Team:UGA-Georgia/Team

From 2013.igem.org

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(Who we are)
(Who we are)
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= Tunable Synthetic Ecology =
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| [[File:Bsas2012-banderaArgentina.png|100px|]]
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| align = "center" | '''We are the first Argentinian team to participate in iGEM competition, so everything is new for us! Here we are, ready to work hard on our project!'''. '''Meet us, and also check [[Team:Buenos_Aires/Team/BsAs | where we come from]] '''
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| [[File:Bsas2012-banderaArgentina.png|100px|]]
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We aimed to create a stable community of microorganisms that could be used as a standard tool in lab and industry.
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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.
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== Students ==
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In order to do this we´ve come up with [[Team:Buenos_Aires/Project/Schemes | several plausible circuits designs]] and made  [[Team:Buenos_Aires/Project/Model | 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 [[Team:Buenos_Aires/Results/Strains | characterized]] two auxotrophic yeast strains (for tryptophan and histidine) and designed novel [[Team:Buenos_Aires/Results/Bb1 | biobricks]] that regulate the export of Trp and His rich peptides, therefore regulating the growth rate of the complementary strain.
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{| style="width:90%"
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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 may have a specific function, that it is in you to decide.  
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| rowspan="2" style="width: 20%; text-align: center;" | [[Image:Basas2012-Vero.jpg]]
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| style="width: 75%;" | '''Veronica Parasco - Physics Student'''
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|I guess I like to study too much, or the stress and making summaries. I'm about to finish my Licentiate in Physics, when I started to think about what was next, I realized that several of the decision I made took me closer to other career fields. So here I am, learning biology and opening new doors.
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{| style="width:90%"
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== Motivation ==
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|- valign="top"
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| rowspan="2" style="width: 20%; text-align: center;" | [[Image:Bsas2012-mario.jpg|160px]]
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| rowspan="2" style="width: 3%; background: #009ee1;" |
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| style="width: 75%;" | '''Mario J. Rugiero - Chemistry and Computer Sciece Student'''
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|Initially, the iGEM grabbed my attention through its idea of free thought and colaboration, which I consider should be general rule if we want society to progress. Even though my career choice seems remotely related to synthetic biology, curiosity moves me to learn a little bit about everything, which is never a bad idea.
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I'd like to see that this initiative will bring together all the synthetic biology investigation groups in Argentina because I see in this discipline an opportunity to develop and resolve many local, and very important, problems.
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{| style="width: 100%"
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| align="center" | [[File:BsasHow.JPG‎|300px]]
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| rowspan="2" style="width: 20%; text-align: center;" | [[Image:Bsas2012-ale.jpg|160px]]
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| rowspan="2" style="width: 3%; background: #009ee1;" |
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| style="width: 75%;" | '''María Alejandra Parreño - Biology Student'''
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|Within the vast field of Biology, i like ALL study subjects, but my specialty at the moment Biological Ecology of Populations (previously known as Population Genetics). Since 2009, I work in conserving the genetic variability of fruit flies which are a plague in Latin America, and I also do so with other insects with economical importance.
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I have another two very strong interests: on one hand, the spread of science, and on the other, the sustainable development of societies and handling of natural and biological resources. So I try to match my academic ocupations with relevant participations in congress and with activities in these two areas.
I love competition, innovation, and challenges, which is what attracted me to participate in iGEM and test my abilities. I see an unexplored synthetic biology field in Argentina, with a great potential to solve scientific and social problems, using wits and creativity as the main tools. As a first iGEM group, we want to impulse these ideas into reality and start off with the right foot!
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This is the key question that drives our motivation. How much of what we want to do are we actually able to do with the tools that we have now a days? And more important, how can we increase our possibilities so that much more of what we can imagine becomes plausible?
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{| style="width:90%"
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As we get more ambitious with our objectives in the field of synthetic biology, the complexity of biological circuits increases. This translates into an increase in the number and variety of different components that our designs must carry in order to be capable of the useful and interesting behaviors we want to achieve.  
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| rowspan="2" style="width: 20%; text-align: center;" | [[Image:Bsas2012-lucho.jpg|160px]]
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| style="width: 75%;" | '''Luciano G. Morosi- Biology Student'''
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| I am currently going through my last year, and i've been an intern in an investigation laboratory.
 From a very young age, I was passionate and interested in natural sciences, and I decided to study biology because you must have knowledge in all sciences to be able to understand it. In this way, my interest in synthetic biology comes from the fact that it is multidisciplinary, and I'm immensely attracted to the idea of being able to create biological systems – or based in biological parts- that are innovative, which carry out specifically designed actions, using and creating standarized and combined parts. I'm not only attracted to science, but also literature, theater, music and sports: for a very long time I participated in gymnastics and swimming, and I still do the latter. I love to write, I feel comfortable writing social, political and cultural papers, and also stories.
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In our imagination, the increase in complexity may be only bounded to our creativity but experimentally there are several constrains and obstacles we can stump into in the way to our objective.
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{| style="width:90%"
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# Firstly, the greater the number of elements the system has, the greater the chances that they interact with each other, or with the chassis, in unexpected ways.  
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|- valign="top"
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# 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 while keeping it viable'''
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| rowspan="2" style="width: 20%; text-align: center;" | [[Image:Bsas2012-manugimenez.jpg|160px]]
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| style="width: 75%;" | '''Manuel Giménez - Computer Science Student'''
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| I'm writing my licentiate thesis on building a tool to automatically reason about regulations. Yes yes, nothing relates to biology. Weird, right? Nevertheless, I've always had an interest for this discipline, mainly molecular biology. When I found out about the existance of iGEM – a couple of months ago- I said ''we have to assemble a team in Argentina''. Coincidence or not, a week later I got an email inviting me to be a part of the first Argentinian iGEM team, and now I find myself taking my first steps in synthetic biology as a member of iGEM BsAs. I'm mainly interested in the engineering vision that synthetic biology has, and I believe from the computer science standpoint, I have several ideas I can bring to this new subject. I love scientific dissemination and teaching; I consider myself a straightforward communicator, and my natural way of working is in groups. I'm part of a political movements in my university, and I try to make my passing through this world the most transforming and engaging possible.
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== Advisors ==
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These problems impose a limitation to the increase of the complexity in the synthetic system(Purnick and Weiss 2009).
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{| style="width:90%"
 
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| rowspan="2" style="width: 20%; text-align: center;" | [[Image:Bsas2012-alan.jpg|160px]]
 
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| rowspan="2" style="width: 3%; background: #004a99;" |
 
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| style="width: 75%;" | '''Alan Bush - M.Sc. in Biology'''
 
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|I'm M. Sc. in Biology and I'm currently doing my Ph. D. in Biology. My studies are in the field of "systems biology", an area which attempts to give a more quantitative and integrative approach to molecular biology, through the use of mathematic modeling tools. It is related to synthetic biology since it uses the same kind of tools and model organisms. However, the focus is radically different; while systems biology aims to understand how the cells work,  the main objective of synthetic biology is to design and produce "biological devices" with a given behavior. My main motivation for participating as advisor for UBA's iGEM team is precisely this approach switch. I'm fascinated with the idea of using our knowledge to develop useful devices which can help to solve concrete problems.
 
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|}
 
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'''One solution to this problem is to physically isolate sub-systems in different cells''', creating a “division of labor”.
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{| style="width:90%"
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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.  
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|- valign="top"
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| rowspan="2" style="width: 20%; text-align: center;" | [[Image:Bsas2012-german.jpg|160px]]
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| style="width: 75%;" | '''German Sabio - M.Sc. in Biology'''
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|I'm an extremely curious person, and I've always had a passion for all kind of "bugs" (a highly academic and very complex concept, which includes everything from cell and virus to mammal and aliens) and how "life" works. A M. Sc. in Biology didn't gave me the answer just yet, but kept my curiosity appeased for some years and gave me a big ammount of tools to keep asking new questions.
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Currently, I work on a branch of biology dedicated to living being's development: how, from a single cell, or from a group of similar cells, a differentiated organism gets developed. My Ph. D. discipline is systems biology, which basically studies several biology areas looking for math patterns and predicting (or, actually, modelling) different living systems. Synthetic biology would be the other face of the same coin: while systems biology tries to find and define mechanisms in nature to understand how they work, synthetic biology tries to reproduce or generate new systems with a predetermined function.
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I'm very interested in this chance to take part on an iGEM team, not just for the ammount of tools it represents, but because it's a good and interesting experience to start a group and discuss and work together.
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== Instructors==
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This 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 the complexity of the system
-
{| style="width:90%"
+
However, there are two major obstacles to overcome for the proposed approach to work.  
-
|- valign="top"
+
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| rowspan="2" style="width: 20%; text-align: center;" | [[Image:Bsas2012-nacho.jpg|160px]]
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| rowspan="2" style="width: 3%; background: #0B2161;" |
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| style="width: 75%;" | '''Ignacio E. Sánchez - M.Sc. in Chemistry and Ph.D. in Biophysics'''
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|I'm a spanish bioinformatics specialist, currently living in Argentina. Before, I was an in training biophysicist on northern Europe, and before, a chemist, fascinated by biological molecules. Day by day, I study oncogenic virus in the Department of Biological Chemistry's Protein's Physiology Laboratory.
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I think synthetic biology is an excellent chance for developing countries to acquire new capabilities in the subject of biotechnology. Because of that reason, I joined Dr. Nadra in 2011 to promote the formation of synthetic biologists in Argentina. By now, the experience is being highly fun and rewarding.
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# 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 are already of common use in the synthetic biology community.
-
{| style="width:90%"
+
In order to overcome these obstacles, we decided to construct a system in which two or more strain could be grown in stable, defined and tunable proportions.
-
|- valign="top"
+
 
-
| rowspan="2" style="width: 20%; text-align: center;" | [[Image:Bsas2012-alenadra.jpg|160px]]
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'''Now you are free to imagine without restrain! We`ll make it happen.'''
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| rowspan="2" style="width: 3%; background: #0B2161;" |
+
 
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| style="width: 75%;" | '''Alejandro D. Nadra - M.Sc. in Biology and Ph.D. in Chemistry'''
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'''We call this ''Tunable Synthetic Ecology''.'''
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|- valign="top"
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|I'm M. Sc. in Biology (2001) and Ph. D. in Chemistry (2005). My subjects of interest are protein's structure/function/folding and their interaction with nucleic acids. Also hemoproteins, evolution and synthetic biology, and others. I did a post-doc with the molecular modelling group in the FCEyN and another one in the systems biology program of the Genomic Regulation Center from Barcelona.
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== Design ==
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I started to work as teacher in the FCEyN in 2000 and I'm currently Assistant Professor in the Department of Biological Chemistry and researcher at CONICET. I'm also a member of the Structural Biochemistry Laboratory, Department of Biological Chemistry.
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Given the lack of Synthetic Biology in Argentina, we are promoting the area with Ignacio Sánchez since 2011. For this effort, we organized the first post-graduate course in the subject in 2011, and conducted a course with featured international referents in April 2012. I'm convinced that from my place and given my formation, I can boost Synthetic Biology and begin formation of pure strain Synthetic Biologists. I believe the iGEM competitions are an excellente tool for the education and motivation of students. I'm convinced that from Argentina we can contribute to the subject and be on pair with teams from everywhere in the world. Even though we count on less resources and funds, this lack is compensated with a huge motivation and creativity.
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{|
 +
|After considering [[Team:Buenos_Aires/Project/Schemes | several possible schemes]], we decided to construct a [[Team:Buenos_Aires/Project/Schemes#Crossfeeding | crossfeeding system]], in which each strain produces and secretes a metabolite the other strain is incapable of producing and requires for growth (Figure 1). The main advantage of this scheme is that it maintains strain proportions in a way independent of signaling molecules, which will be required to couple sub-systems.  
 +
|
 +
[[File:Bsas2012-schemes-crossfeeding.png|450px]]
|}
|}
 +
 +
Some examples of synthetic crossfeeding systems have been reported in the literature. For example two auxotrophic strains of ''E. coli'' (one for isoleucine and an other for leucine) were coevolved and selected for their ability to grow in a co-culture (Hosoda et al 2011). In another work two auxotrophic yeast strains were used, one for adenine and the other for lysine. In this case they were genetically modified to overproduce the relevant metabolite by elimination of the end-product feedback inhibition normally present in biosynthetic pathways (Shou et al. 2007). The co-culture could grow, but the metabolites were only released to the medium by cell lysis, therefore producing a lag phase in the growth of the culture and oscillatory dynamics.
 +
 +
Our design is based on aminoacid auxotrophy, allowing to engineer the secretion of the crossfeeding metabolite in the form of a peptide with a secretion tag. Furthermore, the production rate of this peptide can be easily regulated by controlling the strength of the promoter of its coding gene (see [[Team:Buenos_Aires/Results/Bb1 | biobrick design section]] for details). The fact that the produced aminoacid is exported would effectively eliminate the end-product feedback inhibition, not by mutation, but by avoiding the accumulation of the aminoacid within the cell. 
 +
 +
As a proof of principle we decided to construct a crossfeeding system with two ''Saccharomyces cerevisiae'' yeast strains. We choose the tryptophan (Trp) and histidine (His) auxotrophs because they are common markers for yeast genetics and they are fairly rare aminoacid in proteins, therefore we reasoned that a small amount in the medium may have a big effect in the growth rate. An other advantage of using Trp is that its abundance can be easily measured in the medium by its fluorescence properties. 
 +
 +
To gain some insight into the expected behavior of the system we build a [[Team:Buenos_Aires/Project/Model|mathematical model]]. With this model we tried to asses the feasibility of the design by estimating the parameters required for the system to work, and compare them with estimations for the parameters we could actually achieve. We had to do some [[Team:Buenos_Aires/Results/Strains|initial characterization of the strains]] and estimation of relevant parameter.
 +
 +
== Possible applications ==
 +
 +
The main purpose of the system is to allow a new modularity level, circuit isolation, parts reutilization and scale-up properties, as discussed above. Nevertheless, the “tunable synthetic ecology” could also be use in some specific application:
 +
 +
* '''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.
 +
 +
* '''Color Palette''': By controlling the proportions of three strains, each of which produces a primary color pigment (or bioluminescence), one can control the color of the culture. If grown as a lawn on an agar plate one can then paint on a “cell canvas”.  In the future this could be combined with light sensing parts to create a bacterial color photographic paper.
 +
 +
== References ==
 +
 +
 +
# Purnick, P.E. and R. Weiss, The second wave of synthetic biology: from modules to systems. Nat Rev Mol Cell Biol, 2009. 10(6): p. 410-22.
 +
# Hosoda, K., et al., Cooperative adaptation to establishment of a synthetic bacterial mutualism. PLoS One, 2011. 6(2): p. e17105.
 +
# Shou, W., S. Ram, and J.M. Vilar, Synthetic cooperation in engineered yeast populations. Proc Natl Acad Sci U S A, 2007. 104(6): p. 1877-82.

Revision as of 22:11, 27 June 2013

iGem Buenos Aires

Background

Buenos AiresiGem

Objectives

The project will focus on the development of a specific water biosensor , but with a modular and scalable approach. Thereby you could easily afford multiple measurements with the very same device.

The device will be designed in a way that its collected data will be easily accessible via a web interface, and later it could be transferred to the relevant agencies upon request.

At the end of this project we expect to have a prototype of measuring device and a diagram of a system for the distribution, collection and centralization of data.

The project will aim on the measurement of a primary pollutant: arsenic . However, its modular and scalable design provides an easy way to incorporate various contaminants already identified, such as nitrate / nitrite, lead and hydrocarbons.

With the data collected is expected that any user with minimal training (using an image-based Instruction given) could easily and quickly determine the presence and level of the contaminant on his water. Also with the systematic use of this tool by the enforcement authorities, specific public policies could be implemented based on current and reliable information.

Motivation

Limited access to clean water is a deep problem and tends to worsen with time. The pollution that converts water in non-drinkable can vary from just a single toxic (eg arsenic) to a highly complex mixture of types of substances such as those found in various river basins (eg Sali-Dulce, Matanza-Riachuelo among others).

Depending on the type of contamination (complexity and abundance), making the water to be drinkable could be easy and inexpensive. Even if it weren't possible to make it drinkable, information on pollutant levels could be easily used to modify consumption patterns and seek alternative sources of water.

At present, the spatial and temporal quantification of contaminants is limited by the difficulty in processing the samples and associated costs. Moreover, the lack of centralization and systematization of data does the task of obtain them by decision makers, stakeholders and the general public, very difficult.

Apli

Contents

Tunable Synthetic 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 may have a specific function, that it is in you to decide.


Motivation

300px

This is the key question that drives our motivation. How much of what we want to do are we actually able to do with the tools that we have now a days? And more important, how can we increase our possibilities so that much more of what we can imagine becomes plausible?

As we get more ambitious with our objectives in the field of synthetic biology, the complexity of biological circuits increases. This translates into an increase in the number and variety of different components that our designs must carry in order to be capable of the useful and interesting behaviors we want to achieve.

In our imagination, the increase in complexity may be only bounded to our creativity but experimentally there are several constrains and obstacles we can stump into in the way to our objective.

  1. Firstly, the greater the number of elements the system has, the greater the chances that they interact with each other, or with the chassis, in unexpected ways.
  2. 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 while keeping it viable

These problems impose a limitation to the increase of the complexity in the synthetic system(Purnick and Weiss 2009).


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 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 the complexity of the system

However, there are two major obstacles to overcome for the proposed approach to work.

  1. 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”.
  2. 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 are already of common use in the synthetic biology community.

In order to overcome these obstacles, we decided to construct a system in which two or more strain could be grown in stable, defined and tunable proportions.

Now you are free to imagine without restrain! We`ll make it happen.

We call this Tunable Synthetic Ecology.

Design

After considering several possible schemes, we decided to construct a crossfeeding system, in which each strain produces and secretes a metabolite the other strain is incapable of producing and requires for growth (Figure 1). The main advantage of this scheme is that it maintains strain proportions in a way independent of signaling molecules, which will be required to couple sub-systems.

450px

Some examples of synthetic crossfeeding systems have been reported in the literature. For example two auxotrophic strains of E. coli (one for isoleucine and an other for leucine) were coevolved and selected for their ability to grow in a co-culture (Hosoda et al 2011). In another work two auxotrophic yeast strains were used, one for adenine and the other for lysine. In this case they were genetically modified to overproduce the relevant metabolite by elimination of the end-product feedback inhibition normally present in biosynthetic pathways (Shou et al. 2007). The co-culture could grow, but the metabolites were only released to the medium by cell lysis, therefore producing a lag phase in the growth of the culture and oscillatory dynamics.

Our design is based on aminoacid auxotrophy, allowing to engineer the secretion of the crossfeeding metabolite in the form of a peptide with a secretion tag. Furthermore, the production rate of this peptide can be easily regulated by controlling the strength of the promoter of its coding gene (see biobrick design section for details). The fact that the produced aminoacid is exported would effectively eliminate the end-product feedback inhibition, not by mutation, but by avoiding the accumulation of the aminoacid within the cell.

As a proof of principle we decided to construct a crossfeeding system with two Saccharomyces cerevisiae yeast strains. We choose the tryptophan (Trp) and histidine (His) auxotrophs because they are common markers for yeast genetics and they are fairly rare aminoacid in proteins, therefore we reasoned that a small amount in the medium may have a big effect in the growth rate. An other advantage of using Trp is that its abundance can be easily measured in the medium by its fluorescence properties.

To gain some insight into the expected behavior of the system we build a mathematical model. With this model we tried to asses the feasibility of the design by estimating the parameters required for the system to work, and compare them with estimations for the parameters we could actually achieve. We had to do some initial characterization of the strains and estimation of relevant parameter.

Possible applications

The main purpose of the system is to allow a new modularity level, circuit isolation, parts reutilization and scale-up properties, as discussed above. Nevertheless, the “tunable synthetic ecology” could also be use in some specific application:

  • 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.
  • Color Palette: By controlling the proportions of three strains, each of which produces a primary color pigment (or bioluminescence), one can control the color of the culture. If grown as a lawn on an agar plate one can then paint on a “cell canvas”. In the future this could be combined with light sensing parts to create a bacterial color photographic paper.

References

  1. Purnick, P.E. and R. Weiss, The second wave of synthetic biology: from modules to systems. Nat Rev Mol Cell Biol, 2009. 10(6): p. 410-22.
  2. Hosoda, K., et al., Cooperative adaptation to establishment of a synthetic bacterial mutualism. PLoS One, 2011. 6(2): p. e17105.
  3. Shou, W., S. Ram, and J.M. Vilar, Synthetic cooperation in engineered yeast populations. Proc Natl Acad Sci U S A, 2007. 104(6): p. 1877-82.