Team:Grenoble

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Ne laissez pas le carré trop longtemps si vous n'éditez pas, chaque fois reprenez ce qui est sur internet plutôt que ce que vous avez sur votre PC
Ne laissez pas le carré trop longtemps si vous n'éditez pas, chaque fois reprenez ce qui est sur internet plutôt que ce que vous avez sur votre PC
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ps: dans Geany selectionner une ou plusieurs lignes et appuyer sur "Ctrl + E" pour commenter ou décommenter
 
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  Characterisation of the RsmA post-transcriptional regulation system components
 
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    <h2 id="Necessity">Justification of the need of a regulation system</h2>
 
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  In our project, we developed a translation regulation system in order to keep the system OFF until the pollutant is added.
 
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  With modelling, we show that this system is really important and without it, the measure is really disturbed.
 
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  In all of our simulation, we considered the initial concentrations of both repressors equal to zero. This will be the case
 
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  with the regulation system. Without this system, the initial concentrations of the repressors are higher. In the following
 
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  figure, we performed a simulation for an aTc concentration of $1.10^{-6}$ with initial concentrations equal to zero and
 
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  one with initial concentrations equal to $5\%$ of the concentrations in the steady state of the previous simulation.
 
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Figure 1:
 
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Observation of the interface when the regulation system works and when it doesn't for an aTc concentration of $1x10^{-6}$.
 
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When the regulation system doesn't work, the interface is not at the place it supposed to be. Because we can't havemeasures of the initial concentration of both repressors, to well predict where the interface will appear, we need to control these concentrations. That's why we developed the translation regulation system.
 
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    <h2>
 
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    Can rsmA be transferred into E. coli?
 
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The RsmA system from Pseudomonas has a homologue in Escherichia coli, named "CsrA". We know that these two systems are extremely similar. Consequently we ask ourselves whether the synthesis of RsmA in E. coli interferes with its survival. Figure 1 shows growth curves of E.coli cells transformed by a plasmid containing an IPTG-inducible rsmA sequence from Pseudomonas and control cells carrying the same plasmid without rsmA. The superimposed curves demonstrate that the synthesis of RsmA does not interfere with the growth of E. coli.
 
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Figure 2
 
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Influence of RsmA production on the growth of E coli DH5α. Bacteria transformed with pVLT31-rsmA or empty pVLT31 from overnight culture were re-suspended in rich medium supplemented with tetracycline at 20mg/ml. Induction of rsmA transcription is induced by IPTG at the concentrations given in the legend. Curves are normalized to their first OD value.
 
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Diaporama
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Characterisation of the leader sequences
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We cloned several leader sequences that contain a ribosome-binding site (RBS) in front of a reporter gene, GFP, in order to:
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<a href="https://static.igem.org/mediawiki/2011/1/17/RBS_strenght.png" alt="figure4">
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Characterise their RBS strength
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Use them for the translational control of downstream genes by the RsmA/rsmY system.
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The leader sequences used were mag and fha from Pseudomonas as well as the biobrick BBa_K256003, which was used as a reference. All constructs of Figure 2 have identical promoters, GFP reporter gene and terminators and are carried by the same plasmid.
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<a href="https://static.igem.org/mediawiki/2011/1/1a/Contructions_test.png">
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Figure 3:
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:Constructions used to characterise RBS included sequences strength of magA and fha leader sequences.
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Flow cytometry test
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We used a FACSCalibur flow cytometer to measure the GFP fluorescence emitted by cells containing the constructs shown in Fig.3. Two negative controls were set up: a brick having the GFP reporter gene but no promoter (
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BBa_E0840
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) and a cell culture containing no plasmid.
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50 μl of LB containing cells were diluted into 500 μl of filtered PBS (OD600 of inoculum was 3± 0,3 for all samples) and then introduced into the FACS ten minutes after dilution.
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The cytometer counts each particle that passes through the light beam. Therefore it is necessary to select an analysis window that corresponds to the size of the bacteria (see Fig. 3).
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Figure 4:
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Dot plots of particle size distributions obtained with a water sample containing no bacteria (left) and a sample containing bacteria (right). 40000 events were counted. FACS measurements were realised in duplicate.
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We then analysed the basal fluorescence recorded from control bacteria expressing no GFP. This allows to define two windows: the M1 window referring to basal fluorescence levels and containing fluorescence values obtained for all negative controls; the M2 window comprising fluorescence signals that are greater than the basal level. We show for each construction the average fluorescence (in red) within the window that contains most of the cells (percentages indicated in black, see Fig. 5).
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<a href="https://2011.igem.org/Team:Grenoble/Projet/Intro" title="Introduction"><img src="https://static.igem.org/mediawiki/2011/d/d3/Slide_home2.png"></img></a>
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Figure 5:
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Individual flow cytometry results (at the right) for different constructs (at the left). The red number indicates an average of GFP fluorescence. The percentage in black indicates the fraction of the bacterial population within the respective analysis window M1 or M2.
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The two negative controls (cells containing no plasmid (1) or plasmid without promoter (2)) show a basal fluorescence signal as expected. Cells containing the reference brick BBa_K25003 (5) show a maximum amount of GFP fluorescence with 77 % of the cell population that fluoresce more than the negative controls. The average fluorescent signal calculated for construct 5 is 1030 (vs 2,5 (neg control)).  The fluorescence signal obtained with cells containing the brick with the maga leader sequence (3) does not differ very much from the negative controls (4,4 vs 2,5). Four per cent of this cell population fluoresces more than the negative control populations.
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<img src="https://static.igem.org/mediawiki/2011/a/a6/Slide_home3.png" usemap="#genetic_network"></img>
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<area href="https://2011.igem.org/Team:Grenoble/Projet/Design" title="Overview of the genetic network" coords="81,15,422,349" shape="rect">
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90 % of cells containing the fha leader sequence (4) present a fluorescence signal that is higher than control cells. Their average fluorescence is 98.
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<area href="https://2011.igem.org/Team:Grenoble/Projet/Design/quorum" title="Quorum sensing + coloration" coords="670,214,867,332" shape="rect">
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<area href="https://2011.igem.org/Team:Grenoble/Projet/regulation" title="RsmA: post-transcriptional regulation system" coords="447,214,669,332" shape="rect">
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<a href="https://static.igem.org/mediawiki/2011/0/03/Superposition_RBS_strenght.png">
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Figure 6:
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<a href="https://2011.igem.org/Team:Grenoble/Projet/regulation" title="RsmA: post-transcriptional regulation system"><img src="https://static.igem.org/mediawiki/2011/1/1a/Slide_home4.png"/></a>
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Compilation of the flow cytometer measurements. 1and 2 are the negative controls, 3 : maga leader sequence, 4: fha leader sequence, 5 : reference biobrick BBa_K256003.
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<a href="https://2011.igem.org/Team:Grenoble/Projet/Modelling" title="Model"><img src="https://static.igem.org/mediawiki/2011/1/19/Slide_home5.png"></img></a>
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<a href="https://2011.igem.org/Team:Grenoble/Projet/Device" title="Device prototype"><img src="https://static.igem.org/mediawiki/2011/5/51/Slide_home6.png"></img></a>
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<a href="https://2011.igem.org/Team:Grenoble/HumanPractice" title="Human Practice"><img src="https://static.igem.org/mediawiki/2011/8/85/Slide_home7.png"/></a>
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Figure 6 summarises the cytometry results. We can see that both leader sequences mag and fha allow the translation of the gfp mRNA. They can therefore be used for further characterisation of the system.  The GFP fluorescence signal is much higher using the fha leader sequence when compared to mag. Figure 7 focuses on the RBS strength of those two gene leader sequences, and compares them to the strongest RBS of the part registry: BBa_0034. Mag has got very week RBS binding site strength, whereas fha is stronger (10% of the maximum value obtained for BBa_0034).
 
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<a href="https://static.igem.org/mediawiki/2011/a/a7/Digrame_rbs_strengh.png">
 
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Figure 7:
 
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Relative strength of maga (BBa_K545006) and fha (BBa_K545005) leader sequences compared to BBa_K25003 RBS. The latter is the strongest RBS used to compare RBS sequences of the registry.
 
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<h2>Ready for take-off !<span>By the Team</span></h2>
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Effect of rsmA  and rsmY on the mag and fha reporter genes
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<p>We are proud to say we obtained our pass for the iGEM World Championship at the MIT (Boston) and got a gold medal !
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We will double our efforts to acomplish our goals until Boston in early November. Many thanks to all our
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<h2>Mercuro-Coli: A new way to quantify heavy metals.<span>By JB</span></h2>
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<h2>Mercuro-Coli: A new way to quantify heavy metals.</h2>
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<div class="noindent">
<p>
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After having established the RBS strength of fha and mag leader sequences using the GFP reporter gene constructs, we quantified the translational inhibition effect of the RsmA protein and the relieve of this inhibition in presence of the rsmY RNA. In order to do these experiments, E. coli cells were co-transformed with a combination of 3 different plasmids containing:
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Our project aims at constructing an easy to use, transportable sensor capable of quantifying the concentration of mercury, in an aqueous sample.  
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A leader sequence or the reference RBS upstream of GFP (constructs 3, 4 and 5 on Fig. 4)
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Our system is based on a comparison between an unknown mercury concentration and a known IPTG concentration. A linear IPTG gradient is present on a test-strip containing the engineered bacteria. When the mercury solution is added, the regulatory network will switch to one of two states depending on the IPTG/mercury ratio.  
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A leader sequence or RBS plus an other plasmid containing rsmA  (figure 7)
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Bacteria become either “sender” or “receiver”. The bacteria sensing a predominance of mercury over IPTG, the “senders”, will release a quorum sensing molecule which is detected by the nearby “receivers”. The reception of quorum sensing molecules will induce the expression of a red dye in the “receivers”. In this way, a red line emerges at a position in the IPTG gradient from which the unknown mercury concentration can be deduced.
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A leader sequence or RBS plus rsmA plus rsmY (figure 7)
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Figure 7:
 
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The IPTG-inducible rsmA gene is cloned in plasmid pSB3C5 and the Tet-inducible rsmY in plasmid pSB4K5.
 
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The GFP fluorescence obtained from the constructs with fha, mag and reference RBS were analysed in the absence of RsmA and rsmY (single transformants) in the presence of RsmA (double transformants) and in the presence of both RsmA and rsmY (triple transformants). Note that in our experiments rsmA and rsmY were constitutively expressed because the repressors LacI and TetR were absent from our strains.
 
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As expected, no inhibitory effect of RsmA was observed when the reference RBS (
 
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<a Href="http://partsregistry.org/Part:BBa_B0034">
 
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BBa_0034
 
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) was used as this leader sequence does not have a binding site for RsmA (data not shown).
 
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In contrast, RsmA decreased the GFP fluorescence level when mag or fha leader sequences were provided upstream of GFP and this decrease could be partially relieved in the presence of rsmY for fha (Figs 8 and 9).
 
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Effect of rsmA and rsmY on fha-GFP constructs
 
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<h2 class="lien"id="Paragraphe2">iGEM and the future of synthetic biology<span>By Geoffrey</span></h2>
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Now booming, synthetic biology is a scientific field that divides the world into 2 parts. On the one hand, people who believe it will  science and our life. On the over hand, people who believe that synthetic biology is dangerous and it simply means playing God.
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Like all new domains of science, many questions may be raised about synthetic biology. Albert Einstein said: “It is strange that science which before seemed to be inoffensive will be turned into a nightmare that frightens everyone”. The goal of iGEM competition is to promote synthetic biology in the scientific community, but also to inform the public about this new science. Synthetic biology is a very controlled science to prevent side-effects and many countries such as France move to a strong legislative supervision in order to respect the ethical issues related to the modification of life.
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According to the <a href="http://www.bulletins-electroniques.com/actualites/66814.htm">newsletter of the French Embassy to the United States</a>, the international scientific community recognizes the dangers of synthetic biology and it must identify them precisely. It has especially given to the competition iGEM an important educational role for the scientific community of tomorrow.
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However, synthetic biology is a science with an extraordinary potential. Technological advances it could bring are huge. The fields of application are vast, from agriculture to medicine through the environment and energy, synthetic biology offers new possibilities and new alternatives to current technologies. The iGEM competition directly contributes to advance research in all these fields by enabling student teams to contribute in <a href="http://ung.igem.org/Team_Tracks?year=2011">these categories</a>.
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"Open source" policy conveyed by iGEM allows the world to take advantage of advances in this field and thus to improve every year the level of competition and projects. And we can say that the objective is on track, because the number of teams participating in this competition is <a href="http://ung.igem.org/Previous_iGEM_Competitions">constantly increasing</a>.
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<h2> A great human adventure </h2>
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So far only the characterisation of the leader sequences has been completed. Fha allows a better expression of GFP than mag.The next experiment will be to look at how these leader sequences react to the presence of the RsmA protein.
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iGEM competition includes about 160 teams (more or less 2000 students) over the world. There will be a first regional jamboree in different regions such as Europe, Asia, America,… and then a third of us will go to Boston for the last jamboree. This will be the first opportunities for most of students to meet people from so many universities at once ! We ‘ll be hosting at the same places, sharing breakfast and even partying together !... We ‘ll be able to discuss abouts many topics related to our fields, share ideas, and to see how others managed their work by watching their presentations.
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We did a few tests with ptet rsma and pcons-fha-gfp or pcons-mag-gfp.  The first result showed an extremely low gfp signal, which suggests a repressive effect of Rsma on fha and mag. We cannot conclude yet because some controls are lacking. This however gives very promising prospects.
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iGEM competition is not just about doing scientific work in order to get a medal, but is also about doing all of what an engineer or researcher needs to do ! Each team needs to set up contacts and collaboration with other groups. Sharing bricks or helping others is a criteria for winning a gold medal! We also need to get funds in order to finance our work…. So we must make a project that fits some challenge of our society ! As every one knows, new technologies rise up new ethical issues. This aspect has to be considered on every single project !
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Now that we have found an interesting  project and can be useful for the society, we are on the way for 4 month of intense work ! Our group is made of 12 people with very different education , that have to understand and synchronise each other. Since we are spread out in multiple places, we already meet once a week in order to coordinate or work. This need of an efficient communication within the team is an exiting part of the project !
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{{:Team:Grenoble/Design/pied}}

Latest revision as of 22:11, 28 October 2011

Grenoble 2011, Mercuro-Coli iGEM

Mercuro-Coli: A new way to quantify heavy metals.

Our project aims at constructing an easy to use, transportable sensor capable of quantifying the concentration of mercury, in an aqueous sample.

Our system is based on a comparison between an unknown mercury concentration and a known IPTG concentration. A linear IPTG gradient is present on a test-strip containing the engineered bacteria. When the mercury solution is added, the regulatory network will switch to one of two states depending on the IPTG/mercury ratio.

Bacteria become either “sender” or “receiver”. The bacteria sensing a predominance of mercury over IPTG, the “senders”, will release a quorum sensing molecule which is detected by the nearby “receivers”. The reception of quorum sensing molecules will induce the expression of a red dye in the “receivers”. In this way, a red line emerges at a position in the IPTG gradient from which the unknown mercury concentration can be deduced.

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