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Team/CINVESTAV-IPN-UNAM MX/Rule-Based.htm
2012-10-27T04:04:02Z
<p>Ao.patricia: </p>
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<h1><em>Simulation of Promoters behavior using a <br /><br />Rule-Based model.!</em></h1><br />
<p>Like we said before the proteins in our regulatory system PpsR, AppA and PrrA belong to<br />
acomplex regulatory network and they all work together to control genetic expression and<br />
induce all the metabolic changes in Rhodobacter sphaeroides.<br><br><br />
<br />
<br />
We already developed one model based on ODEs, but since this is the first time there has<br />
been an attempted to build BioBricks using photosynthetic bacteria, we wanted to try<br />
another model to simulate promoters behavior under the same conditions that were tested<br />
in the lab and see if they correlated.<br><br><br />
<br />
<br />
Using a rule-based model we were able to evaluate site specific details about protein-<br />
protein interaction and for this part we focused mainly on interactions of the domains<br />
between PpsR, AppA and PrrA.<br><br><br />
<br />
<br />
The following scheme, Figure 1, shows a complex network, in where our main proteins are<br />
involved and the intermediate molecules that help establish the interactions. And the<br />
Figure 2, shows the reduced and oxidized form of AppA and PpsR.</p><br />
<img src="https://static.igem.org/mediawiki/2012/9/9a/Rule01.jpg" width="284" height="296"><img src="https://static.igem.org/mediawiki/2012/4/4e/Rule02.jpg" width="284" height="296"><br />
<br />
The tetramers with intramolecular disulfide bonds (S-S)</strong> and thiol groups (SH) denote the<br />
oxidized and the reduced form of the PpsR repressor, respectively. The AppA protein has<br />
an FAD and a heme cofactor attached where h+ and h–denote the oxidized and reduced<br />
form of the heme cofactor (TspO).</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/08/Rule03.jpg" width="566" height="379"><br />
<p>Aerobically, the volume of electron flow through the cbb3 oxidase is sufficient to generate an<br />
inhibitory signal keeping the PrrBA system inactive and no GFP expression, because the Quinone<br />
Pool is maximally oxidized, which is mirrored in the redox state of AppA, keeping PpsR active.<br><br />
<br><br />
<br />
As O2 tensions diminish, the volume of electron flow through the cbb3 oxidase decreases as well<br />
and the PrrBA system becomes active through an autophosphorylation of PrrB and transfer of this<br />
phosphate group to PrrA.</p><br />
<p id="text2">Parameters:</p><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2012/4/4e/Rule04.jpg" width="566"><br />
</div><br />
<p>Reference: <a href="http://bionumbers.hms.harvard.edu/" target="_blank">http://bionumbers.hms.harvard.edu/</a> and literature, the rest of the constants were<br />
assumed second-order rate constants.</p><br />
<p id="text2">Model Construction</p><br />
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<p><em><strong>Rhodobacter spahaeroides</em></strong></p><br />
<object classid="clsid:d27cdb6e-ae6d-11cf-96b8-444553540000" codebase="http://fpdownload.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=8,0,0,0" width="560" height="450" id="graficas" align="middle"><br />
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<p><em><strong>Rhodopseudomonas palustris</em></strong></p><br />
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<p id="text2">Discussion.</p><br />
<p>The graphs shows the concentration of our four main proteins in their different sate shuch as, PrrA Active/Inactive, PpsR reduced(-)/oxidize(+) and AP2 (complex formation), all this states in a certain condition (aerobic/darkness, anaerobic/light) over time. Our simulations predict that in aerobic/ darkness condition we can see high concentration of inactive PrrA and in an anaerobic/light condition active PrrA shows high concentration. PpsR it will remain mostly oxidize in aerobic/ darkness and reduced PpsR will be in higher concentration in anaerobic/light, allowing complex formation (AP2).<br />
<br><br />
<p id="text2">Overall conclusion about the models.</p><br />
<p>Well, but we have two almost completely different models, the question is why use two<br />
models for the same phenomenon? Well, that's because the perspective of each model,<br />
the pros and cons of them. Using two models gives us the opportunity to see two<br />
perspectives that help us understand the phenomenon.<br><br />
<br><br />
<br />
On one hand, the differential equations describing protein concentrations and its<br />
relationship to oxygen and light, while the rule-based model allows us to observe<br />
interactions between molecules, considering agents in agents in a confined space. In other<br />
words, the first model allows us to see the system as a whole while the latter sees it as the<br />
aggregation of various agents.</p><br />
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<h2>Modeling</h2><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Cellular.htm
Team/CINVESTAV-IPN-UNAM MX/Cellular.htm
2012-10-27T03:57:32Z
<p>Ao.patricia: </p>
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<h1><em>Strange variant of cellular automata for <br /><br />the simulation of a bio-reactor!</em></h1><br />
<br />
<p id="text2">Introduction</p><br />
<p><br />
First of all: why use cellular automata if any linear models are easier to analyze? First, it looks great.<br />
</p><center><br />
<img src="https://static.igem.org/mediawiki/2012/5/50/CA1.gif" alt="This look great, isn't it?"></center><br />
<p>Second, depending on the configuration of the Cellular Automaton (CA) "how does it look", the CA is more realistic, and it can provide enough data for other models. In fact, is just what took place in this model.<br />
</p><p>Imagine that we are scientists with more resources and that we can generate hundreds of experiments in the conditions we want to feed our prediction models. The CA allows us to do this with the only drawback that we hate our CPU.<br />
</p><h2>Brief History</h2><br />
<p>Cellular automata are a mathematical model developed by Konrad Zuse and Stanislaw Ulam, but was better known and developed by John Vonn Neumann who loved parallel computing, history remembers him as the father of sequential computing.<br />
</p><p>Later during the 60's, a crazy mathematical said "oh, it would be great to do a math game where no players" (you know, the time before tetris and farmville), and created the world famous "Game of Life". The name of this cool mathematician was John Horton Conway. The really curious thing about this game was that, unlike tetris and Farmville, is a universal machine,is that you can solve almost any problem with it.<br />
</p><p>For this reason, a lot of scientists began to study de about the CA. First a physicist named Steven Wolfram, he emphasize the importance of simulations and modeling computer as unquestionable substitute of the traditional experimentation.<br />
</p><h2>How does a Cellurar Automata work?</h2><br />
<center><img src="https://static.igem.org/mediawiki/2012/b/b8/Resistentbacter.gif" alt="A very Resistent bacterium"></center><br />
<p>In the near future, our project can be used to produce biofuels, so we decided to create a software that allows us to generate Pilot-Plant simulations. <br />
<br />
To perform this software, we are taking the two-dimensional Cellular Automata developed by John Conway called "GAME OF LIFE", and extending it over a living cell type, in where there are several stages of the cell and different scope ranges. Our goal is to generate enough simulations at different concentrations of reactants or products under distinct environmental conditions; until the program shows what inputs (concentrations) are optimal to maximize the production.<br />
At this point, the program runs simulations based on precompiled transition rules, but due to the inherent complexity of cellular automata, a single simulation isn´t enough. So the next step is to vary the concentrations and apply an evolutionary algorithm to modify the starting conditions of the "game" in each iteration cycle.<br />
<br />
<br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Rule-Based.htm
Team/CINVESTAV-IPN-UNAM MX/Rule-Based.htm
2012-10-27T03:54:17Z
<p>Ao.patricia: </p>
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<h1><em>Simulation of Promoters behavior using a <br /><br />Rule-Based model.!</em></h1><br />
<p>Like we said before the proteins in our regulatory system PpsR, AppA and PrrA belong to<br />
acomplex regulatory network and they all work together to control genetic expression and<br />
induce all the metabolic changes in Rhodobacter sphaeroides.<br><br><br />
<br />
<br />
We already developed one model based on ODEs, but since this is the first time there has<br />
been an attempted to build BioBricks using photosynthetic bacteria, we wanted to try<br />
another model to simulate promoters behavior under the same conditions that were tested<br />
in the lab and see if they correlated.<br><br><br />
<br />
<br />
Using a rule-based model we were able to evaluate site specific details about protein-<br />
protein interaction and for this part we focused mainly on interactions of the domains<br />
between PpsR, AppA and PrrA.<br><br><br />
<br />
<br />
The following scheme, Figure 1, shows a complex network, in where our main proteins are<br />
involved and the intermediate molecules that help establish the interactions. And the<br />
Figure 2, shows the reduced and oxidized form of AppA and PpsR.</p><br />
<img src="https://static.igem.org/mediawiki/2012/9/9a/Rule01.jpg" width="284" height="296"><img src="https://static.igem.org/mediawiki/2012/4/4e/Rule02.jpg" width="284" height="296"><br />
<br />
The tetramers with intramolecular disulfide bonds (S-S)</strong> and thiol groups (SH) denote the<br />
oxidized and the reduced form of the PpsR repressor, respectively. The AppA protein has<br />
an FAD and a heme cofactor attached where h+ and h–denote the oxidized and reduced<br />
form of the heme cofactor (TspO).</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/08/Rule03.jpg" width="566" height="379"><br />
<p>Aerobically, the volume of electron flow through the cbb3 oxidase is sufficient to generate an<br />
inhibitory signal keeping the PrrBA system inactive and no GFP expression, because the Quinone<br />
Pool is maximally oxidized, which is mirrored in the redox state of AppA, keeping PpsR active.<br><br />
<br><br />
<br />
As O2 tensions diminish, the volume of electron flow through the cbb3 oxidase decreases as well<br />
and the PrrBA system becomes active through an autophosphorylation of PrrB and transfer of this<br />
phosphate group to PrrA.</p><br />
<p id="text2">Parameters:</p><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2012/4/4e/Rule04.jpg" width="566"><br />
</div><br />
<p>Reference: <a href="http://bionumbers.hms.harvard.edu/" target="_blank">http://bionumbers.hms.harvard.edu/</a> and literature, the rest of the constants were<br />
assumed second-order rate constants.</p><br />
<p id="text2">Model Construction</p><br />
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<p><em><strong>Rhodobacter spahaeroides</em></strong></p><br />
<object classid="clsid:d27cdb6e-ae6d-11cf-96b8-444553540000" codebase="http://fpdownload.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=8,0,0,0" width="560" height="450" id="graficas" align="middle"><br />
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</div><br />
<div align="center"><br><br />
<p><em><strong>Rhodopseudomonas palustris</em></strong></p><br />
<object classid="clsid:d27cdb6e-ae6d-11cf-96b8-444553540000" codebase="http://fpdownload.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=8,0,0,0" width="560" height="450" id="graficaspalustris" align="middle"><br />
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<p id="text2">Disscusion.</p><br />
<p>The graphs shows the concentration of our four main proteins in their different sate shuch as, PrrA Active/Inactive, PpsR reduced(-)/oxidize(+) and AP2 (complex formation), all this states in a certain condition (aerobic/darkness, anaerobic/light) over time. Our simulations predict that in aerobic/ darkness condition we can see high concentration of inactive PrrA and in an anaerobic/light condition active PrrA shows high concentration. PpsR it will remain mostly oxidize in aerobic/ darkness and reduced PpsR will be in higher concentration in anaerobic/light, allowing complex formation (AP2).<br />
<br><br />
<p id="text2">Overall conclusion about the models.</p><br />
<p>Well, but we have two almost completely different models, the question is why use two<br />
models for the same phenomenon? Well, that's because the perspective of each model,<br />
the pros and cons of them. Using two models gives us the opportunity to see two<br />
perspectives that help us understand the phenomenon.<br><br />
<br><br />
<br />
On one hand, the differential equations describing protein concentrations and its<br />
relationship to oxygen and light, while the rule-based model allows us to observe<br />
interactions between molecules, considering agents in agents in a confined space. In other<br />
words, the first model allows us to see the system as a whole while the latter sees it as the<br />
aggregation of various agents.</p><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Rule-Based.htm
Team/CINVESTAV-IPN-UNAM MX/Rule-Based.htm
2012-10-27T03:17:11Z
<p>Ao.patricia: </p>
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<h1><em>Simulation of Promoters behavior using a <br /><br />Rule-Based model.!</em></h1><br />
<p>Like we said before the proteins in our regulatory system PpsR, AppA and PrrA belong to<br />
acomplex regulatory network and they all work together to control genetic expression and<br />
induce all the metabolic changes in Rhodobacter sphaeroides.<br><br><br />
<br />
<br />
We already developed one model based on ODEs, but since this is the first time there has<br />
been an attempted to build BioBricks using photosynthetic bacteria, we wanted to try<br />
another model to simulate promoters behavior under the same conditions that were tested<br />
in the lab and see if they correlated.<br><br><br />
<br />
<br />
Using a rule-based model we were able to evaluate site specific details about protein-<br />
protein interaction and for this part we focused mainly on interactions of the domains<br />
between PpsR, AppA and PrrA.<br><br><br />
<br />
<br />
The following scheme, Figure 1, shows a complex network, in where our main proteins are<br />
involved and the intermediate molecules that help establish the interactions. And the<br />
Figure 2, shows the reduced and oxidized form of AppA and PpsR.</p><br />
<img src="https://static.igem.org/mediawiki/2012/9/9a/Rule01.jpg" width="284" height="296"><img src="https://static.igem.org/mediawiki/2012/4/4e/Rule02.jpg" width="284" height="296"><br />
<br />
The tetramers with intramolecular disulfide bonds (S-S)</strong> and thiol groups (SH) denote the<br />
oxidized and the reduced form of the PpsR repressor, respectively. The AppA protein has<br />
an FAD and a heme cofactor attached where h+ and h–denote the oxidized and reduced<br />
form of the heme cofactor (TspO).</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/08/Rule03.jpg" width="566" height="379"><br />
<p>Aerobically, the volume of electron flow through the cbb3 oxidase is sufficient to generate an<br />
inhibitory signal keeping the PrrBA system inactive and no GFP expression, because the Quinone<br />
Pool is maximally oxidized, which is mirrored in the redox state of AppA, keeping PpsR active.<br><br />
<br><br />
<br />
As O2 tensions diminish, the volume of electron flow through the cbb3 oxidase decreases as well<br />
and the PrrBA system becomes active through an autophosphorylation of PrrB and transfer of this<br />
phosphate group to PrrA.</p><br />
<p id="text2">Parameters:</p><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2012/4/4e/Rule04.jpg" width="566"><br />
</div><br />
<p>Reference: <a href="http://bionumbers.hms.harvard.edu/" target="_blank">http://bionumbers.hms.harvard.edu/</a> and literature, the rest of the constants were<br />
assumed second-order rate constants.</p><br />
<p id="text2">Model Construction</p><br />
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<p><em><strong>Rhodobacter spahaeroides</em></strong></p><br />
<object classid="clsid:d27cdb6e-ae6d-11cf-96b8-444553540000" codebase="http://fpdownload.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=8,0,0,0" width="560" height="450" id="graficas" align="middle"><br />
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</object><br><br />
</div><br />
<div align="center"><br><br />
<p><em><strong>Rhodopseudomonas palustris</em></strong></p><br />
<object classid="clsid:d27cdb6e-ae6d-11cf-96b8-444553540000" codebase="http://fpdownload.macromedia.com/pub/shockwave/cabs/flash/swflash.cab#version=8,0,0,0" width="560" height="450" id="graficaspalustris" align="middle"><br />
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</object></div><br />
<p id="text2">Overall conclusion about the models.</p><br />
<p>Well, but we have two almost completely different models, the question is why use two<br />
models for the same phenomenon? Well, that's because the perspective of each model,<br />
the pros and cons of them. Using two models gives us the opportunity to see two<br />
perspectives that help us understand the phenomenon.<br><br />
<br><br />
<br />
On one hand, the differential equations describing protein concentrations and its<br />
relationship to oxygen and light, while the rule-based model allows us to observe<br />
interactions between molecules, considering agents in agents in a confined space. In other<br />
words, the first model allows us to see the system as a whole while the latter sees it as the<br />
aggregation of various agents.</p><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Cellular.htm
Team/CINVESTAV-IPN-UNAM MX/Cellular.htm
2012-10-27T03:13:08Z
<p>Ao.patricia: </p>
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<h1><em>Strange variant of cellular automata for <br /><br />the simulation of a bio-reactor!</em></h1><br />
<br />
<p id="text2">Introduction</p><br />
<p><br />
First of all: why use cellular automata if any linear models are easier to analyze? First, it looks great.<br />
</p><center><br />
<img src="https://static.igem.org/mediawiki/2012/5/50/CA1.gif" alt="This look great, isn't it?"></center><br />
<p>Second, depending on the configuration of the Cellular Automaton (CA) "how does it look", the CA is more realistic, and it can provide enough data for other models. In fact, is just what took place in this model.<br />
</p><p>Imagine that we are scientists with more resources and that we can generate hundreds of experiments in the conditions we want to feed our prediction models. The CA allows us to do this with the only drawback that we hate our CPU.<br />
</p><h2>Brief History</h2><br />
<p>Cellular automata are a mathematical model developed by Konrad Zuse and Stanislaw Ulam, but was better known and developed by John Vonn Neumann who loved parallel computing, history remembers him as the father of sequential computing.<br />
</p><p>Later during the 60's, a crazy mathematical said "oh, it would be great to do a math game where no players" (you know, the time before tetris and farmville), and created the world famous "Game of Life". The name of this cool mathematician was John Horton Conway. The really curious thing about this game was that, unlike tetris and Farmville, is a universal machine,is that you can solve almost any problem with it.<br />
</p><p>For this reason, a lot of scientists began to study de about the CA. First a physicist named Steven Wolfram, he emphasize the importance of simulations and modeling computer as unquestionable substitute of the traditional experimentation.<br />
</p><h2>How does a Cellurar Automata work?</h2><br />
<center><img src="https://static.igem.org/mediawiki/2012/b/b8/Resistentbacter.gif" alt="A very Resistent bacterium"></center><br />
<p>Imagine a group of guys in a dance floor. One of these says, "If I'm the only one who is dancing or there is only one person dancing, I think that i'm don't i will not be a laughing stock" and "If more than 4 people are dancing, the better is stop of dance, it's uncomfortable dancing among many people ". And of course "else, i dancing all the night". Now, imagine that all people think the same track. A CA is a group of individuals, in this case cells, that act in a similar way and at the same time within a specifc environment.</p><br />
<p>But, think more applied: if instead of teenage dancers, bacteria were willing to eat? Instead of a dance floor, outside a bioreactor? There are a more couple of questions to answer: If you want to feed the bacteria, the amount of food that is around you is also important, and we should consider adding it, and if they are fed well, they can not go to the bathroom to leave their waste , so we must take into account their waste.<br />
</p><p>This is a brief description of our model, we have a bioreactor cells, bacteria, food and debris. That is, a lattice and three types of cells that fill it.<br />
</p><p>What else do we need to know? In the past example we said, "I need two or three people so that I can dance", but as we speak now of bacteria and food, we say "I need amount n of food and amout m of other bacteria that can feed with me", because , you know, no one likes to eat alone.<br />
</p><p>But we can go further in this model. Suppose the bioreactor gives us ideal conditions every so often and eliminates food waste produced and they distributed throughout the lattice.<br />
</p><p>In our model, we decided to have two lattices: one for Rhodobacter, where there may be a bacterium or may be not. And another lattice with the same number of squares, but with x percent of food, "y" percent of wastes and z percent for other resources. In this way we can generate simple rules:<br />
</p><ul><br />
<li>If, for example, among all the cells I have around, sum at least 2 complete squares, the bacter will survive.</li><br />
<li>However, If there more than 5 boxes of waste, the bacterium die.</li><br />
<li>If at least two bacteria and exist enough food, namely more than 2 squares of food, grow a new bacteria.</li><br />
</ul><br />
<p>Thus, the CA showed a more realistic simulation, but mostly we will get data from simulation models can feed others.<br />
</p><p>The two main disadvantages of the CA is the CPU time needed to calculate the simulation and randomness that can have its outcome, in other words, depending on how initialize living cells and the percentage that we decide to add for each compound in the second lattice.<br />
</p><p>The first problem is easy to solve: the current CPU is fast enough to avoid problems in these calculations.<br />
</p><p>The second problem is harder to solve. There are several methods, our favorite, another mathematical model: perform multiple simulations with different distributions of bacteria and its results are added to a probability function that characterizes certain experiment.<br />
<br />
<br />
<br />
</p><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Light_Response.htm
Team/CINVESTAV-IPN-UNAM MX/Light Response.htm
2012-10-27T02:19:39Z
<p>Ao.patricia: </p>
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<h1><em>Light & Oxygen Response: AppA/PpsR <br /><br />Regulation System! </em></h1><br />
<p>This is a repressor/antirepressor system, which under high oxygen tension; PpsR represses GFP expression by avoiding RNA polymerase binding <br />
the promoter sequence.<br />
When oxygen concentration decreases and in presence of light AppA has a conformational change and can bind with PpsR, this complex prevents the union of<br />
PpsR to its target sequence, thus GFP expression can begin.<br><br />
<br />
(See the next video for a visual explanation).</p><br />
<div align="center"><iframe width="480" height="360" src="http://www.youtube.com/embed/XBNRLq9pL8c" frameborder="0" allowfullscreen></iframe></div><br><br />
<p id="text2">Our biobricks </p><br />
<p>The first biobrick consists in the complete light-oxygen dependent system, AppA and PpsR, each one with Ribosome Binding Site, under a<br />
Medium strength promoter (J23104), this first biobrick also it has the PpsR dependent promoter and GFP as a reporter gene.</p><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Rodo02.jpg" alt="rodo02" width="568" height="107"><br><br />
<p><br><br />
The second circuit is just the PpsR dependent promoter and GFP as a reporter gene.</p><br />
<img src="https://static.igem.org/mediawiki/2012/a/a1/Rodo03.jpg" alt="rodo03" width="562" height="252"><br />
<p id="text2">We were inspired in:</p><br />
<p><br />
This system is inspired in AppA/PpsR repressor/antirepressor system from Rhodobacter sphaeroides. The PpsR protein is a master repressor of<br />
Photosynthesis (PS) genes (Moskvin and Gomelsky 2005). Inactivation of the ppsR gene is enough to turn on PS gene expression and formation<br />
of the photosynthetic apparatus even at a high oxygen concentration, whereas ppsR overexpression is sufficient to block PS development even<br />
in the absence of oxygen. PpsR directly represses transcription of most carotenoid and pigment synthesis genes, photosystems operons, and<br />
genes involved in tetrapyrrole biosynthesis (Gomelsky and Kaplan 1995). The upstream regions of these genes contain two PpsR binding sites,<br />
TGTcN10gACA.<br><br />
<br />
A second protein called AppA, which has no known homologues, plays a role in controlling gene expression in <em>R. sphaeroides</em> in response to both<br />
light and O2 by acting as an antirepressor of PpsR. Our parts (appa, ppsr and ppsr-promoter) were synthesized by Genescript, and are codon<br />
optimized for <em>R. sphaeroides.</em></p><br />
<hr><br />
<p id="refe">References<br><br />
<br />
1. Gomelsky L., Moskvin L., Stenzel A., Jones D., Donohue T. and Gomelsky M.(2008) <strong>Hierarchical Regulation of Photosynthesis<br />
Gene Expression by the Oxygen-Responsive PrrBA and AppA-PpsR Systems of <em>Rhodobacter sphaeroides.</em></strong> J. Bacteriol.<br />
Dec. 2008, p. 8106–8114 Vol. 190, No. 24<br><br />
<br />
2. Moskvin, O. V., L. Gomelsky, and M. Gomelsky. (2005). <strong>Transcriptome analysis of the <em>Rhodobacter sphaeroides</em> PpsR<br />
regulon: PpsR as a master regulator of photosystem development.</strong> J. Bacteriol. 187:2148–2156.<br><br />
<br />
3. Gomelsky, M., and S. Kaplan. (1995). <strong>Genetic evidence that PpsR from <em>Rhodobacter sphaeroides.</em> 2.4.1 functions as a<br />
repressor of puc and bchF expression.</strong> J. Bacteriol. 177:1634–1637. <br><br />
4. Gomelsky, M., and S. Kaplan. (1995). <strong>AppA, a novel gene encoding a transacting factor involved in the regulation of<br />
photosynthesis gene expression in <em>Rhodobacter sphaeroides.</em> 2.4.1.</strong> J. Bacteriol. 177:4609–4618.<br />
</p><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Light_Response.htm
Team/CINVESTAV-IPN-UNAM MX/Light Response.htm
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<p>Ao.patricia: </p>
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<h1><em>Light & Oxygen Response: AppA/PpsR <br /><br />Regulation System! </em></h1><br />
<p>This is a repressor/antirepressor system, which under high oxygen tension; PpsR represses GFP expression by avoiding RNA polymerase binding <br />
the promoter sequence.<br><br />
<br />
When oxygen concentration decreases and in presence of light AppA has a conformational change and can bind with PpsR, this complex prevents the union of<br />
PpsR to its target sequence, thus GFP expression can begin.<br><br />
<br />
(See the next video for a visual explanation).</p><br />
<div align="center"><iframe width="480" height="360" src="http://www.youtube.com/embed/XBNRLq9pL8c" frameborder="0" allowfullscreen></iframe></div><br><br />
<p id="text2">Our biobricks </p><br />
<p>The first biobrick consists in the complete light-oxygen dependent system, AppA and PpsR, each one with Ribosome Binding Site, under a<br />
Medium strength promoter (J23104), this first biobrick also it has the PpsR dependent promoter and GFP as a reporter gene.</p><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Rodo02.jpg" alt="rodo02" width="568" height="107"><br><br />
<p><br><br />
The second circuit is just the PpsR dependent promoter and GFP as a reporter gene.</p><br />
<img src="https://static.igem.org/mediawiki/2012/a/a1/Rodo03.jpg" alt="rodo03" width="562" height="252"><br />
<p id="text2">We were inspired in:</p><br />
<p><br />
This system is inspired in AppA/PpsR repressor/antirepressor system from Rhodobacter sphaeroides. The PpsR protein is a master repressor of<br />
Photosynthesis (PS) genes (Moskvin and Gomelsky 2005). Inactivation of the ppsR gene is enough to turn on PS gene expression and formation<br />
of the photosynthetic apparatus even at a high oxygen concentration, whereas ppsR overexpression is sufficient to block PS development even<br />
in the absence of oxygen. PpsR directly represses transcription of most carotenoid and pigment synthesis genes, photosystems operons, and<br />
genes involved in tetrapyrrole biosynthesis (Gomelsky and Kaplan 1995). The upstream regions of these genes contain two PpsR binding sites,<br />
TGTcN10gACA.<br><br />
<br />
A second protein called AppA, which has no known homologues, plays a role in controlling gene expression in <em>R. sphaeroides</em> in response to both<br />
light and O2 by acting as an antirepressor of PpsR. Our parts (appa, ppsr and ppsr-promoter) were synthesized by Genescript, and are codon<br />
optimized for <em>R. sphaeroides.</em></p><br />
<hr><br />
<p id="refe">References<br><br />
<br />
1. Gomelsky L., Moskvin L., Stenzel A., Jones D., Donohue T. and Gomelsky M.(2008) <strong>Hierarchical Regulation of Photosynthesis<br />
Gene Expression by the Oxygen-Responsive PrrBA and AppA-PpsR Systems of <em>Rhodobacter sphaeroides.</em></strong> J. Bacteriol.<br />
Dec. 2008, p. 8106–8114 Vol. 190, No. 24<br><br />
<br />
2. Moskvin, O. V., L. Gomelsky, and M. Gomelsky. (2005). <strong>Transcriptome analysis of the <em>Rhodobacter sphaeroides</em> PpsR<br />
regulon: PpsR as a master regulator of photosystem development.</strong> J. Bacteriol. 187:2148–2156.<br><br />
<br />
3. Gomelsky, M., and S. Kaplan. (1995). <strong>Genetic evidence that PpsR from <em>Rhodobacter sphaeroides.</em> 2.4.1 functions as a<br />
repressor of puc and bchF expression.</strong> J. Bacteriol. 177:1634–1637. <br><br />
4. Gomelsky, M., and S. Kaplan. (1995). <strong>AppA, a novel gene encoding a transacting factor involved in the regulation of<br />
photosynthesis gene expression in <em>Rhodobacter sphaeroides.</em> 2.4.1.</strong> J. Bacteriol. 177:4609–4618.<br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Overview.htm
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2012-10-27T02:05:38Z
<p>Ao.patricia: </p>
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<h1><em>Overview! </em></h1><br />
<p>The purple non-sulfur photosynthetic bacteria (PNSB) belong to the alpha-proteobacteria group, because of their genetic regulatory systems<br />
which coordinate different metabolic states, these microorganisms are able to grow under a wide variety of environmental conditions (1).<br><br />
<br />
Specifically, our project aims to isolate two genetic control systems based on <em>Rhodobacter sphaeroides</em> photosynthesis cluster regulation. The first one is the<br />
oxygen dependant system PrrA/PrrB, when oxygen tension is high it remains inactive, and when the oxygen is low it activates gene expression<br />
(2). The second system is the light and oxygen mediated system AppA/PpsR that represses gene expression under aerobic conditions and allows<br />
transcription in the absence of oxygen and presence light (3).<br><br />
<br />
To achieve this goal we designed two genetic biobrick in which GFP expression is oxygen and light-dependent by the antirepression of PpsR and<br />
oxygen dependent by the activation of PrrA/B system. The lab work is accompanied by a computational model, which will provide a way of<br />
testing our knowledge of these systems.<br><br />
<br />
Once, we have characterized the functionality of these regulatory systems we aim to take advantage of <em>Rhodopseudomonas palustris’</em> metabolic versatility, and use<br />
this bacteria as a microbial factory, that could work for the production of metabolites with economic value products using CO2 as carbon source.<br><br />
<br />
We are planning to use the S04147 clostridial butanol production operon (University of Alberta iGEM Team 2007) to evaluate the synthesis of<br />
this biofuel, linking it to our control systems. This would provide an interesting way to produce butanol using CO2 as carbon source under<br />
anaerobic photosynthetic conditions.</p><br />
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<li><a href="Oxigen_Response.htm">Oxygen Response</a></li><br />
<li><a href="Chassis.htm">Chassis</a></li><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Members.htm
Team/CINVESTAV-IPN-UNAM MX/Members.htm
2012-10-27T01:13:12Z
<p>Ao.patricia: </p>
<hr />
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<h1><em>Members!</em></h1><br />
<p id="text2"> Students</p></br><br />
<img src="https://static.igem.org/mediawiki/2012/9/95/Diez.jpg" alt="diez" width="172" height="235" class="left"><br />
<br><br />
<br><br />
<br><br />
<p>Lissania Guerra-Calderas Contribution: Butanol Production System and BioBrick Designer, outreach activities. 4th year undergraduate of Pharmaceutical and Biological Chemistry, at Faculty of Chemistry. National Autonomous University of Mexico (UNAM) lissxgc.89@gmail.com.</p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/9/98/Ocho.jpg" width="172" height="235" class="right"></p><br />
<br><br />
<br />
<br />
<p>Jhonatan Alejandro Hernández-Valdés Contribution: Project Management, design and development of regulatory systems, BioBrick Designer, outreach activities. 4th year undergraduate of Pharmaceutical and Biological Chemistry, at Faculty of Chemistry. National Autonomous University of Mexico (UNAM) jhonatanhv@hotmail.com.</p><br />
<p>&nbsp;</p><br />
<p><br><br />
<img src="https://static.igem.org/mediawiki/2012/8/84/Once.jpg" alt="once" width="164" height="211" class="left"><br><br />
Maritere Uriostegui-Arcos Contribution: Characterization and measurement protocols. BioBrick Designer, outreach activities. 4th year undergraduate of Pharmaceutical and Biological Chemistry, at Faculty of Chemistry. National Autonomous University of Mexico (UNAM) maritere1011@hotmail.com.<br><br />
</p><br />
<p><br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/f/f6/Nueve.jpg" alt="nueve" width="163" height="237" class="right"><br><br />
</p><br />
<p>&nbsp;</p><br />
<p>Anna Karen Hernández-Gallardo Contribution: Mathematical modeling, 4th year undergraduate Biotechnological Engineering, Interdisciplinary Unit for Biotechnology<br />
National Polytechnic Institute (IPN).<br />
.</p><br />
<p>&nbsp;</p><br />
<p><br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/1/16/Tre.jpg" alt="tre" width="171" height="246" class="left"></p><br />
<p>Patricia Arias-Orozco Contribution: Design and development of regulatory systems, BioBrick Designer, outreach activities. 4th year undergraduate Biotechnological Engineering, Interdisciplinary Unit for Biotechnology National Polytechnic Institute (IPN). ao.patricia@hotmail.com </p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/4/45/Uno.jpg" alt="uno" width="174" height="243" class="right"></p><br />
<p>&nbsp;</p><br />
<p>Daniel Domínguez-Gómez <br><br />
<p>Contribution: Outreach activities.<br> <br />
4th year undergraduate Biotechnological Engineering, Interdisciplinary Unit for Biotechnology, National Polytechnic Institute.<br><br />
<p></p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/4/43/Siete.jpg" alt="siete" width="167" height="234" class="left"></p><br />
<p>&nbsp;</p><br />
<p>Ivan Lopez-Flores Mathematical Team: Mathematical modeling Computational Systems Engineering, Superior School for Computation National Polytechnic Institute National Polytechnic Institute <br><br />
<br><br />
<br><br />
<br><br />
<br />
</p><br />
<br />
<h1>Advisors</h1><br />
<br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/5/59/Catorce.jpg" alt="catorce" width="168" height="244" class="left"><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>Karen Nava Castro PhD Contribution: Flow Cytometry Expert. Researcher at Institute of Biomedical Research, Field of study: Immunology. National Autonomous University of Mexico, UNAM. </p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/c/c8/Quince.jpg" alt="quince" width="161" height="221" class="right"></p><br />
<p>&nbsp;</p><br />
<p>Jorge Morales Montor PhD Contribution: Dry lab support Researcher at Institute of Biomedical Research (IIB, UNAM) Field of study: Neuoimmunoendocrinology National Autonomous University of Mexico, UNAM. </p><br />
<p>&nbsp;</p><br />
<img src="https://static.igem.org/mediawiki/2012/6/67/Dos.jpg" alt="dos" width="194" height="270" class="left"><br />
<p>Daniel Federico Hernandez Gardiol Contribution: Mathematical model Systems Biologist: Universidad de la Republica (Montevideo, Uruguay) Bioengineering Masters Student: Ecole Polytechnique Federal de Lausanne (Lausanne, Switzerland) National Polytechnic Institute.</p><br />
<p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><br />
<p>&nbsp;</p><img src="https://static.igem.org/mediawiki/2012/0/0d/Cinco1.jpg" alt="cinco" width="173" height="205" class="right"><p>&nbsp;</p><br />
<p>Fernando G. Bastida-González Contribution: Wet lab support, Master in Health science at Superior Medicine School at National Polytechnic Institute (IPN).</p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<h1>Instructors</h1><br><br />
<br />
<p><img src="https://static.igem.org/mediawiki/2012/e/e9/Tres.jpg" alt="tres" width="166" height="227" class="left"></p><br />
<p>&nbsp;</p><br />
<p>Agustino Martínez-Antonio PhD Resarcher 3A, SNI I Genetic Engineering Department, CINVESTAV Irapuato.</p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><br><br />
<img src="https://static.igem.org/mediawiki/2012/6/67/Seis.jpg" alt="seis" width="161" height="209" class="right"></p><br />
<p>&nbsp;</p><br />
<p>Fernando Suaste-Olmos PhD Researcher at Institute of Cell Physiology IFC-UNAM.<br />
Expert in Photosynthetic bacteria. </p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/9/98/Doce.jpg" alt="dice" width="173" height="205" class="left"></p><br />
<p>&nbsp;</p><br />
<p>Nuria Sánchez-Puig Researcher at Institute of Chemistry UNAM Dr. Sanchez-Puig has a PhD from the Center for Protein Engineering, Cambridge, UK. </p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/a/ad/Dieciseis.jpg" alt="dieciseis" width="150" height="204" class="right"></p><br />
<p>&nbsp;</p><br />
<p>Paola Zárate-Segura PhD Researcher, at IPN. Dra. Zárate Segura has a PhD in Bioprocess from Interdisciplinary Unit for Biotechnology, National Polytechnic Institute. </p><br />
<p>&nbsp;</p><br />
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<ul><br />
<li><a href="Members.htm" target="_parent">Members</a></li><br />
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<li><a href="Gallery.htm">Gallery</a></li><br />
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<p align="center"><strong>Rhodofactory 2012</strong></p><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Members.htm
Team/CINVESTAV-IPN-UNAM MX/Members.htm
2012-10-27T01:03:27Z
<p>Ao.patricia: </p>
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<br />
<h1><em>Members!</em></h1><br />
<p id="text2"> Students</p></br><br />
<img src="https://static.igem.org/mediawiki/2012/9/95/Diez.jpg" alt="diez" width="172" height="235" class="left"><br />
<br><br />
<br><br />
<br><br />
<p>Lissania Guerra-Calderas Contribution: Butanol Production System and BioBrick Designer, outreach activities. 4th year undergraduate of Pharmaceutical and Biological Chemistry, at Faculty of Chemistry. National Autonomous University of Mexico (UNAM) lissxgc.89@gmail.com.</p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/9/98/Ocho.jpg" width="172" height="235" class="right"></p><br />
<br><br />
<br />
<br />
<p>Jhonatan Alejandro Hernández-Valdés Contribution: Project Management, design and development of regulatory systems, BioBrick Designer, outreach activities. 4th year undergraduate of Pharmaceutical and Biological Chemistry, at Faculty of Chemistry. National Autonomous University of Mexico (UNAM) jhonatanhv@hotmail.com.</p><br />
<p>&nbsp;</p><br />
<p><br><br />
<img src="https://static.igem.org/mediawiki/2012/8/84/Once.jpg" alt="once" width="164" height="211" class="left"><br><br />
Maritere Uriostegui-Arcos Contribution: Characterization and measurement protocols. BioBrick Designer, outreach activities. 4th year undergraduate of Pharmaceutical and Biological Chemistry, at Faculty of Chemistry. National Autonomous University of Mexico (UNAM) maritere1011@hotmail.com.<br><br />
</p><br />
<p><br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/f/f6/Nueve.jpg" alt="nueve" width="163" height="237" class="right"><br><br />
</p><br />
<p>&nbsp;</p><br />
<p>Anna Karen Hernández-Gallardo Contribution: Mathematical modeling, 4th year undergraduate Biotechnological Engineering, Interdisciplinary Unit for Biotechnology<br />
National Polytechnic Institute<br />
.</p><br />
<p>&nbsp;</p><br />
<p><br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/1/16/Tre.jpg" alt="tre" width="171" height="246" class="left"></p><br />
<p>Patricia Arias-Orozco Contribution: Design and development of regulatory systems, BioBrick Designer, outreach activities. 4th year undergraduate Biotechnological Engineering, Interdisciplinary Unit for Biotechnology National Polytechnic Institute. </p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/4/45/Uno.jpg" alt="uno" width="174" height="243" class="right"></p><br />
<p>&nbsp;</p><br />
<p>Daniel Domínguez-Gómez <br><br />
<p>Contribution: Outreach activities.<br> <br />
4th year undergraduate Biotechnological Engineering, Interdisciplinary Unit for Biotechnology, National Polytechnic Institute.<br><br />
<p></p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/4/43/Siete.jpg" alt="siete" width="167" height="234" class="left"></p><br />
<p>&nbsp;</p><br />
<p>Ivan Lopez-Flores Mathematical Team: Mathematical modeling Computational Systems Engineering, Superior School for Computation National Polytechnic Institute National Polytechnic Institute <br><br />
<br><br />
<br><br />
<br><br />
<br />
</p><br />
<br />
<h1>Advisors</h1><br />
<br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/5/59/Catorce.jpg" alt="catorce" width="168" height="244" class="left"><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>Karen Nava Castro PhD Contribution: Flow Cytometry Expert. Researcher at Institute of Biomedical Research, Field of study: Immunology. National Autonomous University of Mexico, UNAM. </p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/c/c8/Quince.jpg" alt="quince" width="161" height="221" class="right"></p><br />
<p>&nbsp;</p><br />
<p>Jorge Morales Montor PhD Contribution: Dry lab support Researcher at Institute of Biomedical Research (IIB, UNAM) Field of study: Neuoimmunoendocrinology National Autonomous University of Mexico, UNAM. </p><br />
<p>&nbsp;</p><br />
<img src="https://static.igem.org/mediawiki/2012/6/67/Dos.jpg" alt="dos" width="194" height="270" class="left"><br />
<p>Daniel Federico Hernandez Gardiol Contribution: Mathematical model Systems Biologist: Universidad de la Republica (Montevideo, Uruguay) Bioengineering Masters Student: Ecole Polytechnique Federal de Lausanne (Lausanne, Switzerland) National Polytechnic Institute.</p><br />
<p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><br />
<p>&nbsp;</p><img src="https://static.igem.org/mediawiki/2012/0/0d/Cinco1.jpg" alt="cinco" width="173" height="205" class="right"><p>&nbsp;</p><br />
<p>Fernando G. Bastida-González Contribution: Wet lab support, Master in Health science at Superior Medicine School at National Polytechnic Institute (IPN).</p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<h1>Instructors</h1><br><br />
<br />
<p><img src="https://static.igem.org/mediawiki/2012/e/e9/Tres.jpg" alt="tres" width="166" height="227" class="left"></p><br />
<p>&nbsp;</p><br />
<p>Agustino Martínez-Antonio PhD Resarcher 3A, SNI I Genetic Engineering Department, CINVESTAV Irapuato.</p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><br><br />
<img src="https://static.igem.org/mediawiki/2012/6/67/Seis.jpg" alt="seis" width="161" height="209" class="right"></p><br />
<p>&nbsp;</p><br />
<p>Fernando Suaste-Olmos PhD Researcher at Institute of Cell Physiology IFC-UNAM.<br />
Expert in Photosynthetic bacteria. </p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/9/98/Doce.jpg" alt="dice" width="173" height="205" class="left"></p><br />
<p>&nbsp;</p><br />
<p>Nuria Sánchez-Puig Researcher at Institute of Chemistry UNAM Dr. Sanchez-Puig has a PhD from the Center for Protein Engineering, Cambridge, UK. </p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/a/ad/Dieciseis.jpg" alt="dieciseis" width="150" height="204" class="right"></p><br />
<p>&nbsp;</p><br />
<p>Paola Zárate-Segura PhD Researcher, at IPN. Dra. Zárate Segura has a PhD in Bioprocess from Interdisciplinary Unit for Biotechnology, National Polytechnic Institute. </p><br />
<p>&nbsp;</p><br />
</div><br />
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<h2>About us </h2><br />
<ul><br />
<li><a href="Members.htm" target="_parent">Members</a></li><br />
<li><a href="Institute.htm">Institute</a></li><br />
<li><a href="Gallery.htm">Gallery</a></li><br />
<li><a href="Acknowledgments.htm">Acknowledgments</a></li><br />
</ul><br />
<br />
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</div> <br />
<div style="clear: both;">&nbsp;</div><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Members.htm
Team/CINVESTAV-IPN-UNAM MX/Members.htm
2012-10-27T01:00:21Z
<p>Ao.patricia: </p>
<hr />
<div><html xmlns="http://www.w3.org/1999/xhtml"><br />
<head><br />
<meta http-equiv="content-type" content="text/html; charset=utf-8" /><br />
<title>Rho</title><br />
<br />
<style type="text/css"><br />
<br />
#top-section {<br />
position: relative;<br />
height: 20px;<br />
width: 975px;<br />
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border-bottom: hidden;<br />
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<h2><span></span></h2><br />
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<div id="context"> <br />
<br />
<h1><em>Members!</em></h1><br />
<p id="text2"> Students</p></br><br />
<img src="https://static.igem.org/mediawiki/2012/9/95/Diez.jpg" alt="diez" width="172" height="235" class="left"><br />
<br><br />
<br><br />
<br><br />
<p>Lissania Guerra-Calderas Contribution: Butanol Production System and BioBrick Designer, outreach activities. 4th year undergraduate of Pharmaceutical and Biological Chemistry, at Faculty of Chemistry. National Autonomous University of Mexico (UNAM) lissxgc.89@gmail.com.</p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/9/98/Ocho.jpg" width="172" height="235" class="right"></p><br />
<br><br />
<br />
<br />
<p>Jhonatan Alejandro Hernández-Valdés Contribution: Project Management, design and development of regulatory systems, BioBrick Designer, outreach activities. 4th year undergraduate of Pharmaceutical and Biological Chemistry, at Faculty of Chemistry. National Autonomous University of Mexico (UNAM) jhonatanhv@hotmail.com.</p><br />
<p>&nbsp;</p><br />
<p><br><br />
<img src="https://static.igem.org/mediawiki/2012/8/84/Once.jpg" alt="once" width="164" height="211" class="left"><br><br />
Maritere Uriostegui-Arcos Contribution: Characterization and measurement protocols. BioBrick Designer, outreach activities. 4th year undergraduate of Pharmaceutical and Biological Chemistry, at Faculty of Chemistry. National Autonomous University of Mexico (UNAM) maritere1011@hotmail.com.<br><br />
</p><br />
<p><br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/f/f6/Nueve.jpg" alt="nueve" width="163" height="237" class="right"><br><br />
</p><br />
<p>&nbsp;</p><br />
<p>Anna Karen Hernández-Gallardo Contribution: Mathematical modeling, 4th year undergraduate Biotechnological Engineering, Interdisciplinary Unit for Biotechnology<br />
National Polytechnic Institute<br />
.</p><br />
<p>&nbsp;</p><br />
<p><br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/1/16/Tre.jpg" alt="tre" width="171" height="246" class="left"></p><br />
<p>Patricia Arias-Orozco Contribution: Design and development of regulatory systems, BioBrick Designer, outreach activities. 4th year undergraduate Biotechnological Engineering, Interdisciplinary Unit for Biotechnology National Polytechnic Institute. </p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/4/45/Uno.jpg" alt="uno" width="174" height="243" class="right"></p><br />
<p>&nbsp;</p><br />
<p>Daniel Domínguez-Gómez <br><br />
<p>Contribution: Outreach activities.<br> <br />
4th year undergraduate Biotechnological Engineering, Interdisciplinary Unit for Biotechnology, National Polytechnic Institute.<br><br />
<p></p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/4/43/Siete.jpg" alt="siete" width="167" height="234" class="left"></p><br />
<p>&nbsp;</p><br />
<p>Ivan Lopez-Flores Mathematical Team: Mathematical modeling Computational Systems Engineering, Superior School for Computation National Polytechnic Institute National Polytechnic Institute <br><br />
<br><br />
<br><br />
<br><br />
<br />
</p><br />
<br />
<h1>Advisors</h1><br />
<p>&nbsp;</p><br />
<img src="https://static.igem.org/mediawiki/2012/6/67/Dos.jpg" alt="dos" width="194" height="270" class="left"><br />
<p>Daniel Federico Hernandez Gardiol Contribution: Mathematical model Systems Biologist: Universidad de la Republica (Montevideo, Uruguay) Bioengineering Masters Student: Ecole Polytechnique Federal de Lausanne (Lausanne, Switzerland) National Polytechnic Institute.</p><br />
<p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2012/5/59/Catorce.jpg" alt="catorce" width="168" height="244" class="left"><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>Karen Nava Castro PhD Contribution: Flow Cytometry Expert. Researcher at Institute of Biomedical Research, Field of study: Immunology. National Autonomous University of Mexico, UNAM. </p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/c/c8/Quince.jpg" alt="quince" width="161" height="221" class="right"></p><br />
<p>&nbsp;</p><br />
<p>Jorge Morales Montor PhD Contribution: Dry lab support Researcher at Institute of Biomedical Research (IIB, UNAM) Field of study: Neuoimmunoendocrinology National Autonomous University of Mexico, UNAM. </p><br />
<p>&nbsp;</p><img src="https://static.igem.org/mediawiki/2012/0/0d/Cinco1.jpg" alt="cinco" width="173" height="205" class="right"><p>&nbsp;</p><br />
<p>Fernando G. Bastida-González Contribution: Wet lab support, Master in Health science at Superior Medicine School at National Polytechnic Institute (IPN).</p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<h1>Instructors</h1><br><br />
<br />
<p><img src="https://static.igem.org/mediawiki/2012/e/e9/Tres.jpg" alt="tres" width="166" height="227" class="left"></p><br />
<p>&nbsp;</p><br />
<p>Agustino Martínez-Antonio PhD Resarcher 3A, SNI I Genetic Engineering Department, CINVESTAV Irapuato.</p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><br><br />
<img src="https://static.igem.org/mediawiki/2012/6/67/Seis.jpg" alt="seis" width="161" height="209" class="right"></p><br />
<p>&nbsp;</p><br />
<p>Fernando Suaste-Olmos PhD Researcher at Institute of Cell Physiology IFC-UNAM.<br />
Expert in Photosynthetic bacteria. </p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/9/98/Doce.jpg" alt="dice" width="173" height="205" class="left"></p><br />
<p>&nbsp;</p><br />
<p>Nuria Sánchez-Puig Researcher at Institute of Chemistry UNAM Dr. Sanchez-Puig has a PhD from the Center for Protein Engineering, Cambridge, UK. </p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/a/ad/Dieciseis.jpg" alt="dieciseis" width="150" height="204" class="right"></p><br />
<p>&nbsp;</p><br />
<p>Paola Zárate-Segura PhD Researcher, at IPN. Dra. Zárate Segura has a PhD in Bioprocess from Interdisciplinary Unit for Biotechnology, National Polytechnic Institute. </p><br />
<p>&nbsp;</p><br />
</div><br />
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<ul><br />
<li><a href="Members.htm" target="_parent">Members</a></li><br />
<li><a href="Institute.htm">Institute</a></li><br />
<li><a href="Gallery.htm">Gallery</a></li><br />
<li><a href="Acknowledgments.htm">Acknowledgments</a></li><br />
</ul><br />
<br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/oxigenresponse.htm
Team/CINVESTAV-IPN-UNAM MX/oxigenresponse.htm
2012-10-27T00:03:45Z
<p>Ao.patricia: </p>
<hr />
<div><html xmlns="http://www.w3.org/1999/xhtml"><br />
<head><br />
<meta http-equiv="content-type" content="text/html; charset=utf-8" /><br />
<title>Rho</title><br />
<br />
<style type="text/css"><br />
<br />
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position: relative;<br />
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<br />
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<br />
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margin: 0 auto;<br />
padding: 20px 0;<br />
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padding-top:5px;<br />
} <br />
/* menudecontenido */<br />
<br />
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<div id="page"><br />
<div id="context"> <br />
<br />
<h1><em>Oxygen Control System!</em></h1><br />
<p id="text2"> PrrA/PrrB two component system</p><br />
<p>This system remains inactive under high oxygen tension, when oxygen<br />
concentration decreases, it is possible the GFP transcription. (See Rhodofactory section for<br />
a complete explanation).<br><br />
<br />
We made two BioBricks (BBa_K776019 y BBa_K776021) to test the Oxygen<br />
Control System, each one has GFP as a reporter gene and the functionality was related to<br />
the fluorescence detection.</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c5/Osy01.jpg"><br />
<p>Figure 1. This BioBrick will show if our dependent promoter is functional, using the<br />
constitutive (or natural) system from <em>R. sphaeroides</em> or the orthologue system from <em>R.<br />
palustris.</em></p><br />
</div><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2012/0/06/Osy02.jpg" width="562" height="192"><br />
<p>Figure 2. This BioBrick will show if our complete system is functional because probably<br />
we need a synthetic system to promote GFP expression by binding its target sequence<br />
(dependent promoter) in <em>R. palustris.</em></p><br />
</div><br />
<p>Both systems were cloned in pRK415 because this is a vector for Purple Non-Sulfur<br />
Photosynthetic Bacteria, the plasmids were introduced in <em>R. sphaeroides</em> and <em>R. palustris</em>,<br />
by biparental and triparental conjugation.</p><br />
<p>The measurement approach we used was:<br />
<li>Fluorescence Microscopy: To have a qualitative detection of GFP in these bacteria.</li><br />
<li>Flow Cytometry: To have a quantitative detection of GFP expression, we calculated the<br />
percentage of bacterial population expressing GFP (GFP+) in 1000 bacteria.</li><br><br />
</p><br />
<p>We used 3 environmental growing conditions:<br />
<li>Aerobic/Darkness</li><br />
<li>Anaerobic/Light</li><br />
<li>Anaeroibic/darkness</li></p><br />
<p>For all data results, we considered a negative control: <em>Rhodobacter sphaeroides</em> or <em>Rhodopseudomonas palustris</em>, conjugated bacteria with pRK415 vector without BioBrick.</p> <br />
<div align="center"><img src="https://static.igem.org/mediawiki/2012/7/73/Osy03.jpg" width="563" height="452"><br />
<img src="https://static.igem.org/mediawiki/2012/e/ec/Osy04.jpg" width="563" height="452"><br />
<p>Figure 3. Percentage of bacterial population expressing GFP.<br><br />
</p><br />
</div><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/6/6b/Osy05.jpg" width="556" height="384"><br />
<p>Figure 4. Representative images obtained by fluorescence microscopy, where our systems<br />
were functional in the expected conditions.</p><br />
</div><br />
<p id="text2">Discussion</p><br />
<p>In <em>R. sphaeroides</em>, as we can see in figure 3 and 4, there was low GFP expression, probably<br />
because growing conditions were microaerophilic instead of extrictly anaerobic. In<br />
anaerobic conditions PrrB autophosphorylates and passes a phosphate group to PrrA, this<br />
activated PrrA binds its promoter sequence to start GFP expression. Furthermore, when<br />
we introduced the complete system (BBa_K776021), actually we are overexpressing the<br />
regulatory proteins and the signaling could not be fully controlled.<br><br />
<br><br />
<br />
In <em>R. palustris</em>, we had GFP expression in PrrA dependent promoter (BBa_K776019), maybe<br />
because orthologous proteins activated it. The complete system (BBa_K776021) also<br />
was functional but in a lower level, assumably due to the interference of other proteins that regulate photosynthetic genes.<br><br />
<br> <br />
The GFP expression that we did not expected was in aerobic condition in the complete system, probably<br />
is due to the complexity of the regulatory network where this system is involved.<br />
</p><br />
<p id="text2">Conclusion</p><br />
<p> Our two BioBricks (K776019 and BBa_K776021) are functional in two photosynthetic<br />
bacteria <em>R. palustris</em> and <em>R. sphaeroides</em>, both in anaerobic/light expected condition. This is a functional system for controlling genetic<br />
expression with Oxygen tension.</p><br />
<br />
</div><br />
<br />
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<h2>Results</h2><br />
<ul><br />
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<li><a href="Lightandoxre.htm">Light and oxygen response</a></li><br />
<li><a href="Notebook.htm">Notebook</a></li><br />
<li><a href="oxigenresponse.htm">Oxygen response</a></li><br />
<li><a href="Protocol.htm">Protocols</a></li><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/oxigenresponse.htm
Team/CINVESTAV-IPN-UNAM MX/oxigenresponse.htm
2012-10-27T00:03:31Z
<p>Ao.patricia: </p>
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<br />
<h1><em>Oxygen Control System!</em></h1><br />
<p id="text2"> PrrA/PrrB two component system</p><br />
<p>This system remains inactive under high oxygen tension, when oxygen<br />
concentration decreases, it is possible the GFP transcription. (See Rhodofactory section for<br />
a complete explanation).<br><br />
<br />
We made two BioBricks (BBa_K776019 y BBa_K776021) to test the Oxygen<br />
Control System, each one has GFP as a reporter gene and the functionality was related to<br />
the fluorescence detection.</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c5/Osy01.jpg"><br />
<p>Figure 1. This BioBrick will show if our dependent promoter is functional, using the<br />
constitutive (or natural) system from <em>R. sphaeroides</em> or the orthologue system from <em>R.<br />
palustris.</em></p><br />
</div><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2012/0/06/Osy02.jpg" width="562" height="192"><br />
<p>Figure 2. This BioBrick will show if our complete system is functional because probably<br />
we need a synthetic system to promote GFP expression by binding its target sequence<br />
(dependent promoter) in <em>R. palustris.</em></p><br />
</div><br />
<p>Both systems were cloned in pRK415 because this is a vector for Purple Non-Sulfur<br />
Photosynthetic Bacteria, the plasmids were introduced in <em>R. sphaeroides</em> and <em>R. palustris</em>,<br />
by biparental and triparental conjugation.</p><br />
<p>The measurement approach we used was:<br />
<li>Fluorescence Microscopy: To have a qualitative detection of GFP in these bacteria.</li><br />
<li>Flow Cytometry: To have a quantitative detection of GFP expression, we calculated the<br />
percentage of bacterial population expressing GFP (GFP+) in 1000 bacteria.</li><br><br />
</p><br />
<p>We used 3 environmental growing conditions:<br />
<li>Aerobic/Darkness</li><br />
<li>Anaerobic/Light</li><br />
<li>Anaeroibic/darkness</li></p><br />
<p>For all data results, we considered a negative control: <em>Rhodobacter sphaeroides</em> or <em>Rhodopseudomonas palustris</em>, conjugated bacteria with pRK415 vector without BioBrick.</p> <br />
<div align="center"><img src="https://static.igem.org/mediawiki/2012/7/73/Osy03.jpg" width="563" height="452"><br />
<img src="https://static.igem.org/mediawiki/2012/e/ec/Osy04.jpg" width="563" height="452"><br />
<p>Figure 3. Percentage of bacterial population expressing GFP.<br><br />
</p><br />
</div><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/6/6b/Osy05.jpg" width="556" height="384"><br />
<p>Figure 4. Representative images obtained by fluorescence microscopy, where our systems<br />
were functional in the expected conditions.</p><br />
</div><br />
<p id="text2">Discussion</p><br />
<p>In <em>R. sphaeroides</em>, as we can see in figure 3 and 4, there was low GFP expression, probably<br />
because growing conditions were microaerophilic instead of extrictly anaerobic. In<br />
anaerobic conditions PrrB autophosphorylates and passes a phosphate group to PrrA, this<br />
activated PrrA binds its promoter sequence to start GFP expression. Furthermore, when<br />
we introduced the complete system (BBa_K776021), actually we are overexpressing the<br />
regulatory proteins and the signaling could not be fully controlled.<br><br />
<br><br />
<br />
In <em>R. palustris</em>, we had GFP expression in PrraA dependent promoter (BBa_K776019), maybe<br />
because orthologous proteins activated it. The complete system (BBa_K776021) also<br />
was functional but in a lower level, assumably due to the interference of other proteins that regulate photosynthetic genes.<br><br />
<br> <br />
The GFP expression that we did not expected was in aerobic condition in the complete system, probably<br />
is due to the complexity of the regulatory network where this system is involved.<br />
</p><br />
<p id="text2">Conclusion</p><br />
<p> Our two BioBricks (K776019 and BBa_K776021) are functional in two photosynthetic<br />
bacteria <em>R. palustris</em> and <em>R. sphaeroides</em>, both in anaerobic/light expected condition. This is a functional system for controlling genetic<br />
expression with Oxygen tension.</p><br />
<br />
</div><br />
<br />
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<h2>Results</h2><br />
<ul><br />
<li><a href="Biobricks.htm" target="_parent">Biobricks</a></li><br />
<li><a href="Lightandoxre.htm">Light and oxygen response</a></li><br />
<li><a href="Notebook.htm">Notebook</a></li><br />
<li><a href="oxigenresponse.htm">Oxygen response</a></li><br />
<li><a href="Protocol.htm">Protocols</a></li><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/oxigenresponse.htm
Team/CINVESTAV-IPN-UNAM MX/oxigenresponse.htm
2012-10-26T23:58:34Z
<p>Ao.patricia: </p>
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<br />
<h1><em>Oxygen Control System!</em></h1><br />
<p id="text2"> PrrA/PrrB two component system</p><br />
<p>This system remains inactive under high oxygen tension, when oxygen<br />
concentration decreases, it is possible the GFP transcription. (See Rhodofactory section for<br />
a complete explanation).<br><br />
<br />
We made two BioBricks (BBa_K776019 y BBa_K776021) to test the Oxygen<br />
Control System, each one has GFP as a reporter gene and the functionality was related to<br />
the fluorescence detection.</p><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/c/c5/Osy01.jpg"><br />
<p>Figure 1. This BioBrick will show if our dependent promoter is functional, using the<br />
constitutive (or natural) system from <em>R. sphaeroides</em> or the orthologue system from <em>R.<br />
palustris.</em></p><br />
</div><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2012/0/06/Osy02.jpg" width="562" height="192"><br />
<p>Figure 2. This BioBrick will show if our complete system is functional because probably<br />
we need a synthetic system to promote GFP expression by binding its target sequence<br />
(dependent promoter) in <em>R. palustris.</em></p><br />
</div><br />
<p>Both systems were cloned in pRK415 because this is a vector for Purple Non-Sulfur<br />
Photosynthetic Bacteria, the plasmids were introduced in <em>R. sphaeroides</em> and <em>R. palustris</em>,<br />
by biparental and triparental conjugation.</p><br />
<p>The measurement approach we used was:<br />
<li>Fluorescence Microscopy: To have a qualitative detection of GFP in these bacteria.</li><br />
<li>Flow Cytometry: To have a quantitative detection of GFP expression, we calculated the<br />
percentage of bacterial population expressing GFP (GFP+) in 1000 bacteria.</li><br><br />
</p><br />
<p>We used 3 environmental growing conditions:<br />
<li>Aerobic/Darkness</li><br />
<li>Anaerobic/Light</li><br />
<li>Anaeroibic/darkness</li></p><br />
<p>For all data results, we considered a negative control: <em>Rhodobacter sphaeroides</em> or <em>Rhodopseudomonas palustris</em>, conjugated bacteria with pRK415 vector without BioBrick.</p> <br />
<div align="center"><img src="https://static.igem.org/mediawiki/2012/7/73/Osy03.jpg" width="563" height="452"><br />
<img src="https://static.igem.org/mediawiki/2012/e/ec/Osy04.jpg" width="563" height="452"><br />
<p>Figure 3. Percentage of bacterial population expressing GFP.<br><br />
</p><br />
</div><br />
<div align="center"><br />
<img src="https://static.igem.org/mediawiki/2012/6/6b/Osy05.jpg" width="556" height="384"><br />
<p>Figure 4. Representative images obtained by fluorescence microscopy, where our systems<br />
were functional in the expected conditions.</p><br />
</div><br />
<p id="text2">Discussion</p><br />
<p>In <em>R. sphaeroides</em>, as we can see in figure 3 and 4, there was low GFP expression, probably<br />
because growing conditions were microaerophilic instead of extrictly anaerobic. In<br />
anaerobic conditions PrrB autophosphorylates and passes a phosphate group to PrrA, this<br />
activated PrrA binds its promoter sequence to start GFP expression. Furthermore, when<br />
we introduced the complete system (BBa_K776021), actually we are overexpressing the<br />
regulatory proteins and the signaling could not be fully controlled.<br><br />
<br><br />
<br />
In <em>R. palustris</em>, we had GFP expression in PrraA dependent promoter (BBa_K776019), maybe<br />
because orthologous proteins activated it. The complete system (BBa_K776021) also<br />
was functional but in a lower level, assumably due to the interference of other proteins that regulate photosynthetic genes.<br><br />
<br> <br />
The GFP expression that we did not expected was in aerobic condition in the complete system, probably<br />
is due to the complexity of the regulatory network where this system is involved and we have to we have to consider that our system is synthetic and the behavior can be different than expected.<br />
</p><br />
<p id="text2">Conclusion</p><br />
<p> Our two BioBricks (K776019 and BBa_K776021) are functional in two photosynthetic<br />
bacteria <em>R. palustris</em> and <em>R. sphaeroides</em>. This is a functional system for controlling genetic<br />
expression with Oxygen tension.</p><br />
<br />
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<h2>Results</h2><br />
<ul><br />
<li><a href="Biobricks.htm" target="_parent">Biobricks</a></li><br />
<li><a href="Lightandoxre.htm">Light and oxygen response</a></li><br />
<li><a href="Notebook.htm">Notebook</a></li><br />
<li><a href="oxigenresponse.htm">Oxygen response</a></li><br />
<li><a href="Protocol.htm">Protocols</a></li><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Lightandoxre.htm
Team/CINVESTAV-IPN-UNAM MX/Lightandoxre.htm
2012-10-26T22:59:56Z
<p>Ao.patricia: </p>
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<h1>Light and oxygen response!</h1><br />
<p id="text2">AppA/PpsR Regulation System</p><br />
<p>In this repressor/antirepressor system, under low oxygen tension, the GFP<br />
expression is possible because the repressor PpsR is forming a complex with the<br />
antirepresssor protein AppA. Moreover, when blue light fall upon the complex, a<br />
conformational change in AppA breaks the complex, and AppA avoid GFP expression. (See<br />
Rhodofactory section for a complete explanation).<br><br><br />
<br />
<br />
We made two BioBricks (BBa_K776018 y BBa_K776020) to test the Light &<br />
Oxygen Control System, each one has GFP as a reporter gene and the functionality was<br />
related to the fluorescence detection.</p><br />
<img src="https://static.igem.org/mediawiki/2012/4/40/Oxy01.jpg" alt="oxy01" width="561" height="241"><br />
<p>Figure 1. This BioBrick will show if our dependent promoter is functional, using the<br />
constitutive (or natural) system from <em>R. sphaeroides</em> or the orthologue system from <em>R.palustris.</em></p><br />
<img src="https://static.igem.org/mediawiki/2012/1/1c/Oxy02.jpg" width="563" height="183"><br />
<p>Figure 2. This BioBrick will show if our complete system is functional because probably<br />
we need a synthetic system to promote GFP expression by binding its target sequence<br />
(dependent promoter) in <em>R. palustris.</em></p><br />
<p>Both systems were cloned in pRK415 because this is a vector for Purple Non-Sulfur<br />
Photosynthetic Bacteria, the plasmids were introduced in <em>R. sphaeroides</em> and <em>R.palustris</em>,<br />
by biparental and triparental conjugation.</p><br />
<p>The measurement approach was:<br />
<li>Fluorescence Microscopy: To have a qualitative GFP detection in these bacteria.</li><br />
<li>Flow Cytometry: To have a quantitative GFP detection, we calculated the percentage of<br />
bacterial population expressing GFP (GFP+) in 1000 bacteria.</li></p><br><br />
<br />
<p>We used 3 environmental growing conditions:<br />
<li>Aerobic/Darkness</li><br />
<li>Anaerobic/Light</li><br />
<li>Anaeroibic/darkness</li><br />
</p><br><br />
<br />
<p>For all data results, we considered a negative control: <em>R. sphaeroides</em> and<br />
<em>R.palustris</em>, conjugated bacteria with pRK415 vector without BioBrick.</p><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2012/f/f0/Oxygenres01.jpg" width="563" height="452"><br />
<img src="https://static.igem.org/mediawiki/2012/c/cc/Oxygenres02.jpg" width="563" height="452"><br />
<p>Figure 3. Percentage of bacterial population expressing GFP..<br><br />
</p><br />
<img src="https://static.igem.org/mediawiki/2012/9/9b/Oxy03.jpg" width="561" height="465"></div><br />
<div align="center"><br />
</div><br />
<p>Figure 4. Images obtained by fluorescence microscopy, where our systems<br />
were functional in the expected conditions.</p><br />
<p id="text2">Discussion</p><br />
<p>In <em>R. sphaeroides</em>, the best functionality of our system was in aerobic and darknesss<br />
condition, it was not the expected result, but probably the activation is because to the<br />
incomplete repression of the system because we were overexpressing the constitutive<br />
proteins. Altought, we obtained GFP expression with a lower level, in the expected<br />
condition, both BioBricks (BBa_K776018 y BBa_K776020) were functional.<br><br />
<br><br />
<br />
In <em>R.palustris</em>, our best condition was in anaerobic and light condition, expected result. The low level of GFP in BioBrick BBa_K77608 it could be due to low affinity of orthologous proteins to our promoter sequence, but when we introduced the complete system BBa_K776020 GFP expression increases showing the funcionality of our system. </p><br />
<br />
<p id="text2">Conclusion</p><br />
<p>Both BioBricks (K776018 and BBa_K776020) are functional in two photosynthetic<br />
bacteria <em>R.palustris</em> and <em>R. sphaeroides.</em> The best condicion was aerobic/darknes and anaerobic light respectively.<br />
<br />
<br />
This is a functional system for controlling genetic expression with Light and Oxygen signals.</p><br />
<br />
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<li><a href="Notebook.htm">Notebook</a></li><br />
<li><a href="oxigenresponse.htm">Oxygen response</a></li><br />
<li><a href="Protocol.htm">Protocols</a></li><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Lightandoxre.htm
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2012-10-26T22:50:47Z
<p>Ao.patricia: </p>
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<h1>Light and oxygen response!</h1><br />
<p id="text2">AppA/PpsR Regulation System</p><br />
<p>In this repressor/antirepressor system, under low oxygen tension, the GFP<br />
expression is possible because the repressor PpsR is forming a complex with the<br />
antirepresssor protein AppA. Moreover, when blue light fall upon the complex, a<br />
conformational change in AppA breaks the complex, and AppA avoid GFP expression. (See<br />
Rhodofactory section for a complete explanation).<br><br><br />
<br />
<br />
We made two BioBricks (BBa_K776018 y BBa_K776020) to test the Light &<br />
Oxygen Control System, each one has GFP as a reporter gene and the functionality was<br />
related to the fluorescence detection.</p><br />
<img src="https://static.igem.org/mediawiki/2012/4/40/Oxy01.jpg" alt="oxy01" width="561" height="241"><br />
<p>Figure 1. This BioBrick will show if our dependent promoter is functional, using the<br />
constitutive (or natural) system from <em>R. sphaeroides</em> or the orthologue system from <em>R.palustris.</em></p><br />
<img src="https://static.igem.org/mediawiki/2012/1/1c/Oxy02.jpg" width="563" height="183"><br />
<p>Figure 2. This BioBrick will show if our complete system is functional because probably<br />
we need a synthetic system to promote GFP expression by binding its target sequence<br />
(dependent promoter) in <em>R. palustris.</em></p><br />
<p>Both systems were cloned in pRK415 because this is a vector for Purple Non-Sulfur<br />
Photosynthetic Bacteria, the plasmids were introduced in <em>R. sphaeroides</em> and <em>R.palustris</em>,<br />
by biparental and triparental conjugation.</p><br />
<p>The measurement approach was:<br />
<li>Fluorescence Microscopy: To have a qualitative GFP detection in these bacteria.</li><br />
<li>Flow Cytometry: To have a quantitative GFP detection, we calculated the percentage of<br />
bacterial population expressing GFP (GFP+) in 1000 bacteria.</li></p><br><br />
<br />
<p>We used 3 environmental growing conditions:<br />
<li>Aerobic/Darkness</li><br />
<li>Anaerobic/Light</li><br />
<li>Anaeroibic/darkness</li><br />
</p><br><br />
<br />
<p>For all data results, we considered a negative control: <em>R. sphaeroides</em> and<br />
<em>R.palustris</em>, conjugated bacteria with pRK415 vector without BioBrick.</p><br />
<div align="center"><img src="https://static.igem.org/mediawiki/2012/f/f0/Oxygenres01.jpg" width="563" height="452"><br />
<img src="https://static.igem.org/mediawiki/2012/c/cc/Oxygenres02.jpg" width="563" height="452"><br />
<p>Figure 3. Percentage of bacterial population expressing GFP..<br><br />
</p><br />
<img src="https://static.igem.org/mediawiki/2012/9/9b/Oxy03.jpg" width="561" height="465"></div><br />
<div align="center"><br />
</div><br />
<p>Figure 4. Images obtained by fluorescence microscopy, where our systems<br />
were functional in the expected conditions.</p><br />
<p id="text2">Discussion</p><br />
<p>In <em>R. sphaeroides</em>, the best functionality of our system was in aerobic and darknesss<br />
condition, it was not the expected result, but probably the activation is because to the<br />
incomplete repression of the system because we were overexpressing the constitutive<br />
proteins. Altought, we obtained GFP expression with a lower level, in the expected<br />
condition, both BioBricks (BBa_K776018 y BBa_K776020) were functional.<br><br />
<br><br />
<br />
In <em>R.palustris</em>, our best condition was in anaerobic and light condition, expected result, the low level of GFP in BioBrick BBa_K77608 it could be due to orthologous proteins have a low affinity to our promoter sequence, but when we introduced the complete system BBa_K776020 GFP expression increases showing the funcionality of our system. </p><br />
<br />
<p id="text2">Conclusion</p><br />
<p>Both BioBricks (K776018 and BBa_K776020) are functional in two photosynthetic<br />
bacteria <em>R.palustris</em> and <em>R. sphaeroides.</em> The best condicion was aerobic/darknes and anaerobic light respectively.<br />
<br />
<br />
This is a functional system for controlling genetic expression with Light and Oxygen signals.</p><br />
<br />
</div><br />
<br />
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<br />
<h2>Results</h2><br />
<ul><br />
<li><a href="Biobricks.htm" target="_parent">Biobricks</a></li><br />
<li><a href="Lightandoxre.htm">Light and oxygen response</a></li><br />
<li><a href="Notebook.htm">Notebook</a></li><br />
<li><a href="oxigenresponse.htm">Oxygen response</a></li><br />
<li><a href="Protocol.htm">Protocols</a></li><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Oxigen_Response.htm
Team/CINVESTAV-IPN-UNAM MX/Oxigen Response.htm
2012-10-26T21:59:23Z
<p>Ao.patricia: </p>
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<h1><em>Oxygen Control System: PrrA/PrrB two component regulation system! </em></h1><br />
<p>This regulatory system is able to sense oxygen concentration and send a response, under high oxygen tension, the system remains inactive, when oxygen concentration decreases PrrB (Histidine sensor kinase) turns active through an autophosphorylation with help of PrrC, which receive the signal from electron transport chain. Then PrrB transfer a phosphate group to PrrA that directly binds promoter and recruits RNA polymerase to start GFP transcription.<br />
<br />
<br />
(See the next video for a visual explanation).</p><br />
<div align="center"><iframe width="480" height="360" src="http://www.youtube.com/embed/pgp6DzpNyA0" frameborder="0" allowfullscreen></iframe></div><br><br />
<p id="text2">Our biobricks </p><br />
<p>The first biobrick consists in the complete oxygen dependent system, PrrA, PrrB and PrrC, each one with a Ribosome Binding Site, under a<br />
Medium strength promoter (J23104), this first biobrick also it has the PrrA dependent promoter and GFP as a reporter gene</p><br />
<img src="https://static.igem.org/mediawiki/2012/4/45/Rodo04.jpg" alt="rodo04" width="560" height="116"><br><br />
<p><br><br />
The second biobrick is just the PrrA dependent promoter and GFP as a reporter gene.</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2012/1/16/Rodo05.jpg" alt="rodo05" width="450" height="249"></p><br />
<p id="text2">We were inspired in:</p><br />
<p>This system is inspired in PrrBCA two component system from <em>R. sphaeroides</em>, which is a master regulator involved in expression of<br />
approximately 850 genes, >20% of the genome (Kaplan & Eraso 2005) This system coordinately controls genes involved in the complex switch<br />
between aerobic and anaerobic conditions and the optimum use of reducing power. It also regulates gene expression involved in<br />
photosynthesis, carbon dioxide fixation, nitrogen fixation, hydrogen uptake, aerotaxis, denitrification, electron transport, aerobic and anaerobic<br />
respiration, and heme biosynthesis, and others. Thus emphasizing its global role (Elsen et.al 2004, Kaplan & Eraso 2005, Zeilstra-Ryalls &<br />
Kaplan 2004).</p><br />
<hr><br />
<p id="refe">References<br><br />
1.<br />
Kaplan S, Eraso J, Roh JH. (2005). <strong>Interacting regulatory networks in the facultative<br />
photosynthetic bacterium, <em>Rhodobacter sphaeroides</em> 2.4.1.</strong> Biochem. Soc. Trans. 33:51–55<br><br />
<br />
2.<br />
Zeilstra-Ryalls JH, Kaplan S. (2004). <strong>Oxygen intervention in the regulation of gene xpression: the photosynthetic<br />
bacterial paradigm.</strong> Cell. Mol. Life Sci. 61:417–36<br><br />
<br />
3.<br />
Eraso JM, Kaplan S (2009) <strong>Regulation of gene expression by PrrA in <em>Rhodobacter sphaeroides</em> 2.4.1: role of<br />
polyamines and DNA topology.</strong> J Bacteriol 2009, 191(13):4341-4352.<br />
</p><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Light_Response.htm
Team/CINVESTAV-IPN-UNAM MX/Light Response.htm
2012-10-26T21:55:11Z
<p>Ao.patricia: </p>
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<h1><em>Light & Oxygen Response: AppA/PpsR Regulation System! </em></h1><br />
<p>This is a repressor/antirepressor system, which under high oxygen tension; PpsR represses GFP expression by avoiding RNA polymerase binding <br />
the promoter sequence.<br><br />
<br />
When oxygen concentration decreases AppA has a conformational change and can bind with PpsR, this complex prevents the union of<br />
PpsR to its target sequence, thus GFP expression can begin.<br><br />
<br />
(See the next video for a visual explanation).</p><br />
<div align="center"><iframe width="480" height="360" src="http://www.youtube.com/embed/XBNRLq9pL8c" frameborder="0" allowfullscreen></iframe></div><br><br />
<p id="text2">Our biobricks </p><br />
<p>The first biobrick consists in the complete light-oxygen dependent system, AppA and PpsR, each one with Ribosome Binding Site, under a<br />
Medium strength promoter (J23104), this first biobrick also it has the PpsR dependent promoter and GFP as a reporter gene.</p><br />
<img src="https://static.igem.org/mediawiki/2012/b/b6/Rodo02.jpg" alt="rodo02" width="568" height="107"><br><br />
<p><br><br />
The second circuit is just the PpsR dependent promoter and GFP as a reporter gene.</p><br />
<img src="https://static.igem.org/mediawiki/2012/a/a1/Rodo03.jpg" alt="rodo03" width="562" height="252"><br />
<p id="text2">We were inspired in:</p><br />
<p><br />
This system is inspired in AppA/PpsR repressor/antirepressor system from Rhodobacter sphaeroides. The PpsR protein is a master repressor of<br />
Photosynthesis (PS) genes (Moskvin and Gomelsky 2005). Inactivation of the ppsR gene is enough to turn on PS gene expression and formation<br />
of the photosynthetic apparatus even at a high oxygen concentration, whereas ppsR overexpression is sufficient to block PS development even<br />
in the absence of oxygen. PpsR directly represses transcription of most carotenoid and pigment synthesis genes, photosystems operons, and<br />
genes involved in tetrapyrrole biosynthesis (Gomelsky and Kaplan 1995). The upstream regions of these genes contain two PpsR binding sites,<br />
TGTcN10gACA.<br><br />
<br />
A second protein called AppA, which has no known homologues, plays a role in controlling gene expression in <em>R. sphaeroides</em> in response to both<br />
light and O2 by acting as an antirepressor of PpsR. Our parts (appa, ppsr and ppsr-promoter) were synthesized by Genescript, and are codon<br />
optimized for <em>R. sphaeroides.</em></p><br />
<hr><br />
<p id="refe">References<br><br />
<br />
1. Gomelsky L., Moskvin L., Stenzel A., Jones D., Donohue T. and Gomelsky M.(2008) <strong>Hierarchical Regulation of Photosynthesis<br />
Gene Expression by the Oxygen-Responsive PrrBA and AppA-PpsR Systems of <em>Rhodobacter sphaeroides.</em></strong> J. Bacteriol.<br />
Dec. 2008, p. 8106–8114 Vol. 190, No. 24<br><br />
<br />
2. Moskvin, O. V., L. Gomelsky, and M. Gomelsky. (2005). <strong>Transcriptome analysis of the <em>Rhodobacter sphaeroides</em> PpsR<br />
regulon: PpsR as a master regulator of photosystem development.</strong> J. Bacteriol. 187:2148–2156.<br><br />
<br />
3. Gomelsky, M., and S. Kaplan. (1995). <strong>Genetic evidence that PpsR from <em>Rhodobacter sphaeroides.</em> 2.4.1 functions as a<br />
repressor of puc and bchF expression.</strong> J. Bacteriol. 177:1634–1637. <br><br />
4. Gomelsky, M., and S. Kaplan. (1995). <strong>AppA, a novel gene encoding a transacting factor involved in the regulation of<br />
photosynthesis gene expression in <em>Rhodobacter sphaeroides.</em> 2.4.1.</strong> J. Bacteriol. 177:4609–4618.<br />
</p><br />
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Ao.patricia
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Team/CINVESTAV-IPN-UNAM MX/Members.htm
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<p>Ao.patricia: </p>
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<h1><em>Members!</em></h1><br />
<p id="text2"> Students</p></br><br />
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<br><br />
<br><br />
<br><br />
<p>Lissania Guerra-Calderas Contribution: Butanol Production System and BioBrick Designer, outreach activities. 4th year undergraduate of Pharmaceutical and Biological Chemistry, at Faculty of Chemistry. National Autonomous University of Mexico (UNAM) lissxgc.89@gmail.com.</p><br />
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<br />
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<p>Jhonatan Alejandro Hernández-Valdés Contribution: Project Management, design and development of regulatory systems, BioBrick Designer, outreach activities. 4th year undergraduate of Pharmaceutical and Biological Chemistry, at Faculty of Chemistry. National Autonomous University of Mexico (UNAM) jhonatanhv@hotmail.com.</p><br />
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<img src="https://static.igem.org/mediawiki/2012/8/84/Once.jpg" alt="once" width="164" height="211" class="left"><br><br />
Maritere Uriostegui-Arcos Contribution: Characterization and measurement protocols. BioBrick Designer, outreach activities. 4th year undergraduate of Pharmaceutical and Biological Chemistry, at Faculty of Chemistry. National Autonomous University of Mexico (UNAM) maritere1011@hotmail.com.<br><br />
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<br />
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<p>&nbsp;</p><br />
<p>Anna Karen Hernández-Gallardo Contribution:Mathematical modeling, 4th year undergraduate Biotechnological Engineering, Interdisciplinary Unit for Biotechnology<br />
National Polytechnic Institute<br />
.</p><br />
<p>&nbsp;</p><br />
<p><br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/1/16/Tre.jpg" alt="tre" width="171" height="246" class="left"></p><br />
<p>Patricia Arias-Orozco Contribution: Design and development of regulatory systems, BioBrick Designer, outreach activities. 4th year undergraduate Biotechnological Engineering, Interdisciplinary Unit for Biotechnology National Polytechnic Institute. </p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/4/45/Uno.jpg" alt="uno" width="174" height="243" class="right"></p><br />
<p>&nbsp;</p><br />
<p>Daniel Domínguez-Gómez <br><br />
<p>Contribution: Outreach activities, design regulatory systems.<br> <br />
4th year undergraduate Biotechnological Engineering, Interdisciplinary Unit for Biotechnology, National Polytechnic Institute.<br><br />
<p></p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/4/43/Siete.jpg" alt="siete" width="167" height="234" class="left"></p><br />
<p>&nbsp;</p><br />
<p>Ivan Lopez-Flores Mathematical Team: Mathematical modeling Computational Systems Engineering, Superior School for Computation National Polytechnic Institute National Polytechnic Institute <br><br />
<br><br />
<br><br />
<br><br />
<br />
</p><br />
<br />
<h1>Advisors</h1><br />
<img src="https://static.igem.org/mediawiki/2012/5/59/Catorce.jpg" alt="catorce" width="168" height="244" class="left"><p>&nbsp;</p><br />
<p>&nbsp;</p><br />
<p>Karen Nava Castro PhD Contribution: Flow Cytometry Expert. Researcher at Institute of Biomedical Research, Field of study: Immunology. National Autonomous University of Mexico, UNAM. </p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/c/c8/Quince.jpg" alt="quince" width="161" height="221" class="right"></p><br />
<p>&nbsp;</p><br />
<p>Jorge Morales Montor PhD Contribution: Dry lab support Researcher at Institute of Biomedical Research (IIB, UNAM) Field of study: Neuoimmunoendocrinology National Autonomous University of Mexico, UNAM. </p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<img src="https://static.igem.org/mediawiki/2012/6/67/Dos.jpg" alt="dos" width="194" height="270" class="left"><br />
<p>Daniel Federico Hernandez Gardiol Contribution: Mathematical model Systems Biologist: Universidad de la Republica (Montevideo, Uruguay) Bioengineering Masters Student: Ecole Polytechnique Federal de Lausanne (Lausanne, Switzerland) National Polytechnic Institute.</p><br />
<p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0d/Cinco1.jpg" alt="cinco" width="173" height="205" class="right"><p>&nbsp;</p><br />
<p>Fernando G. Bastida-González Contribution: Wet lab support, Master in Health science at Superior Medicine School at National Polytechnic Institute.</p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<h1>Instructors</h1><br><br />
<br />
<p><img src="https://static.igem.org/mediawiki/2012/e/e9/Tres.jpg" alt="tres" width="166" height="227" class="left"></p><br />
<p>&nbsp;</p><br />
<p>Agustino Martínez-Antonio PhD Resarcher 3A, SNI I Genetic Engineering Department, CINVESTAV Irapuato.</p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><br><br />
<img src="https://static.igem.org/mediawiki/2012/6/67/Seis.jpg" alt="seis" width="161" height="209" class="right"></p><br />
<p>&nbsp;</p><br />
<p>Fernando Suaste-Olmos PhD Researcher at Institute of Cell Physiology IFC-UNAM.<br />
Expert in Photosynthetic bacteria. </p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/9/98/Doce.jpg" alt="dice" width="173" height="205" class="left"></p><br />
<p>&nbsp;</p><br />
<p>Nuria Sánchez-Puig Researcher at Institute of Chemistry UNAM Dr. Sanchez-Puig has a PhD from the Center for Protein Engineering, Cambridge, UK. </p><br />
<p>&nbsp;</p><p>&nbsp;</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/a/ad/Dieciseis.jpg" alt="dieciseis" width="150" height="204" class="right"></p><br />
<p>&nbsp;</p><br />
<p>Paola Zárate-Segura PhD Researcher, at IPN. Dra. Zárate Segura has a PhD in Bioprocess from Interdisciplinary Unit for Biotechnology, National Polytechnic Institute. </p><br />
<p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><p>&nbsp;</p><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Acknowledgments.htm
Team/CINVESTAV-IPN-UNAM MX/Acknowledgments.htm
2012-10-26T04:59:48Z
<p>Ao.patricia: </p>
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<h1><em>Acknowledgments</em></h1><br />
<h3>We would like to thank all the people who helped us through our project and our panic attacks:</h3><br />
</br><br />
<br />
<p align="center"><img src="https://static.igem.org/mediawiki/2012/9/93/Agradecimientos.jpg" alt="agradecimientos" width="516" height="362"><br><br />
MBA .Gerónimo Villanueva<br><br />
For his outstanding support </p><br />
<br />
<p align="center">Juan Carlos Martínez García PhD. <br><br />
For his support in mathematical modeling.</p><br />
<p align="center">Ing. Susana Ruiz. <br><br />
For her support in Molecular Biology Techniques.</p><br />
<br />
<p align="center">Ana Lilia Hernández.<br><br />
For her support in Molecular Biology Techniques.</p><br />
<p align="center">Norma Silvia Sánchez Sánchez QFB, UNAM. <br><br />
For her advices and support.</p><br />
<p align="center">Heliodoro Celis Sandoval PhD. <br><br />
For allow us to work in his Lab at IFC-UNAM.</p><br />
<p align="center">Karen Nava Castro PhD. <br><br />
For her advices and help in Flow Citometry.</p><br />
<p align="center">Jorge Morales Montor PhD. <br><br />
For his support in the project and allow us to work in his lab at IIB, UNAM.</p><br />
<p align="center">Msc. Libertad Pantoja Hernández. <br><br />
For her support in mathematical modeling.</p><br />
<p align="center">Jorge Vázquez Ramos PhD., Perla Castañeda López PhD., Raúl Garza Velasco QFB, Guadalupe Castorena Adame QFB, Luz del Carmen Castellanos Román M.Sc., Rodolfo Pastelín Palacios PhD., Verónica Ramón Lic. <br><br />
For their support in our panic moments Ö.</p><br />
<p align="center">Hector Silva-Gutierrez, Eduardo Moreno-Rodríguez, Cesar Villavicencio-Cordova.<br><br />
For their valuable collaboration.</p><br />
<p align="center">Veronica Jazmin Sánchez Ortiz, Manuel Camacho Zaragoza, Angelica Montes Trujillo, Javier De la Mora, Teresa Ballado, Alejandro "Buds". </br><br />
For helping us in our panic moments Ö </p><br />
<p align="center">Abiel Treviño Garza and Maria Luisa Sanez Jaramillo <br><br />
For his advices about wiki design.</p><br />
<p align="center"> Maria Luisa Saenz Jaramillo <br><br />
For rhodofactory logo design.</p><br />
<br />
<p align="center">Wiki Design:<br><br />
<br />
Ing. Alejandra Bartolo Gervacio<br><br />
<br />
Ing. José Adolfo Martínez Olmedo<br><br />
</p><br />
<br />
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<li><a href="Institute.htm">Institute</a></li><br />
<li><a href="Gallery.htm">Gallery</a></li><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Acknowledgments.htm
Team/CINVESTAV-IPN-UNAM MX/Acknowledgments.htm
2012-10-26T04:58:56Z
<p>Ao.patricia: </p>
<hr />
<div><html xmlns="http://www.w3.org/1999/xhtml"><br />
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<br />
<h1><em>Acknowledgments</em></h1><br />
<h3>We would like to thank all the people who helped us through our project and our panic attacks:</h3><br />
</br><br />
<br />
<p align="center"><img src="https://static.igem.org/mediawiki/2012/9/93/Agradecimientos.jpg" alt="agradecimientos" width="516" height="362"><br><br />
MBA .Gerónimo Villanueva<br><br />
For his outstanding support </p><br />
<br />
<p align="center">Juan Carlos Martínez García PhD. <br><br />
For his support in mathematical modeling.</p><br />
<p align="center">Ing. Susana Ruiz. <br><br />
For her support in Molecular Biology Techniques.</p><br />
<br />
<p align="center">Ana Lilia Hernández.<br><br />
For her support in Molecular Biology Techniques.</p><br />
<p align="center">Norma Silvia Sánchez Sánchez QFB, UNAM. <br><br />
For her advices and support.</p><br />
<p align="center">Heliodoro Celis Sandoval PhD. <br><br />
For allow us to work in his Lab at IFC-UNAM.</p><br />
<p align="center">Karen Nava Castro PhD. <br><br />
For her advices and help in Flow Citometry.</p><br />
<p align="center">Jorge Morales Montor PhD. <br><br />
For his support in the project and allow us to work in his lab at IIB, UNAM.</p><br />
<p align="center">Msc. Libertad Pantoja Hernández. <br><br />
For her support in mathematical modeling.</p><br />
<p align="center">Jorge Vázquez Ramos PhD., Perla Castañeda López PhD., Raúl Garza Velasco QFB, Guadalupe Castorena Adame QFB, Luz del Carmen Castellanos Román M.Sc., Rodolfo Pastelín Palacios PhD., Verónica Ramón Lic. <br><br />
For their support in our panic moments Ö.</p><br />
<p align="center">Hector Silva-Gutierrez, Eduardo Moreno-Rodríguez, Cesar Villavicencio-Cordova.<br><br />
For their valuable collaboration.</p><br />
<p align="center">Veronica Jazmin Sánchez Ortiz, Manuel Camacho Zaragoza, Angelica Montes Trujillo, Javier De la Mora, Teresa Ballado, Alejandro "Buds". </br><br />
For helping us in our panic moments Ö </p><br />
<p align="center">Abiel Treviño Garza and Maria Luisa Sanez Jaramillo <br><br />
For his advices about wiki design.</p><br />
<p align="center"> Maria Luisa Sanez Jaramillo <br><br />
For rhodofactory logo design.</p><br />
<br />
<p align="center">Wiki Design:<br><br />
<br />
Ing. Alejandra Bartolo Gervacio<br><br />
<br />
Ing. José Adolfo Martínez Olmedo<br><br />
</p><br />
<br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Members.htm
Team/CINVESTAV-IPN-UNAM MX/Members.htm
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<p>Ao.patricia: </p>
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<p><strong>Students</strong></p><br />
<p>We are an inter-institutional team formed by 7 undergraduate students from two important academic institutions in Mexico City, National Polytechnic Institute (IPN) and National Autonomous University of Mexico (UNAM). We also represent the Center for Research and Advanced Studies at Irapuato, our home for project development and the institution that have supported our work.</p><br />
<p><img src="images/pic.jpg" width="654" height="872" usemap="#Map" border="0" /></p><br />
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<td width="158"><img src="images/equipo/Patricia Arias.jpg" width="150" height="200" /></td><br />
<td width="477"><p><strong>Patricia Arias-Orozco</strong></p><br />
<p><br /><br />
<strong>Experimental Team: </strong>Construction of the main regulatory systems and BioBrick Designer.<br /><br />
Biotechnological Engineering, Interdisciplinary Unit for Biotechnology</p></td><br />
</tr><br />
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<td><img src="images/equipo/Daniel Domínguez.jpg" width="150" height="201" /></td><br />
<td><p><strong>Daniel Domínguez-Gómez</strong></p><br />
<p><br /><br />
<strong>Human Practices: </strong>Ownership and Feedback.<br /><br />
Biotechnological Engineering, Interdisciplinary Unit for Biotechnology<br /><br />
National Polytechnic Institute</p></td><br />
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<td><img src="images/equipo/Lissania Guerra.jpg" width="150" height="200" /></td><br />
<td><p><strong>Lissania Guerra-Calderas</strong></p><br />
<p><br /><br />
<strong>Experimental Team: </strong>Butanol Production System and BioBrick Designer.<br /><br />
Pharmaceutical and Biological Chemistry, Faculty of Chemistry, UNAM.</p></td><br />
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<td><img src="images/equipo/Karen Hernandez gallardo.jpg" width="150" height="200" /></td><br />
<td><p><strong>Anna Karen Hernández-Gallardo</strong></p><br />
<p><br /><br />
<strong>Mathematical Team: </strong>Mathematical modeling <br /><br />
Biotechnological Engineering, Interdisciplinary Unit for Biotechnology<br /><br />
National Polytechnic Institute</p></td><br />
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<td><p><strong>Jhonatan Alejandro Hernández-Valdés </strong></p><br />
<p><br /><br />
<strong>Experimental Team: </strong>Construction of the main regulatory systems and Biobrick Designer<br /><br />
Pharmaceutical and Biological Chemistry, Faculty of Chemistry, UNAM.</p></td><br />
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<td><img src="images/equipo/Ivan Flores.jpg" width="150" height="200" /></td><br />
<td><p><strong>Ivan Lopez-Flores</strong></p><br />
<p><br /><br />
<strong>Mathematical Team: </strong>Mathematical modeling<br /><br />
Computational Systems Engineering, Superior School for Computation<br /><br />
National Polytechnic Institute</p></td><br />
</tr><br />
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<td><img src="images/equipo/Maritere Uriostegui arcos.jpg" width="150" height="202" /></td><br />
<td><p>&nbsp;</p><br />
<p><strong>Maritere Uriostegui-Arcos</strong></p><br />
<p><br /><br />
<strong>Experimental Team: </strong>Characterization and measurement protocols. BioBrick Designer.<strong> </strong><br /><br />
Pharmaceutical and Biological Chemistry, Faculty of Chemistry, UNAM.</p></td><br />
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<p>&nbsp;</p><br />
<p><strong>Advisors:</strong></p><br />
<br />
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<td width="158"><img src="images/equipo/Daniel Hernandez Gardiol.jpg" width="150" height="200" /></td><br />
<td width="477"><p><strong>Daniel Federico Hernandez Gardiol</strong><strong> </strong></p><br />
<p><br /><br />
<strong>Mathematical Team</strong><strong> </strong><br /><br />
Systems Biologist: Universidad de la Republica (Montevideo, Uruguay)</p><br />
Bioengineering Masters Student: Ecole Federal de Lausanne (Lausanne, Switzerland)</td><br />
</tr><br />
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<td><img src="images/equipo/Fernando bastida.jpg" width="150" height="200" /></td><br />
<td><p><strong>Fernando G. Bastida-González</strong></p><br />
<p><br /><br />
<strong>Experimental Team: </strong>Master in Health science at Superior Medicine School at National Polytechnic Institute. <br /><br />
</p></td><br />
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<p><strong>Instructors</strong></p><br />
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<td width="477"><p><strong>Agustino Martínez-Antonio PhD</strong></p><br />
<p><strong>Resarcher 3A, SNI I</strong><br /><br />
<strong>Genetic Engineering Department, CINVESTAV Irapuato.</strong></p><br />
<p>Dr. Martinez-Antonio is our lead instructor; he has a PhD in biochemical sciences from Institute for Biotechnology of National Autonomus University of Mexico. He is the current leader of Systems and Synthetic Biology Lab in CINVESTAV Irapuato. His research interest is focused in the perception of environmental signals, information processing and transcriptional regulation in bacteria model, topological analysis, dynamic and modeling of biological networks, the design and construction of circuits, modules and genetic and metabolic systems, and the design and construction of self-replicating molecular and autoreproducibles systems.</p></td><br />
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<td><img src="images/equipo/Fernando suaste.jpg" width="150" height="200" /></td><br />
<td><p><strong>Fernando Suaste-Olmos PhD</strong></p><br />
<p><br /><br />
Researcher <br /><br />
Institute of Cell Phisiology. UNAM. DR. Suaste has Ph.D. in Basic Biomedical Research</p></td><br />
<tr><br />
<td><img src="images/equipo/Paola Zarate.jpg" width="150" height="184" /></td><br />
<td><p><strong>Paola Zárate-Segura PhD</strong></p><br />
<p><br /><br />
Researcher, Professor<br /><br />
Dra. Zárate Segura has a PhD in Bioprocess from Interdisciplinary Unit for Biotechnology, National Polytechnic Institute. </p></td><br />
</tr><br />
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<td><img src="images/equipo/Nuria sanchez.jpg" width="150" height="200" /></td><br />
<td><p><strong>Nuria Sánchez-Puig</strong></p><br />
<p><br /><br />
Researcher, Professor<br /><br />
Institute of Chemistry UNAM<br /><br />
Dr. Sanchez-Puig has a PhD from the Center for Protein Engineering.</p></td><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/March.htm
Team/CINVESTAV-IPN-UNAM MX/March.htm
2012-09-27T04:01:14Z
<p>Ao.patricia: </p>
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Safety.htm
Team/CINVESTAV-IPN-UNAM MX/Safety.htm
2012-09-27T03:58:07Z
<p>Ao.patricia: </p>
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<p>&nbsp;</p><br />
<p><strong>Question 1</strong>:<strong>'''Would any of your project ideas raise safety issues in terms of researcher, public or enviromental safety?'''</strong> </p><br />
<p><br /><br />
Purple Non sulfur Bacteria are one of the most metabolically diverse groups known in nature, they are able to grow under different conditions such as, aerobic respiration, anoxigenic photosynthesis, and anaerobic fermentation. Due to its diverse metabolism and their ability to eat aromatic compounds, these bacteria can live in hostile media such as polluted water. Despite all these features, work with these microorganisms in lab conditions is very laborious; especially since its growth medium is a complex mix of metals and vitamins.<br /><br />
The risk in PNSB management in the lab is very low since they are non-pathogenic bacteria. R. sphaeroides and R. palustris´ grow rates are quite slow, thus, our genetic systems do not represent a public safety menace since they are highly specific. Besides, we have designed these systems for its specific functioning in purple photosynthetic bacteria. Nevertheless, it is very important to take special care during our wetlab work, taking into account the basic precautions and methods commonly used in microbiology.<br /><br />
Our project aims to build two regulation systems, and introduce them into Rhodopseudomonas palustris. Since PCR isolation of the five genes that form the system is extremely difficult, gene synthesis technology provided by Genescript was used instead for the obtainment of these BioBricks. All our biobricks </p><br />
<p><br /><br />
<strong>Question 2</strong>: <strong>'''Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'''</strong></p><br />
<p><br /><br />
No, our BioBricks parts came from Rhodobacter sphaeroides, this bacteria is non-pathogenic and it can be worked in a Biosafety level 1 laboratory, based on the CDC Biosafety in Microbiological and Biomedical Laboratories (CDC, 2007). The functionality of our parts do not produce any dangerous compound, we are working in gene expression control. Furthermore, we used BioBricks from iGEM distribution for all our assemblies, for example GFP as reporter, these parts do not represent a biological risk.</p><br />
<p><br /><br />
<strong>Question 3</strong>: <strong>'''Is there a local biosafety group, committee, or review board at your institution? If no, which specific biosafety rules or guidelines do you have to consider in your country?'''</strong></p><br />
<p><br /><br />
No, a Biosafety committee must be integrated, by a multidisciplinary group of members among Research Centers, Industries and Universities. This committee should provide expert advice on laboratory, biosafety and biosecurity issues related to research and other academic activities. <br /><br />
The objectives of this group should be:</p><br />
<ul><br />
<li>Plan, design and implement the necessary instruments for biosafety risk assessment. </li><br />
<li>Establish, disseminate and analyze safety approaches in handling chemicals, genetically modified organisms and potentially infectious pathogens that allow us to make decisions for users in order to minimize potential health risks.</li><br />
<li>Conduct briefings and feedback sessions with the actors involved in biosafety.</li><br />
<li>Propound consistent Biosecurity regulations, which allow the development and promotion of basic and applied research. </li><br />
</ul><br />
<p>Particular problems should be evaluated case by case for the analysis of their solutions , this analysis should also include assessment of the risks of alternative technological options for coping with the specific problem to which the GMO was designed. And finally, it is important to review other laws; this will help to strengthen biosecurity aspects of other organisms that are not GMOs.</p><br />
<p>In Mexico CIBIOGEM, which is a governmental organism, was created to regulate biosafety issues mainly related with transgenic organisms and GMOs. The mentioned committee should work along this institution in order to establish a synthetic biology approach to biosafety.</p><br />
<p><strong>Question 4</strong>: <strong>Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?</strong></p><br />
<p><br /><br />
As a part of our human practices work, we are trying to implement the Quality Function Deployment tool in order to answer the most important questions in terms of safety and biosecurity. The relevance of this work is that through a thorough analysis of the proposed goals and needs of our work, we are going to be able to make different proposals that support the development of synthetic biology-based projects in Mexico</p><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Human_Practice.htm
Team/CINVESTAV-IPN-UNAM MX/Human Practice.htm
2012-09-27T03:54:39Z
<p>Ao.patricia: </p>
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<p>Sometimes is hard for scientist to get involved in Human Practices, we think is important to solve all those economical, ethical, legal and social issues in order to change the public perception of synthetic biology, We have worked in many activities for human practices<br /><br />
</p><br />
<p><strong><a href="https://2012.igem.org/wiki/index.php?title=Team/CINVESTAV-IPN-UNAM_MX/Meet.htm">Collaboration: iGEMMX Meet up!</a></strong><br /><br />
Twelve Mexican teams have participated in iGEM since 2007, they have had very successful participations, but they have never had an efficient communication. If we are connected, we can make things better. If we want to be competitive as a country in synthetic biology development, we have to collaborate. So, with the objective of improve our connections, improve our projects and establish collaboration agreements, we decided to organize the First iGEM MeetUP in our headquarters in Irapuato.<br /><br />
</p><br />
<p><strong>Education: SynBio Summer Workshop</strong></p><br />
<p><br /><br />
Every year, the leader of Systems and Synthetic Biology Group, and our main instructor, Agustino Martinez-Antonio organizes a Synthetic Biology Summer Workshop at the Center for Research and Advanced Studies (CINVESTAV), where undergrad students from all the country take part in the creation, design and development of new synbio projects during the summer. Some members of our team guided the new students in this workshop.<br /><br />
</p><br />
<p><a href="https://static.igem.org/mediawiki/2012/1/1a/Education_SynBio_Summer_Workshop.pdf" target="_blank"><strong>Read more…</strong></a><br /><br />
</p><br />
<p>&nbsp;</p><br />
<p><strong>Ownership: Intellectual Property vs. Open Source</strong><br /><br />
We know that Open source is the strategy to reach a global development in Synthetic Biology, but there´re many questions to answer in this topic. First, we have to decide a strategy to establish a strong Intellectual Property Platform. We asked many experts in IP in Mexico some of these questions<br /><br />
</p><br />
<p><strong><a href="https://static.igem.org/mediawiki/2012/3/36/Intellectual_Property_report.pdf" target="_blank">Read more…</a></strong></p><br />
<p>&nbsp;</p><br />
<p><strong>Sharing: iGEM Experience</strong><br /><br />
Maybe the most difficult part in making a new iGEM team is getting started, how do you get the right idea? How do you design the project? How do you work in teams? How do you get money? etc, . Since this is our first iGEM participation, we have had some little troubles that every team can face. We asked for opinions to answer all these questions in a document that can tell future iGEM teams what they should do in order to have success in the iGEM experience.<br /><br />
</p><br />
<p><strong><a href="https://static.igem.org/mediawiki/2012/e/e4/IGEM_Experience.pdf">Read more…</a></strong></p><br />
<p>&nbsp;</p><br />
<p><br /><br />
<strong>Biosafety…</strong><br /><br />
<a href="https://static.igem.org/mediawiki/2012/6/63/Cartel_BN.pdf" target="_blank"><strong>Poster</strong></a></p><br />
<p><strong>&nbsp;</strong></p><br />
<p><strong>Gaceta UNAM!</strong></p><br />
<p><strong><a href="http://www.quimica.unam.mx/IMG/pdf/flogisto81.pdf" target="_blank">Link1</a></strong></p><br />
<p><strong> <a href="http://www.quimica.unam.mx/IMG/pdf/gacetaseptiembre.pdf" target="_blank">Link 2</a></strong></p><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Modelling.htm
Team/CINVESTAV-IPN-UNAM MX/Modelling.htm
2012-09-27T03:53:48Z
<p>Ao.patricia: </p>
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Importance of Modeling and Simulation<br />
</strong><br />
<p>&nbsp;</p><br />
<p>Characterization of biological systems has reached an unparalleled level of detail. To organize this detail and arrive at a better fundamental understanding of life processes, it is essential that powerful conceptual tools from mathematics and computer science be applied to the frontier problems in biology. </p><br />
<p><br /><br />
Modeling of biological systems is evolving into an important partner of experimental work. They attempt to predict and understand the behavior of complex biological systems before they are actually created. Also models can help to simplify the complexity of data and interactions involved into a more concise form with some measure of predictive ability. This can provide valuable insights into the working and general principles of organization of biological systems. Also it may suggest novel experiments for testing hypotheses, based on the modeling experiences.</p><br />
<p><a href="Tools.htm"><strong>Some of the tools that we used during the modeling process.</strong></a></p><br />
<p><strong><a href="Model.htm">Construction of Model</a></strong></p><br />
<p><strong><a href="intercellular.htm">Modeling the intercellular signaling process during regulation of PS genes expression in BioNetGEn.</a></strong></p><br />
<p><strong><a href="equations.htm">Differential equations modeling the interactions between AppA and PpsR. </a></strong><a href="equations.htm"><br /><br />
<strong>Modeling Steady State</strong></a><strong></strong></p><br />
<p><a href="automata.htm"><strong>Cellular automata approaches to biological modeling.</strong></a></p><br />
<p><strong>References used through this section.</strong></p><br />
<ol><br />
<li>Oh JI, Kaplan S. (2001) <strong>Generalized approach to the regulation and integration of gene expression. </strong>Mol Microbiol. </li><br />
</ol><br />
<ol><br />
<li>Zeilstra-Ryalls JH, Kaplan S. (1995) <strong>Aerobic and anaerobic regulation in Rhodobacter sphaeroides</strong> 2.4.1: the role of the fnrL gene. J Bacteriol. </li><br />
</ol><br />
<p>&nbsp;</p><br />
<ol><br />
<li>Rakesh Pandey, Dietrich Flockerz. (2011) <strong>Modeling the Light- and Redox-Dependent Interaction of PpsR/AppA in Rhodobacter sphaeroides. </strong>Cell Press.</li><br />
</ol><br />
<ol><br />
<li>Shinji Masuda&nbsp;and&nbsp;Carl E. Bauer. (2002) <strong>AppA Is a Blue Light Photoreceptor that Antirepresses Photosynthesis Gene Expression in Rhodobacter sphaeroides.</strong> Cell Press.</li><br />
</ol><br />
<ol><br />
<li>Blinov ML, Faeder JR, Goldstein B, Hlavacek WS. (2004) <strong>BioNetGen: software for rule-based modeling of signal transduction based on the interactions of molecular domains. </strong>Bioinformatics. </li><br />
</ol><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Metods.htm
Team/CINVESTAV-IPN-UNAM MX/Metods.htm
2012-09-27T03:52:48Z
<p>Ao.patricia: </p>
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Construction.htm
Team/CINVESTAV-IPN-UNAM MX/Construction.htm
2012-09-27T03:51:56Z
<p>Ao.patricia: </p>
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<p><strong>CHARACTERIZATION OF IGEM BIOPARTS DISTRIBUTION</strong></p><br />
<p>As part of the points required to perform well in 2012 iGEM competition we are asked to characterize some existing biopartes on the record, so we decided to make the characterization of constitutive promoters belonging to the family isolated from a small combinatorial library (J23101 , J23102, J23104, J23107, J23108, J2311, and J23115) which were attached to GFP to determine promoter activity, using the equipment Victor X3 Multilabel Plate Reader.</p><br />
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<p>Fig 1. General scheme of one construction of the promoter expressing GFP</p><br />
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<p>Fig 2. Final construction</p><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/oxigenresponse.htm
Team/CINVESTAV-IPN-UNAM MX/oxigenresponse.htm
2012-09-27T03:51:10Z
<p>Ao.patricia: </p>
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<p><strong></strong><strong> </strong></p><br />
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<p><br /><br />
As we can see, in figure 1A the PpsR promoter system, in R. sphaeroides, shows a high signal (17.66%) of GFP expression, it implies that constitutive proteins from this bacteria were able to activate our system. The growth conditions were aerobic/darkness, although oxidized PpsR binds its target promoter, it is known that AppA can avoid the binding affinity of PpsR in the dark probably by the interference of an AppA-(PpsR)2 complex (Kim, 2006). In the case of AppA/PpsR complete system, we have a high GFP expression due to activity of the extra AppA and PpsR enzymes that were introduced.</p><br />
<p>The blue columns show the low GFP expression with both PrrA promoter and PrrA/PrrB complete system; in aerobic conditions PrrB autophosphorylates and passes a phosphate group to PrrA, this activated PrrA binds to its promoter region as a transcriptional repressor (Bauer, 2003). All the results show an equivalent result with the images that were obtained by fluorescence microscope.</p><br />
<p>Figure 2A shows that in R. sphaeroides, the AppA/PpsR system promoted the GFP expression, it is possible because reduced PpsR is unable to bind its promoter and AppA is a flavin with a photoreceptor, thus under light, AppA is unable to forma a complex with PpsR and we can see GFP expression in the bacterial population. </p><br />
<br />
<p>In opposite way, PrrA/PrrB system shows less GFP expression, it is probably due to the low growth rate in R. sphaeroides, under photosynthetic conditions (anaerobic/light), both systems were tested in one and a half days of growing, and maybe it was not enough time to have a culture in exponential phase of growing. PrrA/PrrB is supposed to be functional because in anaerobic conditions PrrB autophospholylate and can transfer the phosphate group to PrrA, when PrrA receive the phosphate group, it can bind its target promoter and the transcription is possible (Bauer, 2008).</p><br />
<p>AppA/PpsR system can be sense to signals: oxygen and light, in figure 3A, the GFP expression is high with PpsR promoter because we have the natural R. sphaeroides AppA/PpsR system working on our promoter. In anaerobic conditions the reduced PpsR has low affinity to his target sequence and AppA is forming the complex ApA-(PpsR)2, (Bauer, 2001). We can see it with the level of GFP expression 21.21%, however, when we introduce the complete system the GFP expression is much less, probably because we are introducing extra AppA and PpsR enzymes that can interfere with the constitutive system. PrrA/PrrB show GFP expression higher in PrrA promoter than in complete system, these results probably have the same problem, that bacterial cultures were not in exponential phase of growing or that the complete system can be interfering with the constitutive. </p> <br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Lightandoxre.htm
Team/CINVESTAV-IPN-UNAM MX/Lightandoxre.htm
2012-09-27T03:49:17Z
<p>Ao.patricia: </p>
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<p>In Rhodopseudomonas palustris, we can see in Figure 1A a perfect behavior of AppA/PpsR, the PpsR promoter do not show GFP expression, it indicates that other regulation systems do not bind this promoter, but when we introduce the complete system, we see a GFP expression of 31.89%, because the non natural system we designed is functional. In the case of PrrA/PrrB, the PrrA promoter do not show GFP expression, probably because the RegA/B system (the homologs system in this bacteria) do not have affinity for this sequence, but the complete construction is functional, we have to consider that it is an artificial system and the behavior can be different than expected, the functionality at these conditions is interesting because PrrB is supposed to be inactivated at aerobic conditions, or if the PrrA protein need to be phosphorylated, how it is being inactivated in this chassis. R. palustris has a different PrrA/PrrB system, and its mechanism is quite different, in fact, the main regulator to start photosynthetic metabolism is not PrrA/PrrB system, actually it is FixK enzyme (Rey, 2010).<br />
<br />
<br />
The Anaerobic/Light conditions in R. Palustris (figure 2A) made possible the functionality of our Synthetic AppA/PpsR system, the PpsR promoter show a low (0.169%) GFP expression, but the complete construction also show a little GFP expression.<br />
<p> <br />
<br />
In figure 3, the AppA/PpsR system is not functional, because in this condition, reduced PpsR has not affinity by its target sequence and the transcription is possible, AppA is is forming the complex with PpsR, but we can not see GFP expression. PrrA/PrrB show a low response, probably due to the independence of R. palustris for PrrA/PrrB mechanism, it functions with FixJ-FixK to regulate the change of metabolism aerobic to anaerobic (Metz, 2012).<br />
</p><br />
In R. palustris, the Median Fluorescence intensity (MIF) between complete systems and promoter is quite different. Figure 8 shows that PrrA promoter works better in Anaerobic/light conditions, than in others, it is the expected result considering the participation of homolog proteins, but the complete system is different because we saw a big change of fluorescence in aerobic/light conditions, introducing a synthetic system could affect the functionality of our constructions. AppA/PpsR system is functional because our promoter is being activated by other proteins, but the signal increase with our Synthetic Construction in the complete system, and also, as it is expected, the complete system works in aerobic/light conditions.<br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Protocol.htm
Team/CINVESTAV-IPN-UNAM MX/Protocol.htm
2012-09-27T03:47:22Z
<p>Ao.patricia: </p>
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Notebook.htm
Team/CINVESTAV-IPN-UNAM MX/Notebook.htm
2012-09-27T03:46:19Z
<p>Ao.patricia: </p>
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Biobricks.htm
Team/CINVESTAV-IPN-UNAM MX/Biobricks.htm
2012-09-27T03:45:47Z
<p>Ao.patricia: </p>
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/April.htm
Team/CINVESTAV-IPN-UNAM MX/April.htm
2012-09-27T03:44:41Z
<p>Ao.patricia: </p>
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<p align="justify">With the valuable collaboration of the team iGEM-Alberta 2008, we link the the light-oxygen dependent regulation systems with the butanol production pathway and its electron-transfer-flavoprotein alpha and betha polypeptides, this constructions were sent by Dr. Douglas Ridgway. (Reference: <a href="https://2008.igem.org/Team:The_University_of_Alberta/Butanerd" target="_blank">Butanerd</a>)</p><br />
<p align="justify"><em>Clostridium</em><em> acetobutylicum</em> is an anaerobic, spore-forming bacterium with the ability to ferment starch and sugars into solvents. In the past, it has been used for industrial production of acetone and butanol, until cheap crude oil rendered petrochemical synthesis more economically feasible. Both economic and environmental aspects have caused the pendulum to swing back again. Molecular biology has allowed a detailed understanding of genes and enzymes, required for solventogenesis.(1)<br /><br />
Taking advantage of the metabolic versatility of <em>R. sphearoides</em> and <em>R. palustris</em> and their ability to fix Carbon dioxide, we aim to introduce the following constructions into these both bacteria so that the fixed carbon dioxide becomes into butanol.</p><br />
<p align="justify">We already constructed the following circuits, ready for testing in <em>R. Palustris</em></p><br />
<p>&nbsp;</p><br />
<p> <img src="https://static.igem.org/mediawiki/2012/b/b8/Sistema.JPG" /></p><br />
<br />
<p>&nbsp;</p><br />
<p align="justify">We have to link these constructions into the vector pRK415, and conjugate <em>R. sphearoides</em> and <em>R. palutris</em>, to finally test our system and to produce butanol.</p><br />
<h1>References</h1><br />
<p><strong>&nbsp;</strong></p><br />
<h1 align="justify">1.- <strong>Fermentative butanol production: bulk chemical and biofuel</strong>. Ann N Y Acad Sci., Dürre P. 2008 Mar;1125:353-62.</h1><br />
<h1>2.-Cong T. Trinh,<strong> Elucidating and reprogramming Escherichia coli metabolisms for obligate anaerobic n-butanol and isobutanol production</strong> Appl Microbiol Biotechnol DOI 10.1007/s00253-012-4197-7</h1><br />
<p align="justify">3.-Masayuki Inui &amp; Masako Suda &amp; Sakurako Kimura &amp; Kaori Yasuda &amp; Hiroaki Suzuki &amp; Hiroshi Toda &amp; Shogo Yamamoto &amp; Shohei Okino &amp; Nobuaki Suzuki &amp; Hideaki Yukawa<strong> Expression of <em>Clostridium acetobutylicum</em> butanol synthetic genes in <em>Escherichia coli</em> </strong>Appl Microbiol Biotechnol (2008) 77:1305–1316</p><br />
<p align="justify"><br /><br />
4.-Wolfram Andersch, Hubert Bahl, and Gerhard Gottschalk Eur J <strong>Level of Enzymes Involved in Acetate, Butyrate, Acetone and Butanol Formation by <em>Clostridium acetobutylicum </em></strong>Appl Microbiol Biotechnol (1983) 18:327-332</p><br />
<p align="justify"><br /><br />
5.-Hubert Bahl, Wolfram Andersch, and Gerhard Gottschalk European J <strong>Continuous Production of Acetone and Butanol by <em>Clostridium acetobutylicum </em>in a Two-Stage Phosphate Limited Chemostat</strong> Appl Microbiol Biotechnol (1982) 15:201-205 </p><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Chassis.htm
Team/CINVESTAV-IPN-UNAM MX/Chassis.htm
2012-09-27T03:43:57Z
<p>Ao.patricia: </p>
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<p><em>Rhodopseudomonas palustris</em> has an extraordinary metabolic versatility; this organism can grow in a wide variety of environmental conditions. <em>R palustris</em> obtain energy by different mechanism including <strong>anoxygenic</strong> <strong>photosynthesis, aerobic and anaerobic respiration</strong>. </p><br />
<p>&nbsp;</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/a/af/Cha.jpg" width="640" height="266" /></p><br />
<p><strong>Figure</strong> Metabolic versatility or R. palustris (Harwood et. al. (2004))</p><br />
<p><br /><br />
When O2 is absent or limiting, light energy can be harnessed by a photosynthetic electron transport chain that has features similar to those used by plants and other oxygen-evolving phototrophs <strong>(3)</strong>. During photosynthetic growth, <em>R. palustris</em> is capable of autotrophic or heterotrophic growth using either carbon dioxide (CO2) or organic carbon sources. <br /><br />
<strong>Biotechnological potential</strong><br /><br />
<em>R. palustris </em>is an excellent candidate for use in a wide variety of biotechnological applications because of a wide range of regulation systems that allow it to sense environmental conditions.</p><br />
</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/c/cb/Cha2.jpg" width="640" height="474" /></p><br />
<p><strong>Why synthetic biology in <em>R. palustris</em>?</strong></p><br />
<p>The application of Synthetic Biology in R. palustris could help to exploit the biotechnological potential of this organism. We want to explore the functioning of 2 orthologous regulation systems that respond to oxygen and light inspired in <em>Rhodobacter sphaeroides </em>regulatory systems.</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/thumb/9/97/Cha3.jpg/800px-Cha3.jpg" width="640" height="463" /></p><br />
<p>1. Hunter CN, Daldal F, Thurnauer MC, Beatty JT: (2009) <strong>The Purple Phototrophic Bacteria</strong>. Springer; 200928.<em> pp. 707–725.</em> <br /><br />
2. Harwood et. al. (2004) <strong>Complete genome sequence of the metabolically versatile photosynthetic bacterium <em>Rhodopseudomonas palustris</em></strong>, Nature Biotechnology Volume 2, Number 1, January 2004<br /><br />
3. Imam S., Yilmaz S., Sohmen Y, Gorzalski A., Reed J. Noguera D., Donohue T. (2011)<strong> iRsp1095: A genome-scale reconstruction of theRhodobacter sphaeroides metabolic network </strong>BMC Systems Biology 2011, 5:116 </p><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Oxigen_Response.htm
Team/CINVESTAV-IPN-UNAM MX/Oxigen Response.htm
2012-09-27T03:37:57Z
<p>Ao.patricia: </p>
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<h1><strong>Oxygen Control System: PrrA/PrrB two component system </strong></h1><br />
<p>This regulatory system can sense oxygen concentration and senda response, under high oxygen tension, the system remains inactive, when oxygen concentration decreases PrrB (His sensor kinase) turns active trough an autophosphorylation with help of PrrC, which transmit the signal from electron transport chain with help. Then PrrB transmit the phosphate group to PrrA response regulator that directly binds promoter and recruits RNA polymerase to start GFP transcription.</p><br />
<p><strong>How does it work?</strong></p><br />
<p>This genetic system consists in two modules, the first one is formed by <strong><em>prra, prrb </em></strong><em>and<strong> prrc</strong></em>coupled to <strong>B0030</strong> RBS units, <strong>J23104</strong> Strong constitutive promoter and <strong>B0014</strong> Double terminator to form the transcription unit. We will evaluate the functioning of J23104, B0030 and B0014 in a new chassis. The second module consists in <strong>J54103</strong> GFP generator (B0030 RBS + E1010 GFP + B0014 Double terminator) coupled to PpsR repressible promoter.</p><br />
<p>&nbsp;</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/9/93/Oxi.JPG" width="636" height="231" /></p><br />
<h2>&nbsp;</h2><br />
<p center><p><strong>Inspired in…</strong></p><br />
<p>This system is inspired in PrrBCA two component system from Rhodobacter sphaeroides, which is a master regulator that is involved in expression of approximately 850 genes, <strong><em>&gt;</em>20% of the genome</strong> (Kaplan &amp; Eraso 2005) This system coordinately controls genes involved in the complex switch between aerobic and anaerobic lifestyles and the optimum use of reducing power. It also regulates expression of genes involved in photosynthesis, carbon dioxide fixation, nitrogen fixation, hydrogen uptake, aerotaxis, denitrification, electron transport, aerobic and anaerobic respiration, and heme biosynthesis, among others, thus emphasizing its global role (Elsen et.al 2004, Kaplan &amp; Eraso 2005, Zeilstra-Ryalls &amp; Kaplan 2004). </p><br />
<p>&nbsp;</p><br />
<ol><br />
<li> Kaplan S, Eraso J, Roh JH. (2005). <strong>Interacting regulatory networks in the facultative</strong></li><br />
</ol><br />
<p><strong>photosynthetic bacterium, <em>Rhodobacter sphaeroides </em>2.4.1.</strong> <em>Biochem. Soc. Trans. </em>33:51–55</p><br />
<ol><br />
<li> Zeilstra-Ryalls JH, Kaplan S. (2004). <strong>Oxygen intervention in the regulation of gene xpression: the photosynthetic bacterial paradigm</strong>. <em>Cell. Mol. Life Sci. </em>61:417–36</li><br />
<li>Eraso JM, Kaplan S (2009) <strong>Regulation of gene expression by PrrA in Rhodobacter sphaeroides 2.4.1: role of polyamines and DNA topology</strong>. J Bacteriol 2009, 191(13):4341-4352.</li><br />
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Ao.patricia
http://2012.igem.org/Team/CINVESTAV-IPN-UNAM_MX/Light_Response.htm
Team/CINVESTAV-IPN-UNAM MX/Light Response.htm
2012-09-27T03:30:01Z
<p>Ao.patricia: </p>
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<h2><strong>Light + Redox Response: AppA/PpsR Control System</strong></h2><br />
<p>This is a repressor/antirepressor system, under high oxygen tension <strong>PpsR</strong> strongly repress GFP expression by binding a conserved regulatory sequence that avoid RNA polymerase for promoter binding. When oxygen concentration decreases AppA is activated, and bind PpsR avoiding this DNA binding activity, thus, GFP expression can begin.</p><br />
<p>&nbsp;</p><br />
<p><strong>How does it works?</strong></p><br />
<p>Our genetic system consists in 2 modules, the first one is formed by <em>appa</em> and <em>ppsr </em>coupled to B0030 RBS units, J23104 Strong constitutive promoter and B0014 Double terminator to form the transcription unit. We will evaluate the functioning of J23104, B0030 and B0014 in a new chassis. The second module consists in <strong>J54103</strong> GFP generator (B0030 RBS + E1010 GFP + B0014 Double terminator) coupled to PpsR repressible promoter.</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/e/ed/Light.JPG" width="634" height="299" /></p><br />
<p><strong>Inspired in…</strong></p><br />
<p><br /><br />
This system is inspired in AppA/PpsR repressor/antirepressor system from <em>Rhodobacter sphaeroides</em>, PpsR protein is a master repressor of Photosynthesis (PS) genes (Moskvin and Gomelsky 2005). Inactivation of the <em>ppsR </em>gene is enough to turn on PS gene expression and formation of the photosynthetic apparatus even at a high oxygen concentration, whereas <em>ppsR </em>overexpression is sufficient to block PS development even in the absence of oxygen. PpsR directly represses transcription of most carotenoid and pigment synthesis genes, photosystems operons, and genes involved in tetrapyrrole biosynthesis (Gomelsky and Kaplan 1995). <strong>The upstream regions of these genes contain two PpsR binding sites, TGTcN10gACA.</strong></p><br />
<p>A second protein called AppA, which has no known homologues, plays a role in controlling gene expression in <em>R. sphaeroides </em>in response to both light and O2 by acting as an antirepressor of PpsR. Our parts (appa, ppsr and ppsr-promoter) were synthesized with Genescript, and are codon optimized for <em>R. palustris.</em></p><br />
<ol><br />
<li>Gomelsky L., Moskvin L., Stenzel A., Jones D., Donohue T. and Gomelsky M.(2008) <strong>Hierarchical Regulation of Photosynthesis Gene Expression by the Oxygen-Responsive PrrBA and AppA-PpsR Systems of <em>Rhodobacter sphaeroides.</em></strong> J. Bacteriol. Dec. 2008, p. 8106–8114 Vol. 190, No. 24 </li><br />
<li>Moskvin, O. V., L. Gomelsky, and M. Gomelsky<strong>. (</strong>2005<strong>). Transcriptome analysis of the </strong><strong><em>Rhodobacter sphaeroides </em></strong><strong>PpsR regulon: PpsR as a master regulator of photosystem development</strong>. J. Bacteriol. <strong>187:</strong>2148–2156. </li><br />
<li>Gomelsky, M., and S. Kaplan.<strong> (</strong>1995). <strong>Genetic evidence that PpsR from </strong><strong><em>Rhodobacter sphaeroides </em></strong><strong>2.4.1 functions as a repressor of </strong><strong><em>puc </em></strong><strong>and </strong><strong><em>bchF </em></strong><strong>expression</strong>. J. Bacteriol. <strong>177:</strong>1634–1637.</li><br />
<li>Gomelsky, M., and S. Kaplan. <strong>(</strong>1995). <strong><em>appA</em></strong><strong>, a novel gene encoding a </strong><strong><em>trans</em></strong><strong>acting factor involved in the regulation of photosynthesis gene expression in </strong><strong><em>Rhodobacter sphaeroides </em></strong><strong>2.4.1</strong>. J. Bacteriol. <strong>177:</strong>4609–4618.</li><br />
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Ao.patricia
http://2012.igem.org/File:CINVESTAV-IPN-UNAM_MX_team.png
File:CINVESTAV-IPN-UNAM MX team.png
2012-07-01T17:31:51Z
<p>Ao.patricia: uploaded a new version of &quot;File:CINVESTAV-IPN-UNAM MX team.png&quot;: Reverted to version as of 17:25, 1 July 2012</p>
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Ao.patricia
http://2012.igem.org/File:CINVESTAV-IPN-UNAM_MX_team.png
File:CINVESTAV-IPN-UNAM MX team.png
2012-07-01T17:31:17Z
<p>Ao.patricia: uploaded a new version of &quot;File:CINVESTAV-IPN-UNAM MX team.png&quot;</p>
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Ao.patricia
http://2012.igem.org/File:CINVESTAV-IPN-UNAM_MX_team.png
File:CINVESTAV-IPN-UNAM MX team.png
2012-07-01T17:25:33Z
<p>Ao.patricia: uploaded a new version of &quot;File:CINVESTAV-IPN-UNAM MX team.png&quot;</p>
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Ao.patricia
http://2012.igem.org/File:CINVESTAV-IPN-UNAM_MX_team.png
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2012-07-01T17:19:18Z
<p>Ao.patricia: </p>
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Ao.patricia
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File:CINVESTAV-IPN-UNAM MX logo.png
2012-06-07T05:13:56Z
<p>Ao.patricia: uploaded a new version of &quot;File:CINVESTAV-IPN-UNAM MX logo.png&quot;</p>
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Ao.patricia