Team:Valencia Biocampus/Project

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Latest revision as of 19:18, 24 October 2013

Project Overview

Bacteria are essential in biotechnology, but they can hardly move. Nematodes, such as Caenorhabditis elegans, are fast crawling organisms, but they have limited biotechnological applications. By combining the best from both organisms, we present the first artificial synthetic symbiosis with bacteria engineered to ride on worms, which concentrate in hotspots where bacteria perform a desired biotechnological process, such as bioplastic (PHA) production. We have engineered Pseudomonas putida with a whole operon that allows the formation of a biofilm on the worm. Biofilm formation is switched on and off depending on the media, and thus bacteria get on and off the worm like travellers on a bus. We have also engineered a third partner, Escherichia coli, to express an interference RNA that promotes clumping. Taken together, our artificial symbiosis allows biotechnologically interesting bacteria to travel on nematodes, reach nutrient-rich biomass spots and maximize the efficiency of biotechnological fermentations in heterogeneous substrates.

Why E.coli

Escherichia coli is a model organism widely used in fields such as microbiology, molecular biology and genetics. Because of this, there is a great range of genetic manipulation techniques related with this Gram negative bacterium. There is also a lot of information about the biochemistry and genetics of this microorganism. All this allows us to work easily with it in the lab and perform assays testing different conditions that will be tried out in other organisms unstudied in such depth. Moreover, E.coli is the main food of C.elegans, organism that is able to incorporate nucleic acids from the bacteria, RNA, to its cells (Mello et al., 2004). This is the usual mechanism to transform C.elegans, introducing exogenous genetic material from the bacteria and altering the expression of the worm’s genome in order to make modifications of interest.

Controlling the mechanism

The role of E.coli in our project is to carry out the synthesis of an iRNA to induce the social feeding behaviour of Caenorhabditis elegans , the clumping. To achieve this, we cloned the biobrick Bba_K1112000 in E.coli, XL1-Blue strain (fig. 1).

Fig. 1.

pSB1C3 is a high copy number plasmid (RFC [10]) carrying chloramphenicol resistance.
The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell).
pSB1C3 has terminators bracketing its MCS which are designed to prevent transcription from *inside* the MCS from reading out into the vector. The efficiency of these terminators is known to be < 100%. Ideally we would construct a future set of terminators for bracketing a MCS that were 100% efficient in terminating both into and out of the MCS region.

This construction consists of the E.coli fadA promoter, which is activated in the presence of fatty acids (Clark, 1981), and the antisense sequence of the mRNA FLP-21 from C.elegans which encodes a protein involved in the solitary feeding behaviour of the worm, the formation of the dsRNA complex inhibits the expression of this protein, inducing the social feeding behaviour (fig. 2).

Fig.2

Pseudomonas putida and PHA production

Pseudomonas putida is a gram-negative bacterium that is found in most soil and water habitats where there is oxygen. Its diverse metabolism and its capacity to break down organic harmful solvents (most of its genes are involved in degrading aromatic or aliphatic hydrocarbons) in contaminated soils make this microorganism irreplaceable for research studies in the field of bioremediation but also for biosynthesis of value-added products. In addition, Pseudomonas putida has several strains including KT2440, the one we have worked with. This strain can colonize plant roots, from which they take nutrients, while at the same time it offers protection for the plant against pathogens.

For example, it is capable of converting styrene oil into the biodegradable PHA plastic . This helps the degradation of the polystyrene foam which was thought to be non-biodegradable. Styrene is a major environment toxic pollutant released from industrial sites. The conversion to PHA allows the cure of styrene pollution but it is also beneficial for society because of its applications in tissue engineering.
PHA is also environmentally friendly and has a long self-life therefore it is also used in everyday items. Unlike styrene, PHA can break down in soil or water.

Within Pseudomonas putida, PHA accumulates under unbalanced growth conditions as a means of intracellular storage, storing excess carbon and energy. These PHA polymers are synthesized by the enzyme PHA synthase which is bound to the surface of the PHA granules and uses coenzyme A thioesters of hydroxyalkanoic acids as substrates.

The role of P. putida in the Synthetic symbiosis that we have designed is to be carried by C. elegans to hotspots of interest where it would produce bioplastic, PHA.

An overview about our nematode

One of the main characters of our work is a nematode known as Caenorhabditis elegans, from the Rhabditidae family. It was first used as an experimental model in Developmental Genetics studies and nowadays it is also used in other fields such as Clinical Biology, Neurobiology and Cell Biology, being a good model to study Alzheimer disease, obesity, diabetes and aging, among others.

Another interesting thing is that it feeds on Escherichia coli. Its “favorite” strain is OP50, although we checked that it’s also able to feed on XL1-Blue strain, the one that we used in all our molecular biology experiments.

Some advantages of C. elegans when compared with other model organisms are:

Lifespan ranges between 2 and 3 weeks, so experimentation times are reduced.
Its maintenance and study is cheap and simple (it is transparent, which facilitates microscopic observation).
It is very small (1 mm), so it is possible to carry out experiments with a huge number of worms in a small Petri dish having a great statistical support.

Why use C. elegans as a transport?

When we were considering on creating a new system for the transport of bacteria, we found different key advantages that made the nematode the best option. For example, C. elegans is able to move very fast around solid substrates (soil actually is its natural habitat) and also in agar, so it’s very useful for both lab experiments and real-environment tests.

Its movement, in addition to being very fast, has two modes: random and directed.

When there is no attractant in the medium, C. elegans moves doing uncoordinated movements in several directions in what is known as 'random walk'.

The situation changes when there is an attractant in the medium. Here, our 'transport system' begins to direct its movements to the focus of the substance (volatile or soluble) which acts as an attractant in a process known as 'chemotaxis' thanks to the amazing sense of smell of our nematode. This allows us to ‘guide’ the nematodes towards defined spots in irregular substrates.

Chemotaxis is the foundation to guide the transport of bacteria and is therefore the focus of experimentation with C. elegans, with the aim of finding the best attractant. This ability makes C. elegans a perfect 'bus' for bacteria. (simuelegans online here)

Moreover, we found two pathogens (Yersinia pestis and Xhenorhabus nematophila) with the ability to form biofilms on C. elegans thanks to the proteins of the operon hmsHFRS. When genetically-engineered strains of commonly used bacteria such as Escherichia coli or Pseudomonas putida express this hmsHFRS operon, they have the 'ticket' to travel: they are able to adhere to the worm’s surface by means of forming a synthetic biofilm.

Expanding our knowledge about C. elegans...

Once we knew that C. elegans was the best option, we began to discover interesting things for further study.

There are several strains of our nematode. The one that drew our attention was called 'N2', which had a fairly interesting behaviour: under normal conditions, it eats individually, whereas under certain conditions (such as starvation), a social feeding behaviour known as “clumping” is induced. But this phenomenon can also be induced if the expression of some particular genes is interfered. This fact gave us the opportunity to develop the first artificial symbiosis between worms and bacteria, based on the manipulation of the behaviour of C. elegans by simply nourishing it with transformed E.coli able to synthesize the iRNA.

With it, while C. elegans acts as transport, bacteria return the favour giving it the ability to eat in company.

We have created a Results Section to organise our results depending on the activity they belong to (The Riding, The Calling, The Clumping and The Building) in order to make it more intuitive.

Go to Results section

Parts

These are the BioBricks we have designed, constructed, and characterized. We have submitted them to the Registry of Standard Biological Parts

Name	Type	Description	Designer	Length
BBa_K1112000	Regulatory	fadB promoter + FLP-21 iRNA	Pedro Luis Dorado Morales	344
BBa_K1112001	Coding	pGlnA + hmsHFRS operon	Alba Iglesias Vilches	6342
BBa_K1112002	Coding	Cluster PHA	Alba Iglesias Vilches	4978

BBa_K1112000: fadB promoter + FLP-21 iRNA

This construction is made up by a fatty acid-sensitive promoter that directs the transcription of an iRNA responsible for the social or solitary behavior of C.elegans.

BBa_K1112001: pGlnA + hmsHFRS operon

We reused our nitrogen sensitive promoter from one of our last year constructs and we used it to control the expression of the operon that triggers the formation of a biofilm over C.elegans.

BBa_K1112002: cluster PHA

This BioBrick contains the complete natural sequence that induces the production of PHA in Pseudomonas putida (KT2440).

Notebook

During the whole project, we have been working on a notebook in order to show the world what we have been working on and to collect all our progress.

You'll be able to see our results, the picture of the day, the song that was being played in the lab while we were working and our iGEM News.

Go to Notebook

@@ Line 11: / Line 11: @@
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+padding-bottom:15px;
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@@ Line 23: / Line 24: @@
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+        includeTitle:false,
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@@ Line 29: / Line 33: @@
      <ul class="nav nav-tabs" id="myTab">
-	    <li class="active"><a href="#Project">Project Overview</a></li>
+      <li class="active"><a href="#Project">Project Overview</a></li>
-	    <li><a href="#Coli"><i>Escherichia coli</i></a></li>
+      <li><a href="#Coli"><i>Escherichia coli</i></a></li>
-	    <li><a href="#Pseudomonas"><i>Pseudomonas putida</i></a></li>
+      <li><a href="#Pseudomonas"><i>Pseudomonas putida</i></a></li>
-	    <li><a href="#Elegans"><i>Caenorhabditis elegans</i></a></li>
+      <li><a href="#Elegans"><i>Caenorhabditis elegans</i></a></li>
-	    <li><a href="#Results">Results</a></li>
+      <li><a href="#Results">Results</a></li>
-	    <li><a href="#Parts">Biobricks</a></li>
+      <li><a href="#Parts">Biobricks</a></li>
+      <li><a href="#Notebook">Notebook</a></li>
      </ul>
      <div class="tab-content">
-	    <div class="tab-pane fade active in" id="Project">
+      <div class="tab-pane fade active in" id="Project">
-	    	<div class="pull-left" style="width:900px;text-align:justify;">
+        <div class="pull-left" style="width:900px;text-align:justify;">
 </html>
@@ Line 46: / Line 50: @@
 <html>
 <div style="width:540px;margin-right:15px;float:left;">
-Bacteria are essential in biotechnology, but they can hardly move. Nematodes, such as <i>Caenorhabditis elegans</i>, are fast crawling organisms, but they have limited biotechnological applications. By combining the best from both organisms, we present the first artificial synthetic symbiosis with bacteria engineered to ride on worms, which concentrate in hotspots where bacteria perform a desired biotechnological process, such as bioplastic (PHA) production. We have engineered <i>Pseudomonas putida</i> with a whole operon that allows the formation of a biofilm on the worm. Biofilm formation is switched on and off depending on the media, and thus bacteria get on and off the worm like travellers on a bus. We have also engineered a third partner, <i>Escherichia coli</i>, to express an interference RNA that promotes clumping. Taken together, our artificial symbiosis allows biotechnologically interesting bacteria to travel on nematodes, reach nutrient-rich biomass spots and maximize the efficiency of biotechnological fermentations in heterogenous substrates.
+Bacteria are essential in biotechnology, but they can hardly move. Nematodes, such as <i>Caenorhabditis elegans</i>, are fast crawling organisms, but they have limited biotechnological applications. By combining the best from both organisms, we present the first artificial synthetic symbiosis with bacteria engineered to ride on worms, which concentrate in hotspots where bacteria perform a desired biotechnological process, such as bioplastic (PHA) production. We have engineered <i>Pseudomonas putida</i> with a whole operon that allows the formation of a biofilm on the worm. Biofilm formation is switched on and off depending on the media, and thus bacteria get on and off the worm like travellers on a bus. We have also engineered a third partner, <i>Escherichia coli</i>, to express an interference RNA that promotes clumping. Taken together, our artificial symbiosis allows biotechnologically interesting bacteria to travel on nematodes, reach nutrient-rich biomass spots and maximize the efficiency of biotechnological fermentations in heterogeneous substrates.
 </div>
@@ Line 55: / Line 59: @@
 </html>
-==Escherichia coli==
+==Why ''E.coli''==
 <html>
 <p>
-     <b>Why <i>E.coli</i></b>
+     <b><i>Escherichia coli</i></b>
-     is a model organism widely use in fields as microbiology, molecular biology and genetics. Result of that is the great range of genetic manipulation
+     is a model organism widely used in fields such as microbiology, molecular biology and genetics. Because of this, there is a great range of genetic manipulation
-     techniques that can be found related to this Gram negative bacterium. There is also a lot of information about the biochemistry and genetics of this
+     techniques related with this Gram negative bacterium. There is also a lot of information about the biochemistry and genetics of this
-     microorganism. All this allows to work easily with it in the lab and often perform assays testing different conditions that will be tried out in other
+     microorganism. All this allows us to work easily with it in the lab and perform assays testing different conditions that will be tried out in other
-     organisms unstudied in such depth. Moreover, <i>E.coli</i> is the main food of C.elegans, organism that is able to incorporate nucleic acids from the
+     organisms unstudied in such depth. Moreover, <i>E.coli</i> is the main food of <i>C.elegans</i>, organism that is able to incorporate nucleic acids from the
-     bacteria, RNA, to its cells (Mello et al., 2004). This is the usual mechanism of transformation of the worm, introducing exogenous genetic material from
+     bacteria, RNA, to its cells (Mello et al., 2004). This is the usual mechanism to transform C.elegans, introducing exogenous genetic material from
-     the bacteria altering the expression of the worm’s genome in order to make modifications of interest.
+     the bacteria and altering the expression of the worm’s genome in order to make modifications of interest.
 </p>
 <h2>Controlling the mechanism</h2>
 <p>
-     The role of <i>E.coli</i> in our project is to carry out the synthesis of an iRNA for inducing the social feeding behavior of <i>Caenorhabditis elegans</i>
+     The role of <i>E.coli</i> in our project is to carry out the synthesis of an iRNA to induce the social feeding behaviour of <i>Caenorhabditis elegans</i>
-    , the <b>clumping. </b>To achieve this, we cloned the biobrick Bba_K1112000 in <i>E. coli<b>, </b></i><b>XL1-Blue strain (fig. 1). </b>
+   , the <b>clumping. </b>To achieve this, we cloned the biobrick Bba_K1112000 in <i>E.coli</i>, </b></i><b>XL1-Blue strain (fig. 1). </b>
 </p>
-<div>
+<div style="clear:both;width:100%;height:auto;overflow:auto;">
 <img src="https://static.igem.org/mediawiki/2013/4/46/Vlc_biocampus_ecoli_ov1.png" style="float:left" />
 <div style="float:left;width:650px;padding-left:15px;clear:right;">
@@ Line 98: / Line 102: @@
 <p>
 This<b> construction</b> consists of the <i>E.coli </i>fadA promoter, which is <b>activated in the presence of fatty acids</b> (Clark, 1981), and the
-     antisense sequence of the mRNA <i>FLP-21 </i>from C.elegans which encodes a protein involved in the solitary feeding behaviour of the worm, the formation
+     antisense sequence of the mRNA <i>FLP-21 </i>from <i>C.elegans</i> which encodes a protein involved in the solitary feeding behaviour of the worm, the formation
-     of the dsRNA complex inhibits the expression of this protein, inducing the social feeding behavior (<b>fig. 2)</b>.
+     of the dsRNA complex inhibits the expression of this protein, inducing the social feeding behaviour (<b>fig. 2)</b>.
 </p>
 <p style="text-align:center">
-<img src="https://static.igem.org/mediawiki/2013/f/fa/Vlc_biocampus_ecoli_ov2.png" alt="Fig.2" style="width:700px" />
+   <img src="https://static.igem.org/mediawiki/2013/f/fa/Vlc_biocampus_ecoli_ov2.png" alt="Fig.2" style="width:700px" />
 </p>
@@ Line 114: / Line 118: @@
 <p>
 <b><i>Pseudomonas putida</i> </b> is a gram-negative bacterium that is found in most soil and water habitats where there is oxygen. Its diverse metabolism and its capacity to break down
-     organic harmful solvents (it has most genes involved in degrading aromatic or aliphatic hydrocarbons) in contaminated soils make this microorganism
+     organic harmful solvents (most of its genes are involved in degrading aromatic or aliphatic hydrocarbons) in contaminated soils make this microorganism
-     irreplaceable for research studies in the field of bioremediation but also for biosynthesis of value-added products. In addition, <i>Pseudomonas putida</i> has several strains including KT2440, the one we have worked with. This strain can colonize plant roots, from which they take nutrients, while at the same time it offers protection to the plant from pathogens.
+     irreplaceable for research studies in the field of bioremediation but also for biosynthesis of value-added products. In addition, <i>Pseudomonas putida</i> has several strains including KT2440, the one we have worked with. This strain can colonize plant roots, from which they take nutrients, while at the same time it offers protection for the plant against pathogens.
 </p>
 <html>
@@ Line 121: / Line 125: @@
 <div style="width:550px;float:left;">
 <p>
-For example, it is capable of converting <a href="http://en.wikipedia.org/wiki/Styrene">styrene</a> oil into the <b><a href="http://en.wikipedia.org/wiki/Biodegradable_plastic">biodegradable plastic</a> <a href="http://en.wikipedia.org/wiki/Polyhydroxyalkanoates">PHA</a></b>. This helps to degrade the polystyrene foam which
+For example, it is capable of converting <a href="http://en.wikipedia.org/wiki/Styrene" target="_blank">styrene</a> oil into the <b><a href="http://en.wikipedia.org/wiki/Biodegradable_plastic" target="_blank">biodegradable <a href="http://en.wikipedia.org/wiki/Polyhydroxyalkanoates" target="_blank">PHA</a> plastic</a> </b>. This helps the degradation of the polystyrene foam which
      was thought to be non-biodegradable. Styrene is a major environment toxic pollutant released from industrial sites. The conversion to PHA allows the cure
-     of styrene pollution but it is also beneficial to society because of its applications in tissue engineering.
+     of styrene pollution but it is also beneficial for society because of its applications in tissue engineering.
 <br/>
-  PHA is also environmental friendly and has a long self-life therefore it is also used in everyday items. Unlike styrene, PHA can break down in soil or water.
+  PHA is also environmentally friendly and has a long self-life therefore it is also used in everyday items. Unlike styrene, PHA can break down in soil or water.
 </p>
 <p>
      Within <i>Pseudomonas putida</i>, PHA accumulates under unbalanced growth conditions as a means of intracellular storage, storing excess carbon and energy.
-     These PHA polymers are synthesized by enzyme PHA synthase which is bound to the surface of the PHA granules and uses coenzyme A thioesters of
+     These PHA polymers are synthesized by the enzyme PHA synthase which is bound to the surface of the PHA granules and uses coenzyme A thioesters of
      hydroxyalkanoic acids as substrates.
 </p>
@@ Line 139: / Line 143: @@
 <p>
-     The role of <i>P. putida </i>in the Synthetic symbiosis that we have designed is to be carried by <i>C. elegans </i>to hotspots of interest where would
+     The role of <i>P. putida </i>in the Synthetic symbiosis that we have designed is to be carried by <i>C. elegans </i>to hotspots of interest where it would
-     produce bioplastic PHA.
+     produce bioplastic, PHA.
 </p>
@@ Line 150: / Line 154: @@
 <h2>An overview about our nematode</h2>
 <p>
-	One of the main characters of our work is a nematode known as <b><i>Caenorhabditis elegans, </i></b>from the Rhabditidae family. It was first used as an
+  One of the main characters of our work is a nematode known as <b><i>Caenorhabditis elegans, </i></b>from the Rhabditidae family. It was first used as an
-	experimental model in Developmental Genetics studies and nowadays is also used in other fields such as Clinical Biology, Neurobiology and Cell Biology,
+  experimental model in Developmental Genetics studies and nowadays it is also used in other fields such as Clinical Biology, Neurobiology and Cell Biology,
-	being a good model to study Alzheimer disease, obesity, diabetes and aging, among others.
+  being a good model to study Alzheimer disease, obesity, diabetes and aging, among others.
 </p>
 <p>
-	Another interesting thing is that it feeds on <b><i>Escherichia coli</i></b>. Its “favorite” strain is OP50, although we checked that it’s also able to
+  Another interesting thing is that it feeds on <b><i>Escherichia coli</i></b>. Its “favorite” strain is OP50, although we checked that it’s also able to
-	feed on <b>XL1-Blue strain</b>, the one that we used in all our molecular biology experiments.
+  feed on <b>XL1-Blue strain</b>, the one that we used in all our molecular biology experiments.
 </p>
 <p>
-	Some advantages of C. elegans when compared with other model organisms are:
+  Some advantages of C. elegans when compared with other model organisms are:
 </p>
 <br/>
@@ Line 176: / Line 180: @@
 <div class="clearfix"></div>
 <p>
-	<h2>Why use C. elegans as a transport?</h2>
+  <h2>Why use <i>C. elegans</i> as a transport?</h2>
 </p>
 <p>
-	When we were considering on creating a new system for the transport of bacteria, we found different key advantages that made the nematode the best option.
+  When we were considering on creating a new system for the transport of bacteria, we found different key advantages that made the nematode the best option.
-	For example, <i>C. elegans</i> is able to move very fast around solid substrates (soil is, actually, its natural habitat) and also in agar, so it’s very
+  For example, <i>C. elegans</i> is able to move very fast around solid substrates (soil actually is its natural habitat) and also in agar, so it’s very
-	useful for both lab experiments and real-environment tests.
+  useful for both lab experiments and real-environment tests.
 </p>
 <p>
-	Its movement, in addition to being very fast, has two modes: <b>random </b>and <b>directed.</b>
+  Its movement, in addition to being very fast, has two modes: <b>random </b>and <b>directed.</b>
 </p>
 <p>
-When there is no attractant in the medium, <i>C. elegans</i> moves doing uncoordinated movements in several directions in what is known as	<b>'random walk'.</b>
+When there is no attractant in the medium, <i>C. elegans</i> moves doing uncoordinated movements in several directions in what is known as  <b>'random walk'.</b>
 </p>
 <p>
-	The situation changes when there is an attractant in the medium. Here, our 'transport' begins to direct his movements to the focus of the substance
+  The situation changes when there is an attractant in the medium. Here, our 'transport system' begins to direct its movements to the focus of the substance
-	(volatile or soluble) which acts as an attractant in a process known as <b>'chemotaxis'</b> thanks to the amazing smell of our nematode. This allows us to
+  (volatile or soluble) which acts as an attractant in a process known as <b>'chemotaxis'</b> thanks to the amazing sense of smell of our nematode. This allows us to
-	‘guide’ the nematodes <b>towards defined spots in irregular substrates.</b>
+  ‘guide’ the nematodes <b>towards defined spots in irregular substrates.</b>
 </p>
 <p>
-Chemotaxis is the foundation to guide the transport of bacteria and is therefore the focus of experimentation with <i>C. elegans</i>, with <b>the aim of finding the best attractant.</b> This ability makes <i>C. elegans </i>a perfect 'bus' for bacteria.	<b>(<a href="https://2013.igem.org/Team:Valencia_Biocampus/Simuelegans-Chemotaxis">simuelegans online here</a>)</b>
+Chemotaxis is the foundation to guide the transport of bacteria and is therefore the focus of experimentation with <i>C. elegans</i>, with <b>the aim of finding the best attractant.</b> This ability makes <i>C. elegans </i>a perfect 'bus' for bacteria.  <b>(<a href="https://2013.igem.org/Team:Valencia_Biocampus/Simuelegans-Chemotaxis">simuelegans online here</a>)</b>
 </p>
 <p>
-	Moreover, we found two pathogens (<i>Yersinia pestis</i> and <i>Xhenorhabus nematophila</i>) with the ability to form biofilms on <i>C. elegans</i> thanks
+  Moreover, we found two pathogens (<i>Yersinia pestis</i> and <i>Xhenorhabus nematophila</i>) with the ability to form biofilms on <i>C. elegans</i> thanks
-to the proteins of the <b>operon hmsHFRS.</b> When genetically-engineered strains of commonly used bacteria such as <i>Escherichia coli</i> or	<i>Pseudomonas</i> <i>putida</i> express this hmsHFRS operon, they have the 'ticket' to travel: they are able to adhere to the worm’s surface by means of
+to the proteins of the <b>operon hmsHFRS.</b> When genetically-engineered strains of commonly used bacteria such as <i>Escherichia coli</i> or  <i>Pseudomonas</i> <i>putida</i> express this hmsHFRS operon, they have the 'ticket' to travel: they are able to adhere to the worm’s surface by means of
-	forming a synthetic biofilm.
+  forming a synthetic biofilm.
 </p>
 <p>
-<h2>Expanding our knowledge about C. elegans...</h2>
+<h2>Expanding our knowledge about <i>C. elegans</i>...</h2>
 </p>
 <p>
-	Once we knew that <i>C. elegans</i> was the best option, we began to discover interesting things for further study.
+  Once we knew that <i>C. elegans</i> was the best option, we began to discover interesting things for further study.
 </p>
 <p>
-	There are several strains of our nematode. The one that drew our attention was called 'N2', which had a fairly interesting behavior: under normal
+  There are several strains of our nematode. The one that drew our attention was called 'N2', which had a fairly interesting behaviour: under normal
-	conditions, it eats individually, whereas under certain conditions (such as starvation), a social feeding behavior known as <b>“clumping”</b> is induced.
+  conditions, it eats individually, whereas under certain conditions (such as starvation), a social feeding behaviour known as <b>“clumping”</b> is induced.
-But this phenomenon can also be induced if the expression of some particular genes is interfered. This fact gave us the opportunity to develop the	<b>first artificial symbiosis between worms and bacteria</b>, based on the manipulation of the behaviour of <i>C. elegans</i> by simply nourishing it with
+But this phenomenon can also be induced if the expression of some particular genes is interfered. This fact gave us the opportunity to develop the  <b>first artificial symbiosis between worms and bacteria</b>, based on the manipulation of the behaviour of <i>C. elegans</i> by simply nourishing it with
-	transformed <i>E.coli </i>able to synthesize the iRNA.
+  transformed <i>E.coli </i>able to synthesize the iRNA.
 </p>
 <p>
-	<b>With it, while<i> C. elegans </i>acts as transport, bacteria return the favour giving it the ability to eat in company. </b>
+  <b>With it, while<i> C. elegans </i>acts as transport, bacteria return the favour giving it the ability to eat in company. </b>
 </p>
 <p style="text-align:center">
@@ Line 231: / Line 235: @@
 <div class="tab-pane fade" id="Results">
-</html>
-==Results==
-<html>
-<div class="liquid-slider" id="slider-id">
-     <div>
-          <h2 class="title"><img src="https://static.igem.org/mediawiki/2013/2/20/White_riding_icon.png" alt="" class="title-icon" />The Riding</h2>
-<h1 class="title2 riding-color"><img src="https://static.igem.org/mediawiki/2013/2/20/White_riding_icon.png" alt="" class="title2-icon riding-bg" />The Riding</h1>
-     </div>
-     <div>
-          <h2 class="title"><img src="https://static.igem.org/mediawiki/2013/3/36/White_calling_icon.png" alt="" class="title-icon" />The Calling</h2>
-<h1 class="title2"><img src="https://static.igem.org/mediawiki/2013/3/36/White_calling_icon.png" alt="" class="title2-icon" />The Calling</h1>
 <p>
-<h3>Looking for an attractant</h3>
+We have created a <b>Results Section</b> to organise our results depending on the activity they belong to (<b>The Riding, The Calling, The Clumping and The Building</b>) in order to make it more intuitive.
-When we were considering setting up a transport of bacteria was thought to be necessary to have a 'destination', a place to go. That destiny would be a    <b>'hot spot' on a heterogeneous substrate</b> where <b><i>Caenorhabditis</i> <i>elegans</i></b> should lead right to that point the bacteria.
-    <br/>
-Leveraging the powerful smell of the nematode, it was decided to try a number of attractants from various lists from web <a href="http://www.wormbook.org">www.wormbook.org</a> that could work as 'hot spot' of our experiment. Thus, using the<i> C. elegans</i>    <b>chemotaxis, </b>we could direct transport.
-</p>
-<p>
-<h3>The attractants experiment</h3>
-    The test would be carried creating our own plates on which half would be NGM unmodified and the other half part would be including soluble compounds before
-    solidifying or after solidification in the case of volatiles. The list of modifications can be found in <b>Fig. 1</b>.
 </p>
 <p style="text-align:center">
-<img src="https://static.igem.org/mediawiki/2013/8/83/Calling_table1.png" style="width:700px;" alt="Table 1" />
+  <a href="https://2013.igem.org/Team:Valencia_Biocampus/Results" class="btn btn-primary">Go to Results section</a>
 </p>
-<p>
-    To place <b><i>C. elegans</i> </b>on the plate, different cuts were made on a fresh plate of NGM, the resulting small pieces were placed in the exact
-    center of the 50% -50% plates to determine which side of the nematode preferred, one per Petri plate.
-</p>
-<p>
-    The results after counting 2 replicates per attractant can be seen in <b>Fig. 2</b>.
-</p>
-<p style="text-align:center">
-<img src="https://static.igem.org/mediawiki/2013/c/ce/Calling_table2.png" style="width:700px;" alt="Table 2" />
-</p>
-<p>
-    From <b>Table 2 </b>it could rule out many of the attractants that were thought viable.
-</p>
-<p>
-    Volatile attractants were not a good choice to evaporate quickly (which is also limited to the field experiments).
-</p>
-<p>
-    Another impediment arose. Once had already performed the experiments, it was decided that the promoter that will control the production of RNA interference
-    in <i>Escherichia coli</i> would be controlled by nitrogen, amino acids had to be discard as attractants; it would modify controlled expression of E.coli.
-</p>
-<p>
-    That left the MgSO<sub>4</sub> and hypoosmotic media as potential attractants.
-</p>
-<p>
-<h3>Attractant final choice</h3>
-Because the hyposmotic medium could interfere with the proper growth of bacteria (food of our nematode), the final decision was to choose the MgSO<sub>4</sub> as attractant.
-</p>
-<p>
-    The question that arose at that time was: <i>How we can multiply the amount of MgSO<sub>4</sub> to increase efficiency?</i>
-</p>
-<p>
-    <h3>MgSO<sub>4</sub> efficiency</h3>
-Once we had selected the most feasible attractant to the ‘transport’ for our experiments, we needed to know what could be the largest concentration of MgSO    <sub>4</sub> in order to optimize the attraction of the nematode.
-<br/>
- To choose the concentration, were tested in a battery of increased concentrations regarding the initial medium (1 ml / L). Bearing in mind the results of
-    the factor x2, we decided to test other factor concentrations: x3, x4, x5, x8 and x10; covering a range that does not exceed the concentration at which
-    might affect the life of <i>C. elegans</i> or bacteria.
-<br/>
-    Moreover, approaching experiments with <i>E. coli</i> and <i>Pseudomonas putida</i>, these trials were testing the end media: half plate with NGM
-    non-altered and half as PHA production medium for <i>Pseudomonas</i> and interference of <i>E. coli</i>.
-<br/>
-    Not knowing what fatty acid could activate transcription better, we tested two possibilities: oleic acid (named in the tables as PHAol) and octanoic acid
-    (PHAoc). The concentration for each fatty acid was tested in <i>E. coli</i> choosing as better:
-<ul>
-    <li>1.28 µl/ml of Octanoic acid.</li>
-    <li>2.58 µl/ml of Oleic acid.</li>
-</ul>
-</p>
-<p>
-    You can see our results in<b> figures 3, 4, 5 and 6</b>. The two first counts were made after 3 hours and the next ones after 6 hours. We suppose at that
-    time the worms can select their “favorite” half part gone across and their movement would be always in the same area. We prepare as control plates with the
-    same composition but without MgSO<sub>4</sub> in PHA media.
-</p>
-<p style="text-align:center">
-<img src="https://static.igem.org/mediawiki/2013/5/5b/Calling_table3.png" style="width:700px;" alt="Tables 3, 4, 5, 6" />
-</p>
-<p>
-    <h3>Final concentration choice</h3>
-    Once the test already performed and that the results seen by the factor of PHA and MgSO<sub>4</sub> selected fatty acid give very scattered (there is
-    probably repellent effect at high concentrations in the medium with oleic but reversed in octanoic) we make a selection of MgSO<sub>4</sub> concentration
-    for each medium.
-<ul>
- <li> <b>If it is used oleic acid </b><b>à</b><b> </b>Better results with 4ml/L MgSO<sub>4</sub>.
-</li>
-<li>
-    <b>If octanoic acid used </b><b>à</b><b> </b>Best results to 10 ml/L of MgSO<sub>4</sub>.
-</li>
-</ul>
-    Octanoic was discarded as a transcriptional activator of the iRNA of clumping after other result of <i>E. coli</i>, so finally we selected PHA medium with
-    oleic acid.
-<br/>
-    The biggest problem in trying to have an effective attractant was <b>how effective could be in presence of <i>E. coli</i></b>. It was therefore necessary
-    to test the tradeoff between the value of 4ml / L MgSO<sub>4</sub> and pair it with different concentrations of bacteria, high enough to feed the nematode
-    but low enough to permit the attractive effect of MgSO<sub>4</sub>.
-<br/>
-    To find this point of commitment we prepare experiments in which the concentration of 4ml/L of MgSO<sub>4</sub> is faced against different ODs from serial
-    dilutions of a preculture of E. coli DH5a.
-    <br/>
-    <b>Table 7 </b>
-    shows the results. The count took place at 3h after the passing of fresh nematodes.
-</p>
-<p>
-<h3>Best E. coli OD choice</h3>
-    An OD of 1 (minimum concentration of cells / volume) gives an attractive effect even better than expected (subsequent experiments try to see if there is
-    synergy between <i>E. coli </i>and attractive factors MgSO4).
-    <br/>
-    To improve the approximation was decided to repeat the experiment with MgSO<sub>4</sub> concentration and the chosen bacteria OD showing that the system
-    works well. The results can be seen in <b>figure 8.</b>
-</p>
-<p style="text-align:center">
-<img src="https://static.igem.org/mediawiki/2013/c/cc/Calling_table5.png" style="width:700px;" alt="Table 7" />
-</p>
-     </div>
-     <div>
-          <h2 class="title"><img src="https://static.igem.org/mediawiki/2013/8/8e/White_clumping_icon.png" alt="" class="title-icon" />The Clumping</h2>
-<h1 class="title2 clumping-color"><img src="https://static.igem.org/mediawiki/2013/8/8e/White_clumping_icon.png" alt="" class="title2-icon clumping-bg" />The Clumping</h1>
-     </div>
-     <div>
-          <h2 class="title"><img src="https://static.igem.org/mediawiki/2013/9/97/White_building_icon.png" alt="" class="title-icon" />The Building</h2>
-<h1 class="title2 building-color"><img src="https://static.igem.org/mediawiki/2013/9/97/White_building_icon.png" alt="" class="title2-icon building-bg" />The Building</h1>
-     </div>
-</div>
@@ Line 383: / Line 257: @@
 Remember that the goal of proper part documentation is to describe and define a part such that it can be used without a need to refer to the primary literature. The next iGEM team should be able to read your documentation and be able to use the part successfully. Also, you should provide proper references to acknowledge previous authors and to provide for  users who wish to know more.
 <groupparts>iGEM013 Valencia_Biocampus</groupparts>
 -->
 </html>
 <html>
 <br/>
@@ Line 398: / Line 273: @@
              <th> Designer </th>
              <th> Length </th>
-             <th> Structure </th>
+             <th> Fav </th>
          </tr>
      </thead>
@@ Line 407: / Line 282: @@
          <td> fadB promoter + FLP-21 iRNA </td>
          <td> Pedro Luis Dorado Morales </td>
-         <td> </td>
+         <td> 344 </td>
-         <td> </td>
+         <td> <img src="https://2013.igem.org/common/tablesorter/themes/groupparts/heart13.gif" alt="" /></td>
      </tr>
      <tr>
          <td><img class="table-cross" src="https://static.igem.org/mediawiki/2013/0/01/CheckIcon.gif" alt="Yes" height="24" width="24"/></td>
          <td> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1112001">BBa_K1112001</a> </td>
-         <td> Regulatory </td>
+         <td> Coding </td>
          <td> pGlnA + hmsHFRS operon </td>
          <td> Alba Iglesias Vilches </td>
-         <td> </td>
+         <td> 6342</td>
          <td> </td>
      </tr>
@@ Line 422: / Line 297: @@
          <td><img class="table-check" src="https://static.igem.org/mediawiki/2013/0/01/CheckIcon.gif" alt="Yes" height="24" width="24"/></td>
          <td> <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1112002">BBa_K1112002</a> </td>
-         <td> DNA </td>
+         <td> Coding </td>
          <td> Cluster PHA </td>
          <td> Alba Iglesias Vilches </td>
-         <td> </td>
+         <td> 4978</td>
          <td> </td>
      </tr>
@@ Line 440: / Line 315: @@
 <p>
 <a href="http://parts.igem.org/Part:BBa_K1112001"><h3>BBa_K1112001: pGlnA + hmsHFRS operon</h3></a>
-     We reused our nitrogen sensitive promoter from one of our last year constructs and we employ it to control the expression on the operon that triggers the formation of a biofilm over <i>C.elegans</i>.
+     We reused our nitrogen sensitive promoter from one of our last year constructs and we used it to control the expression of the operon that triggers the formation of a biofilm over <i>C.elegans</i>.
 </p>
@@ Line 447: / Line 322: @@
      This BioBrick contains the complete natural sequence that induces the production of PHA in <i>Pseudomonas putida</i> (KT2440).
 </p>
+</div>
+<div class="tab-pane fade" id="Notebook">
+</html>
+==Notebook==
+<html>
+<p>
+During the whole project, we have been working on a notebook in order to show the world what we have been working on and to collect all our progress.
+</p>
+<p>You'll be able to see our results, the picture of the day, the song that was being played in the lab while we were working and our iGEM News.</p>
+<p style="text-align:center">
+  <a href="https://2013.igem.org/Team:Valencia_Biocampus/Notebook" class="btn btn-primary">Go to Notebook</a>
+</p>
 </div>