Team:Buenos Aires/Project

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- Synthetic ecology
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- Modeling synEcology
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Results and Devices
- Strains characterization
- Strains description
- Fluorescence screening
- Screening of strain proportion
- Auxotrophy confirmation
- Coculture in liquid medium
- Revertant control
- Trp basal production
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Tunable syntethic ecology

We aimed to create a stable community of microorganisms that could be used as a standard tool in lab and industry. Our system would allow the co-culture of several genetically engineered machines in defined and tunable proportions, just like different species coexist in an ecosystem in nature. Hence the engineered organism would be a standard part! This defines a new level of modularity allowing the increase of the complexity of the system by moving to the community level.

In order to do this we´ve come up with several plausible circuits designs and made in silico predictions of their behavior. We decided to build a “crossfeeding” system in which each strain produces and secretes an aminoacid the other strains need to grow. We therefore characterized two auxotrophic yeast strains (for tryptophan and histidine) and designed novel biobricks that regulate the export of Trp and His rich peptides, therefore regulating the growth rate of the complementary strain.

In the future this would allow for other modules to control the proportions of each strain, thus allowing dynamic and stimulus dependent changes in the abundances of each strain. It would also allow to build complex systems by combining different strains, each of which have a specific function.

Motivation

The expected trend for the field of synthetic biology is to design and create biological circuits of increasing complexity, capable of useful and interesting behaviors. As we get more ambitious with our objectives, the number of different components of our designs is bound to increase. The greater the number of elements the system has, the greater the chances that they interact with each other, or with the chassis. Furthermore, each component imposes a “load” on the cell, and there is probably an upper bound to the number of components one can introduce into a cell and keep it viable. The complexity of the synthetic system didn´t increase as expected {Purnick and Weiss 2009}, probably due to these effects.

One solution to this problem is to physically isolate sub-systems in different cells, creating a “division of labor”. In this approach one would coculture different strains, each of which performs a specific task and interact with each other to achieve a desired system level behavior. This has approach would have several advantages: 1) Sub-systems are isolated therefore reducing the number of unintended interactions 2) This isolation allows for the same parts to be reutilized in different subsystems 3) A new level of modularity is introduced; strains with specific functions can become “standard parts” to be combined in a higher level system. 4) This approach should allow to easily scale-up in the complexity of the system

There are two major obstacles to overcome for the proposed approach to work. The first one is how to stably maintain a co-culture of different strains (or species), when they most likely will have different growth rate and therefore one is bound to overtake the culture. Even in the unlikely case in which several strains grow at exactly the same rate, in the long term one will dominate because of “genetic drift”.

The second obstacle to overcome is how to couple the different sub-systems. If each strain does a specific task, it will have to interact in some way with the other strains to achieve the global function. This sort of cell-to-cell communication can be achieved with mechanisms as quorum sensing, that is already of common use in the synthetic biology community.

We therefore decided to construct a system in which two or more strain could be grown in stable, defined and tunable proportions. We call this “tunable synthetic ecology”.

Possible applications

(From the most classic to the wildest!)

Circuit Integration: The possibility to co-culture several strains with different circuits can be used to increase the complexity and amount of components of the overall system. Currently there seems to be an upper limit to the amount of components a device can have. Combining several strains to achieve a function can be a way to overcome this limitation and introduce a new layer of modularity and control.

Circuit Isolation: Dividing a big circuit in two or more strains can allow to build sub-circuits that would be otherwise incompatible in the same cell. This can allow more complex designs with fewer parts, as these can be used for different functions in different strains.

Optimization of Bioreactor Output: If the steps of a complex reaction are carried out by different strains, the overall throughput of the process can be optimized by fine-tuning the proportion of each strain. Our system would allow to easily modify these proportions.

Synthetic Oenology: The ability to use several yeast strains in defined proportions in a fermentation process can allow new varieties of wines with unique properties to be created. This can be of interest to the local wine industry.

@@ Line 11: / Line 11: @@
 == Motivation ==
-Through our project, we aim to design a stable community of microorganisms which interact according to our intentions and that could be used as a standard tool in lab and industry for different purposes. Hence our main biobrick is the actual organism!
+The expected trend for the field of synthetic biology is to design and create biological circuits of increasing complexity, capable of useful and interesting behaviors. As we get more ambitious with our objectives, the number of different components of our designs is bound to increase. The greater the number of elements the system has, the greater the chances that they interact with each other, or with the chassis. Furthermore, each component imposes a “load” on the cell, and there is probably an upper bound to the number of components one can introduce into a cell and keep it viable.  The complexity of the synthetic system didn´t increase as expected {Purnick and Weiss 2009}, probably due to these effects.
-In other words, our objective is to create a system that allows the co-culture of several genetically engineered machines in defined and tunable proportions in synthetic conditions, just like different species coexist in an ecosystem in nature. In order to do this we´ve come up with several plausible circuit designs, each of them that uses a different approach to the same issue and we´ll test the easiest one in order to prove it can work.
+One solution to this problem is to physically isolate sub-systems in different cells, creating a “division of labor”. In this approach one would coculture different strains, each of which performs a specific task and interact with each other to achieve a desired system level behavior. This has approach would have several advantages:
+) Sub-systems are isolated therefore reducing the number of unintended interactions
+) This isolation allows for the same parts to be reutilized in different subsystems
+) A new level of modularity is introduced; strains with specific functions can become “standard parts” to be combined in a higher level system.
+) This approach should allow to easily scale-up in the complexity of the system
-As a starting point, we will use two auxotrophic strains, one for tryptophan and other for histidine and we will tune the amount of these aminoacids that they export so that one cannot live without the other and they regulate each other´s growth.
+There are two major obstacles to overcome for the proposed approach to work. The first one is how to stably maintain a co-culture of different strains (or species), when they most likely will have different growth rate and therefore one is bound to overtake the culture. Even in the unlikely case in which several strains grow at exactly the same rate, in the long term one will dominate because of “genetic drift”.
+The second obstacle to overcome is how to couple the different sub-systems. If each strain does a specific task, it will have to interact in some way with the other strains to achieve the global function. This sort of cell-to-cell communication can be achieved with mechanisms as quorum sensing, that is already of common use in the synthetic biology community.
-Therefore the system is also tunable by a externally controlled variable as it is the concentration of aminoacids, such that the A:B proportion has a defined response curve with respect to the concentration of it.   The culture should reach a plateau because of the action of a built-in regulatory system, as opposed to the depletion of nutrients and accumulation of toxic waste products.  The proportion of the strains should be robust against external perturbations and stochastic fluctuations, given that there is a reciprocal regulation that acts like a moderator of changes in populations parameters.
+We therefore decided to construct a system in which two or more strain could be grown in stable, defined and tunable proportions. We call this “tunable synthetic ecology”.
-This system should control the overall optical density (OD) of the culture at levels below saturation and we´ve run several in silico simulations prior to the actual experiments in order to assess the correct concentration and starting proportion needed for the success of our project.
-In the philosophy of the synthetic biology field, we would like to create re-utilizable and well-characterized parts and modules.  In the future this would allow for other modules to control the proportions of each strain, thus allowing dynamic and stimulus dependent changes in the abundances of each strain.
 == Possible applications ==