Team:Valencia Biocampus/Project

From 2014.igem.org

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         <div class="tab-pane fade" id="Stability">
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            <h1>Orange</h1>
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            <p>orange orange orange orange orange</p>
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<div class="row" style="margin-bottom:-15px">
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<div class="col-sm-5">
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<img src="https://static.igem.org/mediawiki/2014/e/e5/VBT_StabilityDrawing.png" alt="Stability Drawing" />
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</div>
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</html>
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== Stability ==
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<html>
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<div class="col-sm-7">
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<p>
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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    <em>. </em>Stability is determined by subjecting the transformed cells to different types of stress and comparing the output with non-transformed ones
 +
    grown in the same conditions, as well as with cells transformed with an empty plasmid (without Biobrick).
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</p>
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</div>
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</div>
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<h2 style="margin-bottom:0">Temperature Stress</h2>
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<p>
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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
 +
    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
 +
    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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</p>
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<p></p>
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<div class="thumbnail col-sm-5 center-block">
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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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  </a>
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  <div class="caption">
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    <p>four replicas of two strains tested simultaneously at 12 different temperatures</p>
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  </div>
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</div>
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<br clear="ALL"/>
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<p>
 +
    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
 +
    measured and later normalized to O.D.<sub>600</sub>, to compare the expression of the different Biobricks.
 +
</p>
 +
</html>
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== Material Fatigue ==
 +
<html>
 +
<p>
 +
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
 +
    repeatedly applied loads. If the loads are above a certain threshold, microscopic cracks will begin to form at the stress concentrators such as the
 +
    surface, persistent slip bands and grain interfaces. In our case, we chose to induce the fatigue by subjecting the cells to temperature fluctuation, every
 +
    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
 +
    oscillates every minute from 37 to 41ºC for 4 hours. What will be the effect of such thermal fluctuations?
 +
</p>
 +
 
 +
</html>
 +
== pH &amp; Salinity ==
 +
<html>
 +
<p>
 +
    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,
 +
    offers the opportunity to test cell viability as well as the Biobrick behaviour at different pH and salt concentrations. Overnight cultures of both strains
 +
    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
 +
    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
 +
    normal pH of the LB medium for all the tubes.
 +
</p>
 +
 
 +
</html>
 +
== UV Radiation ==
 +
<html>
 +
<p>
 +
    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
 +
    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
 +
    experiment which would answer these questions: overnight cultures of the strains will be diluted and spot-plated on LB agar. Subsequently, the spots will
 +
    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
 +
    plates will be incubated and fluorescence will be measured and normalized to O.D.<sub>600.</sub>
 +
</p>
 +
 
 +
</html>
 +
== Vacuum ==
 +
<html>
 +
<p>
 +
    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
 +
    from these, as always, compared to cells spot-plated and grown in optimal conditions, through OD<sub>600</sub> and fluorescence assays.
 +
</p>
 +
 
 +
 
 +
 
 +
 
         </div>
         </div>
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 +
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         <div class="tab-pane fade" id="Standardization ">
         <div class="tab-pane fade" id="Standardization ">
             <h1>Yellow</h1>
             <h1>Yellow</h1>

Revision as of 13:58, 30 September 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.


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 E. coli 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?

Stability Drawing

Stability

Our subteam aims to test the stability of ten different Biobricks transformed in two different strains of Escherichia coli, 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 grown in the same conditions, as well as with cells transformed with an empty plasmid (without Biobrick).

Temperature Stress

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 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 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 temperatures:

Test at 12 temperatures

four replicas of two strains tested simultaneously at 12 different temperatures


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 measured and later normalized to O.D.600, to compare the expression of the different Biobricks.

Material Fatigue

The idea is analogous to material fatigue in material sciences; in materials science, fatigue is the weakening of a material caused by repeatedly applied loads. If the loads are above a certain threshold, microscopic cracks will begin to form at the stress concentrators such as the surface, persistent slip bands and grain interfaces. In our case, we chose to induce the fatigue by subjecting the cells to temperature fluctuation, every minute the temperature will be changed from 37ºC (E.coli optimum growth temperature) to 41ºC and back to 37ºC again, this way, the temperature oscillates every minute from 37 to 41ºC for 4 hours. What will be the effect of such thermal fluctuations?

pH & Salinity

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, offers the opportunity to test cell viability as well as the Biobrick behaviour at different pH and salt concentrations. Overnight cultures of both strains will be resuspended in different LB media with pH ranging from 5 to 9 and incubated for 90 minutes, after which O.D.600 and fluorescence of each 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 normal pH of the LB medium for all the tubes.

UV Radiation

As per today, high doses of UV light are proven detrimental to life, but, to what extent is it injurious to an E.coli cell? Is transformation a 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 experiment which would answer these questions: overnight cultures of the strains will be diluted and spot-plated on LB agar. Subsequently, the spots will be subjected to timed pulses of UV radiation (intensity of 340 µW/cm2) with a control that receives no UV irradiation at all. Thereafter the plates will be incubated and fluorescence will be measured and normalized to O.D.600.

Vacuum

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 from these, as always, compared to cells spot-plated and grown in optimal conditions, through OD600 and fluorescence assays.

Yellow

yellow yellow yellow yellow yellow

Green

green green green green green

Blue

blue blue blue blue blue

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