters that we can express constitutively  
**[[Cambridge/2008/Turing Pattern_Formation/Promoters|Promoters Page]]
**[[Cambridge/2008/Turing Pattern_Formation/Promoters|Promoters & Vectors Page]]
*Look through Bacillus subtilis Stock Center for strains/promoters
*Look through Bacillus subtilis Stock Center for strains/promoters


Line 59: Line 13:
** use mutant with these knocked out (WB600,WB700,WB800) -- see papers on 2007 subtilis page
** use mutant with these knocked out (WB600,WB700,WB800) -- see papers on 2007 subtilis page


=== Lab Work ===
=== Grow up shuttle vectors in E coli ===
 
 
==== Grow up shuttle vectors in E coli ====
*'''[[User:Daniel Goodman|Daniel Goodman]] 18:11, 22 July 2008 (UTC)''': currently testing shuttle vectors & strains for correct resistances
*'''[[User:Daniel Goodman|Daniel Goodman]] 18:11, 22 July 2008 (UTC)''': currently testing shuttle vectors & strains for correct resistances
*'''[[User:Daniel Goodman|Daniel Goodman]] 18:11, 22 July 2008 (UTC)''': next, check to see if shuttle vectors run correctly on the gel
*'''[[User:Daniel Goodman|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 ====
=== Extract biobricks, pull out the genes from the plasmid ===
*A BBa_I746210 Signalling AIP Sender 3353
*A BBa_I746000 AIP Generator (agrB + agrD+ RBSes)                 759bp
*A   BBa_I746211 Signalling AIP Sender 3353
*A   BBa_I746001 AIP Generator (agrB + agrD+ RBSes) + B0015 terminator 896bp
*A BBa_I746220 Signalling AIP Detector 5504
*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 ====  
=== Make shuttle vectors biobrick compatible ===  
* remove unfriendly restriction sites
* remove unfriendly restriction sites


==== Standardize Bacillus transformation protocol ====
=== 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
* 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: ====  
=== Test individual AIP sender-receiver parts: ===  
*reciever: using supernatant or purified AIP
*reciever: using supernatant or purified AIP
*sender: using some method to detect AIP or using a verified reciever
*sender: using some method to detect AIP or using a verified reciever.
*'''[[User:Kevin Xu Cheng|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 =
 
* For a more comprehensive description, go to the [[IGEM:Cambridge/2008/Turing Pattern_Formation/Modelling | Modelling page]].
 
=== Modelling reaction-diffusion using finite difference method === 
* Implement simple reaction-diffusion systems numerically using finite difference methods. Determine parameter space.
*'''[[User:Xiao-Hu Yan|Xiao-Hu Yan]] 10:29, 1 August 2008 (UTC)''':DONE


=== Modeling/Computation ===
=== Modelling intra-/intercellular behaviour ===
==== look over James' MATLAB code ====
* Need to think about which approach is most appropriate to model agr/lux kinematics.
* think about how to implement the diffusion model
* look at cellular automata tools in matlab


== Protocols ==
=== 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?
* [http://www.nature.com/nature/journal/v403/n6767/abs/403339a0.html A genetic toggle switch] Would be great to run some models using this "memory" device. Possible use of leaky promoter?
 
=Resources=
* Check out this huge [http://dbtbs.hgc.jp/ database on B. subtilis genes, promoters and transcription factors]. Use it in conjunction with the [http://www.bgsc.org/ Bacillus Stock Center] or to check and find papers and references on genes/promoters etc we are considering of using.
 
=== Protocols ===


[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T30-3VCK5Y0-3&_user=1495569&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000053194&_version=1&_urlVersion=0&_userid=1495569&md5=847137ffc5cf267e12b5625aaadecb0e  Electroporation Competence protocol]
[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T30-3VCK5Y0-3&_user=1495569&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000053194&_version=1&_urlVersion=0&_userid=1495569&md5=847137ffc5cf267e12b5625aaadecb0e  Electroporation Competence protocol]
Line 96: Line 65:
* Chris French's [http://openwetware.org/wiki/Cfrench:BacTrans1 protocol on transforming B. subtilis]
* Chris French's [http://openwetware.org/wiki/Cfrench:BacTrans1 protocol on transforming B. subtilis]
* We have last year's protocol; not sure if it's trustworthy, need to compare with online resources
* We have last year's protocol; not sure if it's trustworthy, need to compare with online resources
* [[IGEM:Cambridge/2008/Turing_Pattern_Formation/BCSG_Transformation_Protocol|Protocol from BCSG catalog]]
** used in Cornell bacteriology course, probably should start here


== Resources ==
== Notable Papers/Pages ==
=== Turing Patterns/Reaction Diffusion Patterns ===
=== Turing Patterns/Reaction Diffusion Patterns ===
[http://www.google.co.uk/url?sa=t&ct=res&cd=6&url=http%3A%2F%2Fcsmres.jmu.edu%2Fgeollab%2FFichter%2FGS102%2F2004handouts%2Freactiondiffusion2.PDF&ei=C19_SJuwNpye1gavjeDWCQ&usg=AFQjCNFifmBEne6pl0i767IGFEcmSYztnw&sig2=GC7WmW1b4ufJ-LRvpSFJdw Short introduction to reaction/diffusion systems and pattern formation]
[http://www.google.co.uk/url?sa=t&ct=res&cd=6&url=http%3A%2F%2Fcsmres.jmu.edu%2Fgeollab%2FFichter%2FGS102%2F2004handouts%2Freactiondiffusion2.PDF&ei=C19_SJuwNpye1gavjeDWCQ&usg=AFQjCNFifmBEne6pl0i767IGFEcmSYztnw&sig2=GC7WmW1b4ufJ-LRvpSFJdw Short introduction to reaction/diffusion systems and pattern formation]
[http://www.iop.org/EJ/abstract/0034-4885/55/6/003/ An extensive chapter on pattern formation and mathematical models]
[http://prola.aps.org/abstract/PRE/v59/i6/p7036_1 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 ===
=== Bacillus subtilis ===
Line 119: Line 92:
[http://www.jbc.org/cgi/content/abstract/280/17/16695 Identification of the Putative Staphylococcal AgrB Catalytic Residues Involving the Proteolytic Cleavage of AgrD to Generate Autoinducing Peptide]
[http://www.jbc.org/cgi/content/abstract/280/17/16695 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
* 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 ===
=== lux system ===
[http://www3.interscience.wiley.com/journal/118790052/abstract cyclic dipeptides bind competitively and antagonize AHLs in luxR-based signalling..can we exploit?]  
[http://www3.interscience.wiley.com/journal/118790052/abstract cyclic dipeptides bind competitively and antagonize AHLs in luxR-based signalling..can we exploit?]  
=== Quorum Sensing/Cell-Cell Communication ===
=== Quorum Sensing/Cell-Cell Communication ===
[http://arjournals.annualreviews.org/doi/full/10.1146/annurev.cellbio.21.012704.131001 great general overview of QS in both gram-neg and gram-pos bacteria]
[http://arjournals.annualreviews.org/doi/full/10.1146/annurev.cellbio.21.012704.131001 great general overview of QS in both gram-neg and gram-pos bacteria]


=== links to 2007 wiki ===


[http://www.ccbi.cam.ac.uk/iGEM2007/index.php/Bacillus_subtilis_SynBio_chassis B. subtilis group]
 
[http://www.ccbi.cam.ac.uk/iGEM2007/index.php/Signalling_project_description Peptide signaling group]
== Links to Cambridge 2007 Wiki ==
[http://www.ccbi.cam.ac.uk/iGEM2007/index.php/Detailed_background_information_-_B._subtilis B. subtilis group: more info]
 
*[http://www.ccbi.cam.ac.uk/iGEM2007/index.php/Bacillus_subtilis_SynBio_chassis B. subtilis group]
*[http://www.ccbi.cam.ac.uk/iGEM2007/index.php/Signalling_project_description Peptide signaling group]
*[http://www.ccbi.cam.ac.uk/iGEM2007/index.php/Detailed_background_information_-_B._subtilis B. subtilis group: more info]
 
 
 
=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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Latest revision as of 11:19, 1 August 2008

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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