Team:Valencia Biocampus/Project

From 2014.igem.org

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       Synthetic Biology implies an engineering perspective on biotechnology. Similarly to man-made objects, cells are expected to be decoupled, modified and even built from scratch. However, there is a general assent on the current difficulties for fully -and predictably- engineering living organisms, which are always subjected to strong evolutionary constraints. The St<sup>2</sup>OOL project aims at deeply studying four of the key engineering pillars of Synthetic Biology. St<sup>2</sup>OOL stands for Standardization, Stability, Orthogonality and Open Licence.
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       Synthetic Biology implies an engineering perspective on biotechnology. Similarly to man-made objects, cells are expected to be decoupled, modified and even built from scratch. However, there is a general assent on the current difficulties for fully -and predictably- engineering living organisms, which are always subjected to strong evolutionary constraints. The St<sup>2</sup>OOL project aims at deeply studying four of the key engineering pillars of Synthetic Biology. <b>St<sup>2</sup>OOL</b> stands for <b>St</b>andardization, <b>St</b>ability, <b>O</b>rthogonality and <b>O</b>pen Licence.
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      The first approach, an analytical one, will consist of a vast range of experimental studies to find out how standard, stable, orthogonal and patentable several selected Biobrick are. The second approach, a synthetic one, will include functional metagenomics: several environmental libraries will be set and screened in <em>E. coli</em> in order to select new biological parts -promoters-, not because of their strength but because of their particularly standard, stable or orthogonal behavior. Taken together, the results of our project are expected to contribute in answering this key question: Is life fully engineerable?
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    <strong>We have performed a vast range of experimental studies to find out how standard, stable, orthogonal and patentable ten selected Biobrick parts are</strong>. The results of our work -such as those on standardization and orthogonality-, surprised us, since a huge amount of data from different techniques clearly indicate that engineering principles should not be taken for granted in Synthetic Biology. In a second approach, still under way, we are currently isolating through functional metagenomics new biological parts -promoters-, not because of their strength but because of their particularly standard, stable or orthogonal behavior. Taken together, the results of our project are expected to contribute in answering this key question: <strong>Is life fully engineerable?</strong> and if not, can we improve the "engineerability" of life?
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<img src="https://static.igem.org/mediawiki/2014/e/e5/VBT_StabilityDrawing.png" alt="Stability Drawing" />
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== Stability ==
== Stability ==
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Our subteam aims to test the stability of ten different Biobricks transformed in two different strains of<em> Escherichia coli, </em>XL1 Blue and DH5α. Stability is determined by subjecting the transformed cells to different types of stress and comparing the output with non-transformed ones
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A computer works optimally at 10ºC to 35ºC, but it still works very well in a much wider range of temperatures. What about engineered bacteria? What are the limits of their -expected- behavior? We wanted to test two things: How stable is <em> Escherichia coli </em>when subjected to sub-optimal conditions (stability of the chassis), and what is the behavior of ten different Biobricks in two different strains of<em> E. coli, </em>XL1 Blue and DH5α (stability of the output signals), under those conditions. Stability was determined by subjecting either untransformed or transformed cells to different types of stress, including a range of extreme or cycling temperatures, radiation, pH, salinity or vacuum.  By measuring bacterial growth, we have been able to characterize the robustness of <em> E. coli </em> as a chassis for Synthetic Biology. By subjecting Biobrick-transformed cells to environmental stresses and then measuring their outputs, we have been able to determine which are the boundaries of Biobricks and how narrow or large are the conditions under which they behave as expected.
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    grown in the same conditions, as well as with cells transformed with an empty plasmid (without Biobrick).
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===Temperature Stress===
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    We start out by establishing an overnight culture of each strain, every one of which will be subjected to the different temperatures. The experiment will
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    be carried out in an improvised miniature system we just developed; PCR tubes filled with 75 µL of culture are placed in a thermo cycler, in which a
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    gradient of temperatures from 30-50ºC will be set. In each round, four replicas of two strains are to be tested simultaneously at 12 different
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    temperatures:
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  <a href="https://static.igem.org/mediawiki/2014/4/4f/VBT_StabilityImage_1.png">
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    <img src="https://static.igem.org/mediawiki/2014/4/4f/VBT_StabilityImage_1.png" alt="Test at 12 temperatures" /> 
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    <p>four replicas of two strains tested simultaneously at 12 different temperatures</p>
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    After an incubation period at the given temperatures the tubes containing the culture will be collected to measure the O.D. Fluorescence will also be
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    measured and later normalized to O.D.<sub>600</sub>, to compare the expression of the different Biobricks.
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=== Material Fatigue ===
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The idea is analogous to material fatigue in material sciences; in <a href="http://en.wikipedia.org/wiki/Materials_science" title="Materials science">materials science</a>, fatigue is the weakening of a material caused by
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    repeatedly applied loads. If the loads are above a certain threshold, microscopic cracks will begin to form at the stress concentrators such as the
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    surface, persistent slip bands and grain interfaces. In our case, we chose to induce the fatigue by subjecting the cells to temperature fluctuation, every
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    minute the temperature will be changed from 37ºC (<em>E.coli</em> optimum growth temperature) to 41ºC and back to 37ºC again, this way, the temperature
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    oscillates every minute from 37 to 41ºC for 4 hours. What will be the effect of such thermal fluctuations?
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=== pH &amp; Salinity ===
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    One of the best known factors that conditions bacterial growth is proton and salt concentration in the media. This experiment, as simple as may seem,
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    offers the opportunity to test cell viability as well as the Biobrick behaviour at different pH and salt concentrations. Overnight cultures of both strains
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    will be resuspended in different LB media with pH ranging from 5 to 9 and incubated for 90 minutes, after which O.D.<sub>600 </sub>and fluorescence of each
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    tube is to be measured. The design of the experiment is the same for salts, ranging from LB medium without salt to an extra 4% of salt, maintaining the
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    normal pH of the LB medium for all the tubes.
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=== UV Radiation ===
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    As per today, high doses of UV light are proven detrimental to life, but, to what extent is it injurious to an <em>E.coli</em> cell? Is transformation a
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    factor that enhances the lethality of UV radiation or, contrary to what logic dictates, helps resist the UV radiation? Curiosity led us to design an
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    experiment which would answer these questions: overnight cultures of the strains will be diluted and spot-plated on LB agar. Subsequently, the spots will
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    be subjected to timed pulses of UV radiation (intensity of 340 µW/cm<sup>2) </sup>with a control that receives no UV irradiation at all. Thereafter the
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    plates will be incubated and fluorescence will be measured and normalized to O.D.<sub>600.</sub>
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=== Vacuum ===
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    A simple experiment which mainly consists of exposing the cells, spot-plated on Petri dishes, to vacuum during 48 hours will be carried out; the output
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    from these, as always, compared to cells spot-plated and grown in optimal conditions, through OD<sub>600</sub> and fluorescence assays.
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== Standardization ==
== Standardization ==
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   One should not assume that a functional module working fine in one cell type will work the same way in even a closely related cell type (Renshaw, 1993). Whereas traditional engineering practices typically rely on the standardization of parts, the uncertain and intricate nature of biology makes standardization in the synthetic biology field difficult. Beyond typical circuit design issues, synthetic biologists must also account for cell death, crosstalk, mutations, intracellular and extracellular conditions, noise and other biological phenomena. As the number of system components grows, it becomes increasingly difficult to coordinate component inputs and outputs to produce the overall desired behaviour (Purnick &amp; Weiss, 2009). In the St²OOL Project, we wanted to check how standard biobricks are. For this goal, we are using a set of strains of <i>Escherichia coli</i>  transformed, in single transformation, with <a href="https://2014.igem.org/Team:Valencia_Biocampus/Biobricks">ten different Biobricks</a> whose outputs will be measured by fluorometry, colorimetry or luminometry. The aim of this approach is simple and fits with the answer to the following question:
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   One should not assume that a functional module working fine in one cell type will work the same way in even a closely related cell type <a href="http://books.google.es/books?hl=es&lr=&id=9iKDQEFM0ScC&oi=fnd&pg=PR13&dq=Modelling+Biological+Populations+in+Space+and+Time&ots=eVAhPiiIT9&sig=fGjzEYakHztERdAs9gw5eKfIfbI#v=onepage&q=Modelling%20Biological%20Populations%20in%20Space%20and%20Time&f=false" target="_blank">(Renshaw, 1993)</a>. Whereas traditional engineering practices typically rely on the standardization of parts, the uncertain and intricate nature of biology makes standardization in the synthetic biology field difficult. Beyond typical circuit design issues, synthetic biologists must also account for cell death, crosstalk, mutations, intracellular and extracellular conditions, noise and other biological phenomena. As the number of system components grows, it becomes increasingly difficult to coordinate component inputs and outputs to produce the overall desired behaviour <a href="http://www.nature.com/nrm/journal/v10/n6/pdf/nrm2698.pdf" target="_blank">(Purnick & Weiss, 2009)</a>. In the St²OOL Project, we wanted to check how standard biobricks are. For this goal, we have used a set of strains of <i>Escherichia coli</i>  transformed, in single transformation, with <a href="https://2014.igem.org/Team:Valencia_Biocampus/Biobricks" target="_blank">ten different Biobricks</a> whose outputs have been measured by fluorometry, colorimetry or luminometry. The aim of this approach is simple and fits with the answer to the following question:
</p>
</p>
<p>
<p>
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   Will the Biobricks behave the same way independently of the host strain they have been transformed into? Will we obtain the same output from all of them? The answer to these questions… in our result section!  
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   Will the Biobricks behave the same way independently of the host strain they have been transformed into? The answer to this question, in our result section!  
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=== References ===
 
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<a href="http://www.nature.com/nrm/journal/v10/n6/pdf/nrm2698.pdf">Purnick, P. E. M. & Weiss, R. <i>The second wave of synthetic biology: from modules to systems</i>. Nature Reviews <b>10</b>, 410-422 (2009)</a>.
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<a href="http://books.google.es/books?hl=es&lr=&id=9iKDQEFM0ScC&oi=fnd&pg=PR13&dq=Modelling+Biological+Populations+in+Space+and+Time&ots=eVAhPiiIT9&sig=fGjzEYakHztERdAs9gw5eKfIfbI#v=onepage&q=Modelling%20Biological%20Populations%20in%20Space%20and%20Time&f=false">Purnick, P. E. M. & Weiss, R. Renshaw, E. <i>Modelling Biological Populations in Space and Time</i>. Cambridge University Press (1993)</a>.
 
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<img src="https://static.igem.org/mediawiki/2014/7/7b/VBT_standardizationDrawing.png" alt="Standardization drawing" />
<img src="https://static.igem.org/mediawiki/2014/7/7b/VBT_standardizationDrawing.png" alt="Standardization drawing" />
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     To study this pillar of synthetic biology we are analyzing different combinations of Biobricks in cotransformed cells. That way, we can study the
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     To study this pillar of synthetic biology we have analyzed different combinations of Biobrick parts in cotransformed <em> Escherichia coli </em> cells. We have studied their behavior in comparison with simple transformants, and therefore determined whether their output was the predicted one. We did that at two different levels: the population level -with standard fluorometry assays-, and the cell level -by using flow cytometry-.
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    expression of pairs of Biobricks and compare them with simple transformations, and therefore determine whether the presence of two constructions in the
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    same cell affects their behavior. In order to do so, we will measure the output of each Biobrick (i.e. fluorescense) in both the cotransformants and the
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    controls.
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    We are going to study three combinations, taking in to account that each pair of biobricks is controlled by the same promoter and only differs in the
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But we also studied orthogonality at a third level: the proteome. We have analyzed and compared the whole proteome of three different strains of <i>E. coli</i>: a wild-type strain, a strain expressing a simple Biobrick part, and a strain carrying the empty cloning plasmid. With this experiment, we have determined the orthogonality of a Biobrick part with respect with the other parts naturally present in the chassis.  
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    protein expressed and the antibiotic resistance:
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        Constitutive promoter I (strong) – GFP &amp; Constitutive promoter I (strong) – RFP
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        Constitutive promoter II (less strong) – GFP &amp; Constitutive promoter I (strong) – RFP
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        Ptrc – lacZ &amp; Ptrc – luciferase
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    Besides individually transformed cells, we will use a control consisting of cotransformed cells with a Biobrick-bearing plasmid and an empty one.
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    We are currently working on all this. But we have another, very ambitious experiment. We plan to determine the effect of the production of a single
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    heterologous protein on the proteomic architecture of the host cell. For this endeavor, we just submitted our samples for a proteomic quantification.
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== Open License ==
== Open License ==
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<p>
     We did not want the Human Practices section to be a cosmetic addendum to our iGEM project. In The St<sup>2</sup>OOL, we have performed a critical but
     We did not want the Human Practices section to be a cosmetic addendum to our iGEM project. In The St<sup>2</sup>OOL, we have performed a critical but
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     constructive analysis of four of the pillars of the competition. Three of them are technical, engineering assumptions (stability, standardization,
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     constructive analysis of four of the pillars of the competition. Three of them are technical, engineering assumptions (<strong>stability</strong>, <strong>standardization</strong>,
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     orthogonality). The fourth leg of our St<sup>2</sup>OOL is for us at least as important as the other three, and deals with the answer to this difficult
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     <strong>orthogonality</strong>). The fourth leg of our St<sup>2</sup>OOL is for us at least as important as the other three, and deals with the answer to this difficult
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     question: who owns synthetic bio-constructions?<strong></strong>
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     question: who owns synthetic constructions?<strong></strong>
</p>
</p>
<p>
<p>
     iGEM is linked to the Registry of Standard Biological Parts, a non-profit organization (as iGEM Foundation itself) archiving and distributing thousands of
     iGEM is linked to the Registry of Standard Biological Parts, a non-profit organization (as iGEM Foundation itself) archiving and distributing thousands of
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     BioBrick parts for free (<a href="http://parts.igem.org/Main_Page">http://parts.igem.org/Main_Page</a>). No patents, no licenses, no royalties. Just Open Access (either totally free access or under
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     BioBrick parts for free. No patents, no licenses, no royalties. Just Open Access (either totally free access or under
     certain conditions such as a creative commons subscription). As stated in their Website, it’s “an open community that runs and grows on the Get &amp; Give
     certain conditions such as a creative commons subscription). As stated in their Website, it’s “an open community that runs and grows on the Get &amp; Give
     (&amp; Share) philosophy. Users get parts, samples, data, and tools from the Registry to work on their synthetic biology projects. They will give back to
     (&amp; Share) philosophy. Users get parts, samples, data, and tools from the Registry to work on their synthetic biology projects. They will give back to
     the Registry the new parts they have made, as well as data and experience on new and existing parts”. But of course, the world of Biotechnology/SynBio is
     the Registry the new parts they have made, as well as data and experience on new and existing parts”. But of course, the world of Biotechnology/SynBio is
     far more than iGEM and a range of Intellectual Property protection formulas do exist. Is iGEM an island of Free Access in an ocean of patentable SynBio
     far more than iGEM and a range of Intellectual Property protection formulas do exist. Is iGEM an island of Free Access in an ocean of patentable SynBio
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     achievements? How can one deal with the variety of IP forms?
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     achievements?
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     By checking how standard, stable, orthogonal or patentable Biobricks are we have performed an analysis of four engineering pillars. But we wanted to
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     By checking how standard, stable, orthogonal or patentable Biobricks are we have performed an analysis of the four engineering pillars of Synthetic Biology. But we wanted to
     combine these pillars, these four legs of our St<sup>2</sup>OOL, with a truly synthetic and constructive approach.
     combine these pillars, these four legs of our St<sup>2</sup>OOL, with a truly synthetic and constructive approach.
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     In The Seat of the St<sup>2</sup>OOL we are developing a functional metagenomic strategy in which we aim at isolating promoter sequences from natural
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     In The Seat of the St<sup>2</sup>OOL we designed a functional metagenomic strategy aiming at isolating promoter sequences from natural
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     environments. We will then characterize these sequences in order to select those with the highest stability, standard and orthogonal behaviors. We are
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     environments. The goal was to characterize the promoter sequences in order to select those with the highest stability, standard and orthogonal behaviors. We have
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     working with soil and compost samples, from which we will isolate total metagenomic DNA, which will then be digested, ligated into an expression vector and
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     worked with soil and compost samples, from which we have isolated total metagenomic DNA, which was then digested, ligated into an expression vector and
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     used to built a library in <em>E. coli</em>. Transformants –clones with a promoter-like natural sequence- will be subjected to a range of stresses and
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     used to build a library in <em>E. coli</em>. We planned to subject transformants –clones with a promoter-like natural sequence- to a range of stresses and
     subtransformation steps to find out their stability and standard/orthogonal behavior. To the best of our knowledge, this is the first Synthetic Biology
     subtransformation steps to find out their stability and standard/orthogonal behavior. To the best of our knowledge, this is the first Synthetic Biology
     functional metagenomic approach aiming at selecting biological parts not because of their “strength” but because of their compatibility with other parts
     functional metagenomic approach aiming at selecting biological parts not because of their “strength” but because of their compatibility with other parts
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     Finally, one technical –yet important- detail: we are not using neither commercial kits nor manual protocols for isolating metagenomic DNA. We are building
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     Finally, one technical –yet important- detail: we have not used neither commercial kits nor manual protocols for isolating metagenomic DNA. We have built our own robot, <a href="https://2014.igem.org/Team:Valencia_Biocampus/Results#TheSeatSection" target="_blank">"the TOOL"</a>, able to automatically isolate metagenomic DNA from environmental samples.
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    our own robot…
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      <img src="https://static.igem.org/mediawiki/2014/1/18/VBT_seatofstool.png" alt="Robot" />   
 
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Latest revision as of 00:21, 18 October 2014

The St2OOL project

Overview

Overview

Synthetic Biology implies an engineering perspective on biotechnology. Similarly to man-made objects, cells are expected to be decoupled, modified and even built from scratch. However, there is a general assent on the current difficulties for fully -and predictably- engineering living organisms, which are always subjected to strong evolutionary constraints. The St2OOL project aims at deeply studying four of the key engineering pillars of Synthetic Biology. St2OOL stands for Standardization, Stability, Orthogonality and Open Licence.


We have performed a vast range of experimental studies to find out how standard, stable, orthogonal and patentable ten selected Biobrick parts are. The results of our work -such as those on standardization and orthogonality-, surprised us, since a huge amount of data from different techniques clearly indicate that engineering principles should not be taken for granted in Synthetic Biology. In a second approach, still under way, we are currently isolating through functional metagenomics new biological parts -promoters-, not because of their strength but because of their particularly standard, stable or orthogonal behavior. Taken together, the results of our project are expected to contribute in answering this key question: Is life fully engineerable? and if not, can we improve the "engineerability" of life?

Stability Drawing

Stability

A computer works optimally at 10ºC to 35ºC, but it still works very well in a much wider range of temperatures. What about engineered bacteria? What are the limits of their -expected- behavior? We wanted to test two things: How stable is Escherichia coli when subjected to sub-optimal conditions (stability of the chassis), and what is the behavior of ten different Biobricks in two different strains of E. coli, XL1 Blue and DH5α (stability of the output signals), under those conditions. Stability was determined by subjecting either untransformed or transformed cells to different types of stress, including a range of extreme or cycling temperatures, radiation, pH, salinity or vacuum. By measuring bacterial growth, we have been able to characterize the robustness of E. coli as a chassis for Synthetic Biology. By subjecting Biobrick-transformed cells to environmental stresses and then measuring their outputs, we have been able to determine which are the boundaries of Biobricks and how narrow or large are the conditions under which they behave as expected.

Standardization

One should not assume that a functional module working fine in one cell type will work the same way in even a closely related cell type (Renshaw, 1993). Whereas traditional engineering practices typically rely on the standardization of parts, the uncertain and intricate nature of biology makes standardization in the synthetic biology field difficult. Beyond typical circuit design issues, synthetic biologists must also account for cell death, crosstalk, mutations, intracellular and extracellular conditions, noise and other biological phenomena. As the number of system components grows, it becomes increasingly difficult to coordinate component inputs and outputs to produce the overall desired behaviour (Purnick & Weiss, 2009). In the St²OOL Project, we wanted to check how standard biobricks are. For this goal, we have used a set of strains of Escherichia coli transformed, in single transformation, with ten different Biobricks whose outputs have been measured by fluorometry, colorimetry or luminometry. The aim of this approach is simple and fits with the answer to the following question:

Will the Biobricks behave the same way independently of the host strain they have been transformed into? The answer to this question, in our result section!

Standardization drawing

Orthogonality

Orthogonality Drawing

Do biobricks behave independently when cotransformed in a cell? In other words, can we consider them to be orthogonal? Orthogonality is a mathematical concept that refers to the independence of behavior. Translating this concept into the biological realm, two constructions can be considered orthogonal when they only interact at specific and predictable interphases and do not disturb each other. This is a desirable feature of Biobricks if one wants to build synthetic circuits with predictable and robust behaviors.

To study this pillar of synthetic biology we have analyzed different combinations of Biobrick parts in cotransformed Escherichia coli cells. We have studied their behavior in comparison with simple transformants, and therefore determined whether their output was the predicted one. We did that at two different levels: the population level -with standard fluorometry assays-, and the cell level -by using flow cytometry-.

But we also studied orthogonality at a third level: the proteome. We have analyzed and compared the whole proteome of three different strains of E. coli: a wild-type strain, a strain expressing a simple Biobrick part, and a strain carrying the empty cloning plasmid. With this experiment, we have determined the orthogonality of a Biobrick part with respect with the other parts naturally present in the chassis.

Open License

We did not want the Human Practices section to be a cosmetic addendum to our iGEM project. In The St2OOL, we have performed a critical but constructive analysis of four of the pillars of the competition. Three of them are technical, engineering assumptions (stability, standardization, orthogonality). The fourth leg of our St2OOL is for us at least as important as the other three, and deals with the answer to this difficult question: who owns synthetic constructions?

iGEM is linked to the Registry of Standard Biological Parts, a non-profit organization (as iGEM Foundation itself) archiving and distributing thousands of BioBrick parts for free. No patents, no licenses, no royalties. Just Open Access (either totally free access or under certain conditions such as a creative commons subscription). As stated in their Website, it’s “an open community that runs and grows on the Get & Give (& Share) philosophy. Users get parts, samples, data, and tools from the Registry to work on their synthetic biology projects. They will give back to the Registry the new parts they have made, as well as data and experience on new and existing parts”. But of course, the world of Biotechnology/SynBio is far more than iGEM and a range of Intellectual Property protection formulas do exist. Is iGEM an island of Free Access in an ocean of patentable SynBio achievements?

The Seat of the St²OOL

Tool robot

By checking how standard, stable, orthogonal or patentable Biobricks are we have performed an analysis of the four engineering pillars of Synthetic Biology. But we wanted to combine these pillars, these four legs of our St2OOL, with a truly synthetic and constructive approach.

In The Seat of the St2OOL we designed a functional metagenomic strategy aiming at isolating promoter sequences from natural environments. The goal was to characterize the promoter sequences in order to select those with the highest stability, standard and orthogonal behaviors. We have worked with soil and compost samples, from which we have isolated total metagenomic DNA, which was then digested, ligated into an expression vector and used to build a library in E. coli. We planned to subject transformants –clones with a promoter-like natural sequence- to a range of stresses and subtransformation steps to find out their stability and standard/orthogonal behavior. To the best of our knowledge, this is the first Synthetic Biology functional metagenomic approach aiming at selecting biological parts not because of their “strength” but because of their compatibility with other parts and suitability for engineering purposes.

Finally, one technical –yet important- detail: we have not used neither commercial kits nor manual protocols for isolating metagenomic DNA. We have built our own robot, "the TOOL", able to automatically isolate metagenomic DNA from environmental samples.