http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=500&target=Joelkuiper&year=&month=2010.igem.org - User contributions [en]2024-03-29T13:27:16ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Groningen/Biofilm_modelTeam:Groningen/Biofilm model2010-11-17T01:54:53Z<p>Joelkuiper: /* Results */</p>
<hr />
<div>__NOTOC__<br />
==Biofilm Dynamics==<br />
<br />
===Introduction===<br />
Biofilms are multicellular conglomerates which attach to surfaces. The formation of biofilms is triggered by high cell density and limited resources. The sensing is of these conditions is often mediated by an extracellular signaling compound which increases in concentration and triggers regulating circuitry. This process is called quorum sensing and it plays an important role in the dynamics of multicellular systems. <br />
<br />
Quorum sensing systems can cause complicated effects including cell differentiation within single species conglomerates. A recent example of this can be seen in the difference of expression in the [http://subtiwiki.uni-goettingen.de/wiki/index.php/TasA TasA protein] which plays a major role in the formation of the biofilm matrix by forming amyloid fibers. Expression of this protein is mediated by competitive inhibitive systems by, amongst others, the [http://subtiwiki.uni-goettingen.de/wiki/index.php/YqxM yqxM gene](<partinfo>BBa_K305006</partinfo>) which linked to the [https://2010.igem.org/Team:Groningen#/expression_model ComXPA quorum sensing] system. <br />
<br />
However it has been found that not all bacteria respond to the signaling molecules in the same way. It was shown that biofilm formation in ''Bacillus subtilis'' involves paracrine signaling in which most cells produce and secrete the signaling molecule ComX but only a sub population of the cells is triggered to make surfactin. Surfactin serves as paracrine signaling molecule and the cells which are not able to make surfactin, respond to surfactin by making extracellular matrix components for the biofilm. <br />
<br />
It is hypothesized that the extracellular matrix interferes with the interaction between ComX and the trans membrane histidine kinase ComP and therefore prevents surfactin production in extracellular matrix producing cells. In a mutant which did not express extracellular matrix proteins, the surfactin expression was more than three times higher. <br />
<br />
While differentiation requires interaction between several genes and complicated effects as described above we believe even simple extra-cellular accumulation of a pheromone can be responsible. <br />
<br />
A very simple model is proposed based on 2D growth of bacteria by cellular automata on a restricted plane. Each of the cells (a pixel in the model) secretes an unnamed compound which is allowed to accumulate. The accumulated compound is diffused at each discrete time step by applying a Gaussian Kernel. Finally at each cell a sigmoidal response curve is mapped, simplifying an all-or-nothing response similar to signal amplification through auto-phosphorylation of transmembrane histidine kinases. Cell differentiation was demonstrated within this theoretical model and variation of the parameters change the response drastically.<br />
<br />
===Growth by cellular automata===<br />
Cellular Automata are discrete computational models. The model consist of a finite grid (of any dimension) in which each of the cells can be in one of a finite set of states. The model behaves discretely in time by evaluating the number of neighbors of each cell and updating its state by some algebraic rule. A popular example of such a model with only two possible states, on and off, and a 2 dimensional grid is Conways Game of life. A possible instantiation of this "game" (it is a zero player game in the sense that it plays itself) is shown below. <br />
<br />
[[Image:Groningen-wiki-Gospers_glider_gun.gif|frame|none|Conways Game of life(from [http://en.wikipedia.org/wiki/Conway_game_of_life wikipedia])]]<br />
<br />
Simpler one dimensional models are an important subject of study in theoretical computer science. We modeled unrestricted bacterial growth by Cellular Automata. The model is a 2D grid of 400x400 pixels, the states are on and off indicating the presence of biomass. The grid is initialized randomly with a fixed distribution of "on" states. At every time step the following rule is applied by evaluating each of the cells 8 neighbors, if a cell has 3 or 4 neighbors with the "on" state the cell itself becomes "on" (a growth step, or birth), if a cell has 4, 5, or 6 neighbors which are on the cell is allowed to live and thus remains on. If any cell does not meet these criteria the cell will remain or become off. An animation of this model is shown below.<br />
<br />
[[Image:Groningen-CAModel.gif]]<br />
<br />
Due to the random initialization the first time steps are trimmed to allow for the distribution to stabilize. The result of the simulation is a binary matrix C for each time step representing biomass occupation. It can be shown by summing the total amount of occupied cell increases exponentially at each step and finally reaching a stationary phase because of spatial limitations.<br />
<br />
[[Image:Groningen-model-cellDensity.png|300px]]<br />
<br />
===Quorum sensing pheromones===<br />
Quorum sensing pheromones are modeled by sampling a random number from the normal distribution for each cell which in on resulting in a new matrix P. Because the sensing pheromones are allowed to accumulate each next time step is the sum of the previous matrices. Quorum sensing requires diffusion of the pheromone from one occupied cell to another. Plotting the density of pheromone gives the following graph:<br />
<br />
[[Image:Groningen-model-subsNoDiff.png]]<br />
<br />
Continuous diffusion can be modeled using the Heat equation, a two dimensional Partial Differential Equation (PDE) as shown below. Fick's second law is a a common biological and chemical model and is analogous to the two-dimensional Heat equation. This would generate a discrete-differential model.<br />
<br />
[[Image:Groningen-heat-model.png]]<br />
<br />
Solving this equation allows for diffusion continuous in both space and time. However for simplicity we applied a discrete Gaussian filter, equivalent to a convoluting a Gaussian matrix to our substance matrix P generating P'. The Gaussian Filter in 2D has the following form: <br />
<br />
[[Image:Groningen-GaussianFilter.png]]<br />
<br />
Where &sigma; is the standard deviation. The result of this process with &sigma; = 3 is demonstrated at a single time step below: <br />
<br />
[[Image:Groningen-model-subsDiff.png|frame|none|Diffusion applied at t=8]]<br />
<br />
===Activation of genes===<br />
Simplification of the Hill kinetics allowed for modeling of gene activation through simple sigmoidal function:<br />
<br />
[[Image:Groningen-Sigmoid-Model.png]]<br />
<br />
Imputing every element of P' into the function shows which genes are activated. Binarization of the responses allowed to color the activated cells in the original matrix C. Results are discussed below.<br />
<br />
===Results===<br />
The results of the model are shown below in simplified form. Download the interactive code and the [http://www.wolfram.com/products/player/ Mathematica 7 viewer] to play interactively with the model. The cells which have the gene expressed are shown in red. <br />
[[Image:Groningen-Biofilm3.png|150px|frame|none|Early time step with high expression threshold]]<br />
<br />
<br />
[[Image:Groningen-Biofilm1.png|150px|frame|none|Later time step with high expression threshold, some expression shown]]<br />
<br />
<br />
[[Image:Groningen-biofilm2.png|150px|frame|none|Late time step with high expression threshold, cell differentiation clearly visible]]<br />
<br />
<br />
[[Image:Groningen-biofilm4.png|150px|frame|none|Late time step with high expression threshold and high diffusion rate]]<br />
<br />
<br />
Animation of expression based on high threshold and low signaling molecule diffusion, each frame is a 100 steps in the model. <br />
<br />
[[Image:ExpressionTimeFramed.gif]]<br />
<br />
===Future work=== <br />
The model proposed here is a grave oversimplification and many enhancements can be made. First of all the growth of the biofilm is unrestricted by substance availability Loosdrecht et. al. proposed a more realistic model of biofilm formation taking into account oxygen and substance concentrations. The pheromone is diffused discretely, however a larger resolution would be needed to differentiate between cellular and molecular scale. Implementing the Heat equation would increase the resolution and allow for a more realistic diffusion model. It is now assumed that the pheromone concentrations remain stable over time, however to fully account for its presence substance degradation by extracellular proteins would also need to be modeled. <br />
<br />
A more realistic model of [https://2010.igem.org/Team:Groningen#/expression_model gene expression] could be incorporated further increasing its realism by taking into account within-cell dynamics. Also the ''B.subtilis'' quorum sensing system does not rely on a single compound, many different molecules diffuse and influence cell behavior. Sporulation for example is known to be a competitive inhibitory system between CSF and ComX.<br />
<br />
===Sources===<br />
The Mathematica 7 source files are available below:<br />
<br />
Biofilm model ([http://dl.dropbox.com/u/391356/iGEM/biofilm.nb source])(interactive)<br><br />
2D Heat equation ([http://dl.dropbox.com/u/391356/iGEM/diff.nb source])<br><br />
Sigmoid response curve ([http://dl.dropbox.com/u/391356/iGEM/hillSimpl.nb source])([http://dl.dropbox.com/u/391356/iGEM/hillSimpl.nbp interactive])<br />
<br />
<br />
===References===<br />
<small>Comella N, Grossman AD, Conservation of genes and processes controlled by the quorum response in bacteria: characterization of genes controlled by the quorum-sensing transcription factor ComA in Bacillus subtilis, Molecular Microbiology (2005), 57 (4): 1159–1174. PMID 16091051 <br />
<br />
Lemon KP, Earl AM, Vlamakis HC, Aguilar C, Kolter R., Biofilm development with an emphasis on Bacillus subtilis, Current Topics Microbiology and Immunology, 2008, 322:1-16. PMID 18453269 <br />
<br />
López D, Vlamakis H, Kolter R, Biofilms, Cold Spring Harbor Perspectives in Biology, 2010, (2):a000398. PMID 20519345 <br />
<br />
López D, Vlamakis H, Losick R, Kolter R, Paracrine signaling in a bacterium, Genes & Development, 2009 23(14):1631-8. <br />
<br />
Pérez J, Picioreanu C, van Loosdrecht M, Modeling biofilm and floc diffusion processes based on analytical solution of reaction-diffusion equations, Water Research, 2005, 39:1311-1323.<br />
<br />
Picioreanu C, van Loosdrecht MC, Heijnen JJ. A new combined differential-discrete cellular automaton approach for biofilm modeling: application for growth in gel beads, Biotechnology Bioengineering, 1998, 57(6):718-31. PMID 10099251<br />
<br />
Picioreanu C, van Loosdrecht MC, Heijnen JJ. Mathematical modeling of biofilm structure with a hybrid differential-discrete cellular automaton approach, Biotechnology Bioengineering, 1998, 58(1):101-16. PMID <br />
10099266 <br />
<br />
Roggiani M, Dubnau D, ComA, a phosphorylated response regulator protein of Bacillus subtilis, binds to the promoter region of srfA, Journal of Bacteriology, 1993, 175(10): 3182-7. PMID 8387999 <br />
<br />
Shah IM, Dworkin J, Microbial interactions: bacteria talk to (some of) their neighbors, Current Biology, 2009, 19(16): 689-91. PMID 19706277</small></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/Biofilm_modelTeam:Groningen/Biofilm model2010-11-17T01:50:35Z<p>Joelkuiper: /* Results */</p>
<hr />
<div>__NOTOC__<br />
==Biofilm Dynamics==<br />
<br />
===Introduction===<br />
Biofilms are multicellular conglomerates which attach to surfaces. The formation of biofilms is triggered by high cell density and limited resources. The sensing is of these conditions is often mediated by an extracellular signaling compound which increases in concentration and triggers regulating circuitry. This process is called quorum sensing and it plays an important role in the dynamics of multicellular systems. <br />
<br />
Quorum sensing systems can cause complicated effects including cell differentiation within single species conglomerates. A recent example of this can be seen in the difference of expression in the [http://subtiwiki.uni-goettingen.de/wiki/index.php/TasA TasA protein] which plays a major role in the formation of the biofilm matrix by forming amyloid fibers. Expression of this protein is mediated by competitive inhibitive systems by, amongst others, the [http://subtiwiki.uni-goettingen.de/wiki/index.php/YqxM yqxM gene](<partinfo>BBa_K305006</partinfo>) which linked to the [https://2010.igem.org/Team:Groningen#/expression_model ComXPA quorum sensing] system. <br />
<br />
However it has been found that not all bacteria respond to the signaling molecules in the same way. It was shown that biofilm formation in ''Bacillus subtilis'' involves paracrine signaling in which most cells produce and secrete the signaling molecule ComX but only a sub population of the cells is triggered to make surfactin. Surfactin serves as paracrine signaling molecule and the cells which are not able to make surfactin, respond to surfactin by making extracellular matrix components for the biofilm. <br />
<br />
It is hypothesized that the extracellular matrix interferes with the interaction between ComX and the trans membrane histidine kinase ComP and therefore prevents surfactin production in extracellular matrix producing cells. In a mutant which did not express extracellular matrix proteins, the surfactin expression was more than three times higher. <br />
<br />
While differentiation requires interaction between several genes and complicated effects as described above we believe even simple extra-cellular accumulation of a pheromone can be responsible. <br />
<br />
A very simple model is proposed based on 2D growth of bacteria by cellular automata on a restricted plane. Each of the cells (a pixel in the model) secretes an unnamed compound which is allowed to accumulate. The accumulated compound is diffused at each discrete time step by applying a Gaussian Kernel. Finally at each cell a sigmoidal response curve is mapped, simplifying an all-or-nothing response similar to signal amplification through auto-phosphorylation of transmembrane histidine kinases. Cell differentiation was demonstrated within this theoretical model and variation of the parameters change the response drastically.<br />
<br />
===Growth by cellular automata===<br />
Cellular Automata are discrete computational models. The model consist of a finite grid (of any dimension) in which each of the cells can be in one of a finite set of states. The model behaves discretely in time by evaluating the number of neighbors of each cell and updating its state by some algebraic rule. A popular example of such a model with only two possible states, on and off, and a 2 dimensional grid is Conways Game of life. A possible instantiation of this "game" (it is a zero player game in the sense that it plays itself) is shown below. <br />
<br />
[[Image:Groningen-wiki-Gospers_glider_gun.gif|frame|none|Conways Game of life(from [http://en.wikipedia.org/wiki/Conway_game_of_life wikipedia])]]<br />
<br />
Simpler one dimensional models are an important subject of study in theoretical computer science. We modeled unrestricted bacterial growth by Cellular Automata. The model is a 2D grid of 400x400 pixels, the states are on and off indicating the presence of biomass. The grid is initialized randomly with a fixed distribution of "on" states. At every time step the following rule is applied by evaluating each of the cells 8 neighbors, if a cell has 3 or 4 neighbors with the "on" state the cell itself becomes "on" (a growth step, or birth), if a cell has 4, 5, or 6 neighbors which are on the cell is allowed to live and thus remains on. If any cell does not meet these criteria the cell will remain or become off. An animation of this model is shown below.<br />
<br />
[[Image:Groningen-CAModel.gif]]<br />
<br />
Due to the random initialization the first time steps are trimmed to allow for the distribution to stabilize. The result of the simulation is a binary matrix C for each time step representing biomass occupation. It can be shown by summing the total amount of occupied cell increases exponentially at each step and finally reaching a stationary phase because of spatial limitations.<br />
<br />
[[Image:Groningen-model-cellDensity.png|300px]]<br />
<br />
===Quorum sensing pheromones===<br />
Quorum sensing pheromones are modeled by sampling a random number from the normal distribution for each cell which in on resulting in a new matrix P. Because the sensing pheromones are allowed to accumulate each next time step is the sum of the previous matrices. Quorum sensing requires diffusion of the pheromone from one occupied cell to another. Plotting the density of pheromone gives the following graph:<br />
<br />
[[Image:Groningen-model-subsNoDiff.png]]<br />
<br />
Continuous diffusion can be modeled using the Heat equation, a two dimensional Partial Differential Equation (PDE) as shown below. Fick's second law is a a common biological and chemical model and is analogous to the two-dimensional Heat equation. This would generate a discrete-differential model.<br />
<br />
[[Image:Groningen-heat-model.png]]<br />
<br />
Solving this equation allows for diffusion continuous in both space and time. However for simplicity we applied a discrete Gaussian filter, equivalent to a convoluting a Gaussian matrix to our substance matrix P generating P'. The Gaussian Filter in 2D has the following form: <br />
<br />
[[Image:Groningen-GaussianFilter.png]]<br />
<br />
Where &sigma; is the standard deviation. The result of this process with &sigma; = 3 is demonstrated at a single time step below: <br />
<br />
[[Image:Groningen-model-subsDiff.png|frame|none|Diffusion applied at t=8]]<br />
<br />
===Activation of genes===<br />
Simplification of the Hill kinetics allowed for modeling of gene activation through simple sigmoidal function:<br />
<br />
[[Image:Groningen-Sigmoid-Model.png]]<br />
<br />
Imputing every element of P' into the function shows which genes are activated. Binarization of the responses allowed to color the activated cells in the original matrix C. Results are discussed below.<br />
<br />
===Results===<br />
The results of the model are shown below in simplified form. Download the interactive code and the [http://www.wolfram.com/products/player/ Mathematica 7 viewer] to play interactively with the model. The cells which have the gene expressed are shown in red. <br />
[[Image:Groningen-Biofilm3.png|150px|frame|none|Early time step with high expression threshold]]<br />
<br />
<br />
[[Image:Groningen-Biofilm1.png|150px|frame|none|Later time step with high expression threshold, some expression shown]]<br />
<br />
<br />
[[Image:Groningen-biofilm2.png|150px|frame|none|Late time step with high expression threshold, cell differentiation clearly visible]]<br />
<br />
<br />
[[Image:Groningen-biofilm4.png|150px|frame|none|Late time step with high expression threshold and high diffusion rate]]<br />
<br />
<br />
Animation of expression based on high threshold and low signaling molecule diffusion.<br />
<br />
[[Image:ExpressionTimeFramed.gif]]<br />
<br />
===Future work=== <br />
The model proposed here is a grave oversimplification and many enhancements can be made. First of all the growth of the biofilm is unrestricted by substance availability Loosdrecht et. al. proposed a more realistic model of biofilm formation taking into account oxygen and substance concentrations. The pheromone is diffused discretely, however a larger resolution would be needed to differentiate between cellular and molecular scale. Implementing the Heat equation would increase the resolution and allow for a more realistic diffusion model. It is now assumed that the pheromone concentrations remain stable over time, however to fully account for its presence substance degradation by extracellular proteins would also need to be modeled. <br />
<br />
A more realistic model of [https://2010.igem.org/Team:Groningen#/expression_model gene expression] could be incorporated further increasing its realism by taking into account within-cell dynamics. Also the ''B.subtilis'' quorum sensing system does not rely on a single compound, many different molecules diffuse and influence cell behavior. Sporulation for example is known to be a competitive inhibitory system between CSF and ComX.<br />
<br />
===Sources===<br />
The Mathematica 7 source files are available below:<br />
<br />
Biofilm model ([http://dl.dropbox.com/u/391356/iGEM/biofilm.nb source])(interactive)<br><br />
2D Heat equation ([http://dl.dropbox.com/u/391356/iGEM/diff.nb source])<br><br />
Sigmoid response curve ([http://dl.dropbox.com/u/391356/iGEM/hillSimpl.nb source])([http://dl.dropbox.com/u/391356/iGEM/hillSimpl.nbp interactive])<br />
<br />
<br />
===References===<br />
<small>Comella N, Grossman AD, Conservation of genes and processes controlled by the quorum response in bacteria: characterization of genes controlled by the quorum-sensing transcription factor ComA in Bacillus subtilis, Molecular Microbiology (2005), 57 (4): 1159–1174. PMID 16091051 <br />
<br />
Lemon KP, Earl AM, Vlamakis HC, Aguilar C, Kolter R., Biofilm development with an emphasis on Bacillus subtilis, Current Topics Microbiology and Immunology, 2008, 322:1-16. PMID 18453269 <br />
<br />
López D, Vlamakis H, Kolter R, Biofilms, Cold Spring Harbor Perspectives in Biology, 2010, (2):a000398. PMID 20519345 <br />
<br />
López D, Vlamakis H, Losick R, Kolter R, Paracrine signaling in a bacterium, Genes & Development, 2009 23(14):1631-8. <br />
<br />
Pérez J, Picioreanu C, van Loosdrecht M, Modeling biofilm and floc diffusion processes based on analytical solution of reaction-diffusion equations, Water Research, 2005, 39:1311-1323.<br />
<br />
Picioreanu C, van Loosdrecht MC, Heijnen JJ. A new combined differential-discrete cellular automaton approach for biofilm modeling: application for growth in gel beads, Biotechnology Bioengineering, 1998, 57(6):718-31. PMID 10099251<br />
<br />
Picioreanu C, van Loosdrecht MC, Heijnen JJ. Mathematical modeling of biofilm structure with a hybrid differential-discrete cellular automaton approach, Biotechnology Bioengineering, 1998, 58(1):101-16. PMID <br />
10099266 <br />
<br />
Roggiani M, Dubnau D, ComA, a phosphorylated response regulator protein of Bacillus subtilis, binds to the promoter region of srfA, Journal of Bacteriology, 1993, 175(10): 3182-7. PMID 8387999 <br />
<br />
Shah IM, Dworkin J, Microbial interactions: bacteria talk to (some of) their neighbors, Current Biology, 2009, 19(16): 689-91. PMID 19706277</small></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/Biofilm_modelTeam:Groningen/Biofilm model2010-11-17T01:50:07Z<p>Joelkuiper: /* Results */</p>
<hr />
<div>__NOTOC__<br />
==Biofilm Dynamics==<br />
<br />
===Introduction===<br />
Biofilms are multicellular conglomerates which attach to surfaces. The formation of biofilms is triggered by high cell density and limited resources. The sensing is of these conditions is often mediated by an extracellular signaling compound which increases in concentration and triggers regulating circuitry. This process is called quorum sensing and it plays an important role in the dynamics of multicellular systems. <br />
<br />
Quorum sensing systems can cause complicated effects including cell differentiation within single species conglomerates. A recent example of this can be seen in the difference of expression in the [http://subtiwiki.uni-goettingen.de/wiki/index.php/TasA TasA protein] which plays a major role in the formation of the biofilm matrix by forming amyloid fibers. Expression of this protein is mediated by competitive inhibitive systems by, amongst others, the [http://subtiwiki.uni-goettingen.de/wiki/index.php/YqxM yqxM gene](<partinfo>BBa_K305006</partinfo>) which linked to the [https://2010.igem.org/Team:Groningen#/expression_model ComXPA quorum sensing] system. <br />
<br />
However it has been found that not all bacteria respond to the signaling molecules in the same way. It was shown that biofilm formation in ''Bacillus subtilis'' involves paracrine signaling in which most cells produce and secrete the signaling molecule ComX but only a sub population of the cells is triggered to make surfactin. Surfactin serves as paracrine signaling molecule and the cells which are not able to make surfactin, respond to surfactin by making extracellular matrix components for the biofilm. <br />
<br />
It is hypothesized that the extracellular matrix interferes with the interaction between ComX and the trans membrane histidine kinase ComP and therefore prevents surfactin production in extracellular matrix producing cells. In a mutant which did not express extracellular matrix proteins, the surfactin expression was more than three times higher. <br />
<br />
While differentiation requires interaction between several genes and complicated effects as described above we believe even simple extra-cellular accumulation of a pheromone can be responsible. <br />
<br />
A very simple model is proposed based on 2D growth of bacteria by cellular automata on a restricted plane. Each of the cells (a pixel in the model) secretes an unnamed compound which is allowed to accumulate. The accumulated compound is diffused at each discrete time step by applying a Gaussian Kernel. Finally at each cell a sigmoidal response curve is mapped, simplifying an all-or-nothing response similar to signal amplification through auto-phosphorylation of transmembrane histidine kinases. Cell differentiation was demonstrated within this theoretical model and variation of the parameters change the response drastically.<br />
<br />
===Growth by cellular automata===<br />
Cellular Automata are discrete computational models. The model consist of a finite grid (of any dimension) in which each of the cells can be in one of a finite set of states. The model behaves discretely in time by evaluating the number of neighbors of each cell and updating its state by some algebraic rule. A popular example of such a model with only two possible states, on and off, and a 2 dimensional grid is Conways Game of life. A possible instantiation of this "game" (it is a zero player game in the sense that it plays itself) is shown below. <br />
<br />
[[Image:Groningen-wiki-Gospers_glider_gun.gif|frame|none|Conways Game of life(from [http://en.wikipedia.org/wiki/Conway_game_of_life wikipedia])]]<br />
<br />
Simpler one dimensional models are an important subject of study in theoretical computer science. We modeled unrestricted bacterial growth by Cellular Automata. The model is a 2D grid of 400x400 pixels, the states are on and off indicating the presence of biomass. The grid is initialized randomly with a fixed distribution of "on" states. At every time step the following rule is applied by evaluating each of the cells 8 neighbors, if a cell has 3 or 4 neighbors with the "on" state the cell itself becomes "on" (a growth step, or birth), if a cell has 4, 5, or 6 neighbors which are on the cell is allowed to live and thus remains on. If any cell does not meet these criteria the cell will remain or become off. An animation of this model is shown below.<br />
<br />
[[Image:Groningen-CAModel.gif]]<br />
<br />
Due to the random initialization the first time steps are trimmed to allow for the distribution to stabilize. The result of the simulation is a binary matrix C for each time step representing biomass occupation. It can be shown by summing the total amount of occupied cell increases exponentially at each step and finally reaching a stationary phase because of spatial limitations.<br />
<br />
[[Image:Groningen-model-cellDensity.png|300px]]<br />
<br />
===Quorum sensing pheromones===<br />
Quorum sensing pheromones are modeled by sampling a random number from the normal distribution for each cell which in on resulting in a new matrix P. Because the sensing pheromones are allowed to accumulate each next time step is the sum of the previous matrices. Quorum sensing requires diffusion of the pheromone from one occupied cell to another. Plotting the density of pheromone gives the following graph:<br />
<br />
[[Image:Groningen-model-subsNoDiff.png]]<br />
<br />
Continuous diffusion can be modeled using the Heat equation, a two dimensional Partial Differential Equation (PDE) as shown below. Fick's second law is a a common biological and chemical model and is analogous to the two-dimensional Heat equation. This would generate a discrete-differential model.<br />
<br />
[[Image:Groningen-heat-model.png]]<br />
<br />
Solving this equation allows for diffusion continuous in both space and time. However for simplicity we applied a discrete Gaussian filter, equivalent to a convoluting a Gaussian matrix to our substance matrix P generating P'. The Gaussian Filter in 2D has the following form: <br />
<br />
[[Image:Groningen-GaussianFilter.png]]<br />
<br />
Where &sigma; is the standard deviation. The result of this process with &sigma; = 3 is demonstrated at a single time step below: <br />
<br />
[[Image:Groningen-model-subsDiff.png|frame|none|Diffusion applied at t=8]]<br />
<br />
===Activation of genes===<br />
Simplification of the Hill kinetics allowed for modeling of gene activation through simple sigmoidal function:<br />
<br />
[[Image:Groningen-Sigmoid-Model.png]]<br />
<br />
Imputing every element of P' into the function shows which genes are activated. Binarization of the responses allowed to color the activated cells in the original matrix C. Results are discussed below.<br />
<br />
===Results===<br />
The results of the model are shown below in simplified form. Download the interactive code and the [http://www.wolfram.com/products/player/ Mathematica 7 viewer] to play interactively with the model. The cells which have the gene expressed are shown in red. <br />
[[Image:Groningen-Biofilm3.png|150px|frame|none|Early time step with high expression threshold]]<br />
<br />
<br />
[[Image:Groningen-Biofilm1.png|150px|frame|none|Later time step with high expression threshold, some expression shown]]<br />
<br />
<br />
[[Image:Groningen-biofilm2.png|150px|frame|none|Late time step with high expression threshold, cell differentiation clearly visible]]<br />
<br />
<br />
[[Image:Groningen-biofilm4.png|150px|frame|none|Late time step with high expression threshold and high diffusion rate]]<br />
<br />
<br />
Animation of expression based on high threshold en low diffusion<br />
<br />
[[Image:ExpressionTimeFramed.gif]]<br />
<br />
===Future work=== <br />
The model proposed here is a grave oversimplification and many enhancements can be made. First of all the growth of the biofilm is unrestricted by substance availability Loosdrecht et. al. proposed a more realistic model of biofilm formation taking into account oxygen and substance concentrations. The pheromone is diffused discretely, however a larger resolution would be needed to differentiate between cellular and molecular scale. Implementing the Heat equation would increase the resolution and allow for a more realistic diffusion model. It is now assumed that the pheromone concentrations remain stable over time, however to fully account for its presence substance degradation by extracellular proteins would also need to be modeled. <br />
<br />
A more realistic model of [https://2010.igem.org/Team:Groningen#/expression_model gene expression] could be incorporated further increasing its realism by taking into account within-cell dynamics. Also the ''B.subtilis'' quorum sensing system does not rely on a single compound, many different molecules diffuse and influence cell behavior. Sporulation for example is known to be a competitive inhibitory system between CSF and ComX.<br />
<br />
===Sources===<br />
The Mathematica 7 source files are available below:<br />
<br />
Biofilm model ([http://dl.dropbox.com/u/391356/iGEM/biofilm.nb source])(interactive)<br><br />
2D Heat equation ([http://dl.dropbox.com/u/391356/iGEM/diff.nb source])<br><br />
Sigmoid response curve ([http://dl.dropbox.com/u/391356/iGEM/hillSimpl.nb source])([http://dl.dropbox.com/u/391356/iGEM/hillSimpl.nbp interactive])<br />
<br />
<br />
===References===<br />
<small>Comella N, Grossman AD, Conservation of genes and processes controlled by the quorum response in bacteria: characterization of genes controlled by the quorum-sensing transcription factor ComA in Bacillus subtilis, Molecular Microbiology (2005), 57 (4): 1159–1174. PMID 16091051 <br />
<br />
Lemon KP, Earl AM, Vlamakis HC, Aguilar C, Kolter R., Biofilm development with an emphasis on Bacillus subtilis, Current Topics Microbiology and Immunology, 2008, 322:1-16. PMID 18453269 <br />
<br />
López D, Vlamakis H, Kolter R, Biofilms, Cold Spring Harbor Perspectives in Biology, 2010, (2):a000398. PMID 20519345 <br />
<br />
López D, Vlamakis H, Losick R, Kolter R, Paracrine signaling in a bacterium, Genes & Development, 2009 23(14):1631-8. <br />
<br />
Pérez J, Picioreanu C, van Loosdrecht M, Modeling biofilm and floc diffusion processes based on analytical solution of reaction-diffusion equations, Water Research, 2005, 39:1311-1323.<br />
<br />
Picioreanu C, van Loosdrecht MC, Heijnen JJ. A new combined differential-discrete cellular automaton approach for biofilm modeling: application for growth in gel beads, Biotechnology Bioengineering, 1998, 57(6):718-31. PMID 10099251<br />
<br />
Picioreanu C, van Loosdrecht MC, Heijnen JJ. Mathematical modeling of biofilm structure with a hybrid differential-discrete cellular automaton approach, Biotechnology Bioengineering, 1998, 58(1):101-16. PMID <br />
10099266 <br />
<br />
Roggiani M, Dubnau D, ComA, a phosphorylated response regulator protein of Bacillus subtilis, binds to the promoter region of srfA, Journal of Bacteriology, 1993, 175(10): 3182-7. PMID 8387999 <br />
<br />
Shah IM, Dworkin J, Microbial interactions: bacteria talk to (some of) their neighbors, Current Biology, 2009, 19(16): 689-91. PMID 19706277</small></div>Joelkuiperhttp://2010.igem.org/File:ExpressionTimeFramed.gifFile:ExpressionTimeFramed.gif2010-11-17T01:49:21Z<p>Joelkuiper: </p>
<hr />
<div></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/Biofilm_modelTeam:Groningen/Biofilm model2010-11-17T01:44:05Z<p>Joelkuiper: /* References */</p>
<hr />
<div>__NOTOC__<br />
==Biofilm Dynamics==<br />
<br />
===Introduction===<br />
Biofilms are multicellular conglomerates which attach to surfaces. The formation of biofilms is triggered by high cell density and limited resources. The sensing is of these conditions is often mediated by an extracellular signaling compound which increases in concentration and triggers regulating circuitry. This process is called quorum sensing and it plays an important role in the dynamics of multicellular systems. <br />
<br />
Quorum sensing systems can cause complicated effects including cell differentiation within single species conglomerates. A recent example of this can be seen in the difference of expression in the [http://subtiwiki.uni-goettingen.de/wiki/index.php/TasA TasA protein] which plays a major role in the formation of the biofilm matrix by forming amyloid fibers. Expression of this protein is mediated by competitive inhibitive systems by, amongst others, the [http://subtiwiki.uni-goettingen.de/wiki/index.php/YqxM yqxM gene](<partinfo>BBa_K305006</partinfo>) which linked to the [https://2010.igem.org/Team:Groningen#/expression_model ComXPA quorum sensing] system. <br />
<br />
However it has been found that not all bacteria respond to the signaling molecules in the same way. It was shown that biofilm formation in ''Bacillus subtilis'' involves paracrine signaling in which most cells produce and secrete the signaling molecule ComX but only a sub population of the cells is triggered to make surfactin. Surfactin serves as paracrine signaling molecule and the cells which are not able to make surfactin, respond to surfactin by making extracellular matrix components for the biofilm. <br />
<br />
It is hypothesized that the extracellular matrix interferes with the interaction between ComX and the trans membrane histidine kinase ComP and therefore prevents surfactin production in extracellular matrix producing cells. In a mutant which did not express extracellular matrix proteins, the surfactin expression was more than three times higher. <br />
<br />
While differentiation requires interaction between several genes and complicated effects as described above we believe even simple extra-cellular accumulation of a pheromone can be responsible. <br />
<br />
A very simple model is proposed based on 2D growth of bacteria by cellular automata on a restricted plane. Each of the cells (a pixel in the model) secretes an unnamed compound which is allowed to accumulate. The accumulated compound is diffused at each discrete time step by applying a Gaussian Kernel. Finally at each cell a sigmoidal response curve is mapped, simplifying an all-or-nothing response similar to signal amplification through auto-phosphorylation of transmembrane histidine kinases. Cell differentiation was demonstrated within this theoretical model and variation of the parameters change the response drastically.<br />
<br />
===Growth by cellular automata===<br />
Cellular Automata are discrete computational models. The model consist of a finite grid (of any dimension) in which each of the cells can be in one of a finite set of states. The model behaves discretely in time by evaluating the number of neighbors of each cell and updating its state by some algebraic rule. A popular example of such a model with only two possible states, on and off, and a 2 dimensional grid is Conways Game of life. A possible instantiation of this "game" (it is a zero player game in the sense that it plays itself) is shown below. <br />
<br />
[[Image:Groningen-wiki-Gospers_glider_gun.gif|frame|none|Conways Game of life(from [http://en.wikipedia.org/wiki/Conway_game_of_life wikipedia])]]<br />
<br />
Simpler one dimensional models are an important subject of study in theoretical computer science. We modeled unrestricted bacterial growth by Cellular Automata. The model is a 2D grid of 400x400 pixels, the states are on and off indicating the presence of biomass. The grid is initialized randomly with a fixed distribution of "on" states. At every time step the following rule is applied by evaluating each of the cells 8 neighbors, if a cell has 3 or 4 neighbors with the "on" state the cell itself becomes "on" (a growth step, or birth), if a cell has 4, 5, or 6 neighbors which are on the cell is allowed to live and thus remains on. If any cell does not meet these criteria the cell will remain or become off. An animation of this model is shown below.<br />
<br />
[[Image:Groningen-CAModel.gif]]<br />
<br />
Due to the random initialization the first time steps are trimmed to allow for the distribution to stabilize. The result of the simulation is a binary matrix C for each time step representing biomass occupation. It can be shown by summing the total amount of occupied cell increases exponentially at each step and finally reaching a stationary phase because of spatial limitations.<br />
<br />
[[Image:Groningen-model-cellDensity.png|300px]]<br />
<br />
===Quorum sensing pheromones===<br />
Quorum sensing pheromones are modeled by sampling a random number from the normal distribution for each cell which in on resulting in a new matrix P. Because the sensing pheromones are allowed to accumulate each next time step is the sum of the previous matrices. Quorum sensing requires diffusion of the pheromone from one occupied cell to another. Plotting the density of pheromone gives the following graph:<br />
<br />
[[Image:Groningen-model-subsNoDiff.png]]<br />
<br />
Continuous diffusion can be modeled using the Heat equation, a two dimensional Partial Differential Equation (PDE) as shown below. Fick's second law is a a common biological and chemical model and is analogous to the two-dimensional Heat equation. This would generate a discrete-differential model.<br />
<br />
[[Image:Groningen-heat-model.png]]<br />
<br />
Solving this equation allows for diffusion continuous in both space and time. However for simplicity we applied a discrete Gaussian filter, equivalent to a convoluting a Gaussian matrix to our substance matrix P generating P'. The Gaussian Filter in 2D has the following form: <br />
<br />
[[Image:Groningen-GaussianFilter.png]]<br />
<br />
Where &sigma; is the standard deviation. The result of this process with &sigma; = 3 is demonstrated at a single time step below: <br />
<br />
[[Image:Groningen-model-subsDiff.png|frame|none|Diffusion applied at t=8]]<br />
<br />
===Activation of genes===<br />
Simplification of the Hill kinetics allowed for modeling of gene activation through simple sigmoidal function:<br />
<br />
[[Image:Groningen-Sigmoid-Model.png]]<br />
<br />
Imputing every element of P' into the function shows which genes are activated. Binarization of the responses allowed to color the activated cells in the original matrix C. Results are discussed below.<br />
<br />
===Results===<br />
The results of the model are shown below in simplified form. Download the interactive code and the [http://www.wolfram.com/products/player/ Mathematica 7 viewer] to play interactively with the model. The cells which have the gene expressed are shown in red. <br />
[[Image:Groningen-Biofilm3.png|150px|frame|none|Early time step with high expression threshold]]<br />
<br />
<br />
[[Image:Groningen-Biofilm1.png|150px|frame|none|Later time step with high expression threshold, some expression shown]]<br />
<br />
<br />
[[Image:Groningen-biofilm2.png|150px|frame|none|Late time step with high expression threshold, cell differentiation clearly visible]]<br />
<br />
<br />
[[Image:Groningen-biofilm4.png|150px|frame|none|Late time step with high expression threshold and high diffusion rate]]<br />
<br />
===Future work=== <br />
The model proposed here is a grave oversimplification and many enhancements can be made. First of all the growth of the biofilm is unrestricted by substance availability Loosdrecht et. al. proposed a more realistic model of biofilm formation taking into account oxygen and substance concentrations. The pheromone is diffused discretely, however a larger resolution would be needed to differentiate between cellular and molecular scale. Implementing the Heat equation would increase the resolution and allow for a more realistic diffusion model. It is now assumed that the pheromone concentrations remain stable over time, however to fully account for its presence substance degradation by extracellular proteins would also need to be modeled. <br />
<br />
A more realistic model of [https://2010.igem.org/Team:Groningen#/expression_model gene expression] could be incorporated further increasing its realism by taking into account within-cell dynamics. Also the ''B.subtilis'' quorum sensing system does not rely on a single compound, many different molecules diffuse and influence cell behavior. Sporulation for example is known to be a competitive inhibitory system between CSF and ComX.<br />
<br />
===Sources===<br />
The Mathematica 7 source files are available below:<br />
<br />
Biofilm model ([http://dl.dropbox.com/u/391356/iGEM/biofilm.nb source])(interactive)<br><br />
2D Heat equation ([http://dl.dropbox.com/u/391356/iGEM/diff.nb source])<br><br />
Sigmoid response curve ([http://dl.dropbox.com/u/391356/iGEM/hillSimpl.nb source])([http://dl.dropbox.com/u/391356/iGEM/hillSimpl.nbp interactive])<br />
<br />
<br />
===References===<br />
<small>Comella N, Grossman AD, Conservation of genes and processes controlled by the quorum response in bacteria: characterization of genes controlled by the quorum-sensing transcription factor ComA in Bacillus subtilis, Molecular Microbiology (2005), 57 (4): 1159–1174. PMID 16091051 <br />
<br />
Lemon KP, Earl AM, Vlamakis HC, Aguilar C, Kolter R., Biofilm development with an emphasis on Bacillus subtilis, Current Topics Microbiology and Immunology, 2008, 322:1-16. PMID 18453269 <br />
<br />
López D, Vlamakis H, Kolter R, Biofilms, Cold Spring Harbor Perspectives in Biology, 2010, (2):a000398. PMID 20519345 <br />
<br />
López D, Vlamakis H, Losick R, Kolter R, Paracrine signaling in a bacterium, Genes & Development, 2009 23(14):1631-8. <br />
<br />
Pérez J, Picioreanu C, van Loosdrecht M, Modeling biofilm and floc diffusion processes based on analytical solution of reaction-diffusion equations, Water Research, 2005, 39:1311-1323.<br />
<br />
Picioreanu C, van Loosdrecht MC, Heijnen JJ. A new combined differential-discrete cellular automaton approach for biofilm modeling: application for growth in gel beads, Biotechnology Bioengineering, 1998, 57(6):718-31. PMID 10099251<br />
<br />
Picioreanu C, van Loosdrecht MC, Heijnen JJ. Mathematical modeling of biofilm structure with a hybrid differential-discrete cellular automaton approach, Biotechnology Bioengineering, 1998, 58(1):101-16. PMID <br />
10099266 <br />
<br />
Roggiani M, Dubnau D, ComA, a phosphorylated response regulator protein of Bacillus subtilis, binds to the promoter region of srfA, Journal of Bacteriology, 1993, 175(10): 3182-7. PMID 8387999 <br />
<br />
Shah IM, Dworkin J, Microbial interactions: bacteria talk to (some of) their neighbors, Current Biology, 2009, 19(16): 689-91. PMID 19706277</small></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/BiofilmTeam:Groningen/Biofilm2010-11-17T01:41:00Z<p>Joelkuiper: </p>
<hr />
<div>__NOTOC__<br />
==Biofilm==<br />
<br />
===Summary===<br />
<br />
In our project we want our host bacterium to not only produce the coating material, but also apply it. Therefore we chose ''Bacillus subtilis'' as our host bacterium. ''B. subtilis'' can form a rigid biofilm that will cover the target surface before producing the [https://2010.igem.org/Team:Groningen#/hydrophobins hydrophobic proteins]. As part of our project we made a [https://2010.igem.org/Team:Groningen#/biofilm_model model] on the biofilmformation, but furthermore we looked into ways to easily apply ''B. subtilis'' to the surface and let it form a biofilm there. One way to do this is by adding corn starch to regular TY-medium, making it an easily applicable paste.<br />
<br />
<br />
===Introduction===<br />
<html><br />
<div style="text-align: justify"><br />
</html><br />
[[Image:Structure.jpg|right|350px|''B. sub'' Rok biofilm]]<br />
Using biobased materials in the application or manufacturing of coatings has been the topic of many researches. However, using bacteria to make a coating substance and, most importantly, letting it do the coating process for you is something new. In our hydrophobofilm project we aim to use the extracellular fibrous proteins, DNA and polysaccharides that are formed in a biofilm, as a host matrix to embed our coating material, which in our case are hydrophobic proteins. <br />
<br />
Growing a biofilm on a surface as a way of coating it, might seem like a bad idea, since there are quite a lot of coatings out there to prevent biofilms forming in the first place. But why not "fight fire with fire”, and create a biofilm that is non-pathogenic and prevents other biofouling from taking place. <br />
<br />
''Bacillus subtilis'' is an ideal candidate for a biofilm coating. Firstly because it is quickly grows a biofilm which has a smooth extracellular matrix. Secondly, the bacterium is a well known and extensively studied model organism which makes is easier to work with. Finally ''B. subtilis'' is a gram-positive bacterium like ''Streptomyces coelicolor'', the bacterium that naturally produces hydrophobins. This might be an advantage when expressing and assembling the chaplin proteins in our host.<br />
<br />
<br><br />
<br />
===Biology===<br />
<br><br />
''In nature, bacteria occur predominantly in highly organized multicellular communities called biofilms. Biofilm formation involves a complex developmental process, where cells differ from each other spatially and morphologically. The bacterial cells in biofilms are phenotypically different, demonstrating an intriguing example of heterogeneous regulation within an isogenic culture. Gram-positive bacteria have developed different strategies for survival in unfavorable environments, e.g. by getting competent or by sporulating. Biofilms offer an opportunity for the cells to survive extreme conditions as the cells in biofilms are more resistant to antibiotics and other harsh circumstances like physical stress, drought or competing organisms. ''Bacillus'' even forms highly complex biofilms with a large degree of structural complexity and diversification of cell function within the biofilm. There are even channels within the biofilm to allow drainage of waste and diffusion of oxygen deep within the biofilm.(''Akos Kovacs)<br />
<br />
<br><br />
<br />
===Biofilm formation===[[Image:strain rok.jpg|right|500px]]<br />
Biofilm formation usually starts with the accumulation of biomass, next there is the adhesion to a surface by the production of adhesion proteins. Then the production of "extracellular polymeric substances" (EPS) starts and the phenotypic diversification. After maturation of the biofilm sporulation kicks in. Since the pathways involved in biofilm formation in ''B. subtilis'' are just starting to be unravelled, not everything is known about the complex physiological interactions within a biofilm. By using an already existing pathway in ''B. subtilis'' for the auto-induction of our hydrophobic proteins, we try to minimize the amount of tinkering to the existing signaling pathways. Thereby leaving the natural system intact. <br />
<br />
Timing is one the most important factors in successful assembly of our chaplins in EPS. <br />
''B. subtilis'' produces a protein that forms amyloidfibers called TasA. TasA is a very important protein to provide structural integrity in ''B. subtilis'' biofilms and is formed in the late stage of biofilm formation. The amyloid fibers that are formed provide the biofilm with an increased degree of rigidity (Romero et al, 2009). [https://2010.igem.org/Team:Groningen#/hydrophobins Chaplins] also assemble into amyloid fibers and provide a similar function in the hyphae of ''S. coelicolor'' (Cleassen et al, 2009), giving the hyphae the structural ability to grow high up in the air. Incorperating the chaplins at the same moment as TasA is formed would maximize the chance of successful assembly of chaplins in the EPS, while enabling maximum biofilm coverage. For more details on our expression pathway check out our [https://2010.igem.org/Team:Groningen#/expression expression] or [https://2010.igem.org/Team:Groningen#/modeling modeling] page. <br />
<br />
<br><br />
[[Image:agar TY corn starch.jpg|right|300px]]<br />
====Coating surfaces===<br />
<br />
Prevention from our biofilm to grow out of control, is an important aspect when you would apply the hydrophobofilm outside the lab. To deal with these <br />
[https://2010.igem.org/Team:Groningen#/safety safety issues] we modelled a [https://2010.igem.org/Team:Groningen#/killswitch_model kill switch] for our hydrophobofilm. This kill switch relies on the production of a toxin and anti toxin. Where the anti toxin has a slightly shorter half-life than the toxin, thereby eventually resulting in the toxification of the cell itself. This toxification would occur after maturation of the biofilm. After the autotoxification the cells, the EPS with the embedded chaplin proteins will dry out, leaving a hydrophobic EPS layer on the surface.<br />
<br />
<br><br />
<br />
[[Image:biofilm on ceramics.jpg|left|200px]]<br />
Applying our bacteria effectively to a surface poses big challenges. such as, how to coat a surface in a short period of time, with low cost and low tech methods. Furthermore there must be enough nutrients for the organisms to successfully form a biofilm, yet you do not want to smear you surface in to much medium, so to avoid that the organism will only adhere to the medium and not to the surface itself. <br />
<br />
<br><br />
<br />
[[Image:biofilm ceramics total.jpg|right|ceramics]]<br />
<br />
''Biofilm paste''<br />
We attempted to make a medium that could be easily applied to a surface and enable biofilm formation to take place. To achieve this we tried to make our medium more viscous. By adding corn starch to regular TY medium we increased the viscosity of our medium and also made it richer in nutrients. We [https://2010.igem.org/Team:Groningen/20_September_2010 experimented] with different corn starch concentrations. <br />
<br />
We have created an easily applicable paste, to grow our biofilmcoating on all kinds of different surfaces. Another effect of the addition of cornstarch to the medium is an increased growing speed.<br />
<br />
<br><br />
<br />
''A & B: B. subtilis biofilms grown overnight on ceramics coated with the biofilm paste. C: B subtilis biofilms dried out over four days, after formation.''<br />
<br />
<br><br />
<br />
==References==<br />
<small><br />
1. A. Kovacs, Elucidation of the molecular mechanisms underlying the phenotypic heterogeneity of Bacillus subtilis in biofilms<br />
<br />
2. Romero et al, 2009, Amyloid Fibers Provide Structural Integrity to Bacillus<br />
subtilis Biolms<br />
<br />
3. Dennis Claessen, Rick Rink, Wouter de Jong, et al, 2009, A novel class of secreted hydrophobic proteins is involved in aerial hyphae formation in Streptomyces coelicolor by forming amyloid-like fibrils<br />
<br />
</small></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/BiofilmTeam:Groningen/Biofilm2010-11-17T01:40:24Z<p>Joelkuiper: </p>
<hr />
<div>__NOTOC__<br />
<br />
==Biofilm==<br />
<br />
===Summary===<br />
<br />
In our project we want our host bacterium to not only produce the coating material, but also apply it. Therefore we chose ''Bacillus subtilis'' as our host bacterium. ''B. subtilis'' can form a rigid biofilm that will cover the target surface before producing the [https://2010.igem.org/Team:Groningen#/hydrophobins hydrophobic proteins]. As part of our project we made a [https://2010.igem.org/Team:Groningen#/biofilm_model model] on the biofilmformation, but furthermore we looked into ways to easily apply ''B. subtilis'' to the surface and let it form a biofilm there. One way to do this is by adding corn starch to regular TY-medium, making it an easily applicable paste.<br />
<br />
<br />
===Introduction===<br />
<html><br />
<div style="text-align: justify"><br />
</html><br />
[[Image:Structure.jpg|right|350px|''B. sub'' Rok biofilm]]<br />
Using biobased materials in the application or manufacturing of coatings has been the topic of many researches. However, using bacteria to make a coating substance and, most importantly, letting it do the coating process for you is something new. In our hydrophobofilm project we aim to use the extracellular fibrous proteins, DNA and polysaccharides that are formed in a biofilm, as a host matrix to embed our coating material, which in our case are hydrophobic proteins. <br />
<br />
Growing a biofilm on a surface as a way of coating it, might seem like a bad idea, since there are quite a lot of coatings out there to prevent biofilms forming in the first place. But why not "fight fire with fire”, and create a biofilm that is non-pathogenic and prevents other biofouling from taking place. <br />
<br />
''Bacillus subtilis'' is an ideal candidate for a biofilm coating. Firstly because it is quickly grows a biofilm which has a smooth extracellular matrix. Secondly, the bacterium is a well known and extensively studied model organism which makes is easier to work with. Finally ''B. subtilis'' is a gram-positive bacterium like ''Streptomyces coelicolor'', the bacterium that naturally produces hydrophobins. This might be an advantage when expressing and assembling the chaplin proteins in our host.<br />
<br />
<br><br />
<br />
===Biology===<br />
<br><br />
''In nature, bacteria occur predominantly in highly organized multicellular communities called biofilms. Biofilm formation involves a complex developmental process, where cells differ from each other spatially and morphologically. The bacterial cells in biofilms are phenotypically different, demonstrating an intriguing example of heterogeneous regulation within an isogenic culture. Gram-positive bacteria have developed different strategies for survival in unfavorable environments, e.g. by getting competent or by sporulating. Biofilms offer an opportunity for the cells to survive extreme conditions as the cells in biofilms are more resistant to antibiotics and other harsh circumstances like physical stress, drought or competing organisms. ''Bacillus'' even forms highly complex biofilms with a large degree of structural complexity and diversification of cell function within the biofilm. There are even channels within the biofilm to allow drainage of waste and diffusion of oxygen deep within the biofilm.(''Akos Kovacs)<br />
<br />
<br><br />
<br />
===Biofilm formation===[[Image:strain rok.jpg|right|500px]]<br />
Biofilm formation usually starts with the accumulation of biomass, next there is the adhesion to a surface by the production of adhesion proteins. Then the production of "extracellular polymeric substances" (EPS) starts and the phenotypic diversification. After maturation of the biofilm sporulation kicks in. Since the pathways involved in biofilm formation in ''B. subtilis'' are just starting to be unravelled, not everything is known about the complex physiological interactions within a biofilm. By using an already existing pathway in ''B. subtilis'' for the auto-induction of our hydrophobic proteins, we try to minimize the amount of tinkering to the existing signaling pathways. Thereby leaving the natural system intact. <br />
<br />
Timing is one the most important factors in successful assembly of our chaplins in EPS. <br />
''B. subtilis'' produces a protein that forms amyloidfibers called TasA. TasA is a very important protein to provide structural integrity in ''B. subtilis'' biofilms and is formed in the late stage of biofilm formation. The amyloid fibers that are formed provide the biofilm with an increased degree of rigidity (Romero et al, 2009). [https://2010.igem.org/Team:Groningen#/hydrophobins Chaplins] also assemble into amyloid fibers and provide a similar function in the hyphae of ''S. coelicolor'' (Cleassen et al, 2009), giving the hyphae the structural ability to grow high up in the air. Incorperating the chaplins at the same moment as TasA is formed would maximize the chance of successful assembly of chaplins in the EPS, while enabling maximum biofilm coverage. For more details on our expression pathway check out our [https://2010.igem.org/Team:Groningen#/expression expression] or [https://2010.igem.org/Team:Groningen#/modeling modeling] page. <br />
<br />
<br><br />
[[Image:agar TY corn starch.jpg|right|300px]]<br />
====Coating surfaces===<br />
<br />
Prevention from our biofilm to grow out of control, is an important aspect when you would apply the hydrophobofilm outside the lab. To deal with these <br />
[https://2010.igem.org/Team:Groningen#/safety safety issues] we modelled a [https://2010.igem.org/Team:Groningen#/killswitch_model kill switch] for our hydrophobofilm. This kill switch relies on the production of a toxin and anti toxin. Where the anti toxin has a slightly shorter half-life than the toxin, thereby eventually resulting in the toxification of the cell itself. This toxification would occur after maturation of the biofilm. After the autotoxification the cells, the EPS with the embedded chaplin proteins will dry out, leaving a hydrophobic EPS layer on the surface.<br />
<br />
<br><br />
<br />
[[Image:biofilm on ceramics.jpg|left|200px]]<br />
Applying our bacteria effectively to a surface poses big challenges. such as, how to coat a surface in a short period of time, with low cost and low tech methods. Furthermore there must be enough nutrients for the organisms to successfully form a biofilm, yet you do not want to smear you surface in to much medium, so to avoid that the organism will only adhere to the medium and not to the surface itself. <br />
<br />
<br><br />
<br />
[[Image:biofilm ceramics total.jpg|right|ceramics]]<br />
<br />
''Biofilm paste''<br />
We attempted to make a medium that could be easily applied to a surface and enable biofilm formation to take place. To achieve this we tried to make our medium more viscous. By adding corn starch to regular TY medium we increased the viscosity of our medium and also made it richer in nutrients. We [https://2010.igem.org/Team:Groningen/20_September_2010 experimented] with different corn starch concentrations. <br />
<br />
We have created an easily applicable paste, to grow our biofilmcoating on all kinds of different surfaces. Another effect of the addition of cornstarch to the medium is an increased growing speed.<br />
<br />
<br><br />
<br />
''A & B: B. subtilis biofilms grown overnight on ceramics coated with the biofilm paste. C: B subtilis biofilms dried out over four days, after formation.''<br />
<br />
<br><br />
<br />
==References==<br />
<small><br />
1. A. Kovacs, Elucidation of the molecular mechanisms underlying the phenotypic heterogeneity of Bacillus subtilis in biofilms<br />
<br />
2. Romero et al, 2009, Amyloid Fibers Provide Structural Integrity to Bacillus<br />
subtilis Biolms<br />
<br />
3. Dennis Claessen, Rick Rink, Wouter de Jong, et al, 2009, A novel class of secreted hydrophobic proteins is involved in aerial hyphae formation in Streptomyces coelicolor by forming amyloid-like fibrils<br />
<br />
</small></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/HomeTeam:Groningen/Home2010-10-28T01:32:25Z<p>Joelkuiper: </p>
<hr />
<div><html><br />
<div margin-left: 10px"> <br />
<script src="https://2010.igem.org/Team:Groningen/files/slider.js?action=raw&ctype=text/javascript" type="text/javascript"></script><br />
<div style="height: 1330px"><br />
<div id="slider" style="margin-left: 10px; height: 343px;"><br />
<a href="https://2010.igem.org/Team:Groningen#/project"><img src="https://static.igem.org/mediawiki/2010/d/dd/Groningen-Reel-Stage1.jpg" alt="" title="#biofilm-caption" /></a><br />
<a href="https://2010.igem.org/Team:Groningen#/project"><img src="https://static.igem.org/mediawiki/2010/d/d3/Groningen-Reel-Stage2.jpg" alt="" title="#chaplin-caption" /></a><br />
<a href="https://2010.igem.org/Team:Groningen#/project"><img src="https://static.igem.org/mediawiki/2010/6/6f/Groningen-Reel-Stage3.jpg" alt="" title="#killswitch-caption" /></a><br />
</div><br />
<script type="text/javascript"><br />
<br />
$('#slider').nivoSlider({effect:'fade'});<br />
<br />
</script><br />
<div id="biofilm-caption" class="nivo-html-caption"><br />
A self assembling bio-based coating is form, a </html>[https://2010.igem.org/Team:Groningen#/biofilm rigid biofilm]<html>.<br />
</div><br />
<div id="chaplin-caption" class="nivo-html-caption"><br />
</html>[https://2010.igem.org/Team:Groningen#/expression Expression]<html> of hydrophobic proteins called </html>[https://2010.igem.org/Team:Groningen#/hydrophobins chaplins]<html> is induced by the biofilm causing strong surface hydrophobicity. <br />
</div><br />
<div id="killswitch-caption" class="nivo-html-caption"><br />
The strongly hydrophobic biofilm will die off by a </html>[https://2010.igem.org/Team:Groningen#/killswitch_model killswitch]<html>, leaving a nice hydrophobic biological coating. <br />
</div><br />
<br />
</html><br />
==Self assembling hydrophobic biofilm==<br />
We aim to design a biological coating as an alternative to for example chemical coatings. For this we, unconventionally, utilized a ''Bacillus subtilis'' [https://2010.igem.org/Team:Groningen#/biofilm biofilm]. We wanted to enable our biofilm to be equipped with an interesting property which is automatically initiated. So we introduced an [https://2010.igem.org/Team:Groningen#/expression expression trigger] which relies on quorum sensing. Our project was directed at finding an alternate solution to biofouling, since regular, chemical coatings which are widely in use pose a threat to the environment. In nature the lotus leaves show self-cleansing properties ascribed to their extreme surface hydrophobicity. In the prokaryotic domain we stumbled upon [https://2010.igem.org/Team:Groningen#/hydrophobins chaplins], strongly hydrophobic proteins originating from ''Streptomyces coelicolor''. Surface hydrophobicity is a very useful property and is used in many [https://2010.igem.org/Team:Groningen#/applications applications] ranging from not only antifouling coatings but also other applications which require water repellence to applications in the field of medical sciences. During our project we have contributed to the parts registry whit numerous [https://2010.igem.org/Team:Groningen#/biobricks BioBricks].<br />
<br />
<br />
We believe that there is a great future for biological coatings as demonstrated within the project. Hydrophobic biological coatings can provide a greener antifouling solution. However using the underlying mechanisms of biofilm triggered expression for other systems like dynamic painting, sensing of environmental changes or even the integration with silicon chips might be within the realm of possibilities. So we challenge coming iGEM teams to further explore and [https://2010.igem.org/Team:Groningen#/brainstorm brainstorm] about the possibilities of using biofilms as a host for a wide range of applications.<br />
<html><br />
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<a href="https://2010.igem.org/Team:Groningen#/team"><img src="https://static.igem.org/mediawiki/2010/f/f5/Groningen-Home-Team.jpg" alt="Team"></a><br />
<div></html><br />
This year a [https://2010.igem.org/Team:Groningen#/team team] of young inspired undergraduates from the [http://www.rug.nl University of Groningen] participated in the amazing challenge of iGEM. A multi-disciplinary team of Molecular Biologists, Chemists, Computer Scientists, Journalists and others spend the summer creating a wonderful project in the emerging field of synthetic biology<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/modeling"><img src="https://static.igem.org/mediawiki/2010/1/1e/Groningen-Home-Model.jpg" alt="Modeling"></a><div></html><br />
Using computer models we worked on the frontiers of knowledge. [https://2010.igem.org/Team:Groningen#/expression_model Gene expression] was simulated and a simple explanation for [https://2010.igem.org/Team:Groningen#/biofilm_model cell differentiation] was proposed. Also aiding in ethics and practical feasibility a [https://2010.igem.org/Team:Groningen#/killswitch_model kill switch] system was studied. Finally a [https://2010.igem.org/Team:Groningen#/info_standard new standard] was proposed for characterizing Biobrick parts so future can be streamlined.<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/practices"><img src="https://static.igem.org/mediawiki/2010/7/77/Groningen-Home-Human.jpg" alt="Human Practices"></a><div></html><br />
Because we believe that synthetic biology can better the lives of people and ensure long term prosperity for all humans we spend time [https://2010.igem.org/Team:Groningen#/practices educating] high school students. But not all is perfect so [https://2010.igem.org/Team:Groningen#/safety risks] were assessed and we philosophized on the [https://2010.igem.org/Team:Groningen#/ethics ethical] aspects of synthetic biology.<br />
<html></div></div><br />
</div><br />
</html><br />
<br />
----<br />
<br />
=== Our sponsors === <br />
{{Team:Groningen/sponsors}}<br />
<html><br />
</div><br />
</div><br />
</html></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/HomeTeam:Groningen/Home2010-10-28T01:30:54Z<p>Joelkuiper: </p>
<hr />
<div><html><br />
<div margin-left: 10px"> <br />
<script src="https://2010.igem.org/Team:Groningen/files/slider.js?action=raw&ctype=text/javascript" type="text/javascript"></script><br />
<div style="height: 1330px"><br />
<div id="slider" style="margin-left: 10px; height: 343px;"><br />
<a href="https://2010.igem.org/Team:Groningen#/project"><img src="https://static.igem.org/mediawiki/2010/d/dd/Groningen-Reel-Stage1.jpg" alt="" title="#biofilm-caption" /></a><br />
<a href="https://2010.igem.org/Team:Groningen#/project"><img src="https://static.igem.org/mediawiki/2010/d/d3/Groningen-Reel-Stage2.jpg" alt="" title="#chaplin-caption" /></a><br />
<a href="https://2010.igem.org/Team:Groningen#/project"><img src="https://static.igem.org/mediawiki/2010/6/6f/Groningen-Reel-Stage3.jpg" alt="" title="#killswitch-caption" /></a><br />
</div><br />
<script type="text/javascript"><br />
<br />
$('#slider').nivoSlider({effect:'fade'});<br />
<br />
</script><br />
<div id="biofilm-caption" class="nivo-html-caption"><br />
A self assembling bio-based coating is form, a </html>[https://2010.igem.org/Team:Groningen#/biofilm rigid biofilm]<html>.<br />
</div><br />
<div id="chaplin-caption" class="nivo-html-caption"><br />
</html>[https://2010.igem.org/Team:Groningen#/expression Expression]<html> of hydrophobic proteins called </html>[https://2010.igem.org/Team:Groningen#/hydrophobins chaplins]<html> is induced by the biofilm causing strong surface hydrophobicity. <br />
</div><br />
<div id="killswitch-caption" class="nivo-html-caption"><br />
The strongly hydrophobic biofilm will die off by a </html>[https://2010.igem.org/Team:Groningen#/killswitch_model killswitch]<html>, leaving a nice hydrophobic biological coating. <br />
</div><br />
<br />
</html><br />
==Self assembling hydrophobic biofilm==<br />
We aim to design a biological coating as an alternative to for example chemical coatings. For this we, unconventionally, utilized a ''Bacillus subtilis'' [https://2010.igem.org/Team:Groningen#/biofilm biofilm]. We wanted to enable our biofilm to be equipped with an interesting property which is automatically initiated. So we introduced an [https://2010.igem.org/Team:Groningen#/expression expression trigger] which relies on quorum sensing. Our project was directed at finding an alternate solution to biofouling, since regular, chemical coatings which are widely in use pose a threat to the environment. In nature the lotus leaves show self-cleansing properties ascribed to their extreme surface hydrophobicity. In the prokaryotic domain we stumbled upon [https://2010.igem.org/Team:Groningen#/hydrophobins chaplins], strongly hydrophobic proteins originating from ''Streptomyces coelicolor''. Surface hydrophobicity is a very useful property and is used in many [https://2010.igem.org/Team:Groningen#/applications applications] ranging from not only antifouling coatings but also other applications which require water repellence to applications in the field of medical sciences. During our project we have contributed to the parts registry whit numerous [https://2010.igem.org/Team:Groningen#/biobricks BioBricks].<br />
<br />
<br />
We believe that there is a great future for biological coatings as demonstrated within the project. Hydrophobic biological coatings can provide a greener antifouling solution. However using the underlying mechanisms of biofilm triggered expression for other systems like dynamic painting, sensing of environmental changes or even the integration with silicon chips might be within the realm of possibilities. So we challenge coming iGEM teams to further explore and [https://2010.igem.org/Team:Groningen#/brainstorm brainstorm] about the possibilities of using biofilms as a host for a wide range of applications.<br />
<html><br />
</div><br />
<br />
<style type="text/css"><br />
#home-boxes {<br />
margin: 2em 0 2em 10px; <br />
width: 751px;<br />
clear:both;<br />
height: 400px;<br />
}<br />
#home-boxes div {<br />
height: 400px;<br />
background-color: #fff4bc;<br />
width: 232px;<br />
float: left; <br />
padding: 0em;<br />
border: 1px solid #efe28d;<br />
margin-right: 15px;<br />
}<br />
#home-boxes div div {<br />
margin: 0 0 0 15px;<br />
height: auto;<br />
text-align: justify;<br />
border: none!important;<br />
width: 200px;<br />
}<br />
#home-boxes img { <br />
padding: 0; margin: 0;<br />
}<br />
<br />
#home-boxes div:hover {<br />
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<br />
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<div id="home-boxes"><div><br />
<a href="https://2010.igem.org/Team:Groningen#/team"><img src="https://static.igem.org/mediawiki/2010/f/f5/Groningen-Home-Team.jpg" alt="Team"></a><br />
<div></html><br />
This year a [https://2010.igem.org/Team:Groningen#/team team] of young inspired undergraduates from the [http://www.rug.nl University of Groningen] participated in the amazing challenge of iGEM. A multi-disciplinary team of Molecular Biologists, Chemists, Computer Scientists, Journalists and others spend the summer creating a wonderful project in the emerging field of synthetic biology<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/modeling"><img src="https://static.igem.org/mediawiki/2010/1/1e/Groningen-Home-Model.jpg" alt="Modeling"></a><div></html><br />
Using computer models we worked on the frontiers of knowledge. [https://2010.igem.org/Team:Groningen#/expression_model Gene expression] was simulated and a simple explanation for [https://2010.igem.org/Team:Groningen#/biofilm_model cell differentiation] was proposed. Also aiding in ethics and practical feasibility a [https://2010.igem.org/Team:Groningen#/killswitch_model kill switch] system was studied. Finally a [https://2010.igem.org/Team:Groningen#/info_standard new standard] was proposed for characterizing Biobrick parts so future can be streamlined.<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/practices"><img src="https://static.igem.org/mediawiki/2010/7/77/Groningen-Home-Human.jpg" alt="Human Practices"></a><div></html><br />
Because we believe that synthetic biology can better the lives of people and ensure long term prosperity for all humans we spend time [https://2010.igem.org/Team:Groningen#/practices educating] high school students. But not all is perfect so [https://2010.igem.org/Team:Groningen#/safety risks] were assessed and we philosophized on the [https://2010.igem.org/Team:Groningen#/ethics ethical] aspects of synthetic biology.<br />
<html></div></div><br />
</div><br />
</html><br />
<br />
----<br />
<br />
=== Our sponsors === <br />
{{Team:Groningen/sponsors}}<br />
<html><br />
</div><br />
</html></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/HomeTeam:Groningen/Home2010-10-28T01:25:15Z<p>Joelkuiper: </p>
<hr />
<div><html><br />
<div margin-left: 10px"> <br />
<br />
<br />
<script src="https://2010.igem.org/Team:Groningen/files/slider.js?action=raw&ctype=text/javascript" type="text/javascript"></script><br />
<div style="height: 1330px"><br />
<div id="slider" style="margin-left: 10px; height: 343px;"><br />
<a href="https://2010.igem.org/Team:Groningen#/project"><img src="https://static.igem.org/mediawiki/2010/d/dd/Groningen-Reel-Stage1.jpg" alt="" title="#biofilm-caption" /></a><br />
<a href="https://2010.igem.org/Team:Groningen#/project"><img src="https://static.igem.org/mediawiki/2010/d/d3/Groningen-Reel-Stage2.jpg" alt="" title="#chaplin-caption" /></a><br />
<a href="https://2010.igem.org/Team:Groningen#/project"><img src="https://static.igem.org/mediawiki/2010/6/6f/Groningen-Reel-Stage3.jpg" alt="" title="#killswitch-caption" /></a><br />
</div><br />
<script type="text/javascript"><br />
<br />
$('#slider').nivoSlider({effect:'fade'});<br />
<br />
</script><br />
<div id="biofilm-caption" class="nivo-html-caption"><br />
A self assembling bio-based coating is form, a </html>[https://2010.igem.org/Team:Groningen#/biofilm rigid biofilm]<html>.<br />
</div><br />
<div id="chaplin-caption" class="nivo-html-caption"><br />
</html>[https://2010.igem.org/Team:Groningen#/expression Expression]<html> of hydrophobic proteins called </html>[https://2010.igem.org/Team:Groningen#/hydrophobins chaplins]<html> is induced by the biofilm causing strong surface hydrophobicity. <br />
</div><br />
<div id="killswitch-caption" class="nivo-html-caption"><br />
The strongly hydrophobic biofilm will die off by a </html>[https://2010.igem.org/Team:Groningen#/killswitch_model killswitch]<html>, leaving a nice hydrophobic biological coating. <br />
</div><br />
<br />
</html><br />
==Self assembling hydrophobic biofilm==<br />
We aim to design a biological coating as an alternative to for example chemical coatings. For this we, unconventionally, utilized a ''Bacillus subtilis'' [https://2010.igem.org/Team:Groningen#/biofilm biofilm]. We wanted to enable our biofilm to be equipped with an interesting property which is automatically initiated. So we introduced an [https://2010.igem.org/Team:Groningen#/expression expression trigger] which relies on quorum sensing. Our project was directed at finding an alternate solution to biofouling, since regular, chemical coatings which are widely in use pose a threat to the environment. In nature the lotus leaves show self-cleansing properties ascribed to their extreme surface hydrophobicity. In the prokaryotic domain we stumbled upon [https://2010.igem.org/Team:Groningen#/hydrophobins chaplins], strongly hydrophobic proteins originating from ''Streptomyces coelicolor''. Surface hydrophobicity is a very useful property and is used in many [https://2010.igem.org/Team:Groningen#/applications applications] ranging from not only antifouling coatings but also other applications which require water repellence to applications in the field of medical sciences. During our project we have contributed to the parts registry whit numerous [https://2010.igem.org/Team:Groningen#/biobricks BioBricks].<br />
<br />
<br />
We believe that there is a great future for biological coatings as demonstrated within the project. Hydrophobic biological coatings can provide a greener antifouling solution. However using the underlying mechanisms of biofilm triggered expression for other systems like dynamic painting, sensing of environmental changes or even the integration with silicon chips might be within the realm of possibilities. So we challenge coming iGEM teams to further explore and [https://2010.igem.org/Team:Groningen#/brainstorm brainstorm] about the possibilities of using biofilms as a host for a wide range of applications.<br />
<html><br />
</div><br />
<br />
<style type="text/css"><br />
#home-boxes {<br />
margin: 2em 0 2em 10px; <br />
width: 751px;<br />
clear:both;<br />
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background-color: #fff4bc;<br />
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<a href="https://2010.igem.org/Team:Groningen#/team"><img src="https://static.igem.org/mediawiki/2010/f/f5/Groningen-Home-Team.jpg" alt="Team"></a><br />
<div></html><br />
This year a [https://2010.igem.org/Team:Groningen#/team team] of young inspired undergraduates from the [http://www.rug.nl University of Groningen] participated in the amazing challenge of iGEM. A multi-disciplinary team of Molecular Biologists, Chemists, Computer Scientists, Journalists and others spend the summer creating a wonderful project in the emerging field of synthetic biology<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/modeling"><img src="https://static.igem.org/mediawiki/2010/1/1e/Groningen-Home-Model.jpg" alt="Modeling"></a><div></html><br />
Using computer models we worked on the frontiers of knowledge. [https://2010.igem.org/Team:Groningen#/expression_model Gene expression] was simulated and a simple explanation for [https://2010.igem.org/Team:Groningen#/biofilm_model cell differentiation] was proposed. Also aiding in ethics and practical feasibility a [https://2010.igem.org/Team:Groningen#/killswitch_model kill switch] system was studied. Finally a [https://2010.igem.org/Team:Groningen#/info_standard new standard] was proposed for characterizing Biobrick parts so future can be streamlined.<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/practices"><img src="https://static.igem.org/mediawiki/2010/7/77/Groningen-Home-Human.jpg" alt="Human Practices"></a><div></html><br />
Because we believe that synthetic biology can better the lives of people and ensure long term prosperity for all humans we spend time [https://2010.igem.org/Team:Groningen#/practices educating] high school students. But not all is perfect so [https://2010.igem.org/Team:Groningen#/safety risks] were assessed and we philosophized on the [https://2010.igem.org/Team:Groningen#/ethics ethical] aspects of synthetic biology.<br />
<html></div></div><br />
</div><br />
</html><br />
<br />
----<br />
<br />
=== Our sponsors === <br />
{{Team:Groningen/sponsors}}<br />
<br />
<br />
<html><br />
</div><br />
</html></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/files/overall.cssTeam:Groningen/files/overall.css2010-10-28T01:21:09Z<p>Joelkuiper: </p>
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<br />
#headerWrapper {<br />
font-family: Sertig, Futura, Impact, sans-serif;<br />
-moz-border-radius-topleft: 8px; -webkit-border-top-left-radius: 8px; border-top-left-radius: 8px; -moz-border-radius-topright: 8px; -webkit-border-top-right-radius: 8px; border-top-right-radius: 8px;<br />
font-size: 21pt; color: white; font-weight: bold;<br />
line-height: 1;<br />
background: url("https://static.igem.org/mediawiki/2010/4/42/94acce-50.png") repeat;<br />
height: 100px;<br />
border: 1px solid #c3bfa8;<br />
border-bottom: none;<br />
padding-top: 50px;<br />
}<br />
#headerWrapper img {<br />
margin-left: 20px;<br />
margin-top: -40px;<br />
}<br />
#otherContent { <br />
font-family: Platino, Garmond, Georgia, serif;<br />
text-align: left;<br />
font-size: 9pt;<br />
padding: 60px 0 2em 50px;<br />
width: 750px;<br />
height: auto;<br />
}<br />
#otherContent img {<br />
padding: 1em 1em 1em 1em;<br />
}<br />
#otherContent .head, .mw-headline {<br />
font-family: "Lucida Grande", Tahoma, Helvetica, Verdana, Arial, sans-serif;<br />
color: #a1bf29;<br />
line-height: 1!important;<br />
font-weight: bold;<br />
font-size: 16pt;<br />
margin: 0important;<br />
}<br />
h3 .mw-headline { <br />
font-size: 13pt !important;<br />
}<br />
.textWrapper {<br />
padding-bottom: 2em;<br />
width: 750px;<br />
margin: 0 0 4em 0;<br />
height: auto;<br />
}<br />
#footerBox { <br />
border-top: 1px solid #bdbdbd;<br />
margin: 0 auto;<br />
height: 400px; <br />
clear: both;<br />
width: 100%<br />
}<br />
/* Menu from http://www.sohtanaka.com/web-design/examples/horizontal-subnav/ */<br />
#tab_nav { <br />
position: absolute;<br />
z-index: 1000;<br />
}<br />
ul#topnav {<br />
list-style-type: none;<br />
float: left;<br />
width: 870px;<br />
list-style: none;<br />
position: relative;<br />
background: url("https://static.igem.org/mediawiki/2010/8/80/Groningen_topnav_stretch.gif") repeat-x;<br />
}<br />
ul#topnav li {<br />
line-height: 1;<br />
float: left;<br />
border-right: 1px solid #555;<br />
}<br />
ul#topnav li a {<br />
padding: 10px 15px;<br />
font-weight: bold;<br />
display: block;<br />
color: #f0f0f0;<br />
text-decoration: none;<br />
}<br />
ul#topnav li a:hover {<br />
text-decoration: underline;<br />
}<br />
ul#topnav li:hover { background: #b5cc86 url("https://static.igem.org/mediawiki/2010/2/29/Groningen_topnav_active.gif") repeat-x; }<br />
ul#topnav li span {<br />
float: left;<br />
padding: 20px 0;<br />
position: absolute;<br />
left: 0; top:30px;<br />
display: none;<br />
width:870px;<br />
background: #b5cc86;<br />
color: #fff;<br />
-moz-border-radius-bottomright: 5px;<br />
-khtml-border-radius-bottomright: 5px;<br />
-webkit-border-bottom-right-radius: 5px;<br />
-moz-border-radius-bottomleft: 5px;<br />
-khtml-border-radius-bottomleft: 5px;<br />
-webkit-border-bottom-left-radius: 5px;<br />
}<br />
/* ul#topnav li:hover span { display: block; } */<br />
ul#topnav li span a { display: inline; font-weight; normal; }<br />
ul#topnav li span a:hover {text-decoration: underline;}<br />
#groupparts {<br />
width: 650px!important;<br />
}</div>Joelkuiperhttp://2010.igem.org/Team:Groningen/files/overall.cssTeam:Groningen/files/overall.css2010-10-28T01:19:27Z<p>Joelkuiper: </p>
<hr />
<div>/* BEGIN CSS RESET v1.0 | 20080212 */<br />
html, body, div, span, applet, object, iframe,<br />
h1, h2, h3, h4, h5, h6, p, blockquote, pre,<br />
a, abbr, acronym, address, big, cite, code,<br />
del, dfn, em, font, img, ins, kbd, q, s, samp,<br />
small, strike, strong, sub, sup, tt, var,<br />
b, u, i, center,<br />
dl, dt, dd, ol, ul, li,<br />
fieldset, form, label, legend,<br />
table, caption, tbody, tfoot, thead, tr, th, td {<br />
margin: 0;<br />
padding: 0;<br />
outline: 0;<br />
vertical-align: baseline;<br />
background: transparent;<br />
}<br />
html {<br />
height: 100%;<br />
}<br />
ol, ul {<br />
list-style: none;<br />
}<br />
blockquote, q {<br />
quotes: none;<br />
}<br />
blockquote:before, blockquote:after,<br />
q:before, q:after {<br />
content: '';<br />
content: none;<br />
}<br />
ins {<br />
text-decoration: none;<br />
}<br />
del {<br />
text-decoration: line-through;<br />
}<br />
pre { <br />
border: none;<br />
}<br />
/* END CSS RESET */<br />
/* Wiki Hacks - START */<br />
/* Author: Pieter van Boheemen */<br />
/* Team: TU Delft */<br />
#globalWrapper { background-color: transparent; border: none; margin: 0; padding: 0; width: 100%; height:100%;}<br />
#content { background-color: transparent; border: none; padding: 0; margin: 0; width: 100%; overflow: hidden; height:100%;}<br />
#bodyContent { border: none; padding:0; margin:0; width:100%; height:100%;}<br />
#top-section { height: 15px; margin: 0px; margin-left: auto; margin-right: auto; margin-bottom: 0 !important; padding:0; border: none; font-size: 10px;}<br />
#p-logo { height:1px; overflow:hidden; display: none;}<br />
#search-controls { overflow:hidden; display:block; background: none; position: absolute; top: 100px; right: 40px;}<br />
.left-menu { width: 500px !important; display:block; margin-top:-80px; border: none; text-align: right;}<br />
.left-menu ul { border: none; }<br />
#menubar.right-menu { width:300px; display:block; float:left; margin-top:-80px; border: none;}<br />
.right-menu ul { border: none; width: 300px;}<br />
#footer-box, #footerBox { background-color: #282828; border: none; width: 100%; margin: -10px auto 0 auto; padding: 20px 0; color: #666;}<br />
.visualClear { display: none; }<br />
#footer { border: none; width: 965px; margin: 0 auto; padding: 0;}<br />
.firstHeading { display: none;}<br />
#f-list a, #footerBox a { color: #666; font-size: 10px;}<br />
#f-list a:hover, #footerBox a:hover { color: #fff;}<br />
.printfooter { display: none; }<br />
#footer ul { margin: 0; padding: 0;}<br />
#footer ul li { margin-top: 0; margin-bottom: 0; margin-left: 10px; margin-right: 10px; padding: 0;}<br />
h3#siteSub { display: none;}<br />
#contentSub {display: none;}<br />
<br />
.thumb, .tright, .tleft, .tnone {<br />
border: none!important <br />
background: transparent!important;<br />
}<br />
#search-controls { display: none; } <br />
<br />
table td { vertical-align: top } <br />
<br />
/* Wiki Hacks - END */<br />
<br />
#overallMask {<br />
position: relative;<br />
margin: 2% auto 2em auto;<br />
width: 870px;<br />
}<br />
<br />
#contentWrapper {<br />
height: 100%;<br />
padding-bottom: 3em;<br />
min-height: 700px;<br />
-moz-box-shadow: rgba(0, 0, 0, 0.3) 8px 8px 8px;<br />
-webkit-box-shadow: rgba(0, 0, 0, 0.3) 8px 8px 8px;<br />
box-shadow: rgba(0, 0, 0, 0.3) 8px 8px 8px;;<br />
background-color: #fffbe7;<br />
}<br />
@font-face {<br />
font-family: Sertig;<br />
src: url('http://casualconnect.net/iGEM/Sertig.otf');<br />
}<br />
<br />
#headerWrapper {<br />
font-family: Sertig, Futura, Impact, sans-serif;<br />
-moz-border-radius-topleft: 8px; -webkit-border-top-left-radius: 8px; border-top-left-radius: 8px; -moz-border-radius-topright: 8px; -webkit-border-top-right-radius: 8px; border-top-right-radius: 8px;<br />
font-size: 21pt; color: white; font-weight: bold;<br />
line-height: 1;<br />
background: url("https://static.igem.org/mediawiki/2010/4/42/94acce-50.png") repeat;<br />
height: 100px;<br />
border: 1px solid #c3bfa8;<br />
border-bottom: none;<br />
padding-top: 50px;<br />
}<br />
#headerWrapper img {<br />
margin-left: 20px;<br />
margin-top: -40px;<br />
}<br />
#otherContent { <br />
font-family: Platino, Garmond, Georgia, serif;<br />
text-align: left;<br />
font-size: 9pt;<br />
padding: 60px 0 2em 50px;<br />
width: 750px;<br />
height: auto;<br />
}<br />
#otherContent img {<br />
padding: 1em 1em 1em 1em;<br />
}<br />
#otherContent .head, .mw-headline {<br />
font-family: "Lucida Grande", Tahoma, Helvetica, Verdana, Arial, sans-serif;<br />
color: #a1bf29;<br />
line-height: 1!important;<br />
font-weight: bold;<br />
font-size: 16pt;<br />
margin: 0important;<br />
}<br />
h3 .mw-headline { <br />
font-size: 13pt !important;<br />
}<br />
.textWrapper {<br />
padding-bottom: 2em;<br />
width: 750px;<br />
margin: 0 0 4em 0;<br />
height: auto;<br />
}<br />
#footerBox { <br />
border-top: 1px solid #bdbdbd;<br />
margin: 0 auto;<br />
height: 400px; <br />
clear: both;<br />
width: 100%<br />
}<br />
/* Menu from http://www.sohtanaka.com/web-design/examples/horizontal-subnav/ */<br />
#tab_nav { <br />
position: absolute;<br />
z-index: 1000;<br />
}<br />
ul#topnav {<br />
list-style-type: none;<br />
float: left;<br />
width: 870px;<br />
list-style: none;<br />
position: relative;<br />
background: url("https://static.igem.org/mediawiki/2010/8/80/Groningen_topnav_stretch.gif") repeat-x;<br />
}<br />
ul#topnav li {<br />
line-height: 1;<br />
float: left;<br />
border-right: 1px solid #555;<br />
}<br />
ul#topnav li a {<br />
padding: 10px 15px;<br />
font-weight: bold;<br />
display: block;<br />
color: #f0f0f0;<br />
text-decoration: none;<br />
}<br />
ul#topnav li a:hover {<br />
text-decoration: underline;<br />
}<br />
ul#topnav li:hover { background: #b5cc86 url("https://static.igem.org/mediawiki/2010/2/29/Groningen_topnav_active.gif") repeat-x; }<br />
ul#topnav li span {<br />
float: left;<br />
padding: 20px 0;<br />
position: absolute;<br />
left: 0; top:30px;<br />
display: none;<br />
width:870px;<br />
background: #b5cc86;<br />
color: #fff;<br />
-moz-border-radius-bottomright: 5px;<br />
-khtml-border-radius-bottomright: 5px;<br />
-webkit-border-bottom-right-radius: 5px;<br />
-moz-border-radius-bottomleft: 5px;<br />
-khtml-border-radius-bottomleft: 5px;<br />
-webkit-border-bottom-left-radius: 5px;<br />
}<br />
/* ul#topnav li:hover span { display: block; } */<br />
ul#topnav li span a { display: inline; font-weight; normal; }<br />
ul#topnav li span a:hover {text-decoration: underline;}<br />
#groupparts {<br />
width: 650px!important;<br />
}</div>Joelkuiperhttp://2010.igem.org/Team:Groningen/files/overall.cssTeam:Groningen/files/overall.css2010-10-28T01:17:39Z<p>Joelkuiper: </p>
<hr />
<div>/* BEGIN CSS RESET v1.0 | 20080212 */<br />
html, body, div, span, applet, object, iframe,<br />
h1, h2, h3, h4, h5, h6, p, blockquote, pre,<br />
a, abbr, acronym, address, big, cite, code,<br />
del, dfn, em, font, img, ins, kbd, q, s, samp,<br />
small, strike, strong, sub, sup, tt, var,<br />
b, u, i, center,<br />
dl, dt, dd, ol, ul, li,<br />
fieldset, form, label, legend,<br />
table, caption, tbody, tfoot, thead, tr, th, td {<br />
margin: 0;<br />
padding: 0;<br />
outline: 0;<br />
vertical-align: baseline;<br />
background: transparent;<br />
}<br />
html {<br />
height: 100%;<br />
}<br />
ol, ul {<br />
list-style: none;<br />
}<br />
blockquote, q {<br />
quotes: none;<br />
}<br />
blockquote:before, blockquote:after,<br />
q:before, q:after {<br />
content: '';<br />
content: none;<br />
}<br />
ins {<br />
text-decoration: none;<br />
}<br />
del {<br />
text-decoration: line-through;<br />
}<br />
pre { <br />
border: none;<br />
}<br />
/* END CSS RESET */<br />
/* Wiki Hacks - START */<br />
/* Author: Pieter van Boheemen */<br />
/* Team: TU Delft */<br />
#globalWrapper { background-color: transparent; border: none; margin: 0; padding: 0; width: 100%; height:100%;}<br />
#content { background-color: transparent; border: none; padding: 0; margin: 0; width: 100%; overflow: hidden; height:100%;}<br />
#bodyContent { border: none; padding:0; margin:0; width:100%; height:100%;}<br />
#top-section { height: 15px; margin: 0px; margin-left: auto; margin-right: auto; margin-bottom: 0 !important; padding:0; border: none; font-size: 10px;}<br />
#p-logo { height:1px; overflow:hidden; display: none;}<br />
#search-controls { overflow:hidden; display:block; background: none; position: absolute; top: 100px; right: 40px;}<br />
.left-menu { width: 500px !important; display:block; margin-top:-80px; border: none; text-align: right;}<br />
.left-menu ul { border: none; }<br />
#menubar.right-menu { width:300px; display:block; float:left; margin-top:-80px; border: none;}<br />
.right-menu ul { border: none; width: 300px;}<br />
#footer-box, #footerBox { background-color: #282828; border: none; width: 100%; margin: -10px auto 0 auto; padding: 20px 0; color: #666;}<br />
.visualClear { display: none; }<br />
#footer { border: none; width: 965px; margin: 0 auto; padding: 0;}<br />
.firstHeading { display: none;}<br />
#f-list a, #footerBox a { color: #666; font-size: 10px;}<br />
#f-list a:hover, #footerBox a:hover { color: #fff;}<br />
.printfooter { display: none; }<br />
#footer ul { margin: 0; padding: 0;}<br />
#footer ul li { margin-top: 0; margin-bottom: 0; margin-left: 10px; margin-right: 10px; padding: 0;}<br />
h3#siteSub { display: none;}<br />
#contentSub {display: none;}<br />
div.thumb { background: transparent!important; } <br />
.tright, .tleft, .tnone {<br />
border: none!important <br />
margin: 0.2em; <br />
}<br />
#search-controls { display: none; } <br />
<br />
table td { vertical-align: top } <br />
<br />
/* Wiki Hacks - END */<br />
<br />
#overallMask {<br />
position: relative;<br />
margin: 2% auto 2em auto;<br />
width: 870px;<br />
}<br />
<br />
#contentWrapper {<br />
height: 100%;<br />
padding-bottom: 3em;<br />
min-height: 700px;<br />
-moz-box-shadow: rgba(0, 0, 0, 0.3) 8px 8px 8px;<br />
-webkit-box-shadow: rgba(0, 0, 0, 0.3) 8px 8px 8px;<br />
box-shadow: rgba(0, 0, 0, 0.3) 8px 8px 8px;;<br />
background-color: #fffbe7;<br />
}<br />
@font-face {<br />
font-family: Sertig;<br />
src: url('http://casualconnect.net/iGEM/Sertig.otf');<br />
}<br />
<br />
#headerWrapper {<br />
font-family: Sertig, Futura, Impact, sans-serif;<br />
-moz-border-radius-topleft: 8px; -webkit-border-top-left-radius: 8px; border-top-left-radius: 8px; -moz-border-radius-topright: 8px; -webkit-border-top-right-radius: 8px; border-top-right-radius: 8px;<br />
font-size: 21pt; color: white; font-weight: bold;<br />
line-height: 1;<br />
background: url("https://static.igem.org/mediawiki/2010/4/42/94acce-50.png") repeat;<br />
height: 100px;<br />
border: 1px solid #c3bfa8;<br />
border-bottom: none;<br />
padding-top: 50px;<br />
}<br />
#headerWrapper img {<br />
margin-left: 20px;<br />
margin-top: -40px;<br />
}<br />
#otherContent { <br />
font-family: Platino, Garmond, Georgia, serif;<br />
text-align: left;<br />
font-size: 9pt;<br />
padding: 60px 0 2em 50px;<br />
width: 750px;<br />
height: auto;<br />
}<br />
#otherContent img {<br />
padding: 1em 1em 1em 1em;<br />
}<br />
#otherContent .head, .mw-headline {<br />
font-family: "Lucida Grande", Tahoma, Helvetica, Verdana, Arial, sans-serif;<br />
color: #a1bf29;<br />
line-height: 1!important;<br />
font-weight: bold;<br />
font-size: 16pt;<br />
margin: 0important;<br />
}<br />
h3 .mw-headline { <br />
font-size: 13pt !important;<br />
}<br />
.textWrapper {<br />
padding-bottom: 2em;<br />
width: 750px;<br />
margin: 0 0 4em 0;<br />
height: auto;<br />
}<br />
#footerBox { <br />
border-top: 1px solid #bdbdbd;<br />
margin: 0 auto;<br />
height: 400px; <br />
clear: both;<br />
width: 100%<br />
}<br />
/* Menu from http://www.sohtanaka.com/web-design/examples/horizontal-subnav/ */<br />
#tab_nav { <br />
position: absolute;<br />
z-index: 1000;<br />
}<br />
ul#topnav {<br />
list-style-type: none;<br />
float: left;<br />
width: 870px;<br />
list-style: none;<br />
position: relative;<br />
background: url("https://static.igem.org/mediawiki/2010/8/80/Groningen_topnav_stretch.gif") repeat-x;<br />
}<br />
ul#topnav li {<br />
line-height: 1;<br />
float: left;<br />
border-right: 1px solid #555;<br />
}<br />
ul#topnav li a {<br />
padding: 10px 15px;<br />
font-weight: bold;<br />
display: block;<br />
color: #f0f0f0;<br />
text-decoration: none;<br />
}<br />
ul#topnav li a:hover {<br />
text-decoration: underline;<br />
}<br />
ul#topnav li:hover { background: #b5cc86 url("https://static.igem.org/mediawiki/2010/2/29/Groningen_topnav_active.gif") repeat-x; }<br />
ul#topnav li span {<br />
float: left;<br />
padding: 20px 0;<br />
position: absolute;<br />
left: 0; top:30px;<br />
display: none;<br />
width:870px;<br />
background: #b5cc86;<br />
color: #fff;<br />
-moz-border-radius-bottomright: 5px;<br />
-khtml-border-radius-bottomright: 5px;<br />
-webkit-border-bottom-right-radius: 5px;<br />
-moz-border-radius-bottomleft: 5px;<br />
-khtml-border-radius-bottomleft: 5px;<br />
-webkit-border-bottom-left-radius: 5px;<br />
}<br />
/* ul#topnav li:hover span { display: block; } */<br />
ul#topnav li span a { display: inline; font-weight; normal; }<br />
ul#topnav li span a:hover {text-decoration: underline;}<br />
#groupparts {<br />
width: 650px!important;<br />
}</div>Joelkuiperhttp://2010.igem.org/Team:Groningen/files/overall.cssTeam:Groningen/files/overall.css2010-10-28T01:16:46Z<p>Joelkuiper: </p>
<hr />
<div>/* BEGIN CSS RESET v1.0 | 20080212 */<br />
html, body, div, span, applet, object, iframe,<br />
h1, h2, h3, h4, h5, h6, p, blockquote, pre,<br />
a, abbr, acronym, address, big, cite, code,<br />
del, dfn, em, font, img, ins, kbd, q, s, samp,<br />
small, strike, strong, sub, sup, tt, var,<br />
b, u, i, center,<br />
dl, dt, dd, ol, ul, li,<br />
fieldset, form, label, legend,<br />
table, caption, tbody, tfoot, thead, tr, th, td {<br />
margin: 0;<br />
padding: 0;<br />
outline: 0;<br />
vertical-align: baseline;<br />
background: transparent;<br />
}<br />
html {<br />
height: 100%;<br />
}<br />
ol, ul {<br />
list-style: none;<br />
}<br />
blockquote, q {<br />
quotes: none;<br />
}<br />
blockquote:before, blockquote:after,<br />
q:before, q:after {<br />
content: '';<br />
content: none;<br />
}<br />
ins {<br />
text-decoration: none;<br />
}<br />
del {<br />
text-decoration: line-through;<br />
}<br />
pre { <br />
border: none;<br />
}<br />
/* END CSS RESET */<br />
/* Wiki Hacks - START */<br />
/* Author: Pieter van Boheemen */<br />
/* Team: TU Delft */<br />
#globalWrapper { background-color: transparent; border: none; margin: 0; padding: 0; width: 100%; height:100%;}<br />
#content { background-color: transparent; border: none; padding: 0; margin: 0; width: 100%; overflow: hidden; height:100%;}<br />
#bodyContent { border: none; padding:0; margin:0; width:100%; height:100%;}<br />
#top-section { height: 15px; margin: 0px; margin-left: auto; margin-right: auto; margin-bottom: 0 !important; padding:0; border: none; font-size: 10px;}<br />
#p-logo { height:1px; overflow:hidden; display: none;}<br />
#search-controls { overflow:hidden; display:block; background: none; position: absolute; top: 100px; right: 40px;}<br />
.left-menu { width: 500px !important; display:block; margin-top:-80px; border: none; text-align: right;}<br />
.left-menu ul { border: none; }<br />
#menubar.right-menu { width:300px; display:block; float:left; margin-top:-80px; border: none;}<br />
.right-menu ul { border: none; width: 300px;}<br />
#footer-box, #footerBox { background-color: #282828; border: none; width: 100%; margin: -10px auto 0 auto; padding: 20px 0; color: #666;}<br />
.visualClear { display: none; }<br />
#footer { border: none; width: 965px; margin: 0 auto; padding: 0;}<br />
.firstHeading { display: none;}<br />
#f-list a, #footerBox a { color: #666; font-size: 10px;}<br />
#f-list a:hover, #footerBox a:hover { color: #fff;}<br />
.printfooter { display: none; }<br />
#footer ul { margin: 0; padding: 0;}<br />
#footer ul li { margin-top: 0; margin-bottom: 0; margin-left: 10px; margin-right: 10px; padding: 0;}<br />
h3#siteSub { display: none;}<br />
#contentSub {display: none;}<br />
div.thumb { background: transparent!important; } <br />
.tright, .tleft, .tnone {<br />
border: none!important <br />
padding: 0.5em; <br />
}<br />
#search-controls { display: none; } <br />
<br />
table td { vertical-align: top } <br />
<br />
/* Wiki Hacks - END */<br />
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#overallMask {<br />
position: relative;<br />
margin: 2% auto 2em auto;<br />
width: 870px;<br />
}<br />
<br />
#contentWrapper {<br />
height: 100%;<br />
padding-bottom: 3em;<br />
min-height: 700px;<br />
-moz-box-shadow: rgba(0, 0, 0, 0.3) 8px 8px 8px;<br />
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background-color: #fffbe7;<br />
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-moz-border-radius-topleft: 8px; -webkit-border-top-left-radius: 8px; border-top-left-radius: 8px; -moz-border-radius-topright: 8px; -webkit-border-top-right-radius: 8px; border-top-right-radius: 8px;<br />
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padding-bottom: 2em;<br />
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border-top: 1px solid #bdbdbd;<br />
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color: #fff;<br />
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<hr />
<div>/* BEGIN CSS RESET v1.0 | 20080212 */<br />
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/* Author: Pieter van Boheemen */<br />
/* Team: TU Delft */<br />
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position: relative;<br />
margin: 2% auto 2em auto;<br />
width: 870px;<br />
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src: url('http://casualconnect.net/iGEM/Sertig.otf');<br />
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<hr />
<div>/* BEGIN CSS RESET v1.0 | 20080212 */<br />
html, body, div, span, applet, object, iframe,<br />
h1, h2, h3, h4, h5, h6, p, blockquote, pre,<br />
a, abbr, acronym, address, big, cite, code,<br />
del, dfn, em, font, img, ins, kbd, q, s, samp,<br />
small, strike, strong, sub, sup, tt, var,<br />
b, u, i, center,<br />
dl, dt, dd, ol, ul, li,<br />
fieldset, form, label, legend,<br />
table, caption, tbody, tfoot, thead, tr, th, td {<br />
margin: 0;<br />
padding: 0;<br />
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height: 100%;<br />
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/* Wiki Hacks - START */<br />
/* Author: Pieter van Boheemen */<br />
/* Team: TU Delft */<br />
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position: relative;<br />
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width: 870px;<br />
}<br />
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#contentWrapper {<br />
height: 100%;<br />
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min-height: 700px;<br />
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box-shadow: rgba(0, 0, 0, 0.3) 8px 8px 8px;;<br />
background-color: #fffbe7;<br />
}<br />
@font-face {<br />
font-family: Sertig;<br />
src: url('http://casualconnect.net/iGEM/Sertig.otf');<br />
}<br />
<br />
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font-family: Sertig, Futura, Impact, sans-serif;<br />
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line-height: 1;<br />
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}<br />
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color: #a1bf29;<br />
line-height: 1!important;<br />
font-weight: bold;<br />
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margin: 0important;<br />
}<br />
h3 .mw-headline { <br />
font-size: 13pt !important;<br />
}<br />
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width: 750px;<br />
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height: auto;<br />
}<br />
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border-top: 1px solid #bdbdbd;<br />
margin: 0 auto;<br />
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clear: both;<br />
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}<br />
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#tab_nav { <br />
position: absolute;<br />
z-index: 1000;<br />
}<br />
ul#topnav {<br />
list-style-type: none;<br />
float: left;<br />
width: 870px;<br />
list-style: none;<br />
position: relative;<br />
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}<br />
ul#topnav li {<br />
line-height: 1;<br />
float: left;<br />
border-right: 1px solid #555;<br />
}<br />
ul#topnav li a {<br />
padding: 10px 15px;<br />
font-weight: bold;<br />
display: block;<br />
color: #f0f0f0;<br />
text-decoration: none;<br />
}<br />
ul#topnav li a:hover {<br />
text-decoration: underline;<br />
}<br />
ul#topnav li:hover { background: #b5cc86 url("https://static.igem.org/mediawiki/2010/2/29/Groningen_topnav_active.gif") repeat-x; }<br />
ul#topnav li span {<br />
float: left;<br />
padding: 20px 0;<br />
position: absolute;<br />
left: 0; top:30px;<br />
display: none;<br />
width:870px;<br />
background: #b5cc86;<br />
color: #fff;<br />
-moz-border-radius-bottomright: 5px;<br />
-khtml-border-radius-bottomright: 5px;<br />
-webkit-border-bottom-right-radius: 5px;<br />
-moz-border-radius-bottomleft: 5px;<br />
-khtml-border-radius-bottomleft: 5px;<br />
-webkit-border-bottom-left-radius: 5px;<br />
}<br />
/* ul#topnav li:hover span { display: block; } */<br />
ul#topnav li span a { display: inline; font-weight; normal; }<br />
ul#topnav li span a:hover {text-decoration: underline;}<br />
#groupparts {<br />
width: 650px!important;<br />
}</div>Joelkuiperhttp://2010.igem.org/Team:Groningen/ExpressionTeam:Groningen/Expression2010-10-28T01:06:59Z<p>Joelkuiper: /* Expression of chaplins */</p>
<hr />
<div>__NOTOC__<br />
==Expression of chaplins==<br />
<br />
'''Summary'''<br />
<br />
The goal of our project is to let ''Bacillus subtilis'' make a hydrophobic coating by forming a [https://2010.igem.org/Team:Groningen#/biofilm biofilm] and then expressing and secreting [https://2010.igem.org/Team:Groningen/Hydrophobins#Chaplins chaplins]. However, first we needed to test whether ''B. subtilis'' was capable of expressing chaplins, since they could impair the cellgrowth due to their hydrophobic and self assembling properties. We succesfully expressed chaplins C, E and H in ''B. subtilis'' using a tightly regulated subtilin inducable system called "SURE". Furthermore we tested the SURE system for optimal subtilin concentration with GFP. We want ''B. subtilis'' to auto-induce the expression of the chaplins after biofilmformation. Therefore we looked into two operons in ''B. subtilis''; one that gets triggered in late exponential growth (''srfA'' operon) and one that is involved in the formation of biofilm (''yqxM-sipW-tasA'' operon). Using the ''srfA'' promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K305007 BBa_K305007]), we succesfully expressed GFP demonstrating that this promoter could be used to auto-induce the expression chaplins.<br />
<br />
<br><br />
<br />
===Subtilin induced expression of chaplins===<br />
<br />
The biofilm forming capacity of ''Bacillus subtilis'' makes it a good host for our application. In addition, ''B. subtilis'' is known for its ability to produce and secrete large amounts of protein at high cell densities. However, despite its track record as an efficient production organism and the fact that both ''B. subtilis'' and ''Streptomyces coelicolor'' are gram-positive bacteria, it is not certain wether chaplins can be heterologously expressed in ''B. subtilis''. Improper folding, unsuccessful export, or even the very nature of the chaplins, could still lead to hampered expression.<br />
We took several steps to ensure optimal expression. The coding sequences of the chaplins were codon optimized for ''B. subtilis'' and synthesized. We placed a ribosome binding site in front of the coding sequences that is known to work well in ''B. subtilis'', and flanked these constructs with the biobrick prefix and suffix. <br />
<br />
'''SURE expression system'''<br />
<br><br />
[[Image:SURE-gfp-gn.jpg|250px|thumb|right|Subtilin induction of GFP by the SURE system (Bongers ''et al'', 2005)]]<br />
Because it is uncertain how chaplin expression will affect ''B. subtilis'', the initial expression attempts were performed with the stringently controlled, subtilin-regulated gene expression (SURE) system (Bongers ''et al'', 2005). This system uses the subtilin sensing machinery present in a strain of ''B. subtilis'' that autoinduces the production of more of the [http://en.wikipedia.org/wiki/Lantibiotics lantibiotic] subtilin. The subtilin sensor histidine kinase SpaK phosphorylates the response regulator SpaR, which can then bind to so-called ''spa'' boxes in the promoter regions of genes involved in subtilin biosynthesis (Kleerebezem ''et al'', 2004). In the SURE system, a ''B. subtilis'' strain naturally lacking the subtilin biosynthesis genes has the ''spaRK'' genes introduced into its genome. A plasmid carrying a ''spa'' box promoter that is transformed to this strain can then drive the expression of proteins upon subtilin induction of SpaRK signalling. <br />
<br />
[[Image:Sub-ind-gn.jpg|250px|thumb|right|Subtilin induced GFP expression in Bacillus subtilis using the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305011 BBa_K305011] backbone]]<br />
<br />
We have adapted this system to make it BioBrick compatible for easy expression of our chaplins, combinations of chaplins, or any other biobrick part that is composed of an RBS followed by a protein coding sequence. We introduced the BioBrick prefix and suffix into the expression plasmid, downstream of the mutated ''spaS'' promoter, producing our subtilin inducible expression backbone part, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305011 BBa_K305011]. To test the expression and find a suitable subtilin concentration for induction of the chaplins we made use of GFP fluorescence measurements. We inserted the part [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0240 BBa_E0240] into the BioBrick site and induced liquid cultures of ''B. subtilis'' carrying this plasmid (and the ''spaRK'' genes) with different volumes of subtilin-containing culture supernatant of a subtilin producing strain of ''B. subtilis''. These results demonstrate that addition of 0.5 to 1%(vol/vol) of subtilin to the culture is sufficient to reach optimal induction. > Chaplins<br />
<br />
<br><br />
'''Chaplin detection'''<br />
<br><br />
Streptomyces secretes the chaplin proteins into the medium, after which they can serve to lower surface tension or self assemble into amyloid fibers on the cell walls, thus in our first expression tests focused on detecting the chaplins in either the medium in which our supposedly chaplin producing population grew, or on the cells of the B. subtilis.<br />
<br><br />
Using the same methods that were used to dissolve and monomerize chaplins in from Streptomyces, we treated cell pellets and [http://en.wikipedia.org/wiki/Trichloricacetic_acid TCA] precipitated supernatant with [http://en.wikipedia.org/wiki/Trifluoroacetic_acid TFA] to purify chaplin proteins. [http://en.wikipedia.org/wiki/Trifluoroacetic_acid TFA] treatment with 99% pure TFA demolishes most proteins and monomerises assembled chaplin fibers, this enables us to detect the chaplins on SDS gel. Using such a harsh method, we hope to denaturate most proteins to prevent their interference in chaplin detection and highten the relative concentration of chaplin proteins in tested samples.<br />
<br />
<br><br />
Since our early expression experiments didn't yield conclusive results regarding the detection of our chaplins, we we tried staining our samples with an amyloid specific stain called [http://en.wikipedia.org/wiki/Thioflavin Thioflavin T]. Initial testing with the supernatant and washed pellet gave intriguing results yet not clear. Our emission graphs showed some irregularities with the subtilin induced samples, but seemed to be distorted by background noise caused by other materials in the sample. To further purify our samples we decided to [extractioncellwallsGR disrupt] our liquid culture and boil it two times in 2% SDS, before treating the freeze dried sample with 99%. TFA This turned out to be a more successful method.<br />
<br />
Using this method we succesfully detected chaplins C, E and H in purified cell walls from induced B. subtilis cultures, confirmation of these results was provided by malditov mass spectrometry.<br />
<br><br />
<html><br />
<table><br />
<tr><br />
<td></html>[[Image:Chgr.jpg|300px|thumb|Purified chaplins from S. coelicolor exitate at 482 when stained with THT. Different dilutions were used to set up a ladder.]]<html></td><br />
<td></html>[[Image:EHgr.jpg|300px|thumb|Purified E and H chaplins from B subtilis exitate at 482 when stained with THT]] <html></td><br />
</tr><br />
<tr><br />
<td></html>[[Image:Cgr.jpg|300px|thumb|Purified C chaplins from B subtilis exitate at 482 when stained with THT]]<html></td><br />
<td></html>[[Image:Egr.jpg|300px|thumb|Purified E chaplins from B subtilis exitate at 482 when stained with THT]]<html></td><br />
</tr><br />
</table><br />
</html><br />
<br><br />
[[Image:MassSpec.jpg|700px|thumb|none|Mass spectometry results confirm the production of Chaplin H (5128) and E (5144) in Bacillus (green). Peaks is Streptomyces (blue) corrispond, although the peak of chaplin E (5144) indicates that somewhere during translation or in post-translational processes a 21kb threonine group is lost. The non induced B. subtilis strain serves as a control (red). Chaplin C has not been detected this way, but that was not to be expected (Claessen et al. (2009).]]<br />
<br />
===Timed expression of chaplins in a biofilm ===<br />
<br />
An important question is which promoter we should use to control the chaplin expression. We assume that an ideal promoter would not be active until the biofilm has formed because the expression of hydrophobic proteins might influence the formation of it. Two promoters where found that are active in biofilms but not during normal growth. <br />
<br />
[[Image:Groningen-Promotors-sketch.png|300px|left]]<br />
<br />
[[image:igemgroningen_srfa_Promotoractivity.jpg|right|200px|srfA|thumb|srfA promotor activity during cell growth (Nakano MM. 1991)]]<br />
<br />
'''''srfA'''''<br />
<br />
The [http://dbtbs.hgc.jp/COG/prom/srfAA-srfAB-comS-srfAC-srfAD.html ''srfA'' operon] has been reported to be important for natural competence and sporulation in ''Bacillus subtilis''. All these activities occur in biofilms, the promoter is not active until the end of exponential growth. It is controlled by the [https://2010.igem.org/Team:Groningen/Expression_model#ComXPA_quorum_sensing_system ComXPA quorum sensing system] and hence active in states of high cell densities. Therefore the ''srfA'' promoter would be suitable for chaplin expression. Two different lengths of the ''srfA'' promoter where chosen due to uncertainties concerning the region between the response element and the transcription start side of the SrfAA protein. In the original promoter this region is unusually long, by shortening it 190bp’s we hope to achieve a higher transcription efficiency. So we came up with two different promoters, the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305008 original] one and the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305007 shortened] one. Promoter studies using GFP as a reporter confirmed our assumption that the short ''srfA''-promoter variant leads to a higher expression. While the fluorescence of the short variant was clearly above background levels, the long variant did not give convincing results. <br />
[[Image:florescence_srfA.jpg]]<br />
<br />
<br />
<br />
'''''yqxM'''''<br />
[[Image:igemgroningen_yqxm_prmoteractivity.jpg|right|200px|yqxm|thumb|yqxM promotor activity during cell growth of different mutants (Axel G. 1999)]]<br />
<br />
The [http://dbtbs.hgc.jp/COG/prom/yqxM-sipW-tasA.html ''yqxM-sipW-tasA''] operon is controlled by the ''yqxM'' promoter. It is needed for biofilm formation because TasA is a key protein of the extracellular matrix. The promotor gets activated via a cascade of other regulatory elements, including SrfA, in response to quorum sensing. Since the chaplins should work in a similar way to TasA we think the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305006 ''yqxM''] promoter would be very suitable for chaplin expression during the stationary phase. We fused the yqxM promoter with GFP but could not observe any expression, since the GFP worked with the srfA promoter we conclude that the yqxM promoter does not work.<br />
<br />
===References===<br />
<small>Bongers RS, Veening JW, Van Wieringen M, Kuipers OP, and Kleerebezem M. Development and characterization of a subtilin-regulated expression system in Bacillus subtilis: strict control of gene expression by addition of subtilin. [http://aem.asm.org/cgi/content/short/71/12/8818Appl Environ Microbiol 2005 Dec; 71(12) 8818-24. pmid:16332878]<br />
<br />
Kleerebezem, M., R. Bongers, G. Rutten, W. M. de Vos, and O. P. Kuipers.<br />
2004. Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of<br />
the spa-box in subtilin-responsive promoters. [http://gbb.eldoc.ub.rug.nl/FILES/root/2004/PeptidesKleerebezem/2004PeptidesKleerebezem.pdf Peptides 25:1415–1424]<br />
<br />
Stöver AG, Driks A. Regulation of synthesis of the ''Bacillus subtilis'' transition-phase, spore-associated antibacterial protein TasA. [http://jb.asm.org/cgi/content/short/181/17/5476 J. Bacteriol. Sept. 1999, p. 5476-5481, Vol. 181, No. 17]<br />
<br />
Nakano MM, Xia LA, Zuber P. Transcription initiation region of the srfA operon, which is controlled by the comP-comA signal transduction system in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC208261/ PMC208261]<br />
<br />
Frances Chu, Daniel B. Kearns, Anna McLoon, Yunrong Chai, Roberto Kolter and Richard Losicka, A Novel Regulatory Protein Governing Biofilm Formation in ''Bacillus subtilis'' [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430766/ PMC2430766]<br />
<br />
Hayashi K, Ohsawa T, Kobayashi K, Ogasawara N, Ogura M. The H2O2 stress-responsive regulator PerR positively regulates srfA expression in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1251593/ PMC1251593]</small></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/ExpressionTeam:Groningen/Expression2010-10-28T01:05:54Z<p>Joelkuiper: /* Expression of chaplins */</p>
<hr />
<div>__NOTOC__<br />
==Expression of chaplins==<br />
<br />
'''Summary'''<br />
<br />
The goal of our project is to let ''Bacillus subtilis'' make a hydrophobic coating by forming a [https://2010.igem.org/Team:Groningen#/biofilm biofilm] and then expressing and secreting [https://2010.igem.org/Team:Groningen/Hydrophobins#Chaplins chaplins]. However, first we needed to test whether ''B. subtilis'' was capable of expressing chaplins, since they could impair the cellgrowth due to their hydrophobic and self assembling properties. We succesfully expressed chaplins C, E and H in ''B. subtilis'' using a tightly regulated subtilin inducable system called "SURE". Furthermore we tested the SURE system for optimal subtilin concentration with GFP. We want ''B. subtilis'' to auto-induce the expression of the chaplins after biofilmformation. Therefore we looked into two operons in ''B. subtilis''; one that gets triggered in late exponential growth (''srfA'' operon) and one that is involved in the formation of biofilm (''yqxM-sipW-tasA'' operon). Using the ''srfA'' promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K305007 BBa_K305007]), we succesfully expressed GFP demonstrating that this promoter could be used to auto-induce the expression chaplins.<br />
<br />
<br><br />
<br />
===Subtilin induced expression of chaplins===<br />
<br />
The biofilm forming capacity of ''Bacillus subtilis'' makes it a good host for our application. In addition, ''B. subtilis'' is known for its ability to produce and secrete large amounts of protein at high cell densities. However, despite its track record as an efficient production organism and the fact that both ''B. subtilis'' and ''Streptomyces coelicolor'' are gram-positive bacteria, it is not certain wether chaplins can be heterologously expressed in ''B. subtilis''. Improper folding, unsuccessful export, or even the very nature of the chaplins, could still lead to hampered expression.<br />
We took several steps to ensure optimal expression. The coding sequences of the chaplins were codon optimized for ''B. subtilis'' and synthesized. We placed a ribosome binding site in front of the coding sequences that is known to work well in ''B. subtilis'', and flanked these constructs with the biobrick prefix and suffix. <br />
<br />
'''SURE expression system'''<br />
<br><br />
[[Image:SURE-gfp-gn.jpg|250px|thumb|right|Subtilin induction of GFP by the SURE system (Bongers ''et al'', 2005)]]<br />
Because it is uncertain how chaplin expression will affect ''B. subtilis'', the initial expression attempts were performed with the stringently controlled, subtilin-regulated gene expression (SURE) system (Bongers ''et al'', 2005). This system uses the subtilin sensing machinery present in a strain of ''B. subtilis'' that autoinduces the production of more of the [http://en.wikipedia.org/wiki/Lantibiotics lantibiotic] subtilin. The subtilin sensor histidine kinase SpaK phosphorylates the response regulator SpaR, which can then bind to so-called ''spa'' boxes in the promoter regions of genes involved in subtilin biosynthesis (Kleerebezem ''et al'', 2004). In the SURE system, a ''B. subtilis'' strain naturally lacking the subtilin biosynthesis genes has the ''spaRK'' genes introduced into its genome. A plasmid carrying a ''spa'' box promoter that is transformed to this strain can then drive the expression of proteins upon subtilin induction of SpaRK signalling. <br />
<br />
[[Image:Sub-ind-gn.jpg|250px|thumb|right|Subtilin induced GFP expression in Bacillus subtilis using the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305011 BBa_K305011] backbone]]<br />
<br />
We have adapted this system to make it BioBrick compatible for easy expression of our chaplins, combinations of chaplins, or any other biobrick part that is composed of an RBS followed by a protein coding sequence. We introduced the BioBrick prefix and suffix into the expression plasmid, downstream of the mutated ''spaS'' promoter, producing our subtilin inducible expression backbone part, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305011 BBa_K305011]. To test the expression and find a suitable subtilin concentration for induction of the chaplins we made use of GFP fluorescence measurements. We inserted the part [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0240 BBa_E0240] into the BioBrick site and induced liquid cultures of ''B. subtilis'' carrying this plasmid (and the ''spaRK'' genes) with different volumes of subtilin-containing culture supernatant of a subtilin producing strain of ''B. subtilis''. These results demonstrate that addition of 0.5 to 1%(vol/vol) of subtilin to the culture is sufficient to reach optimal induction. > Chaplins<br />
<br />
<br><br />
'''Chaplin detection'''<br />
<br><br />
Streptomyces secretes the chaplin proteins into the medium, after which they can serve to lower surface tension or self assemble into amyloid fibers on the cell walls, thus in our first expression tests focused on detecting the chaplins in either the medium in which our supposedly chaplin producing population grew, or on the cells of the B. subtilis.<br />
<br><br />
Using the same methods that were used to dissolve and monomerize chaplins in from Streptomyces, we treated cell pellets and [http://en.wikipedia.org/wiki/Trichloricacetic_acid TCA] precipitated supernatant with [http://en.wikipedia.org/wiki/Trifluoroacetic_acid TFA] to purify chaplin proteins. [http://en.wikipedia.org/wiki/Trifluoroacetic_acid TFA] treatment with 99% pure TFA demolishes most proteins and monomerises assembled chaplin fibers, this enables us to detect the chaplins on SDS gel. Using such a harsh method, we hope to denaturate most proteins to prevent their interference in chaplin detection and highten the relative concentration of chaplin proteins in tested samples.<br />
<br />
<br><br />
Since our early expression experiments didn't yield conclusive results regarding the detection of our chaplins, we we tried staining our samples with an amyloid specific stain called [http://en.wikipedia.org/wiki/Thioflavin Thioflavin T]. Initial testing with the supernatant and washed pellet gave intriguing results yet not clear. Our emission graphs showed some irregularities with the subtilin induced samples, but seemed to be distorted by background noise caused by other materials in the sample. To further purify our samples we decided to [extractioncellwallsGR disrupt] our liquid culture and boil it two times in 2% SDS, before treating the freeze dried sample with 99%. TFA This turned out to be a more successful method.<br />
<br />
Using this method we succesfully detected chaplins C, E and H in purified cell walls from induced B. subtilis cultures, confirmation of these results was provided by malditov mass spectrometry.<br />
<br><br />
<html><br />
<table><br />
<tr><br />
<td></html>[[Image:Chgr.jpg|300px|thumb|Purified chaplins from S. coelicolor exitate at 482 when stained with THT. Different dilutions were used to set up a ladder.]]<html></td><br />
<td></html>[[Image:EHgr.jpg|300px|thumb|Purified E and H chaplins from B subtilis exitate at 482 when stained with THT]] <html></td><br />
</tr><br />
<tr><br />
<td></html>[[Image:Cgr.jpg|300px|thumb|Purified C chaplins from B subtilis exitate at 482 when stained with THT]]>html></td><br />
<td></html>[[Image:Egr.jpg|300px|thumb|Purified E chaplins from B subtilis exitate at 482 when stained with THT]]<html></td><br />
</tr><br />
</table><br />
</html><br />
<br><br />
[[Image:MassSpec.jpg|700px|thumb|none|Mass spectometry results confirm the production of Chaplin H (5128) and E (5144) in Bacillus (green). Peaks is Streptomyces (blue) corrispond, although the peak of chaplin E (5144) indicates that somewhere during translation or in post-translational processes a 21kb threonine group is lost. The non induced B. subtilis strain serves as a control (red). Chaplin C has not been detected this way, but that was not to be expected (Claessen et al. (2009).]]<br />
<br />
===Timed expression of chaplins in a biofilm ===<br />
<br />
An important question is which promoter we should use to control the chaplin expression. We assume that an ideal promoter would not be active until the biofilm has formed because the expression of hydrophobic proteins might influence the formation of it. Two promoters where found that are active in biofilms but not during normal growth. <br />
<br />
[[Image:Groningen-Promotors-sketch.png|300px|left]]<br />
<br />
[[image:igemgroningen_srfa_Promotoractivity.jpg|right|200px|srfA|thumb|srfA promotor activity during cell growth (Nakano MM. 1991)]]<br />
<br />
'''''srfA'''''<br />
<br />
The [http://dbtbs.hgc.jp/COG/prom/srfAA-srfAB-comS-srfAC-srfAD.html ''srfA'' operon] has been reported to be important for natural competence and sporulation in ''Bacillus subtilis''. All these activities occur in biofilms, the promoter is not active until the end of exponential growth. It is controlled by the [https://2010.igem.org/Team:Groningen/Expression_model#ComXPA_quorum_sensing_system ComXPA quorum sensing system] and hence active in states of high cell densities. Therefore the ''srfA'' promoter would be suitable for chaplin expression. Two different lengths of the ''srfA'' promoter where chosen due to uncertainties concerning the region between the response element and the transcription start side of the SrfAA protein. In the original promoter this region is unusually long, by shortening it 190bp’s we hope to achieve a higher transcription efficiency. So we came up with two different promoters, the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305008 original] one and the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305007 shortened] one. Promoter studies using GFP as a reporter confirmed our assumption that the short ''srfA''-promoter variant leads to a higher expression. While the fluorescence of the short variant was clearly above background levels, the long variant did not give convincing results. <br />
[[Image:florescence_srfA.jpg]]<br />
<br />
<br />
<br />
'''''yqxM'''''<br />
[[Image:igemgroningen_yqxm_prmoteractivity.jpg|right|200px|yqxm|thumb|yqxM promotor activity during cell growth of different mutants (Axel G. 1999)]]<br />
<br />
The [http://dbtbs.hgc.jp/COG/prom/yqxM-sipW-tasA.html ''yqxM-sipW-tasA''] operon is controlled by the ''yqxM'' promoter. It is needed for biofilm formation because TasA is a key protein of the extracellular matrix. The promotor gets activated via a cascade of other regulatory elements, including SrfA, in response to quorum sensing. Since the chaplins should work in a similar way to TasA we think the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305006 ''yqxM''] promoter would be very suitable for chaplin expression during the stationary phase. We fused the yqxM promoter with GFP but could not observe any expression, since the GFP worked with the srfA promoter we conclude that the yqxM promoter does not work.<br />
<br />
===References===<br />
<small>Bongers RS, Veening JW, Van Wieringen M, Kuipers OP, and Kleerebezem M. Development and characterization of a subtilin-regulated expression system in Bacillus subtilis: strict control of gene expression by addition of subtilin. [http://aem.asm.org/cgi/content/short/71/12/8818Appl Environ Microbiol 2005 Dec; 71(12) 8818-24. pmid:16332878]<br />
<br />
Kleerebezem, M., R. Bongers, G. Rutten, W. M. de Vos, and O. P. Kuipers.<br />
2004. Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of<br />
the spa-box in subtilin-responsive promoters. [http://gbb.eldoc.ub.rug.nl/FILES/root/2004/PeptidesKleerebezem/2004PeptidesKleerebezem.pdf Peptides 25:1415–1424]<br />
<br />
Stöver AG, Driks A. Regulation of synthesis of the ''Bacillus subtilis'' transition-phase, spore-associated antibacterial protein TasA. [http://jb.asm.org/cgi/content/short/181/17/5476 J. Bacteriol. Sept. 1999, p. 5476-5481, Vol. 181, No. 17]<br />
<br />
Nakano MM, Xia LA, Zuber P. Transcription initiation region of the srfA operon, which is controlled by the comP-comA signal transduction system in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC208261/ PMC208261]<br />
<br />
Frances Chu, Daniel B. Kearns, Anna McLoon, Yunrong Chai, Roberto Kolter and Richard Losicka, A Novel Regulatory Protein Governing Biofilm Formation in ''Bacillus subtilis'' [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430766/ PMC2430766]<br />
<br />
Hayashi K, Ohsawa T, Kobayashi K, Ogasawara N, Ogura M. The H2O2 stress-responsive regulator PerR positively regulates srfA expression in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1251593/ PMC1251593]</small></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/ExpressionTeam:Groningen/Expression2010-10-28T01:03:32Z<p>Joelkuiper: /* Expression of chaplins */</p>
<hr />
<div>__NOTOC__<br />
==Expression of chaplins==<br />
<br />
'''Summary'''<br />
<br />
The goal of our project is to let ''Bacillus subtilis'' make a hydrophobic coating by forming a [https://2010.igem.org/Team:Groningen#/biofilm biofilm] and then expressing and secreting [https://2010.igem.org/Team:Groningen/Hydrophobins#Chaplins chaplins]. However, first we needed to test whether ''B. subtilis'' was capable of expressing chaplins, since they could impair the cellgrowth due to their hydrophobic and self assembling properties. We succesfully expressed chaplins C, E and H in ''B. subtilis'' using a tightly regulated subtilin inducable system called "SURE". Furthermore we tested the SURE system for optimal subtilin concentration with GFP. We want ''B. subtilis'' to auto-induce the expression of the chaplins after biofilmformation. Therefore we looked into two operons in ''B. subtilis''; one that gets triggered in late exponential growth (''srfA'' operon) and one that is involved in the formation of biofilm (''yqxM-sipW-tasA'' operon). Using the ''srfA'' promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K305007 BBa_K305007]), we succesfully expressed GFP demonstrating that this promoter could be used to auto-induce the expression chaplins.<br />
<br />
<br><br />
<br />
===Subtilin induced expression of chaplins===<br />
<br />
The biofilm forming capacity of ''Bacillus subtilis'' makes it a good host for our application. In addition, ''B. subtilis'' is known for its ability to produce and secrete large amounts of protein at high cell densities. However, despite its track record as an efficient production organism and the fact that both ''B. subtilis'' and ''Streptomyces coelicolor'' are gram-positive bacteria, it is not certain wether chaplins can be heterologously expressed in ''B. subtilis''. Improper folding, unsuccessful export, or even the very nature of the chaplins, could still lead to hampered expression.<br />
We took several steps to ensure optimal expression. The coding sequences of the chaplins were codon optimized for ''B. subtilis'' and synthesized. We placed a ribosome binding site in front of the coding sequences that is known to work well in ''B. subtilis'', and flanked these constructs with the biobrick prefix and suffix. <br />
<br />
'''SURE expression system'''<br />
<br><br />
[[Image:SURE-gfp-gn.jpg|250px|thumb|right|Subtilin induction of GFP by the SURE system (Bongers ''et al'', 2005)]]<br />
Because it is uncertain how chaplin expression will affect ''B. subtilis'', the initial expression attempts were performed with the stringently controlled, subtilin-regulated gene expression (SURE) system (Bongers ''et al'', 2005). This system uses the subtilin sensing machinery present in a strain of ''B. subtilis'' that autoinduces the production of more of the [http://en.wikipedia.org/wiki/Lantibiotics lantibiotic] subtilin. The subtilin sensor histidine kinase SpaK phosphorylates the response regulator SpaR, which can then bind to so-called ''spa'' boxes in the promoter regions of genes involved in subtilin biosynthesis (Kleerebezem ''et al'', 2004). In the SURE system, a ''B. subtilis'' strain naturally lacking the subtilin biosynthesis genes has the ''spaRK'' genes introduced into its genome. A plasmid carrying a ''spa'' box promoter that is transformed to this strain can then drive the expression of proteins upon subtilin induction of SpaRK signalling. <br />
<br />
[[Image:Sub-ind-gn.jpg|250px|thumb|right|Subtilin induced GFP expression in Bacillus subtilis using the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305011 BBa_K305011] backbone]]<br />
<br />
We have adapted this system to make it BioBrick compatible for easy expression of our chaplins, combinations of chaplins, or any other biobrick part that is composed of an RBS followed by a protein coding sequence. We introduced the BioBrick prefix and suffix into the expression plasmid, downstream of the mutated ''spaS'' promoter, producing our subtilin inducible expression backbone part, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305011 BBa_K305011]. To test the expression and find a suitable subtilin concentration for induction of the chaplins we made use of GFP fluorescence measurements. We inserted the part [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0240 BBa_E0240] into the BioBrick site and induced liquid cultures of ''B. subtilis'' carrying this plasmid (and the ''spaRK'' genes) with different volumes of subtilin-containing culture supernatant of a subtilin producing strain of ''B. subtilis''. These results demonstrate that addition of 0.5 to 1%(vol/vol) of subtilin to the culture is sufficient to reach optimal induction. > Chaplins<br />
<br />
<br><br />
'''Chaplin detection'''<br />
<br><br />
Streptomyces secretes the chaplin proteins into the medium, after which they can serve to lower surface tension or self assemble into amyloid fibers on the cell walls, thus in our first expression tests focused on detecting the chaplins in either the medium in which our supposedly chaplin producing population grew, or on the cells of the B. subtilis.<br />
<br><br />
Using the same methods that were used to dissolve and monomerize chaplins in from Streptomyces, we treated cell pellets and [http://en.wikipedia.org/wiki/Trichloricacetic_acid TCA] precipitated supernatant with [http://en.wikipedia.org/wiki/Trifluoroacetic_acid TFA] to purify chaplin proteins. [http://en.wikipedia.org/wiki/Trifluoroacetic_acid TFA] treatment with 99% pure TFA demolishes most proteins and monomerises assembled chaplin fibers, this enables us to detect the chaplins on SDS gel. Using such a harsh method, we hope to denaturate most proteins to prevent their interference in chaplin detection and highten the relative concentration of chaplin proteins in tested samples.<br />
<br />
<br><br />
Since our early expression experiments didn't yield conclusive results regarding the detection of our chaplins, we we tried staining our samples with an amyloid specific stain called [http://en.wikipedia.org/wiki/Thioflavin Thioflavin T]. Initial testing with the supernatant and washed pellet gave intriguing results yet not clear. Our emission graphs showed some irregularities with the subtilin induced samples, but seemed to be distorted by background noise caused by other materials in the sample. To further purify our samples we decided to [extractioncellwallsGR disrupt] our liquid culture and boil it two times in 2% SDS, before treating the freeze dried sample with 99%. TFA This turned out to be a more successful method.<br />
<br />
Using this method we succesfully detected chaplins C, E and H in purified cell walls from induced B. subtilis cultures, confirmation of these results was provided by malditov mass spectrometry.<br />
<br><br />
<br />
<table><br />
<tr><br />
<td></html>[[Image:Chgr.jpg|300px|thumb|Purified chaplins from S. coelicolor exitate at 482 when stained with THT. Different dilutions were used to set up a ladder.]]<html></td><br />
<td></html>[[Image:EHgr.jpg|300px|thumb|Purified E and H chaplins from B subtilis exitate at 482 when stained with THT]] <html></td><br />
</tr><br />
<tr><br />
<td></html>[[Image:Cgr.jpg|300px|thumb|Purified C chaplins from B subtilis exitate at 482 when stained with THT]]>html></td><br />
<td></html>[[Image:Egr.jpg|300px|thumb|Purified E chaplins from B subtilis exitate at 482 when stained with THT]]<html></td><br />
</tr><br />
</table><br />
<br />
<br><br />
[[Image:MassSpec.jpg|700px|thumb|none|Mass spectometry results confirm the production of Chaplin H (5128) and E (5144) in Bacillus (green). Peaks is Streptomyces (blue) corrispond, although the peak of chaplin E (5144) indicates that somewhere during translation or in post-translational processes a 21kb threonine group is lost. The non induced B. subtilis strain serves as a control (red). Chaplin C has not been detected this way, but that was not to be expected (Claessen et al. (2009).]]<br />
<br />
===Timed expression of chaplins in a biofilm ===<br />
<br />
An important question is which promoter we should use to control the chaplin expression. We assume that an ideal promoter would not be active until the biofilm has formed because the expression of hydrophobic proteins might influence the formation of it. Two promoters where found that are active in biofilms but not during normal growth. <br />
<br />
[[Image:Groningen-Promotors-sketch.png|300px|left]]<br />
<br />
[[image:igemgroningen_srfa_Promotoractivity.jpg|right|200px|srfA|thumb|srfA promotor activity during cell growth (Nakano MM. 1991)]]<br />
<br />
'''''srfA'''''<br />
<br />
The [http://dbtbs.hgc.jp/COG/prom/srfAA-srfAB-comS-srfAC-srfAD.html ''srfA'' operon] has been reported to be important for natural competence and sporulation in ''Bacillus subtilis''. All these activities occur in biofilms, the promoter is not active until the end of exponential growth. It is controlled by the [https://2010.igem.org/Team:Groningen/Expression_model#ComXPA_quorum_sensing_system ComXPA quorum sensing system] and hence active in states of high cell densities. Therefore the ''srfA'' promoter would be suitable for chaplin expression. Two different lengths of the ''srfA'' promoter where chosen due to uncertainties concerning the region between the response element and the transcription start side of the SrfAA protein. In the original promoter this region is unusually long, by shortening it 190bp’s we hope to achieve a higher transcription efficiency. So we came up with two different promoters, the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305008 original] one and the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305007 shortened] one. Promoter studies using GFP as a reporter confirmed our assumption that the short ''srfA''-promoter variant leads to a higher expression. While the fluorescence of the short variant was clearly above background levels, the long variant did not give convincing results. <br />
[[Image:florescence_srfA.jpg]]<br />
<br />
<br />
<br />
'''''yqxM'''''<br />
[[Image:igemgroningen_yqxm_prmoteractivity.jpg|right|200px|yqxm|thumb|yqxM promotor activity during cell growth of different mutants (Axel G. 1999)]]<br />
<br />
The [http://dbtbs.hgc.jp/COG/prom/yqxM-sipW-tasA.html ''yqxM-sipW-tasA''] operon is controlled by the ''yqxM'' promoter. It is needed for biofilm formation because TasA is a key protein of the extracellular matrix. The promotor gets activated via a cascade of other regulatory elements, including SrfA, in response to quorum sensing. Since the chaplins should work in a similar way to TasA we think the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305006 ''yqxM''] promoter would be very suitable for chaplin expression during the stationary phase. We fused the yqxM promoter with GFP but could not observe any expression, since the GFP worked with the srfA promoter we conclude that the yqxM promoter does not work.<br />
<br />
===References===<br />
<small>Bongers RS, Veening JW, Van Wieringen M, Kuipers OP, and Kleerebezem M. Development and characterization of a subtilin-regulated expression system in Bacillus subtilis: strict control of gene expression by addition of subtilin. [http://aem.asm.org/cgi/content/short/71/12/8818Appl Environ Microbiol 2005 Dec; 71(12) 8818-24. pmid:16332878]<br />
<br />
Kleerebezem, M., R. Bongers, G. Rutten, W. M. de Vos, and O. P. Kuipers.<br />
2004. Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of<br />
the spa-box in subtilin-responsive promoters. [http://gbb.eldoc.ub.rug.nl/FILES/root/2004/PeptidesKleerebezem/2004PeptidesKleerebezem.pdf Peptides 25:1415–1424]<br />
<br />
Stöver AG, Driks A. Regulation of synthesis of the ''Bacillus subtilis'' transition-phase, spore-associated antibacterial protein TasA. [http://jb.asm.org/cgi/content/short/181/17/5476 J. Bacteriol. Sept. 1999, p. 5476-5481, Vol. 181, No. 17]<br />
<br />
Nakano MM, Xia LA, Zuber P. Transcription initiation region of the srfA operon, which is controlled by the comP-comA signal transduction system in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC208261/ PMC208261]<br />
<br />
Frances Chu, Daniel B. Kearns, Anna McLoon, Yunrong Chai, Roberto Kolter and Richard Losicka, A Novel Regulatory Protein Governing Biofilm Formation in ''Bacillus subtilis'' [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430766/ PMC2430766]<br />
<br />
Hayashi K, Ohsawa T, Kobayashi K, Ogasawara N, Ogura M. The H2O2 stress-responsive regulator PerR positively regulates srfA expression in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1251593/ PMC1251593]</small></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/ExpressionTeam:Groningen/Expression2010-10-28T00:57:26Z<p>Joelkuiper: </p>
<hr />
<div>__NOTOC__<br />
==Expression of chaplins==<br />
<br />
'''Summary'''<br />
<br />
The goal of our project is to let ''Bacillus subtilis'' make a hydrophobic coating by forming a [https://2010.igem.org/Team:Groningen#/biofilm biofilm] and then expressing and secreting [https://2010.igem.org/Team:Groningen/Hydrophobins#Chaplins chaplins]. However, first we needed to test whether ''B. subtilis'' was capable of expressing chaplins, since they could impair the cellgrowth due to their hydrophobic and self assembling properties. We succesfully expressed chaplins C, E and H in ''B. subtilis'' using a tightly regulated subtilin inducable system called "SURE". Furthermore we tested the SURE system for optimal subtilin concentration with GFP. We want ''B. subtilis'' to auto-induce the expression of the chaplins after biofilmformation. Therefore we looked into two operons in ''B. subtilis''; one that gets triggered in late exponential growth (''srfA'' operon) and one that is involved in the formation of biofilm (''yqxM-sipW-tasA'' operon). Using the ''srfA'' promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K305007 BBa_K305007]), we succesfully expressed GFP demonstrating that this promoter could be used to auto-induce the expression chaplins.<br />
<br />
<br><br />
<br />
===Subtilin induced expression of chaplins===<br />
<br />
The biofilm forming capacity of ''Bacillus subtilis'' makes it a good host for our application. In addition, ''B. subtilis'' is known for its ability to produce and secrete large amounts of protein at high cell densities. However, despite its track record as an efficient production organism and the fact that both ''B. subtilis'' and ''Streptomyces coelicolor'' are gram-positive bacteria, it is not certain wether chaplins can be heterologously expressed in ''B. subtilis''. Improper folding, unsuccessful export, or even the very nature of the chaplins, could still lead to hampered expression.<br />
We took several steps to ensure optimal expression. The coding sequences of the chaplins were codon optimized for ''B. subtilis'' and synthesized. We placed a ribosome binding site in front of the coding sequences that is known to work well in ''B. subtilis'', and flanked these constructs with the biobrick prefix and suffix. <br />
<br />
'''SURE expression system'''<br />
<br><br />
[[Image:SURE-gfp-gn.jpg|250px|thumb|right|Subtilin induction of GFP by the SURE system (Bongers ''et al'', 2005)]]<br />
Because it is uncertain how chaplin expression will affect ''B. subtilis'', the initial expression attempts were performed with the stringently controlled, subtilin-regulated gene expression (SURE) system (Bongers ''et al'', 2005). This system uses the subtilin sensing machinery present in a strain of ''B. subtilis'' that autoinduces the production of more of the [http://en.wikipedia.org/wiki/Lantibiotics lantibiotic] subtilin. The subtilin sensor histidine kinase SpaK phosphorylates the response regulator SpaR, which can then bind to so-called ''spa'' boxes in the promoter regions of genes involved in subtilin biosynthesis (Kleerebezem ''et al'', 2004). In the SURE system, a ''B. subtilis'' strain naturally lacking the subtilin biosynthesis genes has the ''spaRK'' genes introduced into its genome. A plasmid carrying a ''spa'' box promoter that is transformed to this strain can then drive the expression of proteins upon subtilin induction of SpaRK signalling. <br />
<br />
[[Image:Sub-ind-gn.jpg|300px|thumb|right|Subtilin induced GFP expression in Bacillus subtilis using the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305011 BBa_K305011] backbone]]<br />
<br />
We have adapted this system to make it BioBrick compatible for easy expression of our chaplins, combinations of chaplins, or any other biobrick part that is composed of an RBS followed by a protein coding sequence. We introduced the BioBrick prefix and suffix into the expression plasmid, downstream of the mutated ''spaS'' promoter, producing our subtilin inducible expression backbone part, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305011 BBa_K305011]. To test the expression and find a suitable subtilin concentration for induction of the chaplins we made use of GFP fluorescence measurements. We inserted the part [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0240 BBa_E0240] into the BioBrick site and induced liquid cultures of ''B. subtilis'' carrying this plasmid (and the ''spaRK'' genes) with different volumes of subtilin-containing culture supernatant of a subtilin producing strain of ''B. subtilis''. These results demonstrate that addition of 0.5 to 1%(vol/vol) of subtilin to the culture is sufficient to reach optimal induction. > Chaplins<br />
<br />
<br><br />
'''Chaplin detection'''<br />
<br><br />
Streptomyces secretes the chaplin proteins into the medium, after which they can serve to lower surface tension or self assemble into amyloid fibers on the cell walls, thus in our first expression tests focused on detecting the chaplins in either the medium in which our supposedly chaplin producing population grew, or on the cells of the B. subtilis.<br />
<br><br />
Using the same methods that were used to dissolve and monomerize chaplins in from Streptomyces, we treated cell pellets and [http://en.wikipedia.org/wiki/Trichloricacetic_acid TCA] precipitated supernatant with [http://en.wikipedia.org/wiki/Trifluoroacetic_acid TFA] to purify chaplin proteins. [http://en.wikipedia.org/wiki/Trifluoroacetic_acid TFA] treatment with 99% pure TFA demolishes most proteins and monomerises assembled chaplin fibers, this enables us to detect the chaplins on SDS gel. Using such a harsh method, we hope to denaturate most proteins to prevent their interference in chaplin detection and highten the relative concentration of chaplin proteins in tested samples.<br />
<br />
<br><br />
Since our early expression experiments didn't yield conclusive results regarding the detection of our chaplins, we we tried staining our samples with an amyloid specific stain called [http://en.wikipedia.org/wiki/Thioflavin Thioflavin T]. Initial testing with the supernatant and washed pellet gave intriguing results yet not clear. Our emission graphs showed some irregularities with the subtilin induced samples, but seemed to be distorted by background noise caused by other materials in the sample. To further purify our samples we decided to [extractioncellwallsGR disrupt] our liquid culture and boil it two times in 2% SDS, before treating the freeze dried sample with 99%. TFA This turned out to be a more successful method.<br />
<br />
Using this method we succesfully detected chaplins C, E and H in purified cell walls from induced B. subtilis cultures, confirmation of these results was provided by malditov mass spectrometry.<br />
<br />
<br><br />
{|<br />
|[[Image:Chgr.jpg|300px|thumb|Purified chaplins from S. coelicolor exitate at 482 when stained with THT. Different dilutions were used to set up a ladder.]], <br />
|[[Image:EHgr.jpg|300px|thumb|Purified E and H chaplins from B subtilis exitate at 482 when stained with THT]] <br />
|-<br />
|[[Image:Cgr.jpg|300px|thumb|Purified C chaplins from B subtilis exitate at 482 when stained with THT]], |[[Image:Egr.jpg|300px|thumb|Purified E chaplins from B subtilis exitate at 482 when stained with THT]]<br />
|}<br />
<br />
<br><br />
[[Image:MassSpec.jpg|700px|thumb|none|Mass spectometry results confirm the production of Chaplin H (5128) and E (5144) in Bacillus (green). Peaks is Streptomyces (blue) corrispond, although the peak of chaplin E (5144) indicates that somewhere during translation or in post-translational processes a 21kb threonine group is lost. The non induced B. subtilis strain serves as a control (red). Chaplin C has not been detected this way, but that was not to be expected (Claessen et al. (2009).]]<br />
<br />
===Timed expression of chaplins in a biofilm ===<br />
<br />
An important question is which promoter we should use to control the chaplin expression. We assume that an ideal promoter would not be active until the biofilm has formed because the expression of hydrophobic proteins might influence the formation of it. Two promoters where found that are active in biofilms but not during normal growth. <br />
<br />
[[Image:Groningen-Promotors-sketch.png|300px|left]]<br />
<br />
[[image:igemgroningen_srfa_Promotoractivity.jpg|right|200px|srfA|thumb|srfA promotor activity during cell growth (Nakano MM. 1991)]]<br />
<br />
'''''srfA'''''<br />
<br />
The [http://dbtbs.hgc.jp/COG/prom/srfAA-srfAB-comS-srfAC-srfAD.html ''srfA'' operon] has been reported to be important for natural competence and sporulation in ''Bacillus subtilis''. All these activities occur in biofilms, the promoter is not active until the end of exponential growth. It is controlled by the [https://2010.igem.org/Team:Groningen/Expression_model#ComXPA_quorum_sensing_system ComXPA quorum sensing system] and hence active in states of high cell densities. Therefore the ''srfA'' promoter would be suitable for chaplin expression. Two different lengths of the ''srfA'' promoter where chosen due to uncertainties concerning the region between the response element and the transcription start side of the SrfAA protein. In the original promoter this region is unusually long, by shortening it 190bp’s we hope to achieve a higher transcription efficiency. So we came up with two different promoters, the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305008 original] one and the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305007 shortened] one. Promoter studies using GFP as a reporter confirmed our assumption that the short ''srfA''-promoter variant leads to a higher expression. While the fluorescence of the short variant was clearly above background levels, the long variant did not give convincing results. <br />
[[Image:florescence_srfA.jpg]]<br />
<br />
<br />
<br />
'''''yqxM'''''<br />
[[Image:igemgroningen_yqxm_prmoteractivity.jpg|right|200px|yqxm|thumb|yqxM promotor activity during cell growth of different mutants (Axel G. 1999)]]<br />
<br />
The [http://dbtbs.hgc.jp/COG/prom/yqxM-sipW-tasA.html ''yqxM-sipW-tasA''] operon is controlled by the ''yqxM'' promoter. It is needed for biofilm formation because TasA is a key protein of the extracellular matrix. The promotor gets activated via a cascade of other regulatory elements, including SrfA, in response to quorum sensing. Since the chaplins should work in a similar way to TasA we think the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305006 ''yqxM''] promoter would be very suitable for chaplin expression during the stationary phase. We fused the yqxM promoter with GFP but could not observe any expression, since the GFP worked with the srfA promoter we conclude that the yqxM promoter does not work.<br />
<br />
===References===<br />
<small>Bongers RS, Veening JW, Van Wieringen M, Kuipers OP, and Kleerebezem M. Development and characterization of a subtilin-regulated expression system in Bacillus subtilis: strict control of gene expression by addition of subtilin. [http://aem.asm.org/cgi/content/short/71/12/8818Appl Environ Microbiol 2005 Dec; 71(12) 8818-24. pmid:16332878]<br />
<br />
Kleerebezem, M., R. Bongers, G. Rutten, W. M. de Vos, and O. P. Kuipers.<br />
2004. Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of<br />
the spa-box in subtilin-responsive promoters. [http://gbb.eldoc.ub.rug.nl/FILES/root/2004/PeptidesKleerebezem/2004PeptidesKleerebezem.pdf Peptides 25:1415–1424]<br />
<br />
Stöver AG, Driks A. Regulation of synthesis of the ''Bacillus subtilis'' transition-phase, spore-associated antibacterial protein TasA. [http://jb.asm.org/cgi/content/short/181/17/5476 J. Bacteriol. Sept. 1999, p. 5476-5481, Vol. 181, No. 17]<br />
<br />
Nakano MM, Xia LA, Zuber P. Transcription initiation region of the srfA operon, which is controlled by the comP-comA signal transduction system in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC208261/ PMC208261]<br />
<br />
Frances Chu, Daniel B. Kearns, Anna McLoon, Yunrong Chai, Roberto Kolter and Richard Losicka, A Novel Regulatory Protein Governing Biofilm Formation in ''Bacillus subtilis'' [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430766/ PMC2430766]<br />
<br />
Hayashi K, Ohsawa T, Kobayashi K, Ogasawara N, Ogura M. The H2O2 stress-responsive regulator PerR positively regulates srfA expression in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1251593/ PMC1251593]</small></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/ExpressionTeam:Groningen/Expression2010-10-28T00:53:10Z<p>Joelkuiper: /* Subtilin induced expression of chaplins */</p>
<hr />
<div>__NOTOC__<br />
==Expression of chaplins==<br />
<br />
'''Summary'''<br />
<br />
The goal of our project is to let ''Bacillus subtilis'' make a hydrophobic coating by forming a [https://2010.igem.org/Team:Groningen#/biofilm biofilm] and then expressing and secreting [https://2010.igem.org/Team:Groningen/Hydrophobins#Chaplins chaplins]. However, first we needed to test whether ''B. subtilis'' was capable of expressing chaplins, since they could impair the cellgrowth due to their hydrophobic and self assembling properties. We succesfully expressed chaplins C, E and H in ''B. subtilis'' using a tightly regulated subtilin inducable system called "SURE". Furthermore we tested the SURE system for optimal subtilin concentration with GFP. We want ''B. subtilis'' to auto-induce the expression of the chaplins after biofilmformation. Therefore we looked into two operons in ''B. subtilis''; one that gets triggered in late exponential growth (''srfA'' operon) and one that is involved in the formation of biofilm (''yqxM-sipW-tasA'' operon). Using the ''srfA'' promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K305007 BBa_K305007]), we succesfully expressed GFP demonstrating that this promoter could be used to auto-induce the expression chaplins.<br />
<br />
<br><br />
<br />
===Subtilin induced expression of chaplins===<br />
<br />
The biofilm forming capacity of ''Bacillus subtilis'' makes it a good host for our application. In addition, ''B. subtilis'' is known for its ability to produce and secrete large amounts of protein at high cell densities. However, despite its track record as an efficient production organism and the fact that both ''B. subtilis'' and ''Streptomyces coelicolor'' are gram-positive bacteria, it is not certain wether chaplins can be heterologously expressed in ''B. subtilis''. Improper folding, unsuccessful export, or even the very nature of the chaplins, could still lead to hampered expression.<br />
We took several steps to ensure optimal expression. The coding sequences of the chaplins were codon optimized for ''B. subtilis'' and synthesized. We placed a ribosome binding site in front of the coding sequences that is known to work well in ''B. subtilis'', and flanked these constructs with the biobrick prefix and suffix. <br />
<br />
'''SURE expression system'''<br />
<br><br />
[[Image:SURE-gfp-gn.jpg|250px|thumb|right|Subtilin induction of GFP by the SURE system (Bongers ''et al'', 2005)]]<br />
Because it is uncertain how chaplin expression will affect ''B. subtilis'', the initial expression attempts were performed with the stringently controlled, subtilin-regulated gene expression (SURE) system (Bongers ''et al'', 2005). This system uses the subtilin sensing machinery present in a strain of ''B. subtilis'' that autoinduces the production of more of the [http://en.wikipedia.org/wiki/Lantibiotics lantibiotic] subtilin. The subtilin sensor histidine kinase SpaK phosphorylates the response regulator SpaR, which can then bind to so-called ''spa'' boxes in the promoter regions of genes involved in subtilin biosynthesis (Kleerebezem ''et al'', 2004). In the SURE system, a ''B. subtilis'' strain naturally lacking the subtilin biosynthesis genes has the ''spaRK'' genes introduced into its genome. A plasmid carrying a ''spa'' box promoter that is transformed to this strain can then drive the expression of proteins upon subtilin induction of SpaRK signalling. <br />
<br />
[[Image:Sub-ind-gn.jpg|300px|thumb|right|Subtilin induced GFP expression in Bacillus subtilis using the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305011 BBa_K305011] backbone]]<br />
<br />
We have adapted this system to make it BioBrick compatible for easy expression of our chaplins, combinations of chaplins, or any other biobrick part that is composed of an RBS followed by a protein coding sequence. We introduced the BioBrick prefix and suffix into the expression plasmid, downstream of the mutated ''spaS'' promoter, producing our subtilin inducible expression backbone part, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305011 BBa_K305011]. To test the expression and find a suitable subtilin concentration for induction of the chaplins we made use of GFP fluorescence measurements. We inserted the part [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0240 BBa_E0240] into the BioBrick site and induced liquid cultures of ''B. subtilis'' carrying this plasmid (and the ''spaRK'' genes) with different volumes of subtilin-containing culture supernatant of a subtilin producing strain of ''B. subtilis''. These results demonstrate that addition of 0.5 to 1%(vol/vol) of subtilin to the culture is sufficient to reach optimal induction. > Chaplins<br />
<br />
<br><br />
'''Chaplin detection'''<br />
<br><br />
Streptomyces secretes the chaplin proteins into the medium, after which they can serve to lower surface tension or self assemble into amyloid fibers on the cell walls, thus in our first expression tests focused on detecting the chaplins in either the medium in which our supposedly chaplin producing population grew, or on the cells of the B. subtilis.<br />
<br><br />
Using the same methods that were used to dissolve and monomerize chaplins in from Streptomyces, we treated cell pellets and [http://en.wikipedia.org/wiki/Trichloricacetic_acid TCA] precipitated supernatant with [http://en.wikipedia.org/wiki/Trifluoroacetic_acid TFA] to purify chaplin proteins. [http://en.wikipedia.org/wiki/Trifluoroacetic_acid TFA] treatment with 99% pure TFA demolishes most proteins and monomerises assembled chaplin fibers, this enables us to detect the chaplins on SDS gel. Using such a harsh method, we hope to denaturate most proteins to prevent their interference in chaplin detection and highten the relative concentration of chaplin proteins in tested samples.<br />
<br />
<br><br />
Since our early expression experiments didn't yield conclusive results regarding the detection of our chaplins, we we tried staining our samples with an amyloid specific stain called [http://en.wikipedia.org/wiki/Thioflavin Thioflavin T]. Initial testing with the supernatant and washed pellet gave intriguing results yet not clear. Our emission graphs showed some irregularities with the subtilin induced samples, but seemed to be distorted by background noise caused by other materials in the sample. To further purify our samples we decided to [extractioncellwallsGR disrupt] our liquid culture and boil it two times in 2% SDS, before treating the freeze dried sample with 99%. TFA This turned out to be a more successful method.<br />
<br />
Using this method we succesfully detected chaplins C, E and H in purified cell walls from induced B. subtilis cultures, confirmation of these results was provided by malditov mass spectrometry.<br />
<br />
<br><br />
{|<br />
|[[Image:Chgr.jpg|300px|thumb|Purified chaplins from S. coelicolor exitate at 482 when stained with THT. Different dilutions were used to set up a ladder.]], <br />
|[[Image:EHgr.jpg|300px|thumb|Purified E and H chaplins from B subtilis exitate at 482 when stained with THT]] <br />
|-<br />
|[[Image:Cgr.jpg|300px|thumb|Purified C chaplins from B subtilis exitate at 482 when stained with THT]], |[[Image:Egr.jpg|300px|thumb|Purified E chaplins from B subtilis exitate at 482 when stained with THT]]<br />
|}<br />
[[Image:MassSpec.jpg|700px|thumb|none|Mass spectometry results confirm the production of Chaplin H (5128) and E (5144) in Bacillus (green). Peaks is Streptomyces (blue) corrispond, although the peak of chaplin E (5144) indicates that somewhere during translation or in post-translational processes a 21kb threonine group is lost. The non induced B. subtilis strain serves as a control (red). Chaplin C has not been detected this way, but that was not to be expected (Claessen et al. (2009).]]<br />
<br />
===Timed expression of chaplins in a biofilm ===<br />
<br />
An important question is which promoter we should use to control the chaplin expression. We assume that an ideal promoter would not be active until the biofilm has formed because the expression of hydrophobic proteins might influence the formation of it. Two promoters where found that are active in biofilms but not during normal growth. <br />
<br />
[[Image:Groningen-Promotors-sketch.png|300px|left]]<br />
<br />
[[image:igemgroningen_srfa_Promotoractivity.jpg|right|200px|srfA|thumb|srfA promotor activity during cell growth (Nakano MM. 1991)]]<br />
<br />
'''''srfA'''''<br />
<br />
The [http://dbtbs.hgc.jp/COG/prom/srfAA-srfAB-comS-srfAC-srfAD.html ''srfA'' operon] has been reported to be important for natural competence and sporulation in ''Bacillus subtilis''. All these activities occur in biofilms, the promoter is not active until the end of exponential growth. It is controlled by the [https://2010.igem.org/Team:Groningen/Expression_model#ComXPA_quorum_sensing_system ComXPA quorum sensing system] and hence active in states of high cell densities. Therefore the ''srfA'' promoter would be suitable for chaplin expression. Two different lengths of the ''srfA'' promoter where chosen due to uncertainties concerning the region between the response element and the transcription start side of the SrfAA protein. In the original promoter this region is unusually long, by shortening it 190bp’s we hope to achieve a higher transcription efficiency. So we came up with two different promoters, the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305008 original] one and the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305007 shortened] one. Promoter studies using GFP as a reporter confirmed our assumption that the short ''srfA''-promoter variant leads to a higher expression. While the fluorescence of the short variant was clearly above background levels, the long variant did not give convincing results. <br />
[[Image:florescence_srfA.jpg]]<br />
<br />
<br />
<br />
'''''yqxM'''''<br />
[[Image:igemgroningen_yqxm_prmoteractivity.jpg|right|200px|yqxm|thumb|yqxM promotor activity during cell growth of different mutants (Axel G. 1999)]]<br />
<br />
The [http://dbtbs.hgc.jp/COG/prom/yqxM-sipW-tasA.html ''yqxM-sipW-tasA''] operon is controlled by the ''yqxM'' promoter. It is needed for biofilm formation because TasA is a key protein of the extracellular matrix. The promotor gets activated via a cascade of other regulatory elements, including SrfA, in response to quorum sensing. Since the chaplins should work in a similar way to TasA we think the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305006 ''yqxM''] promoter would be very suitable for chaplin expression during the stationary phase. We fused the yqxM promoter with GFP but could not observe any expression, since the GFP worked with the srfA promoter we conclude that the yqxM promoter does not work.<br />
<br />
===References===<br />
<small>Bongers RS, Veening JW, Van Wieringen M, Kuipers OP, and Kleerebezem M. Development and characterization of a subtilin-regulated expression system in Bacillus subtilis: strict control of gene expression by addition of subtilin. [http://aem.asm.org/cgi/content/short/71/12/8818Appl Environ Microbiol 2005 Dec; 71(12) 8818-24. pmid:16332878]<br />
<br />
Kleerebezem, M., R. Bongers, G. Rutten, W. M. de Vos, and O. P. Kuipers.<br />
2004. Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of<br />
the spa-box in subtilin-responsive promoters. [http://gbb.eldoc.ub.rug.nl/FILES/root/2004/PeptidesKleerebezem/2004PeptidesKleerebezem.pdf Peptides 25:1415–1424]<br />
<br />
Stöver AG, Driks A. Regulation of synthesis of the ''Bacillus subtilis'' transition-phase, spore-associated antibacterial protein TasA. [http://jb.asm.org/cgi/content/short/181/17/5476 J. Bacteriol. Sept. 1999, p. 5476-5481, Vol. 181, No. 17]<br />
<br />
Nakano MM, Xia LA, Zuber P. Transcription initiation region of the srfA operon, which is controlled by the comP-comA signal transduction system in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC208261/ PMC208261]<br />
<br />
Frances Chu, Daniel B. Kearns, Anna McLoon, Yunrong Chai, Roberto Kolter and Richard Losicka, A Novel Regulatory Protein Governing Biofilm Formation in ''Bacillus subtilis'' [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430766/ PMC2430766]<br />
<br />
Hayashi K, Ohsawa T, Kobayashi K, Ogasawara N, Ogura M. The H2O2 stress-responsive regulator PerR positively regulates srfA expression in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1251593/ PMC1251593]</small></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/files/overall.cssTeam:Groningen/files/overall.css2010-10-28T00:47:10Z<p>Joelkuiper: </p>
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}</div>Joelkuiperhttp://2010.igem.org/Team:Groningen/ExpressionTeam:Groningen/Expression2010-10-28T00:41:02Z<p>Joelkuiper: /* Subtilin induced expression of chaplins */</p>
<hr />
<div>__NOTOC__<br />
==Expression of chaplins==<br />
<br />
'''Summary'''<br />
<br />
The goal of our project is to let ''Bacillus subtilis'' make a hydrophobic coating by forming a [https://2010.igem.org/Team:Groningen#/biofilm biofilm] and then expressing and secreting [https://2010.igem.org/Team:Groningen/Hydrophobins#Chaplins chaplins]. However, first we needed to test whether ''B. subtilis'' was capable of expressing chaplins, since they could impair the cellgrowth due to their hydrophobic and self assembling properties. We succesfully expressed chaplins C, E and H in ''B. subtilis'' using a tightly regulated subtilin inducable system called "SURE". Furthermore we tested the SURE system for optimal subtilin concentration with GFP. We want ''B. subtilis'' to auto-induce the expression of the chaplins after biofilmformation. Therefore we looked into two operons in ''B. subtilis''; one that gets triggered in late exponential growth (''srfA'' operon) and one that is involved in the formation of biofilm (''yqxM-sipW-tasA'' operon). Using the ''srfA'' promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K305007 BBa_K305007]), we succesfully expressed GFP demonstrating that this promoter could be used to auto-induce the expression chaplins.<br />
<br />
<br><br />
<br />
===Subtilin induced expression of chaplins===<br />
<br />
The biofilm forming capacity of ''Bacillus subtilis'' makes it a good host for our application. In addition, ''B. subtilis'' is known for its ability to produce and secrete large amounts of protein at high cell densities. However, despite its track record as an efficient production organism and the fact that both ''B. subtilis'' and ''Streptomyces coelicolor'' are gram-positive bacteria, it is not certain wether chaplins can be heterologously expressed in ''B. subtilis''. Improper folding, unsuccessful export, or even the very nature of the chaplins, could still lead to hampered expression.<br />
We took several steps to ensure optimal expression. The coding sequences of the chaplins were codon optimized for ''B. subtilis'' and synthesized. We placed a ribosome binding site in front of the coding sequences that is known to work well in ''B. subtilis'', and flanked these constructs with the biobrick prefix and suffix. <br />
<br />
'''SURE expression system'''<br />
<br><br />
[[Image:SURE-gfp-gn.jpg|250px|thumb|right|Subtilin induction of GFP by the SURE system (Bongers ''et al'', 2005)]]<br />
Because it is uncertain how chaplin expression will affect ''B. subtilis'', the initial expression attempts were performed with the stringently controlled, subtilin-regulated gene expression (SURE) system (Bongers ''et al'', 2005). This system uses the subtilin sensing machinery present in a strain of ''B. subtilis'' that autoinduces the production of more of the [http://en.wikipedia.org/wiki/Lantibiotics lantibiotic] subtilin. The subtilin sensor histidine kinase SpaK phosphorylates the response regulator SpaR, which can then bind to so-called ''spa'' boxes in the promoter regions of genes involved in subtilin biosynthesis (Kleerebezem ''et al'', 2004). In the SURE system, a ''B. subtilis'' strain naturally lacking the subtilin biosynthesis genes has the ''spaRK'' genes introduced into its genome. A plasmid carrying a ''spa'' box promoter that is transformed to this strain can then drive the expression of proteins upon subtilin induction of SpaRK signalling. <br />
<br />
[[Image:Groningen-ODvsFluor-GFP.png|right|300px]]<br />
<br />
We have adapted this system to make it BioBrick compatible for easy expression of our chaplins, combinations of chaplins, or any other biobrick part that is composed of an RBS followed by a protein coding sequence. We introduced the BioBrick prefix and suffix into the expression plasmid, downstream of the mutated ''spaS'' promoter, producing our subtilin inducible expression backbone part, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305011 BBa_K305011]. To test the expression and find a suitable subtilin concentration for induction of the chaplins we made use of GFP fluorescence measurements. We inserted the part [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0240 BBa_E0240] into the BioBrick site and induced liquid cultures of ''B. subtilis'' carrying this plasmid (and the ''spaRK'' genes) with different volumes of subtilin-containing culture supernatant of a subtilin producing strain of ''B. subtilis''. These results demonstrate that addition of 0.5 to 1%(vol/vol) of subtilin to the culture is sufficient to reach optimal induction. > Chaplins<br />
<br />
<br><br />
'''Chaplin detection'''<br />
<br><br />
Streptomyces secretes the chaplin proteins into the medium, after which they can serve to lower surface tension or self assemble into amyloid fibers on the cell walls, thus in our first expression tests focused on detecting the chaplins in either the medium in which our supposedly chaplin producing population grew, or on the cells of the B. subtilis.<br />
<br><br />
Using the same methods that were used to dissolve and monomerize chaplins in from Streptomyces, we treated cell pellets and [http://en.wikipedia.org/wiki/Trichloricacetic_acid TCA] precipitated supernatant with [http://en.wikipedia.org/wiki/Trifluoroacetic_acid TFA] to purify chaplin proteins. [http://en.wikipedia.org/wiki/Trifluoroacetic_acid TFA] treatment with 99% pure TFA demolishes most proteins and monomerises assembled chaplin fibers, this enables us to detect the chaplins on SDS gel. Using such a harsh method, we hope to denaturate most proteins to prevent their interference in chaplin detection and highten the relative concentration of chaplin proteins in tested samples.<br />
<br />
<br><br />
Since our early expression experiments didn't yield conclusive results regarding the detection of our chaplins, we we tried staining our samples with an amyloid specific stain called [http://en.wikipedia.org/wiki/Thioflavin Thioflavin T]. Initial testing with the supernatant and washed pellet gave intriguing results yet not clear. Our emission graphs showed some irregularities with the subtilin induced samples, but seemed to be distorted by background noise caused by other materials in the sample. To further purify our samples we decided to [extractioncellwallsGR disrupt] our liquid culture and boil it two times in 2% SDS, before treating the freeze dried sample with 99%. TFA This turned out to be a more successful method.<br />
<br />
Using this method we succesfully detected chaplins C, E and H in purified cell walls from induced B. subtilis cultures, confirmation of these results was provided by malditov mass spectrometry.<br />
<br />
<br><br />
<br />
[[Image:Chgr.jpg|300px|thumb|left|Purified chaplins from S. coelicolor exitate at 482 when stained with THT. Different dilutions were used to set up a ladder.]] <br />
[[Image:EHgr.jpg|300px|thumb|right|Purified E and H chaplins from B subtilis exitate at 482 when stained with THT]] <br />
<br />
<br><br />
<br />
[[Image:Cgr.jpg|300px|thumb|left|Purified C chaplins from B subtilis exitate at 482 when stained with THT]] [[Image:Egr.jpg|300px|thumb|right|Purified E chaplins from B subtilis exitate at 482 when stained with THT]]<br />
<br />
<br><br />
<br />
[[Image:MassSpec.jpg|700px|thumb|none|Mass spectometry results confirm the production of Chaplin H (5128) and E (5144) in Bacillus (green). Peaks is Streptomyces (blue) corrispond, although the peak of chaplin E (5144) indicates that somewhere during translation or in post-translational processes a 21kb threonine group is lost. The non induced B. subtilis strain serves as a control (red). Chaplin C has not been detected this way, but that was not to be expected (Claessen et al. (2009).]]<br />
<br />
===Timed expression of chaplins in a biofilm ===<br />
<br />
An important question is which promoter we should use to control the chaplin expression. We assume that an ideal promoter would not be active until the biofilm has formed because the expression of hydrophobic proteins might influence the formation of it. Two promoters where found that are active in biofilms but not during normal growth. <br />
<br />
[[Image:Groningen-Promotors-sketch.png|300px|left]]<br />
<br />
[[image:igemgroningen_srfa_Promotoractivity.jpg|right|200px|srfA|thumb|srfA promotor activity during cell growth (Nakano MM. 1991)]]<br />
<br />
'''''srfA'''''<br />
<br />
The [http://dbtbs.hgc.jp/COG/prom/srfAA-srfAB-comS-srfAC-srfAD.html ''srfA'' operon] has been reported to be important for natural competence and sporulation in ''Bacillus subtilis''. All these activities occur in biofilms, the promoter is not active until the end of exponential growth. It is controlled by the [https://2010.igem.org/Team:Groningen/Expression_model#ComXPA_quorum_sensing_system ComXPA quorum sensing system] and hence active in states of high cell densities. Therefore the ''srfA'' promoter would be suitable for chaplin expression. Two different lengths of the ''srfA'' promoter where chosen due to uncertainties concerning the region between the response element and the transcription start side of the SrfAA protein. In the original promoter this region is unusually long, by shortening it 190bp’s we hope to achieve a higher transcription efficiency. So we came up with two different promoters, the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305008 original] one and the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305007 shortened] one. Promoter studies using GFP as a reporter confirmed our assumption that the short ''srfA''-promoter variant leads to a higher expression. While the fluorescence of the short variant was clearly above background levels, the long variant did not give convincing results. <br />
[[Image:florescence_srfA.jpg]]<br />
<br />
<br />
<br />
'''''yqxM'''''<br />
[[Image:igemgroningen_yqxm_prmoteractivity.jpg|right|200px|yqxm|thumb|yqxM promotor activity during cell growth of different mutants (Axel G. 1999)]]<br />
<br />
The [http://dbtbs.hgc.jp/COG/prom/yqxM-sipW-tasA.html ''yqxM-sipW-tasA''] operon is controlled by the ''yqxM'' promoter. It is needed for biofilm formation because TasA is a key protein of the extracellular matrix. The promotor gets activated via a cascade of other regulatory elements, including SrfA, in response to quorum sensing. Since the chaplins should work in a similar way to TasA we think the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305006 ''yqxM''] promoter would be very suitable for chaplin expression during the stationary phase. We fused the yqxM promoter with GFP but could not observe any expression, since the GFP worked with the srfA promoter we conclude that the yqxM promoter does not work.<br />
<br />
===References===<br />
<small>Bongers RS, Veening JW, Van Wieringen M, Kuipers OP, and Kleerebezem M. Development and characterization of a subtilin-regulated expression system in Bacillus subtilis: strict control of gene expression by addition of subtilin. [http://aem.asm.org/cgi/content/short/71/12/8818Appl Environ Microbiol 2005 Dec; 71(12) 8818-24. pmid:16332878]<br />
<br />
Kleerebezem, M., R. Bongers, G. Rutten, W. M. de Vos, and O. P. Kuipers.<br />
2004. Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of<br />
the spa-box in subtilin-responsive promoters. [http://gbb.eldoc.ub.rug.nl/FILES/root/2004/PeptidesKleerebezem/2004PeptidesKleerebezem.pdf Peptides 25:1415–1424]<br />
<br />
Stöver AG, Driks A. Regulation of synthesis of the ''Bacillus subtilis'' transition-phase, spore-associated antibacterial protein TasA. [http://jb.asm.org/cgi/content/short/181/17/5476 J. Bacteriol. Sept. 1999, p. 5476-5481, Vol. 181, No. 17]<br />
<br />
Nakano MM, Xia LA, Zuber P. Transcription initiation region of the srfA operon, which is controlled by the comP-comA signal transduction system in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC208261/ PMC208261]<br />
<br />
Frances Chu, Daniel B. Kearns, Anna McLoon, Yunrong Chai, Roberto Kolter and Richard Losicka, A Novel Regulatory Protein Governing Biofilm Formation in ''Bacillus subtilis'' [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430766/ PMC2430766]<br />
<br />
Hayashi K, Ohsawa T, Kobayashi K, Ogasawara N, Ogura M. The H2O2 stress-responsive regulator PerR positively regulates srfA expression in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1251593/ PMC1251593]</small></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/BiofilmTeam:Groningen/Biofilm2010-10-28T00:29:06Z<p>Joelkuiper: /* References */</p>
<hr />
<div>==Biofilm==<br />
<br />
'''Summary'''<br />
<br />
In our project we want our host bacterium to not only produce the coating material, but also apply it. Therefore we chose ''Bacillus subtilis'' as our host bacterium. ''B. subtilis'' can form a rigid biofilm that will cover the target surface before producing the [https://2010.igem.org/Team:Groningen#/hydrophobins hydrophobic proteins]. As part of our project we made a [https://2010.igem.org/Team:Groningen#/biofilm_model model] on the biofilmformation, but furthermore we looked into ways to easily apply ''B. subtilis'' to the surface and let it form a biofilm there. One way to do this is by adding corn starch to regular TY-medium, making it an easily applicable paste.<br />
<br />
<br />
'''Introduction'''<br />
<html><br />
<div style="text-align: justify"><br />
</html><br />
[[Image:Structure.jpg|right|350px|''B. sub'' Rok biofilm]]<br />
Using biobased materials in the application or manufacturing of coatings has been the topic of many researches. However, using bacteria to make a coating substance and, most importantly, letting it do the coating process for you is something new. In our hydrophobofilm project we aim to use the extracellular fibrous proteins, DNA and polysaccharides that are formed in a biofilm, as a host matrix to embed our coating material, which in our case are hydrophobic proteins. <br />
<br />
Growing a biofilm on a surface as a way of coating it, might seem like a bad idea, since there are quite a lot of coatings out there to prevent biofilms forming in the first place. But why not "fight fire with fire”, and create a biofilm that is non-pathogenic and prevents other biofouling from taking place. <br />
<br />
''Bacillus subtilis'' is an ideal candidate for a biofilm coating. Firstly because it is quickly grows a biofilm which has a smooth extracellular matrix. Secondly, the bacterium is a well known and extensively studied model organism which makes is easier to work with. Finally ''B. subtilis'' is a gram-positive bacterium like ''Streptomyces coelicolor'', the bacterium that naturally produces hydrophobins. This might be an advantage when expressing and assembling the chaplin proteins in our host.<br />
<br />
<br><br />
<br />
'''Biology'''<br />
<br><br />
''In nature, bacteria occur predominantly in highly organized multicellular communities called biofilms. Biofilm formation involves a complex developmental process, where cells differ from each other spatially and morphologically. The bacterial cells in biofilms are phenotypically different, demonstrating an intriguing example of heterogeneous regulation within an isogenic culture. Gram-positive bacteria have developed different strategies for survival in unfavorable environments, e.g. by getting competent or by sporulating. Biofilms offer an opportunity for the cells to survive extreme conditions as the cells in biofilms are more resistant to antibiotics and other harsh circumstances like physical stress, drought or competing organisms. ''Bacillus'' even forms highly complex biofilms with a large degree of structural complexity and diversification of cell function within the biofilm. There are even channels within the biofilm to allow drainage of waste and diffusion of oxygen deep within the biofilm.(''Akos Kovacs)<br />
<br />
<br><br />
<br />
'''Biofilm formation'''[[Image:strain rok.jpg|right|500px]]<br />
Biofilm formation usually starts with the accumulation of biomass, next there is the adhesion to a surface by the production of adhesion proteins. Then the production of "extracellular polymeric substances" (EPS) starts and the phenotypic diversification. After maturation of the biofilm sporulation kicks in. Since the pathways involved in biofilm formation in ''B. subtilis'' are just starting to be unravelled, not everything is known about the complex physiological interactions within a biofilm. By using an already existing pathway in ''B. subtilis'' for the auto-induction of our hydrophobic proteins, we try to minimize the amount of tinkering to the existing signaling pathways. Thereby leaving the natural system intact. <br />
<br />
Timing is one the most important factors in successful assembly of our chaplins in EPS. <br />
''B. subtilis'' produces a protein that forms amyloidfibers called TasA. TasA is a very important protein to provide structural integrity in ''B. subtilis'' biofilms and is formed in the late stage of biofilm formation. The amyloid fibers that are formed provide the biofilm with an increased degree of rigidity (Romero et al, 2009). [https://2010.igem.org/Team:Groningen#/hydrophobins Chaplins] also assemble into amyloid fibers and provide a similar function in the hyphae of ''S. coelicolor'' (Cleassen et al, 2009), giving the hyphae the structural ability to grow high up in the air. Incorperating the chaplins at the same moment as TasA is formed would maximize the chance of successful assembly of chaplins in the EPS, while enabling maximum biofilm coverage. For more details on our expression pathway check out our [https://2010.igem.org/Team:Groningen#/expression expression] or [https://2010.igem.org/Team:Groningen#/modeling modeling] page. <br />
<br />
<br><br />
[[Image:agar TY corn starch.jpg|right|300px]]<br />
'''Coating surfaces'''<br />
<br />
Prevention from our biofilm to grow out of control, is an important aspect when you would apply the hydrophobofilm outside the lab. To deal with these <br />
[https://2010.igem.org/Team:Groningen#/safety safety issues] we modelled a [https://2010.igem.org/Team:Groningen#/killswitch_model kill switch] for our hydrophobofilm. This kill switch relies on the production of a toxin and anti toxin. Where the anti toxin has a slightly shorter half-life than the toxin, thereby eventually resulting in the toxification of the cell itself. This toxification would occur after maturation of the biofilm. After the autotoxification the cells, the EPS with the embedded chaplin proteins will dry out, leaving a hydrophobic EPS layer on the surface.<br />
<br />
<br><br />
<br />
[[Image:biofilm on ceramics.jpg|left|200px]]<br />
Applying our bacteria effectively to a surface poses big challenges. such as, how to coat a surface in a short period of time, with low cost and low tech methods. Furthermore there must be enough nutrients for the organisms to successfully form a biofilm, yet you do not want to smear you surface in to much medium, so to avoid that the organism will only adhere to the medium and not to the surface itself. <br />
<br />
<br><br />
<br />
[[Image:biofilm ceramics total.jpg|right|ceramics]]<br />
<br />
''Biofilm paste''<br />
We attempted to make a medium that could be easily applied to a surface and enable biofilm formation to take place. To achieve this we tried to make our medium more viscous. By adding corn starch to regular TY medium we increased the viscosity of our medium and also made it richer in nutrients. We [https://2010.igem.org/Team:Groningen/20_September_2010 experimented] with different corn starch concentrations. <br />
<br />
We have created an easily applicable paste, to grow our biofilmcoating on all kinds of different surfaces. Another effect of the addition of cornstarch to the medium is an increased growing speed.<br />
<br />
<br><br />
<br />
''A & B: B. subtilis biofilms grown overnight on ceramics coated with the biofilm paste. C: B subtilis biofilms dried out over four days, after formation.''<br />
<br />
<br><br />
<br />
==References==<br />
<small><br />
1. A. Kovacs, Elucidation of the molecular mechanisms underlying the phenotypic heterogeneity of Bacillus subtilis in biofilms<br />
<br />
2. Romero et al, 2009, Amyloid Fibers Provide Structural Integrity to Bacillus<br />
subtilis Biolms<br />
<br />
3. Dennis Claessen, Rick Rink, Wouter de Jong, et al, 2009, A novel class of secreted hydrophobic proteins is involved in aerial hyphae formation in Streptomyces coelicolor by forming amyloid-like fibrils<br />
<br />
</small></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/ImagesTeam:Groningen/Images2010-10-28T00:14:42Z<p>Joelkuiper: /* Image of Science */</p>
<hr />
<div>== Image of Science == <br />
<br />
<html><br />
<center><br />
<object width="600" height="475"> <param name="flashvars" value="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fjoelkuiper%2Fsets%2F72157624427289982%2Fshow%2F&page_show_back_url=%2Fphotos%2Fjoelkuiper%2Fsets%2F72157624427289982%2F&set_id=72157624427289982&jump_to="></param> <param name="movie" value="http://www.flickr.com/apps/slideshow/show.swf?v=71649"></param> <param name="allowFullScreen" value="true"></param><embed type="application/x-shockwave-flash" src="http://www.flickr.com/apps/slideshow/show.swf?v=71649" allowFullScreen="true" flashvars="offsite=true&lang=en-us&page_show_url=%2Fphotos%2Fjoelkuiper%2Fsets%2F72157624427289982%2Fshow%2F&page_show_back_url=%2Fphotos%2Fjoelkuiper%2Fsets%2F72157624427289982%2F&set_id=72157624427289982&jump_to=" width="600" height="475"></embed></object><br />
</center><br />
<br />
<a rel="license" href="http://creativecommons.org/licenses/by-nc-sa/3.0/"><img alt="Creative Commons License" style="border-width:0" src="http://i.creativecommons.org/l/by-nc-sa/3.0/88x31.png" /></a><br /><span xmlns:dct="http://purl.org/dc/terms/" href="http://purl.org/dc/dcmitype/StillImage" property="dct:title" rel="dct:type">Image of Science</span> by <a xmlns:cc="http://creativecommons.org/ns#" href="http://www.igemgroningen.com" property="cc:attributionName" rel="cc:attributionURL">iGEM Groningen &amp; Joël Kuiper</a> is licensed under a <a rel="license" href="http://creativecommons.org/licenses/by-nc-sa/3.0/">Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License</a>.<br />
<br><br />
<br><br />
<br />
Also check out the fungi we've grown during the project in our <a href="https://2010.igem.org/Team:Groningen#/gallery">fungal gallery!</a><br />
</html></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/HomeTeam:Groningen/Home2010-10-27T23:59:30Z<p>Joelkuiper: /* Self assembling hydrophobic biofilm */</p>
<hr />
<div><html><br />
<div margin-left: 10px"> <br />
</html><br />
==Self assembling hydrophobic biofilm==<br />
We aim to design a biological coating as an alternative to for example chemical coatings. For this we, unconventionally, utilized a ''Bacillus subtilis'' [https://2010.igem.org/Team:Groningen#/biofilm biofilm]. We wanted to enable our biofilm to be equipped with an interesting property which is automatically initiated. So we introduced an [https://2010.igem.org/Team:Groningen#/expression expression trigger] which relies on quorum sensing. Our project was directed at finding an alternate solution to biofouling, since regular, chemical coatings which are widely in use pose a threat to the environment. In nature the lotus leaves show self-cleansing properties ascribed to their extreme surface hydrophobicity. In the prokaryotic domain we stumbled upon [https://2010.igem.org/Team:Groningen#/hydrophobins chaplins], strongly hydrophobic proteins originating from ''Streptomyces coelicolor''. Surface hydrophobicity is a very useful property and is used in many [https://2010.igem.org/Team:Groningen#/applications applications] ranging from not only antifouling coatings but also other applications which require water repellence to applications in the field of medical sciences. During our project we have contributed to the parts registry whit numerous [https://2010.igem.org/Team:Groningen#/biobricks BioBricks].<br />
<br />
<br />
We believe that there is a great future for biological coatings as demonstrated within the project. Hydrophobic biological coatings can provide a greener antifouling solution. However using the underlying mechanisms of biofilm triggered expression for other systems like dynamic painting, sensing of environmental changes or even the integration with silicon chips might be within the realm of possibilities. So we challenge coming iGEM teams to further explore and [https://2010.igem.org/Team:Groningen#/brainstorm brainstorm] about the possibilities of using biofilms as a host for a wide range of applications.<br />
<html><br />
</div><br />
<br />
<script src="https://2010.igem.org/Team:Groningen/files/slider.js?action=raw&ctype=text/javascript" type="text/javascript"></script><br />
<div style="height: 1330px"><br />
<div id="slider" style="margin-left: 10px; height: 343px;"><br />
<a href="https://2010.igem.org/Team:Groningen#/project"><img src="https://static.igem.org/mediawiki/2010/d/dd/Groningen-Reel-Stage1.jpg" alt="" title="#biofilm-caption" /></a><br />
<a href="https://2010.igem.org/Team:Groningen#/project"><img src="https://static.igem.org/mediawiki/2010/d/d3/Groningen-Reel-Stage2.jpg" alt="" title="#chaplin-caption" /></a><br />
<a href="https://2010.igem.org/Team:Groningen#/project"><img src="https://static.igem.org/mediawiki/2010/6/6f/Groningen-Reel-Stage3.jpg" alt="" title="#killswitch-caption" /></a><br />
</div><br />
<script type="text/javascript"><br />
<br />
$('#slider').nivoSlider({effect:'fade'});<br />
<br />
</script><br />
<div id="biofilm-caption" class="nivo-html-caption"><br />
A self assembling bio-based coating is form, a </html>[https://2010.igem.org/Team:Groningen#/biofilm rigid biofilm]<html>.<br />
</div><br />
<div id="chaplin-caption" class="nivo-html-caption"><br />
</html>[https://2010.igem.org/Team:Groningen#/expression Expression]<html> of hydrophobic proteins called </html>[https://2010.igem.org/Team:Groningen#/hydrophobins chaplins]<html> is induced by the biofilm causing strong surface hydrophobicity. <br />
</div><br />
<div id="killswitch-caption" class="nivo-html-caption"><br />
The strongly hydrophobic biofilm will die off by a </html>[https://2010.igem.org/Team:Groningen#/killswitch_model killswitch]<html>, leaving a nice hydrophobic biological coating. <br />
</div><br />
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<div></html><br />
This year a [https://2010.igem.org/Team:Groningen#/team team] of young inspired undergraduates from the [http://www.rug.nl University of Groningen] participated in the amazing challenge of iGEM. A multi-disciplinary team of Molecular Biologists, Chemists, Computer Scientists, Journalists and others spend the summer creating a wonderful project in the emerging field of synthetic biology<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/modeling"><img src="https://static.igem.org/mediawiki/2010/1/1e/Groningen-Home-Model.jpg" alt="Modeling"></a><div></html><br />
Using computer models we worked on the frontiers of knowledge. [https://2010.igem.org/Team:Groningen#/expression_model Gene expression] was simulated and a simple explanation for [https://2010.igem.org/Team:Groningen#/biofilm_model cell differentiation] was proposed. Also aiding in ethics and practical feasibility a [https://2010.igem.org/Team:Groningen#/killswitch_model kill switch] system was studied. Finally a [https://2010.igem.org/Team:Groningen#/info_standard new standard] was proposed for characterizing Biobrick parts so future can be streamlined.<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/practices"><img src="https://static.igem.org/mediawiki/2010/7/77/Groningen-Home-Human.jpg" alt="Human Practices"></a><div></html><br />
Because we believe that synthetic biology can better the lives of people and ensure long term prosperity for all humans we spend time [https://2010.igem.org/Team:Groningen#/practices educating] high school students. But not all is perfect so [https://2010.igem.org/Team:Groningen#/safety risks] were assessed and we philosophized on the [https://2010.igem.org/Team:Groningen#/ethics ethical] aspects of synthetic biology.<br />
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<br />
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<br />
=== Our sponsors === <br />
{{Team:Groningen/sponsors}}<br />
<br />
<br />
<html><br />
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</html></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/HomeTeam:Groningen/Home2010-10-27T23:57:27Z<p>Joelkuiper: /* Self assembling hydrophobic biofilm */</p>
<hr />
<div><html><br />
<div margin-left: 10px"> <br />
</html><br />
==Self assembling hydrophobic biofilm==<br />
We aim to design a biological coating as an alternative to for example chemical coatings. For this we, unconventionally, utilized a ''Bacillus subtilis'' [https://2010.igem.org/Team:Groningen#/biofilm biofilm]. We wanted to enable our biofilm to be equipped with an interesting property which is automatically initiated. So we introduced an [https://2010.igem.org/Team:Groningen#/expression expression trigger] which relies on quorum sensing. Our project was directed at finding an alternate solution to biofouling, since regular, chemical coatings which are widely in use pose a threat to the environment. In nature the lotus leaves show self-cleansing properties ascribed to their extreme surface hydrophobicity. In the prokaryotic domain we stumbled upon [https://2010.igem.org/Team:Groningen#/hydrophobins chaplins], strongly hydrophobic proteins originating from ''Streptomyces coelicolor''. Surface hydrophobicity is a very useful property and is used in many [https://2010.igem.org/Team:Groningen#/applications applications] ranging from not only antifouling coatings but also other applications which require water repellence to applications in the field of medical sciences. During our project we have contributed to the parts registry whit numerous [https://2010.igem.org/Team:Groningen#/biobricks BioBricks].<br />
<br />
<br />
We believe that there is a great future for biological coatings as demonstrated within the project. Hydrophobic biological coatings can provide a greener antifouling solution. However using the underlying mechanisms of biofilm triggered expression other systems like dynamic coloring, sensing of environmental changes or even the integration with silicon chips might be within the realm of possibilities. So we challenge coming iGEM teams to further explore and [https://2010.igem.org/Team:Groningen#/brainstorm brainstorm] about the possibilities of using biofilms as a host for a wide range of applications.<br />
<html><br />
</div><br />
<br />
<script src="https://2010.igem.org/Team:Groningen/files/slider.js?action=raw&ctype=text/javascript" type="text/javascript"></script><br />
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<a href="https://2010.igem.org/Team:Groningen#/project"><img src="https://static.igem.org/mediawiki/2010/d/d3/Groningen-Reel-Stage2.jpg" alt="" title="#chaplin-caption" /></a><br />
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<script type="text/javascript"><br />
<br />
$('#slider').nivoSlider({effect:'fade'});<br />
<br />
</script><br />
<div id="biofilm-caption" class="nivo-html-caption"><br />
A self assembling bio-based coating is form, a </html>[https://2010.igem.org/Team:Groningen#/biofilm rigid biofilm]<html>.<br />
</div><br />
<div id="chaplin-caption" class="nivo-html-caption"><br />
</html>[https://2010.igem.org/Team:Groningen#/expression Expression]<html> of hydrophobic proteins called </html>[https://2010.igem.org/Team:Groningen#/hydrophobins chaplins]<html> is induced by the biofilm causing strong surface hydrophobicity. <br />
</div><br />
<div id="killswitch-caption" class="nivo-html-caption"><br />
The strongly hydrophobic biofilm will die off by a </html>[https://2010.igem.org/Team:Groningen#/killswitch_model killswitch]<html>, leaving a nice hydrophobic biological coating. <br />
</div><br />
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<br />
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<a href="https://2010.igem.org/Team:Groningen#/team"><img src="https://static.igem.org/mediawiki/2010/f/f5/Groningen-Home-Team.jpg" alt="Team"></a><br />
<div></html><br />
This year a [https://2010.igem.org/Team:Groningen#/team team] of young inspired undergraduates from the [http://www.rug.nl University of Groningen] participated in the amazing challenge of iGEM. A multi-disciplinary team of Molecular Biologists, Chemists, Computer Scientists, Journalists and others spend the summer creating a wonderful project in the emerging field of synthetic biology<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/modeling"><img src="https://static.igem.org/mediawiki/2010/1/1e/Groningen-Home-Model.jpg" alt="Modeling"></a><div></html><br />
Using computer models we worked on the frontiers of knowledge. [https://2010.igem.org/Team:Groningen#/expression_model Gene expression] was simulated and a simple explanation for [https://2010.igem.org/Team:Groningen#/biofilm_model cell differentiation] was proposed. Also aiding in ethics and practical feasibility a [https://2010.igem.org/Team:Groningen#/killswitch_model kill switch] system was studied. Finally a [https://2010.igem.org/Team:Groningen#/info_standard new standard] was proposed for characterizing Biobrick parts so future can be streamlined.<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/practices"><img src="https://static.igem.org/mediawiki/2010/7/77/Groningen-Home-Human.jpg" alt="Human Practices"></a><div></html><br />
Because we believe that synthetic biology can better the lives of people and ensure long term prosperity for all humans we spend time [https://2010.igem.org/Team:Groningen#/practices educating] high school students. But not all is perfect so [https://2010.igem.org/Team:Groningen#/safety risks] were assessed and we philosophized on the [https://2010.igem.org/Team:Groningen#/ethics ethical] aspects of synthetic biology.<br />
<html></div></div><br />
</div><br />
</html><br />
<br />
----<br />
<br />
=== Our sponsors === <br />
{{Team:Groningen/sponsors}}<br />
<br />
<br />
<html><br />
</div><br />
</html></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/HomeTeam:Groningen/Home2010-10-27T23:54:40Z<p>Joelkuiper: /* Self assembling hydrophobic biofilm */</p>
<hr />
<div><html><br />
<div margin-left: 10px"> <br />
</html><br />
==Self assembling hydrophobic biofilm==<br />
We aim to design a biological coating as an alternative to for example chemical coatings. For this we, unconventionally, utilized a ''Bacillus subtilis'' [https://2010.igem.org/Team:Groningen#/biofilm biofilm]. We wanted to enable our biofilm to be equipped with an interesting property which is automatically initiated. So we introduced an [https://2010.igem.org/Team:Groningen#/expression expression trigger] which relies on quorum sensing. Our project was directed at finding an alternate solution to biofouling, since regular, chemical coatings which are widely in use pose a threat to the environment. In nature the lotus leaves show self-cleansing properties ascribed to their extreme surface hydrophobicity. In the prokaryotic domain we stumbled upon [https://2010.igem.org/Team:Groningen#/hydrophobins chaplins], strongly hydrophobic proteins originating from ''Streptomyces coelicolor''. Surface hydrophobicity is a very useful property and is used in many [https://2010.igem.org/Team:Groningen#/applications applications] ranging from not only antifouling coatings but also other applications which require water repellence to applications in the field of medical sciences. During our project we have contributed to the parts registry whit numerous [https://2010.igem.org/Team:Groningen#/biobricks BioBricks].<br />
<br />
<br />
We believe that there is a great future for biological coatings as demonstrated within the project. Hydrophobic biologicalcoatings can provide a greener antifouling solution. However using the underlying mechanisms of biofilm triggered expression other systems like dynamic coloring, sensing of environmental changes or even the integration with silicon chips might be within the realm of possibilities. <br />
<html><br />
</div><br />
<br />
<script src="https://2010.igem.org/Team:Groningen/files/slider.js?action=raw&ctype=text/javascript" type="text/javascript"></script><br />
<div style="height: 1330px"><br />
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Using computer models we worked on the frontiers of knowledge. [https://2010.igem.org/Team:Groningen#/expression_model Gene expression] was simulated and a simple explanation for [https://2010.igem.org/Team:Groningen#/biofilm_model cell differentiation] was proposed. Also aiding in ethics and practical feasibility a [https://2010.igem.org/Team:Groningen#/killswitch_model kill switch] system was studied. Finally a [https://2010.igem.org/Team:Groningen#/info_standard new standard] was proposed for characterizing Biobrick parts so future can be streamlined.<br />
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Because we believe that synthetic biology can better the lives of people and ensure long term prosperity for all humans we spend time [https://2010.igem.org/Team:Groningen#/practices educating] high school students. But not all is perfect so [https://2010.igem.org/Team:Groningen#/safety risks] were assessed and we philosophized on the [https://2010.igem.org/Team:Groningen#/ethics ethical] aspects of synthetic biology.<br />
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==Self assembling hydrophobic biofilm==<br />
We aim to design a biological coating as an alternative to for example chemical coatings. For this we, unconventionally, utilized a ''Bacillus subtilis'' [https://2010.igem.org/Team:Groningen#/biofilm biofilm]. We wanted to enable our biofilm to be equipped with an interesting property which is automatically initiated. So we introduced an [https://2010.igem.org/Team:Groningen#/expression expression trigger] which relies on quorum sensing. Our project was directed at finding an alternate solution to biofouling, since regular, chemical coatings which are widely in use pose a threat to the environment. In nature the lotus leaves show self-cleansing properties ascribed to their extreme surface hydrophobicity. In the prokaryotic domain we stumbled upon [https://2010.igem.org/Team:Groningen#/hydrophobins chaplins], strongly hydrophobic proteins originating from ''Streptomyces coelicolor''. Surface hydrophobicity is a very useful property and is used in many [https://2010.igem.org/Team:Groningen#/applications applications] ranging from not only antifouling coatings but also other applications which require water repellence to applications in the field of medical sciences. During our project we have contributed to the parts registry whit numerous [https://2010.igem.org/Team:Groningen#/biobricks BioBricks].<br />
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This year a [https://2010.igem.org/Team:Groningen#/team team] of young inspired undergraduates from the [http://www.rug.nl University of Groningen] participated in the amazing challenge of iGEM. A multi-disciplinary team of Molecular Biologists, Chemists, Computer Scientists, Journalists and others spend the summer creating a wonderful project in the emerging field of synthetic biology<br />
<html></div></div><br />
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<div><a href="https://2010.igem.org/Team:Groningen#/modeling"><img src="https://static.igem.org/mediawiki/2010/1/1e/Groningen-Home-Model.jpg" alt="Modeling"></a><div></html><br />
Using computer models we worked on the frontiers of knowledge. [https://2010.igem.org/Team:Groningen#/expression_model Gene expression] was simulated and a simple explanation for [https://2010.igem.org/Team:Groningen#/biofilm_model cell differentiation] was proposed. Also aiding in ethics and practical feasibility a [https://2010.igem.org/Team:Groningen#/killswitch_model kill switch] system was studied. Finally a [https://2010.igem.org/Team:Groningen#/info_standard new standard] was proposed for characterizing Biobrick parts so future can be streamlined.<br />
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Because we believe that synthetic biology can better the lives of people and ensure long term prosperity for all humans we spend time [https://2010.igem.org/Team:Groningen#/practices educating] high school students. But not all is perfect so [https://2010.igem.org/Team:Groningen#/safety risks] were assessed and we philosophized on the [https://2010.igem.org/Team:Groningen#/ethics ethical] aspects of synthetic biology.<br />
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=== Our sponsors === <br />
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</html></div>Joelkuiperhttp://2010.igem.org/File:ChpCartoon.jpgFile:ChpCartoon.jpg2010-10-27T23:21:14Z<p>Joelkuiper: uploaded a new version of "Image:ChpCartoon.jpg"</p>
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<div id="otherContent"></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/Biofilm_modelTeam:Groningen/Biofilm model2010-10-27T22:52:36Z<p>Joelkuiper: /* Future work */</p>
<hr />
<div>__NOTOC__<br />
==Biofilm Dynamics==<br />
<br />
===Introduction===<br />
Biofilms are multicellular conglomerates which attach to surfaces. The formation of biofilms is triggered by high cell density and limited resources. The sensing is of these conditions is often mediated by an extracellular signaling compound which increases in concentration and triggers regulating circuitry. This process is called quorum sensing and it plays an important role in the dynamics of multicellular systems. <br />
<br />
Quorum sensing systems can cause complicated effects including cell differentiation within single species conglomerates. A recent example of this can be seen in the difference of expression in the [http://subtiwiki.uni-goettingen.de/wiki/index.php/TasA TasA protein] which plays a major role in the formation of the biofilm matrix by forming amyloid fibers. Expression of this protein is mediated by competitive inhibitive systems by, amongst others, the [http://subtiwiki.uni-goettingen.de/wiki/index.php/YqxM yqxM gene](<partinfo>BBa_K305006</partinfo>) which linked to the [https://2010.igem.org/Team:Groningen#/expression_model ComXPA quorum sensing] system. <br />
<br />
However it has been found that not all bacteria respond to the signaling molecules in the same way. It was shown that biofilm formation in ''Bacillus subtilis'' involves paracrine signaling in which most cells produce and secrete the signaling molecule ComX but only a sub population of the cells is triggered to make surfactin. Surfactin serves as paracrine signaling molecule and the cells which are not able to make surfactin, respond to surfactin by making extracellular matrix components for the biofilm. <br />
<br />
It is hypothesized that the extracellular matrix interferes with the interaction between ComX and the trans membrane histidine kinase ComP and therefore prevents surfactin production in extracellular matrix producing cells. In a mutant which did not express extracellular matrix proteins, the surfactin expression was more than three times higher. <br />
<br />
While differentiation requires interaction between several genes and complicated effects as described above we believe even simple extra-cellular accumulation of a pheromone can be responsible. <br />
<br />
A very simple model is proposed based on 2D growth of bacteria by cellular automata on a restricted plane. Each of the cells (a pixel in the model) secretes an unnamed compound which is allowed to accumulate. The accumulated compound is diffused at each discrete time step by applying a Gaussian Kernel. Finally at each cell a sigmoidal response curve is mapped, simplifying an all-or-nothing response similar to signal amplification through auto-phosphorylation of transmembrane histidine kinases. Cell differentiation was demonstrated within this theoretical model and variation of the parameters change the response drastically.<br />
<br />
===Growth by cellular automata===<br />
Cellular Automata are discrete computational models. The model consist of a finite grid (of any dimension) in which each of the cells can be in one of a finite set of states. The model behaves discretely in time by evaluating the number of neighbors of each cell and updating its state by some algebraic rule. A popular example of such a model with only two possible states, on and off, and a 2 dimensional grid is Conways Game of life. A possible instantiation of this "game" (it is a zero player game in the sense that it plays itself) is shown below. <br />
<br />
[[Image:Groningen-wiki-Gospers_glider_gun.gif|frame|none|Conways Game of life(from [http://en.wikipedia.org/wiki/Conway_game_of_life wikipedia])]]<br />
<br />
Simpler one dimensional models are an important subject of study in theoretical computer science. We modeled unrestricted bacterial growth by Cellular Automata. The model is a 2D grid of 400x400 pixels, the states are on and off indicating the presence of biomass. The grid is initialized randomly with a fixed distribution of "on" states. At every time step the following rule is applied by evaluating each of the cells 8 neighbors, if a cell has 3 or 4 neighbors with the "on" state the cell itself becomes "on" (a growth step, or birth), if a cell has 4, 5, or 6 neighbors which are on the cell is allowed to live and thus remains on. If any cell does not meet these criteria the cell will remain or become off. An animation of this model is shown below.<br />
<br />
[[Image:Groningen-CAModel.gif]]<br />
<br />
Due to the random initialization the first time steps are trimmed to allow for the distribution to stabilize. The result of the simulation is a binary matrix C for each time step representing biomass occupation. It can be shown by summing the total amount of occupied cell increases exponentially at each step and finally reaching a stationary phase because of spatial limitations.<br />
<br />
[[Image:Groningen-model-cellDensity.png|300px]]<br />
<br />
===Quorum sensing pheromones===<br />
Quorum sensing pheromones are modeled by sampling a random number from the normal distribution for each cell which in on resulting in a new matrix P. Because the sensing pheromones are allowed to accumulate each next time step is the sum of the previous matrices. Quorum sensing requires diffusion of the pheromone from one occupied cell to another. Plotting the density of pheromone gives the following graph:<br />
<br />
[[Image:Groningen-model-subsNoDiff.png]]<br />
<br />
Continuous diffusion can be modeled using the Heat equation, a two dimensional Partial Differential Equation (PDE) as shown below. Fick's second law is a a common biological and chemical model and is analogous to the two-dimensional Heat equation. This would generate a discrete-differential model.<br />
<br />
[[Image:Groningen-heat-model.png]]<br />
<br />
Solving this equation allows for diffusion continuous in both space and time. However for simplicity we applied a discrete Gaussian filter, equivalent to a convoluting a Gaussian matrix to our substance matrix P generating P'. The Gaussian Filter in 2D has the following form: <br />
<br />
[[Image:Groningen-GaussianFilter.png]]<br />
<br />
Where &sigma; is the standard deviation. The result of this process with &sigma; = 3 is demonstrated at a single time step below: <br />
<br />
[[Image:Groningen-model-subsDiff.png|frame|none|Diffusion applied at t=8]]<br />
<br />
===Activation of genes===<br />
Simplification of the Hill kinetics allowed for modeling of gene activation through simple sigmoidal function:<br />
<br />
[[Image:Groningen-Sigmoid-Model.png]]<br />
<br />
Imputing every element of P' into the function shows which genes are activated. Binarization of the responses allowed to color the activated cells in the original matrix C. Results are discussed below.<br />
<br />
===Results===<br />
The results of the model are shown below in simplified form. Download the interactive code and the [http://www.wolfram.com/products/player/ Mathematica 7 viewer] to play interactively with the model. The cells which have the gene expressed are shown in red. <br />
[[Image:Groningen-Biofilm3.png|150px|frame|none|Early time step with high expression threshold]]<br />
<br />
<br />
[[Image:Groningen-Biofilm1.png|150px|frame|none|Later time step with high expression threshold, some expression shown]]<br />
<br />
<br />
[[Image:Groningen-biofilm2.png|150px|frame|none|Late time step with high expression threshold, cell differentiation clearly visible]]<br />
<br />
<br />
[[Image:Groningen-biofilm4.png|150px|frame|none|Late time step with high expression threshold and high diffusion rate]]<br />
<br />
===Future work=== <br />
The model proposed here is a grave oversimplification and many enhancements can be made. First of all the growth of the biofilm is unrestricted by substance availability Loosdrecht et. al. proposed a more realistic model of biofilm formation taking into account oxygen and substance concentrations. The pheromone is diffused discretely, however a larger resolution would be needed to differentiate between cellular and molecular scale. Implementing the Heat equation would increase the resolution and allow for a more realistic diffusion model. It is now assumed that the pheromone concentrations remain stable over time, however to fully account for its presence substance degradation by extracellular proteins would also need to be modeled. <br />
<br />
A more realistic model of [https://2010.igem.org/Team:Groningen#/expression_model gene expression] could be incorporated further increasing its realism by taking into account within-cell dynamics. Also the ''B.subtilis'' quorum sensing system does not rely on a single compound, many different molecules diffuse and influence cell behavior. Sporulation for example is known to be a competitive inhibitory system between CSF and ComX.<br />
<br />
===Sources===<br />
The Mathematica 7 source files are available below:<br />
<br />
Biofilm model ([http://dl.dropbox.com/u/391356/iGEM/biofilm.nb source])(interactive)<br><br />
2D Heat equation ([http://dl.dropbox.com/u/391356/iGEM/diff.nb source])<br><br />
Sigmoid response curve ([http://dl.dropbox.com/u/391356/iGEM/hillSimpl.nb source])([http://dl.dropbox.com/u/391356/iGEM/hillSimpl.nbp interactive])<br />
<br />
<br />
===References===<br />
<small>Comella N, Grossman AD, Conservation of genes and processes controlled by the quorum response in bacteria: characterization of genes controlled by the quorum-sensing transcription factor ComA in Bacillus subtilis, Molecular Microbiology (2005), 57 (4): 1159–1174. PMID 16091051 <br />
<br />
Lemon KP, Earl AM, Vlamakis HC, Aguilar C, Kolter R., Biofilm development with an emphasis on Bacillus subtilis, Current Topics Microbiology and Immunology, 2008, 322:1-16. PMID 18453269 <br />
<br />
López D, Vlamakis H, Kolter R, Biofilms, Cold Spring Harbor Perspectives in Biology, 2010, (2):a000398. PMID 20519345 <br />
<br />
López D, Vlamakis H, Losick R, Kolter R, Paracrine signaling in a bacterium, Genes & Development, 2009 23(14):1631-8. <br />
<br />
Pérez J, Picioreanu C, van Loosdrecht M, Modeling biofilm and floc diffusion processes based on analytical solution of reaction-diffusion equations, Water Research, 2005, 39:1311-1323.<br />
<br />
Picioreanu C, van Loosdrecht MC, Heijnen JJ. A new combined differential-discrete cellular automaton approach for biofilm modeling: application for growth in gel beads, Biotechnology Bioengineering, 1998, 57(6):718-31. PMID 10099251<br />
<br />
Picioreanu C, van Loosdrecht MC, Heijnen JJ. Mathematical modeling of biofilm structure with a hybrid differential-discrete cellular automaton approach, Biotechnology Bioengineering, 1998, 58(1):101-16. PMID <br />
10099266 <br />
Roggiani M, Dubnau D, ComA, a phosphorylated response regulator protein of Bacillus subtilis, binds to the promoter region of srfA, Journal of Bacteriology, 1993, 175(10): 3182-7. PMID 8387999 <br />
<br />
Shah IM, Dworkin J, Microbial interactions: bacteria talk to (some of) their neighbors, Current Biology, 2009, 19(16): 689-91. PMID 19706277</small></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/Biofilm_modelTeam:Groningen/Biofilm model2010-10-27T22:52:06Z<p>Joelkuiper: /* Future work */</p>
<hr />
<div>__NOTOC__<br />
==Biofilm Dynamics==<br />
<br />
===Introduction===<br />
Biofilms are multicellular conglomerates which attach to surfaces. The formation of biofilms is triggered by high cell density and limited resources. The sensing is of these conditions is often mediated by an extracellular signaling compound which increases in concentration and triggers regulating circuitry. This process is called quorum sensing and it plays an important role in the dynamics of multicellular systems. <br />
<br />
Quorum sensing systems can cause complicated effects including cell differentiation within single species conglomerates. A recent example of this can be seen in the difference of expression in the [http://subtiwiki.uni-goettingen.de/wiki/index.php/TasA TasA protein] which plays a major role in the formation of the biofilm matrix by forming amyloid fibers. Expression of this protein is mediated by competitive inhibitive systems by, amongst others, the [http://subtiwiki.uni-goettingen.de/wiki/index.php/YqxM yqxM gene](<partinfo>BBa_K305006</partinfo>) which linked to the [https://2010.igem.org/Team:Groningen#/expression_model ComXPA quorum sensing] system. <br />
<br />
However it has been found that not all bacteria respond to the signaling molecules in the same way. It was shown that biofilm formation in ''Bacillus subtilis'' involves paracrine signaling in which most cells produce and secrete the signaling molecule ComX but only a sub population of the cells is triggered to make surfactin. Surfactin serves as paracrine signaling molecule and the cells which are not able to make surfactin, respond to surfactin by making extracellular matrix components for the biofilm. <br />
<br />
It is hypothesized that the extracellular matrix interferes with the interaction between ComX and the trans membrane histidine kinase ComP and therefore prevents surfactin production in extracellular matrix producing cells. In a mutant which did not express extracellular matrix proteins, the surfactin expression was more than three times higher. <br />
<br />
While differentiation requires interaction between several genes and complicated effects as described above we believe even simple extra-cellular accumulation of a pheromone can be responsible. <br />
<br />
A very simple model is proposed based on 2D growth of bacteria by cellular automata on a restricted plane. Each of the cells (a pixel in the model) secretes an unnamed compound which is allowed to accumulate. The accumulated compound is diffused at each discrete time step by applying a Gaussian Kernel. Finally at each cell a sigmoidal response curve is mapped, simplifying an all-or-nothing response similar to signal amplification through auto-phosphorylation of transmembrane histidine kinases. Cell differentiation was demonstrated within this theoretical model and variation of the parameters change the response drastically.<br />
<br />
===Growth by cellular automata===<br />
Cellular Automata are discrete computational models. The model consist of a finite grid (of any dimension) in which each of the cells can be in one of a finite set of states. The model behaves discretely in time by evaluating the number of neighbors of each cell and updating its state by some algebraic rule. A popular example of such a model with only two possible states, on and off, and a 2 dimensional grid is Conways Game of life. A possible instantiation of this "game" (it is a zero player game in the sense that it plays itself) is shown below. <br />
<br />
[[Image:Groningen-wiki-Gospers_glider_gun.gif|frame|none|Conways Game of life(from [http://en.wikipedia.org/wiki/Conway_game_of_life wikipedia])]]<br />
<br />
Simpler one dimensional models are an important subject of study in theoretical computer science. We modeled unrestricted bacterial growth by Cellular Automata. The model is a 2D grid of 400x400 pixels, the states are on and off indicating the presence of biomass. The grid is initialized randomly with a fixed distribution of "on" states. At every time step the following rule is applied by evaluating each of the cells 8 neighbors, if a cell has 3 or 4 neighbors with the "on" state the cell itself becomes "on" (a growth step, or birth), if a cell has 4, 5, or 6 neighbors which are on the cell is allowed to live and thus remains on. If any cell does not meet these criteria the cell will remain or become off. An animation of this model is shown below.<br />
<br />
[[Image:Groningen-CAModel.gif]]<br />
<br />
Due to the random initialization the first time steps are trimmed to allow for the distribution to stabilize. The result of the simulation is a binary matrix C for each time step representing biomass occupation. It can be shown by summing the total amount of occupied cell increases exponentially at each step and finally reaching a stationary phase because of spatial limitations.<br />
<br />
[[Image:Groningen-model-cellDensity.png|300px]]<br />
<br />
===Quorum sensing pheromones===<br />
Quorum sensing pheromones are modeled by sampling a random number from the normal distribution for each cell which in on resulting in a new matrix P. Because the sensing pheromones are allowed to accumulate each next time step is the sum of the previous matrices. Quorum sensing requires diffusion of the pheromone from one occupied cell to another. Plotting the density of pheromone gives the following graph:<br />
<br />
[[Image:Groningen-model-subsNoDiff.png]]<br />
<br />
Continuous diffusion can be modeled using the Heat equation, a two dimensional Partial Differential Equation (PDE) as shown below. Fick's second law is a a common biological and chemical model and is analogous to the two-dimensional Heat equation. This would generate a discrete-differential model.<br />
<br />
[[Image:Groningen-heat-model.png]]<br />
<br />
Solving this equation allows for diffusion continuous in both space and time. However for simplicity we applied a discrete Gaussian filter, equivalent to a convoluting a Gaussian matrix to our substance matrix P generating P'. The Gaussian Filter in 2D has the following form: <br />
<br />
[[Image:Groningen-GaussianFilter.png]]<br />
<br />
Where &sigma; is the standard deviation. The result of this process with &sigma; = 3 is demonstrated at a single time step below: <br />
<br />
[[Image:Groningen-model-subsDiff.png|frame|none|Diffusion applied at t=8]]<br />
<br />
===Activation of genes===<br />
Simplification of the Hill kinetics allowed for modeling of gene activation through simple sigmoidal function:<br />
<br />
[[Image:Groningen-Sigmoid-Model.png]]<br />
<br />
Imputing every element of P' into the function shows which genes are activated. Binarization of the responses allowed to color the activated cells in the original matrix C. Results are discussed below.<br />
<br />
===Results===<br />
The results of the model are shown below in simplified form. Download the interactive code and the [http://www.wolfram.com/products/player/ Mathematica 7 viewer] to play interactively with the model. The cells which have the gene expressed are shown in red. <br />
[[Image:Groningen-Biofilm3.png|150px|frame|none|Early time step with high expression threshold]]<br />
<br />
<br />
[[Image:Groningen-Biofilm1.png|150px|frame|none|Later time step with high expression threshold, some expression shown]]<br />
<br />
<br />
[[Image:Groningen-biofilm2.png|150px|frame|none|Late time step with high expression threshold, cell differentiation clearly visible]]<br />
<br />
<br />
[[Image:Groningen-biofilm4.png|150px|frame|none|Late time step with high expression threshold and high diffusion rate]]<br />
<br />
===Future work=== <br />
The model proposed here is a grave oversimplification and many enhancements can be made. First of all the growth of the biofilm is unrestricted by substance availability Loosdrecht et. al. proposed a more realistic model of biofilm formation taking into account oxygen and substance concentrations. The pheromone is diffused discretely, however a larger resolution would be needed to differentiate between cellular and molecular scale. Implementing the Heat equation would increase the resolution and allow for a more realistic diffusion model. It is now assumed that the pheromone concentrations remain stable over time, however to fully account for its presence substance degradation by extracellular proteins would also need to be modeled. <br />
<br />
A more realistic model of [https://2010.igem.org/Team:Groningen#/expression_model gene expression], could be incorporated further increasing its realism by taking into account within-cell dynamics. Also the ''B.subtilis'' quorum sensing system does not rely on a single compound, many different molecules diffuse and influence cell behavior. Sporulation for example is known to be a competitive inhibitory system between CSF and ComX.<br />
<br />
===Sources===<br />
The Mathematica 7 source files are available below:<br />
<br />
Biofilm model ([http://dl.dropbox.com/u/391356/iGEM/biofilm.nb source])(interactive)<br><br />
2D Heat equation ([http://dl.dropbox.com/u/391356/iGEM/diff.nb source])<br><br />
Sigmoid response curve ([http://dl.dropbox.com/u/391356/iGEM/hillSimpl.nb source])([http://dl.dropbox.com/u/391356/iGEM/hillSimpl.nbp interactive])<br />
<br />
<br />
===References===<br />
<small>Comella N, Grossman AD, Conservation of genes and processes controlled by the quorum response in bacteria: characterization of genes controlled by the quorum-sensing transcription factor ComA in Bacillus subtilis, Molecular Microbiology (2005), 57 (4): 1159–1174. PMID 16091051 <br />
<br />
Lemon KP, Earl AM, Vlamakis HC, Aguilar C, Kolter R., Biofilm development with an emphasis on Bacillus subtilis, Current Topics Microbiology and Immunology, 2008, 322:1-16. PMID 18453269 <br />
<br />
López D, Vlamakis H, Kolter R, Biofilms, Cold Spring Harbor Perspectives in Biology, 2010, (2):a000398. PMID 20519345 <br />
<br />
López D, Vlamakis H, Losick R, Kolter R, Paracrine signaling in a bacterium, Genes & Development, 2009 23(14):1631-8. <br />
<br />
Pérez J, Picioreanu C, van Loosdrecht M, Modeling biofilm and floc diffusion processes based on analytical solution of reaction-diffusion equations, Water Research, 2005, 39:1311-1323.<br />
<br />
Picioreanu C, van Loosdrecht MC, Heijnen JJ. A new combined differential-discrete cellular automaton approach for biofilm modeling: application for growth in gel beads, Biotechnology Bioengineering, 1998, 57(6):718-31. PMID 10099251<br />
<br />
Picioreanu C, van Loosdrecht MC, Heijnen JJ. Mathematical modeling of biofilm structure with a hybrid differential-discrete cellular automaton approach, Biotechnology Bioengineering, 1998, 58(1):101-16. PMID <br />
10099266 <br />
Roggiani M, Dubnau D, ComA, a phosphorylated response regulator protein of Bacillus subtilis, binds to the promoter region of srfA, Journal of Bacteriology, 1993, 175(10): 3182-7. PMID 8387999 <br />
<br />
Shah IM, Dworkin J, Microbial interactions: bacteria talk to (some of) their neighbors, Current Biology, 2009, 19(16): 689-91. PMID 19706277</small></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/HomeTeam:Groningen/Home2010-10-27T22:46:21Z<p>Joelkuiper: </p>
<hr />
<div><html><br />
<div margin-left: 10px"> <br />
</html><br />
==Self assembling hydrophobic biofilm==<br />
We aim to design a biological coating as an alternative to for example chemical coatings. For this we, unconventionally, utilized a ''Bacillus subtilis'' [https://2010.igem.org/Team:Groningen#/biofilm biofilm]. We wanted to enable our biofilm to be equipped with an interesting property which is automatically initiated. So we introduced an [https://2010.igem.org/Team:Groningen#/expression expression trigger] which relies on quorum sensing. Our project was directed at finding an alternate solution to biofouling, since regular, chemical coatings which are widely in use pose a threat to the environment. In nature the lotus leaves show self-cleansing properties ascribed to their extreme surface hydrophobicity. In the prokaryotic domain we stumbled upon [https://2010.igem.org/Team:Groningen#/hydrophobins chaplins], strongly hydrophobic proteins originating from ''Streptomyces coelicolor''. Surface hydrophobicity is a very useful property and is used in many [https://2010.igem.org/Team:Groningen#/applications applications] ranging from not only antifouling coatings but also other applications which require water repellence to applications in the field of medical sciences. During our project we have contributed to the parts registry whit numerous [https://2010.igem.org/Team:Groningen#/biobricks BioBricks].<br />
<html><br />
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A self assembling bio-based coating is form, a </html>[https://2010.igem.org/Team:Groningen#/biofilm rigid biofilm]<html>.<br />
</div><br />
<div id="chaplin-caption" class="nivo-html-caption"><br />
</html>[https://2010.igem.org/Team:Groningen#/expression Expression]<html> of hydrophobic proteins called </html>[https://2010.igem.org/Team:Groningen#/hydrophobins chaplins]<html> is induced by the biofilm causing strong surface hydrophobicity. <br />
</div><br />
<div id="killswitch-caption" class="nivo-html-caption"><br />
The strongly hydrophobic biofilm will die off by a </html>[https://2010.igem.org/Team:Groningen#/killswitch_model killswitch]<html>, leaving a nice hydrophobic biological coating. <br />
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<div></html><br />
This year a [https://2010.igem.org/Team:Groningen#/team team] of young inspired undergraduates from the [http://www.rug.nl University of Groningen] participated in the amazing challenge of iGEM. A multi-disciplinary team of Molecular Biologists, Chemists, Computer Scientists, Journalists and others spend the summer creating a wonderful project in the emerging field of synthetic biology<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/modeling"><img src="https://static.igem.org/mediawiki/2010/1/1e/Groningen-Home-Model.jpg" alt="Modeling"></a><div></html><br />
Using computer models we worked on the frontiers of knowledge. [https://2010.igem.org/Team:Groningen#/expression_model Gene expression] was simulated and a simple explanation for [https://2010.igem.org/Team:Groningen#/biofilm_model cell differentiation] was proposed. Also aiding in ethics and practical feasibility a [https://2010.igem.org/Team:Groningen#/killswitch_model kill switch] system was studied. Finally a [https://2010.igem.org/Team:Groningen#/info_standard new standard] was proposed for characterizing Biobrick parts so future can be streamlined.<br />
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<div><a href="https://2010.igem.org/Team:Groningen#/practices"><img src="https://static.igem.org/mediawiki/2010/7/77/Groningen-Home-Human.jpg" alt="Human Practices"></a><div></html><br />
Because we believe that synthetic biology can better the lives of people and ensure long term prosperity for all humans we spend time [https://2010.igem.org/Team:Groningen#/practices educating] high school students. But not all is perfect so [https://2010.igem.org/Team:Groningen#/safety risks] were assessed and we philosophized on the [https://2010.igem.org/Team:Groningen#/ethics ethical] aspects of synthetic biology.<br />
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</html></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/HomeTeam:Groningen/Home2010-10-27T22:42:09Z<p>Joelkuiper: /* Hydrophobofilm Self assembling hydrophobic biofilm */</p>
<hr />
<div><html><br />
<div margin-left: 10px"> <br />
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==Self assembling hydrophobic biofilm==<br />
We aim to design a biological coating as an alternative to for example chemical coatings. For this we, unconventionally, utilized a ''Bacillus subtilis'' [https://2010.igem.org/Team:Groningen#/biofilm biofilm]. We wanted to enable our biofilm to be equipped with an interesting property which is automatically initiated. So we introduced an [https://2010.igem.org/Team:Groningen#/expression expression trigger] which relies on quorum sensing. Our project was directed at finding an alternate solution to biofouling, since regular, chemical coatings which are widely in use pose a threat to the environment. In nature the lotus leaves show self-cleansing properties ascribed to their extreme surface hydrophobicity. In the prokaryotic domain we stumbled upon [https://2010.igem.org/Team:Groningen#/hydrophobins chaplins], strongly hydrophobic proteins originating from ''Streptomyces coelicolor''. Surface hydrophobicity is a very useful property and is used in many [https://2010.igem.org/Team:Groningen#/applications applications] ranging from not only antifouling coatings but also other applications which require water repellence to applications in the field of medical sciences. During our project we have contributed to the parts registry whit numerous [https://2010.igem.org/Team:Groningen#/biobricks BioBricks].<br />
<html><br />
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$('#slider').nivoSlider({effect:'fade'});<br />
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A self assembling bio-based coating is form, a </html>[https://2010.igem.org/Team:Groningen#/biofilm rigid biofilm]<html>.<br />
</div><br />
<div id="chaplin-caption" class="nivo-html-caption"><br />
</html>[https://2010.igem.org/Team:Groningen#/expression Expression]<html> of hydrophobic proteins called </html>[https://2010.igem.org/Team:Groningen#/hydrophobins chaplins]<html> is induced by the biofilm causing strong surface hydrophobicity. <br />
</div><br />
<div id="killswitch-caption" class="nivo-html-caption"><br />
The strongly hydrophobic biofilm will die off by a </html>[https://2010.igem.org/Team:Groningen#/killswitch_model killswitch]<html>, leaving a nice hydrophobic biological coating. <br />
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<div></html><br />
This year a [https://2010.igem.org/Team:Groningen#/team team] of young inspired undergraduates from the [http://www.rug.nl University of Groningen] participated in the amazing challenge of iGEM. A multi-disciplinary team of Molecular Biologists, Chemists, Computer Scientists, Journalists and others spend the summer creating a wonderful project in the emerging field of synthetic biology<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/modeling"><img src="https://static.igem.org/mediawiki/2010/1/1e/Groningen-Home-Model.jpg" alt="Modeling"></a><div></html><br />
Using computer models we worked on the frontiers of knowledge. [https://2010.igem.org/Team:Groningen#/expression_model Gene expression] was simulated and a simple explanation for [https://2010.igem.org/Team:Groningen#/biofilm_model cell differentiation] was proposed. Also aiding in ethics and practical feasibility a [https://2010.igem.org/Team:Groningen#/killswitch_model kill switch] system was studied. Finally a [https://2010.igem.org/Team:Groningen#/info_standard new standard] was proposed for characterizing Biobrick parts so future can be streamlined.<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/practices"><img src="https://static.igem.org/mediawiki/2010/7/77/Groningen-Home-Human.jpg" alt="Human Practices"></a><div></html><br />
Because we believe that synthetic biology can better the lives of people and ensure long term prosperity for all humans we spend time [https://2010.igem.org/Team:Groningen#/practices educating] high school students. But not all is perfect so [https://2010.igem.org/Team:Groningen#/safety risks] were assessed and we philosophized on the [https://2010.igem.org/Team:Groningen#/ethics ethical] aspects of synthetic biology.<br />
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----<br />
<br />
=== Our sponsors === <br />
{{Team:Groningen/sponsors}}<br />
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<br />
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</div><br />
</html></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/HomeTeam:Groningen/Home2010-10-27T22:40:25Z<p>Joelkuiper: /* Hydrophobofilm Self assembling hydrophobic biofilm */</p>
<hr />
<div><html><br />
<div margin-left: 10px"> <br />
</html><br />
==Hydrophobofilm Self assembling hydrophobic biofilm==<br />
We aim to design a biological coating as an alternative to for example chemical coatings. For this we, unconventionally, utilized a ''Bacillus subtilis'' [https://2010.igem.org/Team:Groningen#/biofilm biofilm]. We wanted to enable our biofilm to be equipped with an interesting property which is automatically initiated. So we introduced an [https://2010.igem.org/Team:Groningen#/expression expression trigger] which relies on quorum sensing. Our project was directed at finding an alternate solution to biofouling, since regular, chemical coatings which are widely in use pose a threat to the environment. In nature the lotus leaves show self-cleansing properties ascribed to their extreme surface hydrophobicity. In the prokaryotic domain we stumbled upon [https://2010.igem.org/Team:Groningen#/hydrophobins chaplins], strongly hydrophobic proteins originating from ''Streptomyces coelicolor''. Surface hydrophobicity is a very useful property and is used in many [https://2010.igem.org/Team:Groningen#/applications applications] ranging from not only antifouling coatings but also other applications which require water repellence to applications in the field of medical sciences. During our project we have contributed to the parts registry whit numerous [https://2010.igem.org/Team:Groningen#/biobricks BioBricks].<br />
<html><br />
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<script src="https://2010.igem.org/Team:Groningen/files/slider.js?action=raw&ctype=text/javascript" type="text/javascript"></script><br />
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<a https://2010.igem.org/Team:Groningen#/project"><img src="https://static.igem.org/mediawiki/2010/d/d3/Groningen-Reel-Stage2.jpg" alt="" title="#chaplin-caption" /></a><br />
<a https://2010.igem.org/Team:Groningen#/project"><img src="https://static.igem.org/mediawiki/2010/6/6f/Groningen-Reel-Stage3.jpg" alt="" title="#killswitch-caption" /></a><br />
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$('#slider').nivoSlider({effect:'fade'});<br />
<br />
</script><br />
<div id="biofilm-caption" class="nivo-html-caption"><br />
A self assembling bio-based coating is form, a </html>[https://2010.igem.org/Team:Groningen#/biofilm rigid biofilm]<html>.<br />
</div><br />
<div id="chaplin-caption" class="nivo-html-caption"><br />
</html>[https://2010.igem.org/Team:Groningen#/expression Expression]<html> of hydrophobic proteins called </html>[https://2010.igem.org/Team:Groningen#/hydrophobins chaplins]<html> is induced by the biofilm causing strong surface hydrophobicity. <br />
</div><br />
<div id="killswitch-caption" class="nivo-html-caption"><br />
The strongly hydrophobic biofilm will die off by a </html>[https://2010.igem.org/Team:Groningen#/killswitch_model killswitch]<html>, leaving a nice hydrophobic biological coating. <br />
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<a href="https://2010.igem.org/Team:Groningen#/team"><img src="https://static.igem.org/mediawiki/2010/f/f5/Groningen-Home-Team.jpg" alt="Team"></a><br />
<div></html><br />
This year a [https://2010.igem.org/Team:Groningen#/team team] of young inspired undergraduates from the [http://www.rug.nl University of Groningen] participated in the amazing challenge of iGEM. A multi-disciplinary team of Molecular Biologists, Chemists, Computer Scientists, Journalists and others spend the summer creating a wonderful project in the emerging field of synthetic biology<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/modeling"><img src="https://static.igem.org/mediawiki/2010/1/1e/Groningen-Home-Model.jpg" alt="Modeling"></a><div></html><br />
Using computer models we worked on the frontiers of knowledge. [https://2010.igem.org/Team:Groningen#/expression_model Gene expression] was simulated and a simple explanation for [https://2010.igem.org/Team:Groningen#/biofilm_model cell differentiation] was proposed. Also aiding in ethics and practical feasibility a [https://2010.igem.org/Team:Groningen#/killswitch_model kill switch] system was studied. Finally a [https://2010.igem.org/Team:Groningen#/info_standard new standard] was proposed for characterizing Biobrick parts so future can be streamlined.<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/practices"><img src="https://static.igem.org/mediawiki/2010/7/77/Groningen-Home-Human.jpg" alt="Human Practices"></a><div></html><br />
Because we believe that synthetic biology can better the lives of people and ensure long term prosperity for all humans we spend time [https://2010.igem.org/Team:Groningen#/practices educating] high school students. But not all is perfect so [https://2010.igem.org/Team:Groningen#/safety risks] were assessed and we philosophized on the [https://2010.igem.org/Team:Groningen#/ethics ethical] aspects of synthetic biology.<br />
<html></div></div><br />
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<br />
----<br />
<br />
=== Our sponsors === <br />
{{Team:Groningen/sponsors}}<br />
<br />
<br />
<html><br />
</div><br />
</html></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/HomeTeam:Groningen/Home2010-10-27T22:39:19Z<p>Joelkuiper: /* Self assembling hydrophobic biofilm */</p>
<hr />
<div><html><br />
<div margin-left: 10px"> <br />
</html><br />
==Hydrophobofilm Self assembling hydrophobic biofilm==<br />
We aim to design a biological coating as an alternative to for example chemical coatings. For this we, unconventionally, utilized a ''Bacillus subtilis'' [https://2010.igem.org/Team:Groningen#/biofilm biofilm]. We wanted to enable our biofilm to be equipped with an interesting property which is automatically initiated. So we introduced an [https://2010.igem.org/Team:Groningen#/expression expression trigger] which relies on quorum sensing. Our project was directed at finding an alternate solution to biofouling, since regular, chemical coatings which are widely in use pose a threat to the environment. In nature the lotus leaves show self-cleansing properties ascribed to their extreme surface hydrophobicity. In the prokaryotic domain we stumbled upon [https://2010.igem.org/Team:Groningen#/hydrophobins chaplins], strongly hydrophobic proteins originating from ''Streptomyces coelicolor''. Surface hydrophobicity is a very useful property and is used in many [https://2010.igem.org/Team:Groningen#/applications applications] ranging from not only antifouling coatings but also other applications which require water repellence to applications in the field of medical sciences. During our project we have contributed to the parts registry whit numerous [https://2010.igem.org/Team:Groningen#/biobricks BioBricks].<br />
<html><br />
</div><br />
<br />
<script src="https://2010.igem.org/Team:Groningen/files/slider.js?action=raw&ctype=text/javascript" type="text/javascript"></script><br />
<div style="height: 1330px"><br />
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<a href="https://2010.igem.org/Team:Groningen#/project"><img src="https://static.igem.org/mediawiki/2010/d/dd/Groningen-Reel-Stage1.jpg" alt="" title="#biofilm-caption" /></a><br />
https://2010.igem.org/Team:Groningen#/project"><img src="https://static.igem.org/mediawiki/2010/d/d3/Groningen-Reel-Stage2.jpg" alt="" title="#chaplin-caption" /></a><br />
https://2010.igem.org/Team:Groningen#/project"><img src="https://static.igem.org/mediawiki/2010/6/6f/Groningen-Reel-Stage3.jpg" alt="" title="#killswitch-caption" /></a><br />
</div><br />
<script type="text/javascript"><br />
<br />
$('#slider').nivoSlider({effect:'fade'});<br />
<br />
</script><br />
<div id="biofilm-caption" class="nivo-html-caption"><br />
A self assembling bio-based coating is form, a </html>[https://2010.igem.org/Team:Groningen#/biofilm rigid biofilm]<html>.<br />
</div><br />
<div id="chaplin-caption" class="nivo-html-caption"><br />
</html>[https://2010.igem.org/Team:Groningen#/expression Expression]<html> of hydrophobic proteins called </html>[https://2010.igem.org/Team:Groningen#/hydrophobins chaplins]<html> is induced by the biofilm causing strong surface hydrophobicity. <br />
</div><br />
<div id="killswitch-caption" class="nivo-html-caption"><br />
The strongly hydrophobic biofilm will die off by a </html>[https://2010.igem.org/Team:Groningen#/killswitch_model killswitch]<html>, leaving a nice hydrophobic biological coating. <br />
</div><br />
<br />
<br />
<br />
<style type="text/css"><br />
#home-boxes {<br />
margin: 2em 0 2em 10px; <br />
width: 751px;<br />
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<div id="home-boxes"><div><br />
<a href="https://2010.igem.org/Team:Groningen#/team"><img src="https://static.igem.org/mediawiki/2010/f/f5/Groningen-Home-Team.jpg" alt="Team"></a><br />
<div></html><br />
This year a [https://2010.igem.org/Team:Groningen#/team team] of young inspired undergraduates from the [http://www.rug.nl University of Groningen] participated in the amazing challenge of iGEM. A multi-disciplinary team of Molecular Biologists, Chemists, Computer Scientists, Journalists and others spend the summer creating a wonderful project in the emerging field of synthetic biology<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/modeling"><img src="https://static.igem.org/mediawiki/2010/1/1e/Groningen-Home-Model.jpg" alt="Modeling"></a><div></html><br />
Using computer models we worked on the frontiers of knowledge. [https://2010.igem.org/Team:Groningen#/expression_model Gene expression] was simulated and a simple explanation for [https://2010.igem.org/Team:Groningen#/biofilm_model cell differentiation] was proposed. Also aiding in ethics and practical feasibility a [https://2010.igem.org/Team:Groningen#/killswitch_model kill switch] system was studied. Finally a [https://2010.igem.org/Team:Groningen#/info_standard new standard] was proposed for characterizing Biobrick parts so future can be streamlined.<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/practices"><img src="https://static.igem.org/mediawiki/2010/7/77/Groningen-Home-Human.jpg" alt="Human Practices"></a><div></html><br />
Because we believe that synthetic biology can better the lives of people and ensure long term prosperity for all humans we spend time [https://2010.igem.org/Team:Groningen#/practices educating] high school students. But not all is perfect so [https://2010.igem.org/Team:Groningen#/safety risks] were assessed and we philosophized on the [https://2010.igem.org/Team:Groningen#/ethics ethical] aspects of synthetic biology.<br />
<html></div></div><br />
</div><br />
</html><br />
<br />
----<br />
<br />
=== Our sponsors === <br />
{{Team:Groningen/sponsors}}<br />
<br />
<br />
<html><br />
</div><br />
</html></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/HomeTeam:Groningen/Home2010-10-27T22:32:40Z<p>Joelkuiper: </p>
<hr />
<div><html><br />
<div margin-left: 10px"> <br />
</html><br />
==Self assembling hydrophobic biofilm==<br />
We aim to design a biological coating as an alternative to for example chemical coatings. For this we, unconventionally, utilized a ''Bacillus subtilis'' [https://2010.igem.org/Team:Groningen#/biofilm biofilm]. We wanted to enable our biofilm to be equipped with an interesting property which is automatically initiated. So we introduced an [https://2010.igem.org/Team:Groningen#/expression expression trigger] which relies on quorum sensing. Our project was directed at finding an alternate solution to biofouling, since regular, chemical coatings which are widely in use pose a threat to the environment. In nature the lotus leaves show self-cleansing properties ascribed to their extreme surface hydrophobicity. In the prokaryotic domain we stumbled upon [https://2010.igem.org/Team:Groningen#/hydrophobins chaplins], strongly hydrophobic proteins originating from ''Streptomyces coelicolor''. Surface hydrophobicity is a very useful property and is used in many [https://2010.igem.org/Team:Groningen#/applications applications] ranging from not only antifouling coatings but also other applications which require water repellence to applications in the field of medical sciences. During our project we have contributed to the parts registry whit numerous [https://2010.igem.org/Team:Groningen#/biobricks BioBricks].<br />
<html><br />
</div><br />
<br />
<script src="https://2010.igem.org/Team:Groningen/files/slider.js?action=raw&ctype=text/javascript" type="text/javascript"></script><br />
<div style="height: 1330px"><br />
<div id="slider" style="margin-left: 10px; height: 343px;"><br />
<img src="https://static.igem.org/mediawiki/2010/d/dd/Groningen-Reel-Stage1.jpg" alt="" title="#biofilm-caption" /><br />
<img src="https://static.igem.org/mediawiki/2010/d/d3/Groningen-Reel-Stage2.jpg" alt="" title="#chaplin-caption" /><br />
<img src="https://static.igem.org/mediawiki/2010/6/6f/Groningen-Reel-Stage3.jpg" alt="" title="#killswitch-caption" /><br />
</div><br />
<script type="text/javascript"><br />
<br />
$('#slider').nivoSlider({effect:'fade'});<br />
<br />
</script><br />
<div id="biofilm-caption" class="nivo-html-caption"><br />
A self assembling bio-based coating is form, a </html>[https://2010.igem.org/Team:Groningen#/biofilm rigid biofilm]<html>.<br />
</div><br />
<div id="chaplin-caption" class="nivo-html-caption"><br />
</html>[https://2010.igem.org/Team:Groningen#/expression Expression]<html> of hydrophobic proteins called </html>[https://2010.igem.org/Team:Groningen#/hydrophobins chaplins]<html> is induced by the biofilm causing strong surface hydrophobicity. <br />
</div><br />
<div id="killswitch-caption" class="nivo-html-caption"><br />
The strongly hydrophobic biofilm will die off by a </html>[https://2010.igem.org/Team:Groningen#/killswitch_model killswitch]<html>, leaving a nice hydrophobic biological coating. <br />
</div><br />
<br />
<br />
<br />
<style type="text/css"><br />
#home-boxes {<br />
margin: 2em 0 2em 10px; <br />
width: 751px;<br />
clear:both;<br />
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}<br />
#home-boxes div {<br />
height: 400px;<br />
background-color: #fff4bc;<br />
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border: 1px solid #efe28d;<br />
margin-right: 15px;<br />
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text-align: justify;<br />
border: none!important;<br />
width: 200px;<br />
}<br />
#home-boxes img { <br />
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<a href="https://2010.igem.org/Team:Groningen#/team"><img src="https://static.igem.org/mediawiki/2010/f/f5/Groningen-Home-Team.jpg" alt="Team"></a><br />
<div></html><br />
This year a [https://2010.igem.org/Team:Groningen#/team team] of young inspired undergraduates from the [http://www.rug.nl University of Groningen] participated in the amazing challenge of iGEM. A multi-disciplinary team of Molecular Biologists, Chemists, Computer Scientists, Journalists and others spend the summer creating a wonderful project in the emerging field of synthetic biology<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/modeling"><img src="https://static.igem.org/mediawiki/2010/1/1e/Groningen-Home-Model.jpg" alt="Modeling"></a><div></html><br />
Using computer models we worked on the frontiers of knowledge. [https://2010.igem.org/Team:Groningen#/expression_model Gene expression] was simulated and a simple explanation for [https://2010.igem.org/Team:Groningen#/biofilm_model cell differentiation] was proposed. Also aiding in ethics and practical feasibility a [https://2010.igem.org/Team:Groningen#/killswitch_model kill switch] system was studied. Finally a [https://2010.igem.org/Team:Groningen#/info_standard new standard] was proposed for characterizing Biobrick parts so future can be streamlined.<br />
<html></div></div><br />
<br />
<div><a href="https://2010.igem.org/Team:Groningen#/practices"><img src="https://static.igem.org/mediawiki/2010/7/77/Groningen-Home-Human.jpg" alt="Human Practices"></a><div></html><br />
Because we believe that synthetic biology can better the lives of people and ensure long term prosperity for all humans we spend time [https://2010.igem.org/Team:Groningen#/practices educating] high school students. But not all is perfect so [https://2010.igem.org/Team:Groningen#/safety risks] were assessed and we philosophized on the [https://2010.igem.org/Team:Groningen#/ethics ethical] aspects of synthetic biology.<br />
<html></div></div><br />
</div><br />
</html><br />
<br />
----<br />
<br />
=== Our sponsors === <br />
{{Team:Groningen/sponsors}}<br />
<br />
<br />
<html><br />
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</html></div>Joelkuiperhttp://2010.igem.org/Template:Team:Groningen/HeaderTemplate:Team:Groningen/Header2010-10-27T22:30:52Z<p>Joelkuiper: </p>
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<p style="float: right; margin-right: 30px"><br />
Hydrophobofilm<br><br />
<span style="margin-left: 2em">pushing coatings into a greener future</span><br />
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<span><br />
<a href="https://2010.igem.org/Team:Groningen#/modeling">Overview</a> |<br />
<a href="https://2010.igem.org/Team:Groningen#/biofilm_model">Biofilm dynamics</a> |<br />
<a href="https://2010.igem.org/Team:Groningen#/expression_model">Expression</a> |<br />
<a href="https://2010.igem.org/Team:Groningen#/killswitch_model">Kill Switch</a> |<br />
<a href="https://2010.igem.org/Team:Groningen#/info_standard">Information standard</a><br />
</span><br />
</li><br />
<li><br />
<a href="https://2010.igem.org/Team:Groningen#/human">Human Practices</a><br />
<span><br />
<a href="https://2010.igem.org/Team:Groningen#/human">Overview</a> |<br />
<a href="https://2010.igem.org/Team:Groningen#/practices">Education</a> |<br />
<a href="https://2010.igem.org/Team:Groningen#/safety">Safety</a> |<br />
<a href="https://2010.igem.org/Team:Groningen#/survey">Survey</a> |<br />
<a href="https://2010.igem.org/Team:Groningen#/ethics">Ethics</a> |<br />
<a href="https://2010.igem.org/Team:Groningen#/for_the_parents"> For Parents </a><br />
</span><br />
<br />
</li><br />
<li><a href="https://2010.igem.org/Team:Groningen#/organization">Organization</a><br />
<span><br />
<a href="https://2010.igem.org/Team:Groningen#/organization">Overview</a> |<br />
<a href="https://2010.igem.org/Team:Groningen#/brainstorm">Brainstorm</a> | <br />
<a href="https://2010.igem.org/Team:Groningen#/flowchart">Flow Chart</a> |<br />
<a href="https://2010.igem.org/Team:Groningen#/protocols">Protocols</a> |<br />
<a href="https://2010.igem.org/Team:Groningen#/notebook">Notebook</a><br />
</span> <br />
</li><br />
<li><a href="https://2010.igem.org/Team:Groningen#/team">Team</a><br />
<span><br />
<a href="https://2010.igem.org/Team:Groningen#/team">Overview</a> |<br />
<a href="https://2010.igem.org/Team:Groningen#/members">Members</a> |<br />
<a href="https://2010.igem.org/Team:Groningen#/collaboration">Collaboration</a> |<br />
<a href="https://2010.igem.org/Team:Groningen#/images">Image of Science</a> |<br />
<a href="https://2010.igem.org/Team:Groningen#/contact">Contact</a><br />
</span><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Groningen#/judging">Judging</a><br />
</li><br />
<br />
</ul><br />
</div><br />
<div id="otherContent"></div>Joelkuiperhttp://2010.igem.org/File:Groningen-ProjectPlan.pngFile:Groningen-ProjectPlan.png2010-10-27T21:14:24Z<p>Joelkuiper: uploaded a new version of "Image:Groningen-ProjectPlan.png"</p>
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<div></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/MembersTeam:Groningen/Members2010-10-27T20:55:04Z<p>Joelkuiper: </p>
<hr />
<div><html><br />
<style><br />
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background-color:#a3ad49;<br />
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<div id="teammembers-container" style="overflow:hidden;"><br />
<h2>Team Members</h2><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Ekkers"><img src="https://static.igem.org/mediawiki/2010/thumb/b/b1/DSCI2647.JPG/180px-DSCI2647.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Ekkers">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>David Ekkers</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Marine Biology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work & President</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">22</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Ptr"><img src="https://static.igem.org/mediawiki/2010/1/13/Peter.jpg" width="120" height="159" /></a><br />
<p><a href="https://2010.igem.org/User:Ptr">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<br />
<h3>Peter Roemers</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Biomedical Science</td><br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work & Vice president</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">22</td><br />
</tr><br />
<br />
</table><br />
</div><br />
</div><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:joelkuiper"><img src="https://static.igem.org/mediawiki/2010/0/0f/Joel.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:joelkuiper">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Joël Kuiper</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Artificial Intelligence</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling & Treasurer</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">22</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:LauravdS"><img src="https://static.igem.org/mediawiki/2010/c/cf/Laura1_gr.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:LauravdS">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Laura van der Straat</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">26</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:aslomp"><img src="https://static.igem.org/mediawiki/2010/4/47/Arend1.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:aslomp">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Arend Slomp</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Computer Science</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">25</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
</div><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:AJ"><img src="https://static.igem.org/mediawiki/2010/thumb/e/ee/Aj_21.jpg/200px-Aj_21.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:AJ">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Arend Jan Suk</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Moleculair Biology & Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab manager & Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">24</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:djokehendriks"><img src="https://static.igem.org/mediawiki/2010/thumb/e/ed/DSC05903.JPG/180px-DSC05903.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:djokehendriks">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Djoke Hendriks</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Journalism & Molecular Biology and Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling & PR</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">25</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Neima"><img src="https://static.igem.org/mediawiki/2010/thumb/6/69/CIMG0399.JPG/180px-CIMG0399.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Neima">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Ezgi</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Biology and Genetics</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">20</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Ramon"><img src="https://static.igem.org/mediawiki/2010/thumb/3/38/Ramon.JPG/180px-Ramon.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Ramon">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Ramon Sieber</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Biology and Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">23</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:MJvdN"><img src="https://static.igem.org/mediawiki/2010/9/95/Maarten1.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:MJvdN">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Maarten<br> van den Nieuwenhof</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Life Sciences</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">25</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Jorrithillebrand"><img src="https://static.igem.org/mediawiki/2010/1/1b/Jorrit1.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Jorrithillebrand">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Jorrit Hillebrand</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Chemistry</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work & Vice Treasurer</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">24</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Geeske"><img src="https://static.igem.org/mediawiki/2010/1/17/Geeske.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Geeske">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Geeske van Heel</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Biology and Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Labwork & Fundraising</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">24</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
</div><br />
<br />
<br />
<br />
</html></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/MembersTeam:Groningen/Members2010-10-27T20:53:39Z<p>Joelkuiper: </p>
<hr />
<div><html><br />
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<br />
</style><br />
<div id="teammembers-container" style="overflow:hidden;"><br />
<h2>Team Members</h2><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Ekkers"><img src="https://static.igem.org/mediawiki/2010/thumb/b/b1/DSCI2647.JPG/180px-DSCI2647.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Ekkers">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>David Ekkers</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Marine Biology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work & President</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">22</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Ptr"><img src="https://static.igem.org/mediawiki/2010/1/13/Peter.jpg" width="120" height="159" /></a><br />
<p><a href="https://2010.igem.org/User:Ptr">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<br />
<h3>Peter Roemers</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Biomedical Science</td><br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work & Vice president</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">22</td><br />
</tr><br />
<br />
</table><br />
</div><br />
</div><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:joelkuiper"><img src="https://static.igem.org/mediawiki/2010/0/0f/Joel.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:joelkuiper">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Joël Kuiper</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Artificial Intelligence</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling & Treasurer</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">22</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:LauravdS"><img src="https://static.igem.org/mediawiki/2010/c/cf/Laura1_gr.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:LauravdS">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Laura van der Straat</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">26</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:aslomp"><img src="https://static.igem.org/mediawiki/2010/4/47/Arend1.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:aslomp">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Arend Slomp</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Computer Science</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">25</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
</div><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:AJ"><img src="https://static.igem.org/mediawiki/2010/thumb/e/ee/Aj_21.jpg/200px-Aj_21.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:AJ">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Arend Jan Suk</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Moleculair Biology & Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab manager & Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">24</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:djokehendriks"><img src="https://static.igem.org/mediawiki/2010/thumb/e/ed/DSC05903.JPG/180px-DSC05903.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:djokehendriks">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Djoke Hendriks</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Journalism & Molecular Biology and Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling & PR</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">25</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Neima"><img src="https://static.igem.org/mediawiki/2010/thumb/6/69/CIMG0399.JPG/180px-CIMG0399.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Neima">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Ezgi</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Biology and Genetics</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">20</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Ramon"><img src="https://static.igem.org/mediawiki/2010/thumb/3/38/Ramon.JPG/180px-Ramon.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Ramon">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Ramon Sieber</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Biology and Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">23</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:MJvdN"><img src="https://static.igem.org/mediawiki/2010/9/95/Maarten1.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:MJvdN">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Maarten<br> van den Nieuwenhof</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Life Sciences</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">25</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Jorrithillebrand"><img src="https://static.igem.org/mediawiki/2010/1/1b/Jorrit1.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Jorrithillebrand">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Jorrit Hillebrand</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Chemistry</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work & Vice Treasurer</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">24</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Geeske"><img src="https://static.igem.org/mediawiki/2010/1/17/Geeske.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Geeske">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Geeske van Heel</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Biology and Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Labwork & Fundraising</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">24</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
</div><br />
<br />
<br />
<br />
</html></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/MembersTeam:Groningen/Members2010-10-27T20:52:15Z<p>Joelkuiper: </p>
<hr />
<div><html><br />
<style><br />
.member-container {<br />
background-color:#a3ad49;<br />
width: 350px;<br />
float: left;<br />
margin: 10px;<br />
}<br />
.member-container .member-picture {<br />
width: 120px;<br />
text-align: center;<br />
float: left;<br />
margin: 1em;<br />
}<br />
<br />
.member-container .member-picture p {<br />
margin-top: 10px;<br />
margin-bottom: 20px;<br />
}<br />
.member-container .member-picture p a {<br />
color: #fff;<br />
}<br />
.member-container .member-picture img {<br />
width: 120px;<br />
height: 159px;<br />
}<br />
.member-container .member-description {<br />
margin-top: 10px;<br />
float: left;<br />
margin-right: 10px;<br />
}<br />
.member-container h3 {<br />
color: #fff;<br />
}<br />
<br />
.member-description table {<br />
margin: 0;<br />
padding: 0;<br />
width: 175px;<br />
font-size: 11px;<br />
color: white;<br />
margin-top: -10px;<br />
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border-collapse:collapse;<br />
}<br />
<br />
.member-description table .name {<br />
font-weight: bold;<br />
}<br />
<br />
.member-description table .story {<br />
font-style: italic;<br />
padding: 10px;<br />
}<br />
<br />
</style><br />
<div id="teammembers-container" style="overflow:hidden;"><br />
<h2>Team Members</h2><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Ekkers"><img src="https://static.igem.org/mediawiki/2010/thumb/b/b1/DSCI2647.JPG/180px-DSCI2647.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Ekkers">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>David Ekkers</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Marine Biology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work & President</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">22</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Ptr"><img src="https://static.igem.org/mediawiki/2010/1/13/Peter.jpg" width="120" height="159" /></a><br />
<p><a href="https://2010.igem.org/User:Ptr">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<br />
<h3>Peter Roemers</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Biomedical Science</td><br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work & Vice president</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">22</td><br />
</tr><br />
<br />
</table><br />
</div><br />
</div><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:joelkuiper"><img src="https://static.igem.org/mediawiki/2010/0/0f/Joel.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:joelkuiper">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Joël Kuiper</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Artificial Intelligence</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling & Treasurer</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">22</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:LauravdS"><img src="https://static.igem.org/mediawiki/2010/c/cf/Laura1_gr.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:LauravdS">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Laura van der Straat</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">26</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:aslomp"><img src="https://static.igem.org/mediawiki/2010/4/47/Arend1.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:aslomp">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Arend Slomp</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Computer Science</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">25</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
</div><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:AJ"><img src="https://static.igem.org/mediawiki/2010/thumb/e/ee/Aj_21.jpg/200px-Aj_21.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:AJ">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Arend Jan Suk</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Moleculair Biology & Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab manager & Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">24</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:djokehendriks"><img src="https://static.igem.org/mediawiki/2010/thumb/e/ed/DSC05903.JPG/180px-DSC05903.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:djokehendriks">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Djoke Hendriks</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Journalism & Molecular Biology and Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling & PR</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">25</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Neima"><img src="https://static.igem.org/mediawiki/2010/thumb/6/69/CIMG0399.JPG/180px-CIMG0399.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Neima">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Ezgi</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Biology and Genetics</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">20</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Ramon"><img src="https://static.igem.org/mediawiki/2010/thumb/3/38/Ramon.JPG/180px-Ramon.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Ramon">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Ramon Sieber</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Biology and Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">23</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:MJvdN"><img src="https://static.igem.org/mediawiki/2010/9/95/Maarten1.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:MJvdN">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Maarten<br> van den Nieuwenhof</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Life Sciences</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">25</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Jorrithillebrand"><img src="https://static.igem.org/mediawiki/2010/1/1b/Jorrit1.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Jorrithillebrand">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Jorrit Hillebrand</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Chemistry</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work & Vice Treasurer</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">24</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Geeske"><img src="https://static.igem.org/mediawiki/2010/1/17/Geeske.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Geeske">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Geeske van Heel</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Biology and Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Labwork & Fundraising</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">24</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
</div><br />
<br />
<br />
<br />
</html></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/MembersTeam:Groningen/Members2010-10-27T20:51:00Z<p>Joelkuiper: </p>
<hr />
<div><html><br />
<style><br />
.member-container {<br />
background-color:#a3ad49;<br />
width: 350px;<br />
float: left;<br />
margin: 10px;<br />
}<br />
.member-container .member-picture {<br />
width: 120px;<br />
text-align: center;<br />
float: left;<br />
margin: 1em;<br />
}<br />
<br />
.member-container .member-picture p {<br />
margin-top: 10px;<br />
margin-bottom: 20px;<br />
}<br />
.member-container .member-picture p a {<br />
color: #fff;<br />
}<br />
.member-container .member-picture img {<br />
width: 120px;<br />
height: 159px;<br />
}<br />
.member-container .member-description {<br />
margin-top: 10px;<br />
float: left; color: white;<br />
margin-right: 10px;<br />
}<br />
.member-container h3 {<br />
color: #fff;<br />
}<br />
<br />
.member-description table {<br />
margin: 0;<br />
padding: 0;<br />
width: 175px;<br />
font-size: 11px;<br />
margin-top: -10px;<br />
background-color: #a3ad49;<br />
border-collapse:collapse;<br />
}<br />
<br />
.member-description table .name {<br />
font-weight: bold;<br />
}<br />
<br />
.member-description table .story {<br />
font-style: italic;<br />
padding: 10px;<br />
}<br />
<br />
</style><br />
<div id="teammembers-container" style="overflow:hidden;"><br />
<h2>Team Members</h2><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Ekkers"><img src="https://static.igem.org/mediawiki/2010/thumb/b/b1/DSCI2647.JPG/180px-DSCI2647.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Ekkers">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>David Ekkers</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Marine Biology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work & President</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">22</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Ptr"><img src="https://static.igem.org/mediawiki/2010/1/13/Peter.jpg" width="120" height="159" /></a><br />
<p><a href="https://2010.igem.org/User:Ptr">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<br />
<h3>Peter Roemers</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Biomedical Science</td><br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work & Vice president</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">22</td><br />
</tr><br />
<br />
</table><br />
</div><br />
</div><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:joelkuiper"><img src="https://static.igem.org/mediawiki/2010/0/0f/Joel.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:joelkuiper">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Joël Kuiper</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Artificial Intelligence</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling & Treasurer</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">22</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:LauravdS"><img src="https://static.igem.org/mediawiki/2010/c/cf/Laura1_gr.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:LauravdS">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Laura van der Straat</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">26</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:aslomp"><img src="https://static.igem.org/mediawiki/2010/4/47/Arend1.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:aslomp">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Arend Slomp</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Computer Science</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">25</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
</div><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:AJ"><img src="https://static.igem.org/mediawiki/2010/thumb/e/ee/Aj_21.jpg/200px-Aj_21.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:AJ">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Arend Jan Suk</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Moleculair Biology & Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab manager & Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">24</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:djokehendriks"><img src="https://static.igem.org/mediawiki/2010/thumb/e/ed/DSC05903.JPG/180px-DSC05903.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:djokehendriks">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Djoke Hendriks</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Journalism & Molecular Biology and Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling & PR</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">25</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Neima"><img src="https://static.igem.org/mediawiki/2010/thumb/6/69/CIMG0399.JPG/180px-CIMG0399.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Neima">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Ezgi</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Biology and Genetics</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">20</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Ramon"><img src="https://static.igem.org/mediawiki/2010/thumb/3/38/Ramon.JPG/180px-Ramon.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Ramon">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Ramon Sieber</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Biology and Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">23</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:MJvdN"><img src="https://static.igem.org/mediawiki/2010/9/95/Maarten1.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:MJvdN">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Maarten<br> van den Nieuwenhof</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Life Sciences</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">25</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Jorrithillebrand"><img src="https://static.igem.org/mediawiki/2010/1/1b/Jorrit1.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Jorrithillebrand">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Jorrit Hillebrand</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Chemistry</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work & Vice Treasurer</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">24</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Geeske"><img src="https://static.igem.org/mediawiki/2010/1/17/Geeske.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Geeske">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Geeske van Heel</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Biology and Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Labwork & Fundraising</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">24</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
</div><br />
<br />
<br />
<br />
</html></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/MembersTeam:Groningen/Members2010-10-27T20:50:33Z<p>Joelkuiper: </p>
<hr />
<div><html><br />
<style><br />
.member-container {<br />
background-color:#a3ad49;<br />
width: 350px;<br />
float: left;<br />
color: white;<br />
margin: 10px;<br />
}<br />
.member-container .member-picture {<br />
width: 120px;<br />
text-align: center;<br />
float: left;<br />
margin: 1em;<br />
}<br />
<br />
.member-container .member-picture p {<br />
margin-top: 10px;<br />
margin-bottom: 20px;<br />
}<br />
.member-container .member-picture p a {<br />
color: #fff;<br />
}<br />
.member-container .member-picture img {<br />
width: 120px;<br />
height: 159px;<br />
}<br />
.member-container .member-description {<br />
margin-top: 10px;<br />
float: left;<br />
margin-right: 10px;<br />
}<br />
.member-container h3 {<br />
color: #fff;<br />
}<br />
<br />
.member-description table {<br />
margin: 0;<br />
padding: 0;<br />
width: 175px;<br />
font-size: 11px;<br />
margin-top: -10px;<br />
background-color: #a3ad49;<br />
border-collapse:collapse;<br />
}<br />
<br />
.member-description table .name {<br />
font-weight: bold;<br />
}<br />
<br />
.member-description table .story {<br />
font-style: italic;<br />
padding: 10px;<br />
}<br />
<br />
</style><br />
<div id="teammembers-container" style="overflow:hidden;"><br />
<h2>Team Members</h2><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Ekkers"><img src="https://static.igem.org/mediawiki/2010/thumb/b/b1/DSCI2647.JPG/180px-DSCI2647.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Ekkers">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>David Ekkers</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Marine Biology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work & President</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">22</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Ptr"><img src="https://static.igem.org/mediawiki/2010/1/13/Peter.jpg" width="120" height="159" /></a><br />
<p><a href="https://2010.igem.org/User:Ptr">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<br />
<h3>Peter Roemers</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Biomedical Science</td><br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work & Vice president</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">22</td><br />
</tr><br />
<br />
</table><br />
</div><br />
</div><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:joelkuiper"><img src="https://static.igem.org/mediawiki/2010/0/0f/Joel.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:joelkuiper">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Joël Kuiper</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Artificial Intelligence</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling & Treasurer</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">22</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:LauravdS"><img src="https://static.igem.org/mediawiki/2010/c/cf/Laura1_gr.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:LauravdS">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Laura van der Straat</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">26</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:aslomp"><img src="https://static.igem.org/mediawiki/2010/4/47/Arend1.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:aslomp">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Arend Slomp</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Computer Science</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">25</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
</div><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:AJ"><img src="https://static.igem.org/mediawiki/2010/thumb/e/ee/Aj_21.jpg/200px-Aj_21.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:AJ">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Arend Jan Suk</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Moleculair Biology & Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab manager & Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">24</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:djokehendriks"><img src="https://static.igem.org/mediawiki/2010/thumb/e/ed/DSC05903.JPG/180px-DSC05903.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:djokehendriks">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Djoke Hendriks</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Journalism & Molecular Biology and Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling & PR</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">25</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Neima"><img src="https://static.igem.org/mediawiki/2010/thumb/6/69/CIMG0399.JPG/180px-CIMG0399.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Neima">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Ezgi</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Biology and Genetics</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">20</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Ramon"><img src="https://static.igem.org/mediawiki/2010/thumb/3/38/Ramon.JPG/180px-Ramon.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Ramon">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Ramon Sieber</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Biology and Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">23</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:MJvdN"><img src="https://static.igem.org/mediawiki/2010/9/95/Maarten1.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:MJvdN">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Maarten<br> van den Nieuwenhof</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Life Sciences</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">25</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Jorrithillebrand"><img src="https://static.igem.org/mediawiki/2010/1/1b/Jorrit1.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Jorrithillebrand">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Jorrit Hillebrand</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Chemistry</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work & Vice Treasurer</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">24</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Geeske"><img src="https://static.igem.org/mediawiki/2010/1/17/Geeske.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Geeske">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Geeske van Heel</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Biology and Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Labwork & Fundraising</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">24</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
</div><br />
<br />
<br />
<br />
</html></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/MembersTeam:Groningen/Members2010-10-27T20:49:50Z<p>Joelkuiper: </p>
<hr />
<div><html><br />
<style><br />
.member-container {<br />
background-color:#a3ad49;<br />
width: 350px;<br />
float: left;<br />
margin: 10px;<br />
}<br />
.member-container .member-picture {<br />
width: 120px;<br />
text-align: center;<br />
float: left;<br />
margin: 1em;<br />
}<br />
<br />
.member-container .member-picture p {<br />
margin-top: 10px;<br />
margin-bottom: 20px;<br />
}<br />
.member-container .member-picture p a {<br />
color: #fff;<br />
}<br />
.member-container .member-picture img {<br />
width: 120px;<br />
height: 159px;<br />
}<br />
.member-container .member-description {<br />
margin-top: 10px;<br />
float: left;<br />
margin-right: 10px;<br />
}<br />
.member-container h3 {<br />
color: #fff;<br />
}<br />
<br />
.member-description table {<br />
margin: 0;<br />
padding: 0;<br />
width: 175px;<br />
font-size: 11px;<br />
margin-top: -10px;<br />
background-color: #a3ad49;<br />
border-collapse:collapse;<br />
}<br />
<br />
.member-description table .name {<br />
font-weight: bold;<br />
}<br />
<br />
.member-description table .story {<br />
font-style: italic;<br />
padding: 10px;<br />
}<br />
<br />
</style><br />
<div id="teammembers-container" style="overflow:hidden;"><br />
<h2>Team Members</h2><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Ekkers"><img src="https://static.igem.org/mediawiki/2010/thumb/b/b1/DSCI2647.JPG/180px-DSCI2647.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Ekkers">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>David Ekkers</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Marine Biology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work & President</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">22</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Ptr"><img src="https://static.igem.org/mediawiki/2010/1/13/Peter.jpg" width="120" height="159" /></a><br />
<p><a href="https://2010.igem.org/User:Ptr">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<br />
<h3>Peter Roemers</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Biomedical Science</td><br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work & Vice president</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">22</td><br />
</tr><br />
<br />
</table><br />
</div><br />
</div><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:joelkuiper"><img src="https://static.igem.org/mediawiki/2010/0/0f/Joel.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:joelkuiper">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Joël Kuiper</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Artificial Intelligence</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling & Treasurer</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">22</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:LauravdS"><img src="https://static.igem.org/mediawiki/2010/c/cf/Laura1_gr.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:LauravdS">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Laura van der Straat</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">26</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:aslomp"><img src="https://static.igem.org/mediawiki/2010/4/47/Arend1.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:aslomp">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Arend Slomp</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Computer Science</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">25</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
</div><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:AJ"><img src="https://static.igem.org/mediawiki/2010/thumb/e/ee/Aj_21.jpg/200px-Aj_21.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:AJ">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Arend Jan Suk</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Moleculair Biology & Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab manager & Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">24</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:djokehendriks"><img src="https://static.igem.org/mediawiki/2010/thumb/e/ed/DSC05903.JPG/180px-DSC05903.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:djokehendriks">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Djoke Hendriks</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Journalism & Molecular Biology and Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling & PR</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">25</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Neima"><img src="https://static.igem.org/mediawiki/2010/thumb/6/69/CIMG0399.JPG/180px-CIMG0399.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Neima">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Ezgi</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Biology and Genetics</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">20</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Ramon"><img src="https://static.igem.org/mediawiki/2010/thumb/3/38/Ramon.JPG/180px-Ramon.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Ramon">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Ramon Sieber</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Biology and Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">23</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:MJvdN"><img src="https://static.igem.org/mediawiki/2010/9/95/Maarten1.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:MJvdN">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Maarten<br> van den Nieuwenhof</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Life Sciences</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">25</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Jorrithillebrand"><img src="https://static.igem.org/mediawiki/2010/1/1b/Jorrit1.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Jorrithillebrand">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Jorrit Hillebrand</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Chemistry</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work & Vice Treasurer</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">24</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Geeske"><img src="https://static.igem.org/mediawiki/2010/1/17/Geeske.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Geeske">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Geeske van Heel</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Biology and Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Labwork & Fundraising</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">24</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
</div><br />
<br />
<br />
<br />
</html></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/MembersTeam:Groningen/Members2010-10-27T20:48:40Z<p>Joelkuiper: </p>
<hr />
<div><html><br />
<style><br />
.member-container {<br />
background-color:#a3ad49;<br />
width: 350px;<br />
float: left;<br />
margin: 10px;<br />
}<br />
.member-container .member-picture {<br />
width: 120px;<br />
text-align: center;<br />
float: left;<br />
}<br />
<br />
.member-container .member-picture p {<br />
margin-top: 10px;<br />
margin-bottom: 20px;<br />
}<br />
.member-container .member-picture p a {<br />
color: #fff;<br />
}<br />
.member-container .member-picture img {<br />
width: 120px;<br />
height: 159px;<br />
}<br />
.member-container .member-description {<br />
margin-top: 10px;<br />
float: left;<br />
margin-right: 10px;<br />
}<br />
.member-container h3 {<br />
color: #fff;<br />
}<br />
<br />
.member-description table {<br />
margin: 0;<br />
padding: 0;<br />
width: 175px;<br />
font-size: 11px;<br />
color: #216085;<br />
margin-top: -10px;<br />
background-color: #a3ad49;<br />
border-collapse:collapse;<br />
}<br />
<br />
.member-description table .name {<br />
font-weight: bold;<br />
}<br />
<br />
.member-description table .story {<br />
font-style: italic;<br />
padding: 10px;<br />
}<br />
<br />
</style><br />
<div id="teammembers-container" style="overflow:hidden;"><br />
<h2>Team Members</h2><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Ekkers"><img src="https://static.igem.org/mediawiki/2010/thumb/b/b1/DSCI2647.JPG/180px-DSCI2647.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Ekkers">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>David Ekkers</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Marine Biology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work & President</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">22</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Ptr"><img src="https://static.igem.org/mediawiki/2010/1/13/Peter.jpg" width="120" height="159" /></a><br />
<p><a href="https://2010.igem.org/User:Ptr">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<br />
<h3>Peter Roemers</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Biomedical Science</td><br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work & Vice president</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">22</td><br />
</tr><br />
<br />
</table><br />
</div><br />
</div><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:joelkuiper"><img src="https://static.igem.org/mediawiki/2010/0/0f/Joel.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:joelkuiper">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Joël Kuiper</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Artificial Intelligence</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling & Treasurer</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">22</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:LauravdS"><img src="https://static.igem.org/mediawiki/2010/c/cf/Laura1_gr.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:LauravdS">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Laura van der Straat</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">26</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:aslomp"><img src="https://static.igem.org/mediawiki/2010/4/47/Arend1.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:aslomp">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Arend Slomp</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Computer Science</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">25</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
</div><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:AJ"><img src="https://static.igem.org/mediawiki/2010/thumb/e/ee/Aj_21.jpg/200px-Aj_21.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:AJ">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Arend Jan Suk</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Moleculair Biology & Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab manager & Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">24</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:djokehendriks"><img src="https://static.igem.org/mediawiki/2010/thumb/e/ed/DSC05903.JPG/180px-DSC05903.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:djokehendriks">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Djoke Hendriks</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Journalism & Molecular Biology and Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Modelling & PR</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">25</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Neima"><img src="https://static.igem.org/mediawiki/2010/thumb/6/69/CIMG0399.JPG/180px-CIMG0399.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Neima">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Ezgi</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Biology and Genetics</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">20</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Ramon"><img src="https://static.igem.org/mediawiki/2010/thumb/3/38/Ramon.JPG/180px-Ramon.JPG" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Ramon">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Ramon Sieber</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Biology and Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">23</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:MJvdN"><img src="https://static.igem.org/mediawiki/2010/9/95/Maarten1.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:MJvdN">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Maarten<br> van den Nieuwenhof</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Life Sciences</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">25</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Jorrithillebrand"><img src="https://static.igem.org/mediawiki/2010/1/1b/Jorrit1.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Jorrithillebrand">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Jorrit Hillebrand</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Chemistry</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Lab work & Vice Treasurer</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">24</td><br />
</tr><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/User:Geeske"><img src="https://static.igem.org/mediawiki/2010/1/17/Geeske.jpg" width="120" height="159" /></a><br />
<br />
<p><a href="https://2010.igem.org/User:Geeske">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Geeske van Heel</h3><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Biology and Biotechnology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Labwork & Fundraising</td><br />
</tr><br />
<tr><br />
<td class="name">Age:</td><td class="value">24</td><br />
</tr><br />
<br />
</table><br />
<br />
</div><br />
</div><br />
<br />
<br />
<br />
</html></div>Joelkuiperhttp://2010.igem.org/Team:Groningen/ApplicationsTeam:Groningen/Applications2010-10-27T20:06:34Z<p>Joelkuiper: </p>
<hr />
<div>__NOTOC__<br />
==Applications==<br />
A biofilm coating can be used for a great number of applications. We developed our hydrophobic coating keeping in mind that future development of our project could focus on adding other properties to a biofilm coating as well, creating functional coatings with different purposes. Utilizing the qualities of different biofilm forming bacteria or micro organisms and combining these coating properties with the ever growing stack of biobricks within the database could give rise to a variety of engineered biofilm machines, capable of transforming surfaces under many conditions.<br />
<br />
===Hydrophobic coatings===<br />
The potential benefits of hydrophobic coatings can be put to use in a wide variety of application areas; hydrophobic clothes, anti-fouling coatings, anti-corrosion coatings, hydrophobic paint and medical coatings for drug delivery or against biofilm formation. For these applications a lot of hydrophobic coatings have been developed. Not all of these coatings have been succesfully used outside the lab, so the search for a cheap, multi-functional hydrophobic coating is still ongoing.<br />
<br />
<br><br />
<br />
===Anti-fouling coatings===<br />
Surface hydrophobicity could also be beneficial in the antifouling industry, for instance when applied on ships. When marine micro-organisms like algae or pocks adhere to the hull of ships, they form a layer which greatly increases drag in the water. This results in higher fuel costs and increased erosion. To prevent organisms to adhere to the hull of ships, chemical antifouling paints, which often contain copper and tin, are used. A lot of these chemicals eventually end up in the oceans ecosystems accumulating in all trophic levels of marine life and contaminating estuarial silt near shipping routes. Estimates show that in the Netherlands alone, approximately 19 tons of organotin and 30 tons of copper end up in the environment every year.<br />
<br />
<br><br />
<br />
Organotin pollution in China: an overview of the current state and potential health risk - Cao et al.<br />
<br />
===Biofilm coatings===<br />
<br />
Although a biofilm is not ideal for all of these coating purposes (imagine a biofilm jacket), an hydrophobic biofilm coating could be applied in fields ranging from anti-fouling coatings on ships, peers and buoys, to coatings used to protect catheters and protheses from pathogenic bacteria. Moreover, with introducing our kill switch and our chaplin coating without living bacteria, a even broader range of applications can be taken into consideration as certain ethicial and safety issues are dealth with.<br />
<br />
<br><br />
The main advantage of a biofilm coating is that it is very cheap, applying it requires no technical pinnacles and it is more environmentally friendly than certain chemical coatings. Next to that, biofilms can grow on a wide variety of surfaces: They are found on your teeth, in catheters, in plumbing, in water cleaning installations (beneficial), in bioreactors and, if you're one of those students, on your bathroom floor (blech).<br />
<br />
<br><br />
So far there have been coatings with biological substances, but bacteria where only used to produce the coating material. In our project the bacteria will form a biofilm on the desired surface, which will then function as a coating. Our bacteria therefore execute the coating process themselves, which could save a lot of effort. In the case of using chaplins as a building blocks for a hydrophobic coating, the biofilm is also used to orient and anchor the proteins in the right way. As chaplins are amphipathic, the orientation in their pure form is determined by the properties of the surface they coat as well: Using a biofilm to structure them the right way is an easy and smart solution that helps us to get around some problems of using the chaplins in their pure form as a coating.<br />
<br />
<br><br />
<br />
===Medical coatings===<br />
Because of their surface modifying abilities, hydrophobic biofilms of non- pathogenic bacteria might be used to prevent pathogenic biofilms from adhering to implants or catheters. Keeping in mind that growing a biofilm, even our good biofilm coating, in a catheter or on prothesis can give serious medical problems (shock, inflammation) we make use of a kill switch to kill off living bacteria. Bacillissubtilis however, is not pathogenic, and we could also make use of some non-pathogenic inhabitants of our body like Lactococcusto form the hydrophobic biofilm. The principle via which we form a Bacillus biofilm coating is of course applicable to a number of hosts. Next to that, using model hosts like Bacillus to produce chaplins also enables us to genetically modify the chaplins. That way they can be used to further functionalize the coating for drug delivery, cell adhesion or anti-bacterial activity.<br />
<br><br />
<br />
===Concrete protection===<br />
As the team of Newcastle enters this years’ competition with a B. subtilis machine that fixes cracks in concrete, preventing corrosion and water- or frost-induced damage to buildings, roads or monuments; we have a great addition to their project (or the other way around). Hydrophobic coatings are used to protect concrete structures, especially when reinforced with metal bars, from corrosion damage. Our B. subtilis biofilm would be able to do the same thing by filling up cracks and reducing water inflow by chaplin production: Thus preventing damage by water or ice.<br />
<br />
<br><br />
A combination of both projects would make for great bacterial machinery: As the crack-filling bacteria repel the water and ice that is damaging concrete structures further damage is prevented and repair is accelerated.</div>Joelkuiperhttp://2010.igem.org/Team:Groningen/ExpressionTeam:Groningen/Expression2010-10-27T20:02:56Z<p>Joelkuiper: </p>
<hr />
<div>__NOTOC__<br />
==Expression of chaplins==<br />
<br />
'''Summary'''<br />
<br />
The goal of our project is to let ''Bacillus subtilis'' make a hydrophobic coating by forming a [https://2010.igem.org/Team:Groningen#/biofilm biofilm] and then expressing and secreting [https://2010.igem.org/Team:Groningen/Hydrophobins#Chaplins chaplins].<br />
<br />
===Subtilin induced expression of chaplins===<br />
<br />
The Biofilm forming capacity of ''Bacillus subtilis'' makes it a good host for our application. In addition, ''B. subtilis'' is known for its ability to produce and secrete large amounts of protein at high cell densities. However, despite its track record as an efficient production organism and the fact that both ''B. subtilis'' and ''Streptomyces coelicolor'' are gram-positive bacteria, it is not certain wether chaplins can be heterologously expressed in ''B. subtilis''. Improper folding, unsuccessful export, or even the very nature of the chaplins, could still lead to hampered expression. <br />
We took several steps to ensure optimal expression. The Coding sequences of the chaplins were codon optimized for ''B. subtilis'' and synthesized. We placed a ribosome binding site in front of the coding sequences that is known to work well in ''B. subtilis'', and flanked these constructs with the biobrick prefix and suffix. <br />
<br />
'''SURE expression system'''<br />
[[Image:SURE-gfp-gn.jpg|250px|thumb|right|Subtilin induction of GFP by the SURE system (Bongers ''et al'', 2005)]]<br />
Because it is uncertain how chaplin expression will affect ''B. subtilis'', the initial expression attempts were performed with the stringently controlled, subtilin-regulated gene expression (SURE) system (Bongers ''et al'', 2005). This system uses the subtilin sensing machinery present in a strain of ''B. subtilis'' that autoinduces the production of more of the [http://en.wikipedia.org/wiki/Lantibiotics lantibiotic] subtilin. The subtilin sensor histidine kinase SpaK phosphorylates the response regulator SpaR, which can then bind to so-called ''spa'' boxes in the promoter regions of genes involved in subtilin biosynthesis (Kleerebezem ''et al'', 2004). In the SURE system, a ''B. subtilis'' strain naturally lacking the subtilin biosynthesis genes has the ''spaRK'' genes introduced into its genome. A plasmid carrying a ''spa'' box promoter that is transformed to this strain can then drive the expression of proteins upon subtilin induction of SpaRK signalling. <br />
<br />
[[Image:Groningen-ODvsFluor-GFP.png|right|300px]]<br />
<br />
We have adapted this system to make it BioBrick compatible for easy expression of our chaplins, combinations of chaplins, or any other biobrick part that is composed of an RBS followed by a protein coding sequence. We introduced the BioBrick prefix and suffix into the expression plasmid, downstream of the mutated ''spaS'' promoter, producing our subtilin inducible expression backbone part, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305011 BBa_K305011]. To test the expression and find a suitable subtilin concentration for induction of the chaplins we made use of GFP fluorescence measurements. We inserted the part [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0240 BBa_E0240] into the BioBrick site and induced liquid cultures of ''B. subtilis'' carrying this plasmid (and the ''spaRK'' genes) with different volumes of subtilin-containing culture supernatant of a subtilin producing strain of ''B. subtilis''. These results demonstrate that addition of 0.5 to 1%(vol/vol) of subtilin to the culture is sufficient to reach optimal induction. > Chaplins<br />
<br />
===Timed expression of chaplins in a biofilm ===<br />
<br />
An important question is which promoter we should use to control the chaplin expression. We assume that an ideal promoter would not be active until the biofilm has formed because the expression of hydrophobic proteins might influence the formation of it. Two promoters where found that are active in biofilms but not during normal growth. <br />
<br />
[[Image:Groningen-Promotors-sketch.png|300px|left]]<br />
<br />
[[image:igemgroningen_srfa_Promotoractivity.jpg|right|200px|srfA|thumb|srfA promotor activity during cell growth (Nakano MM. 1991)]]<br />
<br />
'''''srfA'''''<br />
<br />
The [http://dbtbs.hgc.jp/COG/prom/srfAA-srfAB-comS-srfAC-srfAD.html ''srfA'' operon] has been reported to be important for natural competence and sporulation in ''Bacillus subtilis''. All these activities occur in biofilms, the promoter is not active until the end of exponential growth. It is controlled by the [https://2010.igem.org/Team:Groningen/Expression_model#ComXPA_quorum_sensing_system ComXPA quorum sensing system] and hence active in states of high cell densities. Therefore the ''srfA'' promoter would be suitable for chaplin expression. Two different lengths of the ''srfA'' promoter where chosen due to uncertainties concerning the region between the response element and the transcription start side of the SrfAA protein. In the original promoter this region is unusually long, by shortening it 190bp’s we hope to achieve a higher transcription efficiency. So we came up with two different promoters, the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305008 original] one and the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305007 shortened] one. Fusions with GFP confirmed our assumption that the short srfA variant leads to a higher transcription. While the fluorescence off the short variant was clearly above background levels, the long variant did not give convincing results. <br />
[[Image:florescence_srfA.jpg]]<br />
<br />
<br />
<br />
'''''yqxM'''''<br />
[[Image:igemgroningen_yqxm_prmoteractivity.jpg|right|200px|yqxm|thumb|yqxM promotor activity during cell growth of different mutants (Axel G. 1999)]]<br />
<br />
The [http://dbtbs.hgc.jp/COG/prom/yqxM-sipW-tasA.html ''yqxM-sipW-tasA''] operon is controlled by the ''yqxM'' promoter. It is needed for biofilm formation because TasA is a key protein of the extracellular matrix. The promotor gets activated via a cascade of other regulatory elements, including SrfA, in response to quorum sensing. Since the chaplins should work in a similar way to TasA we think the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K305006 ''yqxM''] promoter would be very suitable for chaplin expression during the stationary phase. We fused the yqxM promoter with GFP but could not observe any expression, since the GFP worked with the srfA promoter we conclude that the yqxM promoter does not work.<br />
<br />
<br />
===References===<br />
<small>Bongers RS, Veening JW, Van Wieringen M, Kuipers OP, and Kleerebezem M. Development and characterization of a subtilin-regulated expression system in Bacillus subtilis: strict control of gene expression by addition of subtilin. [http://aem.asm.org/cgi/content/short/71/12/8818Appl Environ Microbiol 2005 Dec; 71(12) 8818-24. pmid:16332878]<br />
<br />
Kleerebezem, M., R. Bongers, G. Rutten, W. M. de Vos, and O. P. Kuipers.<br />
2004. Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of<br />
the spa-box in subtilin-responsive promoters. [http://gbb.eldoc.ub.rug.nl/FILES/root/2004/PeptidesKleerebezem/2004PeptidesKleerebezem.pdf Peptides 25:1415–1424]<br />
<br />
Stöver AG, Driks A. Regulation of synthesis of the ''Bacillus subtilis'' transition-phase, spore-associated antibacterial protein TasA. [http://jb.asm.org/cgi/content/short/181/17/5476 J. Bacteriol. Sept. 1999, p. 5476-5481, Vol. 181, No. 17]<br />
<br />
Nakano MM, Xia LA, Zuber P. Transcription initiation region of the srfA operon, which is controlled by the comP-comA signal transduction system in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC208261/ PMC208261]<br />
<br />
Frances Chu, Daniel B. Kearns, Anna McLoon, Yunrong Chai, Roberto Kolter and Richard Losicka, A Novel Regulatory Protein Governing Biofilm Formation in ''Bacillus subtilis'' [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430766/ PMC2430766]<br />
<br />
Hayashi K, Ohsawa T, Kobayashi K, Ogasawara N, Ogura M. The H2O2 stress-responsive regulator PerR positively regulates srfA expression in ''Bacillus subtilis''. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1251593/ PMC1251593]</small></div>Joelkuiperhttp://2010.igem.org/Template:Team:Groningen/HeaderTemplate:Team:Groningen/Header2010-10-27T19:49:44Z<p>Joelkuiper: </p>
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