Project X

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=Introduction=
=Introduction=
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=Discussion=
=Discussion=
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Back to the [[Quorum_Sensing_based| Development Group]] (the group that developed the project detailed below).
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Revision as of 11:20, 11 August 2005

Back to ETH Zurich main page.

Back to the Development Group (the group that developed the project detailed below).


Contents

Introduction

Concept

Setup

Two engineered strains of (probably) E.coli, A and B, in a tank (x,y,z).


Stage 1 - Initial Wandering 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 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). Alternatively, if the expression cost is too high or not suitable, specific dyes could be used (that allow to differentiate between the A and B strain).
  • 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).
    • C1.3 Probably, the B population should be much smaller than the A population, so that B does not interfere with the clustering of A (i.e. not hampering the aggregation and no inclusions of B-cells within the A cluster).
  • 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)? What about stirring the contents of the tank? At what speed if any?
    • 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 ([1],[2],[],[],[],[],


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? Another solution could be that the quorum sensing only starts after aggregation, e.g. that cell-to-cell contact triggers the production of a1.
  • Information:
    • Aggregation

There are different candidates for bacterial aggregation: Ag43, Type1 Fimbriae and TibA. They are surface structures that can self-recognize and trigger aggregation and flocculation. Most of the aggregation tests for these factors have been done on plates or in static liquid culture. It is still unclear whether they would be active for stirred solution but there are certainly parameters such as the speed and the temperature to play around in order to improve the aggregation. The time that passes before aggregation is initiated is highly dependent on the cell density of the initial suspension. It seems to be in good agreement with a model following first-order kinetics, where the chance of two bacteria colliding at a given time interval is proportional to the cell density (Hasman et al. 1999). According to the publication of Sherlock et al. (2005), it seems that TibA Adhesin/Invasin would also trigger aggregation in a static culture and then settling of the bacterial culture to the bottom part. One can imagine to start with static culture and to shift slowly to non-static culture to speed up the aggregation process. Working with static culture can also be easier to predict.


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:
    • Replication inhibitors

By impeding DNA replication, there is a possibility to maintain the cell in a non-multiplying state. For example, SeqA competes with initiation complex (binding of dnaA to the oriC then DNA double helix separation then prepriming complex orientation by dnaA) (Torheim et al., 1999). SeqA appears also to release dnaA from the oriC (Taghbalout et al. 2000). The problem with the SeqA approach is that there is little information about the expression that is required to really obtain an inhibitory effect on the initiation of replication. Most of the studies have been done in vitro. This would then be a new challenge if we try to stop bacterial growth with this approach or at least we could probably increase the generation time but then again it is difficult to predict.

    • 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).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

MutantsP-enolpyruvate (in) + carbohydrate (out) ---PTS---> 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 (Postma93). 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.

  • 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?
    • Q3.7 It is important that d1 does not interfere with quorum sensing of A. Are there useful signaling molecules other than oxodecanoyl AHL?
  • Answers:
    • A3.7: There is a range of other AHL known, and they are known to be uncapable of cross stimulation of noncognate systems (miller01) . However, no alternative system is available in the registry of parts.

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? Tuning is necessary, since you don't want the overlap of critical concentrations of a1 and b1 to be too wide. Ideally, this overlap would only consist of a belt of some few layers of A and B cells at the interface.
    • 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?
    • Q4.5 Will the B cells form a layer that is actually thicker than one cell? Why?
  • Answers:
    • A4.1 Assuming that the cells will aggregate properly and thus will be rather tightly packed forming themselves a considerable barrier for diffusion, the overlap could be fairly thin.
    • A4.2 A typical AND gate is e.g. the lac promoter ([3]). 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 (K12 or 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" [4]. Zogaj et al. claim that cellulose synthesis can be turned on by the sole expression of 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: bcsA, bcsB, bcsZ, and 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. However, in order to insure thight bundling of cellulose around the cells, aggregative fimbreiae have to be co-expressed with cellulose. Otherwise bundles of cellulose freely protrude from cell clumps.
    • 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.
    • C5.3 If the A-cells are sufficiently isolated, they could also simply starve to death.
  • 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?


Methods

For now you will find the methods for each solution variant and module we have investigated so far in the corresponding stage section above, either under "Comments" or "Answers". If this project concept will be further developed, then more details will follow, of course.

Discussion

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