From 2006.igem.org

Revision as of 21:56, 10 August 2005

Back to ETH Zurich main page.

Design Group

Design group defined during teammeeting on Thu. 2005.08.04

Purpose

• Discuss those projects assigned to us and develop them further, both conceptually (modularity, coolness, usefulness) and implementation-wise (feasability, availability of components).
• Agree on some favored existing solution or merge current concepts into a new one.

Current Project Variants

• Sorting Bacteria
• Pulser
• Constructor Bacteria
• Controlled Cell Division
• Predator-Prey Behavior

Current Members

• Dominic Frutiger
• Giorgia Valsesia
• Herve Vanderschuren

New Members

New members: please add your name here so that we know that you want to join.

• Urs A. Müller
• Simon Barkow

Discussion Meetings

Everybody is more than welcome to join in!

2005.08.05, Friday, 15:30, Discussion/development of group topics, Giorgia & Dominic
2005.08.07, Sunday, 16:00, Discussion/development of group topics, Herve & Dominic
2005.08.08, Monday, 10:00, Discussion/development of group topics, all (visitors welcome)
2005.08.09, Tuesday, 16:00, Discussion of feasability/implementation regarding modules
2005.08.10, Wednesday, 09:00, Discussion of concept (Dominic, Simon)
2005.08.10, Wednesday, 17:30, Discussion of modules (Giorgia, Dominic) @polyterrasse
2005.08.11, Thursday, 09:00, Discussion of presentation preps (all)

Progress

During the first two meetings we mainly developed the existing concepts further or added variants. On monday we tried to find useful categories to identify what makes the projects similar and what makes them different - in order to reduce the number of true variants and merge interesting functions into a new project idea. We started to divide the existing project ideas further into certain behavior states and their possible expression:

Behavior States

• pattern formation
  • oscillation behavior and/or dynamic equilibria
  • convergence to specific end states with distinct intermediate states

• pattern display
  • fluorescence
  • physical structures

The first step would always be pattern formation. The difficulty with approaches aiming at oscillation behavior and/or dynamic equilibria (such as the Pulser or the Predator-Prey_Behavior) is that we can not break them down to intermediate stages and thus there is no step-wise increase of risk: either all aspects work at once and the interplay is balanced leading to some dynamic stability or the whole concept miserably fails. Also, it is more difficult to prove success.

As a result our discussions more and more converged towards approaches with specific static end states and intermediate layers.

Modules

Also, we tried to break down the existing variants into modules fulfilling a specific function:

Then we discussed the estimated (!) feasability and general usefulness of these modules to get a better feeling of what should be used and what will prove difficult to implement.

Problems with Pred-Prey

We quickly found that the implementation of true Predator-Prey behavior would be very interesting, but that there are serious drawbacks:

• there are no layers: all functions would have to be successfully implemented at the same time (e.g. production of nutrients in pop A, dependence on nutrients of pop B, balancing the killing behavior of B vs. A) otherwise the whole project would fail.
• the resulting population dynamics would be interesting, but neither very useful nor extendable.

Convergence

The remaining project ideas, however, seem to share many modules and can be merged into the following new project variant.

Project X

Basic Concept I

Two engineered strains of E.coli, A and B, in a tank. B's cell division is inhibited. Population A is either much larger than population B in the beginning, or growing much faster.

• Stage 1:
  • 1.1 A constitutively expresses aggregation factor for A
  • 1.2 A and B constitutively express fluorescence genes (different colors)
  • 1.3 A constitutively releases some signaling substance a1, B constitutively expresses b1
• Stage 2:
  • 2.1 A starts to aggregate and form clusters
  • 2.2 B senses concentration of substance a1
• Stage 3:
  • 3.1 The clusters of A reach a critical size. This leads to a certain concentration of a1 in the vicinity of the cluster
  • 3.2 The threshold of a1 is reached and sensed by B. This triggers intensive cell division of B.
  • 3.3 In parallel, B expresses an aggregation factor that makes B attach to A
• Stage 4:
  • 4.1 A shell of B forms around the clusters of A.
  • 4.2 a1 and b1 reach certain concentrations at the B-shell-A-core boundary, which triggers the production of cellulose in both types. Either as AND-implementation or more simply just by b1 triggering A and a1 triggering B.
• Stage 5:
  • 5.1 Due to the growing thickness of the cellulose shell, the A-core gets isolated and starts to express some different fluorescence gene.

That is only a rough status for now, but there are various variations possible, e.g. to reduce complexity if desired or use alternative solutions. Also, not all stages are dependent on each other, i.e. one could simulate the result of a previous stage and still get a result. We are confident that some variant of this concept would be fairly feasable and still lead to interesting results.

Next Steps

Checking out the individual modules and their feasability.

• Herve: aggregation behavior, cell division control
• Giorgia: quorum sensing, AND dependence
• Dominic: cellulose, physical structures

Basic Concept II

This is an attempt to break down the concept into clearer and more elaborate steps, open questions, and pros and cons.

Note: some things have slightly changed. The stages have new, somewhat arbitrary names and assignement of subaspects.

Setup

Two engineered strains of (probably) [http://en.wikipedia.org/wiki/E.coli E.coli], A and B, in a tank (x,y,z).

Stage 1 - Initial Stage

• 1.1 B's cell division is somehow blocked (or naturally very low, but can be increased in a controlled way).
• 1.2 A constitutively expresses aggregation factor for A, i.e. A tends to stick to A when encountered (when cells tumble into each other on their random course).
• 1.3 A constitutively expresses some signaling substance a1 for [http://en.wikipedia.org/wiki/Quorum_sensing Quorum Sensing], i.e. A can sense its own (local) density.
• 1.4 A and B constitutively express fluorescence genes (different colors) for debugging purposes (true for all steps detailed below).

• Comments:
  • C1.1 Note: changed constitutive expression of b1 to Quorum Sensing of A (now 1.3).
  • C1.2 Cell division control: If we can actually achieve cell division control, i.e. blocking at this stage, then this could be an interesting contribution. But obviously, there are simpler solutions to achieve the same goal (i.e. indirect means, see Q1.3).

• Questions:
  • Q1.1 What aggregation factor to use, and how well would it work, i.e. how close do the cells have to be in order to "get into touch" and how strong will the attachment be (e.g. in the case when the contents of the tank are stirred).
  • Q1.2 If the aggregation rate is low, would chemotaxis help? (probably even harder to do)
  • Q1.3 How can the cell division be inhibited in a direct way (as opposed to using indirect means, such as restriction of substrate, poisoning, lack of enzymes)? Is it feasable (for us within the context of iGEM)?
  • Q1.4 What mechanism/part to use for quorum sensing? Are there suitable standard parts in the library?

• Answers:
  • A1.2: I personally think that would be more difficult to achieve. You would have to get just some cells to express an attractant, toward which the others will move. I think that is not easy to implement (or ebvenimpossible?) in a stirred, liquid medium.
  • A1.4: A very well studied quorum-sensing system is that of V. fisheri, the lux operon, that results in bioluminescence. The genes for making luciferase, and therefore bioluminescence, are contained in the lux operon. LuxR, the transcriptional activator, is consistently transcribed at a low level and binds to the lux operon (luxO) near the lux promoter (luxP). AHL (homoserine-lactones) are inducers, which are produced (by gene luxI) at basal level by every cell and can diffuse out of the cell membrane. At low cell density AHL diffuse out of the cells, whereas at high cell density AHL accumulate at a intracellular concentration which equals the extracellular one. At these high local concentrations, AHL binds to LuxR, enabling it to bind to the operator and turn on transcription. The parts for this quorum sening system are already in the registry ([http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=192],[http://parts2.mit.edu/r/parts/partsdb/view.cgi?part_id=193],[],[],[],[],

Stage 2 - Aggregation & Quorum Sensing Stage

• 2.1 A starts to aggregate and form clusters of A-cells (not necessarily spherical).
• 2.2 A senses the increasing concentration of substance a1 within the cluster

• Comments:
  • C2.1 Note: changed former point "2.2 B senses concentration of substance a1" to Quorum Sensing of A
  • C2.2 B is not involved at this point at all (as opposed to before). This might hopefully increase robustness, i.e. make it more likely that we can actually achieve the desired behavior.
  • C2.3 Clustering would be useful in many contexts and it is something we could easily observe (debugging).

• Questions:
  • Q2.1 Tuning of Quorum Sensing, i.e. when the threshold of a1 is reached in A-cells and taking into account where it is reached first (probably in the center of the A-cluster).
  • Q2.2 The aggregation needs to be faster than the increase of a1 OR a1 has to degrade in order to form a gradient (so that A do not start triggering B before proper clustering). How well can this be tuned?

Stage 3 - Generation Control & Shell-forming Stage

• 3.1 The clusters of A reach a critical size. This leads to a certain concentration of a1 within the cluster (Quorum Sensing).
• 3.2 The threshold of a1 is reached and in A a division signal d1 for B is produced. This triggers intensive cell division of B in the vicinity.
• 3.3 In parallel, B expresses an aggregation factor that makes B attach to A, slowly forming a shell of B around A of increasing thickness.
• 3.4 In parallel, B possibly expresses a signalling substance b1.

• Comments:
  • C3.1 Note: changed former point 3.1 where B senses concentration of substance a1 to full dependence on d1 produced by A. The reasons are: i) We have true Quorum Sensing of A, ii) We have the behavior of B fully decoupled from A up to this point (lower risk of B locally sensing too high concentrations of a1 and prematurely starting cell devision).
  • C3.2 The clusters A will tend to have a specific size, which can be tuned over adjusting the sensitivity of A to a1. This is a cool (and hopefully observable) side-effect.

• Information about the modules

Cell division inhibitors

The idea would be to express an inhibitor of the cell division in the population that we want to maintain small in the first phase of the process. It would certainly slow down the bacterial growth but they would keep growing so it cannot be considered then as a tight growth control. It is also difficult to predict but it forms structures (filaments) that might be of interest.

Here are two examples of cell division inhibitors

SulA = SfiA

Part of the SOS response (DNA repair system).(Justice, Garcia-Lara et al. 2000) Inhibition of cell division after DNA damage (Justice, Garcia-Lara et al. 2000) Over expression of SulA prevents the formation of ftz ring at the mid cell (Mukherjee, Cao et al. 1998) SulA appears to block the septation by interfering with FtsZ polymerization and, consequently, preventing FtsZ ring formation.

MinCD MinCD division inhibitor is part of the MinCDE system that determines the correct placement of the division septum. (Justice, Garcia-Lara et al. 2000) Overexpression of MinC and MinD lead to the inhibition of the cell division at all potential division sites (de Boer, Crossley et al. 1989).

Sugar mutants The idea is to use a mutant population B that cannot grow in the medium. The mutant bacteria population lacks an enzyme to process the carbohydrate source. This enzyme would be under an inducible promoter that could be trigger whn the population A is over with its job.

PTS Mutants

                                         pts

P-enolpyruvate (in) + carbohydrate (out) -----> pyruvate (in) + carbohydrate-P (in)

Carbohydrate phosphorylation is coupled to its translocation across the membrane, the energy for these processes being provided by the glycolytic intermediate PEP (Postma, Lengeler et al. 1993). In bacteria, a large number of sugars and hexitols are transported by the phosphorenolpyruvate (PEP)-dependent phosphotransferase system (PTS). The ptsHI genes from Streptococcus mutans and Staphylococcus carnosu were shown to complement E.coli pts mutants (Kohlbrecher, Eisermann et al. 1992; Boyd, Cvitkovitch et al. 1994). Pts E.coli mutants don’t grow on PTS sugars such as glucose, mannose, mannitol.

Boyd, D. A., D. G. Cvitkovitch, et al. (1994). "Sequence and expression of the genes for HPr (ptsH) and enzyme I (ptsI) of the phosphoenolpyruvate-dependent phosphotransferase transport system from Streptococcus mutans." Infect Immun 62(4): 1156-65. de Boer, P. A., R. E. Crossley, et al. (1989). "A division inhibitor and a topological specificity factor coded for by the minicell locus determine proper placement of the division septum in E. coli." Cell 56(4): 641-9. Justice, S. S., J. Garcia-Lara, et al. (2000). "Cell division inhibitors SulA and MinC/MinD block septum formation at different steps in the assembly of the Escherichia coli division machinery." Mol Microbiol 37(2): 410-23. Kohlbrecher, D., R. Eisermann, et al. (1992). "Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: molecular cloning and nucleotide sequence of the Staphylococcus carnosus ptsI gene and expression and complementation studies of the gene product." J Bacteriol 174(7): 2208-14. Mukherjee, A., C. Cao, et al. (1998). "Inhibition of FtsZ polymerization by SulA, an inhibitor of septation in Escherichia coli." Proc Natl Acad Sci U S A 95(6): 2885-90. Postma, P. W., J. W. Lengeler, et al. (1993). "Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria." Microbiol Rev 57(3): 543-94.

• Questions:
  • Q3.1 Tuning of Quorum Sensing, i.e. defining the threshold of a1 and possibly degradation of a1.
  • Q3.2 How to trigger cell division in B over some substance d1? Is it feasable? (otherwise we can use enzyme-based approaches as proposed by Herve).
  • Q3.3 How well can d1 diffuse out of the A-cluster?
  • Q3.4 Will there be enough B cells in the vicinity sensing d1?
  • Q3.5 What shape will the clusters have at this point? Rather spherical or basically random shapes? Will this interfere with the outcome?
  • Q3.6 What will be the behavior of the clusters? Obviously they will have higher inertia. Is this an advantage or a disadvantage? How will this affect the overall behavior? Will they sink to the ground for some reason?

Stage 4 - Cellulose-forming Stage

• 4.1 The shell of B forms around the clusters of A and reaches a certain density at the A-B-interface (shell-core boundary).
• 4.2 a1 and b1 reach certain concentrations at the A-B-interface (or the docking of B to A might trigger some response), which in turn triggers the production of cellulose in both types, A and B. Either as AND-implementation (a1 and b1 have both to be present at certain thresholds) OR - even simpler - just by b1 triggering A and a1 triggering B (while both are degrading rapidly, forming a gradient).

• Comments:
  • C4.1 Note: changing the former steps to Quorum Sensing of A leads to decoupling etc., yes, but it has the clear disadvantage (or is it an advantage?) that now the interface-sensing based on some quickly degrading substances a1 and b1 is also decoupled, i.e. they have to be additionally produced. Thus we would gain in decoupling into independent substages and probably in robustness of the desired behavior, but we also have additional genes and cost.
  • C4.2 The implementation of an AND-module is more complex, but also more sexy.

• Questions:
  • Q4.1 How well can the degradation rate of a1 and b1, respectively, be tuned?
  • Q4.2 How difficult is it to implement an AND-module?
  • Q4.3 Can cellulose be sufficiently excreted by E.coli? Are the genes identified and could be synthesized? Would the parts excreted by different cells sufficiently attach to each other? Would the cellulose matrix be of sufficient strenght?
  • Q4.4 Will cellulose be produced in liquid environment?

• Answers:
  • A4.2 A typical AND gate is e.g. the lac promoter ([http://web.mit.edu/esgbio/www/pge/lac.html]). In order to allow transcription of lacZYA, the repressor LacI has not to be bound to the operator sequence. LacI undergoes a conformational change when interacting with IPTG or lactose and "falls down" from the operator sequence. Moreover, the inducer complex CAP-cAMP trigger docking of the polymerase on the lac promoter. Therefore, the presence of lactose (or IPTG) and cAMP make transcription from lac promoter possible. However, is it still questionable if we can modify such a system to make it compatible with our system. A NOT-AND gate could be assembled from already existing parts (design will follow).
  • A4.3 Several strains with a biosynthesis protein exist ([http://ecocyc.org/ K12] or [http://genome.gen-info.osaka-u.ac.jp/bacteria/o157/ O157:H7], a rather unpleasant germ). Cellulose in bacteria serves to as a means to mechanical stability, protection, and adhesion to host plants. Cellulose seems to be "a significant constituent of the extracellular matrix observed during multicellular morphotype (rdar) growth" [http://biocyc.org/ECOLI/NEW-IMAGE?type=ENZYME&object=EG12259-MONOMER]. Zogaj et al. claim that cellulose synthesis can be turned on by the sole expression of [http://biocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG11257 adrA], leading to the formation of a highly hydrophobic network with tightly packed cells in a rigid matrix. This indicates that the structure might be fairly strong. The bcs (bacterial cellulose synthesis) operon encodes proteins essential for cellulose biosyhtnesis: [http://biocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG12260, bcsA], [http://biocyc.org/ECOLI/NEW-IMAGE?type=ENZYME&object=EG12259-MONOMER bcsB], [http://biocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG12258 bcsZ], and [http://biocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG12257 bcsC] (in this order). Thus, just using some suitable strain (as opposed to synthesizing the bcs genes) in combination with the controlled expression of adrA seems to be a straightforward way to go, no? Or we could just replace the promoter responding to AdrA with a regulated one, so that we could directly switch the transcription of the cluster on/off.
  • A4.4 From römling02 it seems that cellulose is produced at the air-liquid boundary. Anyway, cellulose is highly hydrophilic, so that should not be a problem.

Stage 5 - Encapsulation Stage

• 5.1 Due to the growing thickness of the cellulose shell, the A-core gets isolated, e.g. limiting the diffusion of vital substances, and starts to express some different fluorescence gene (for debugging).

• Comments:
  • C5.1 The formation of closed capsules of a specific size would clearly be a cool thing - and possibly even useful.
  • C5.2 The subsequent fluorescence is just for debugging and to prove the point that the encapsulation could be detected and used for other purposes, e.g. the incapsulation of drugs.

• Questions:
  • Q5.1 How can the isolation be sensed by A? Should we use a different approach?
  • Q5.2 How porous is such a cellulose wall and to what substances?
  • Q5.3 Do we need to actively stop the process at this stage?

Back to ETH Zurich main page.