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2024-03-29T12:46:49Z
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http://2010.igem.org/Team:TU_Delft/Modeling/interaction-mapping
Team:TU Delft/Modeling/interaction-mapping
2010-10-27T23:31:54Z
<p>Jcnossen: </p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
==Homolog Interaction Mapping (HIM)==<br />
Part of our project was development of an application that can generate a list of putative interactions for genes in a new host organism. In our project, the clearest example of this is the insertion of a [[Team:TU_Delft/Project/tolerance|prefoldin (phPFDα + phPFDβ)]] coding plasmid into ''E. coli''. Using this application it was possible to get a list of putative interactions with the prefoldin proteins, and therefore give some hints about how these proteins are actually working.<br />
<br />
<br />
===Problems with introducing new genes===<br />
In our project, we are taking genes from various organisms and putting them as a biobrick plasmid into ''E. coli''.<br />
This may cause various unexpected problems in the newly designed biological system:<br />
* The protein in the original organism may need some other functional partners (proteins or RNAs) in order to behave correctly (I.A.W. a part of the imported system is missing)<br />
* The introduced protein can interact with local proteins that are not ment to interact. <br />
<br />
<br />
<br />
===Application===<br />
An application was developed that can run these queries on a PostgreSQL server running the STRING database. This database can be [http://string-db.org/newstring_cgi/show_download_page.pl downloaded] for free for academic use. Unfortunately, the STRING API does not provide enough functionality to do the homolog mapping, so a PostgreSQL database with the STRING data is always needed.<br />
<br />
More detailed use of the application is described in the [[Team:TU_Delft/Software/im-tutorial|application page]]. <br />
<br />
<br />
===Searching for interactions===<br />
To get an idea of which interactions may occur, interactions within the original organisms where mapped to their ''E. coli'' homologs:<br />
[[Image:IGEMTUDelft_Interactiongraph.PNG]]<br />
<br />
In more detail:<br />
* The sequence of every introduced protein (See the coding [[Team:TU_Delft/project/biobricks|biobricks]]) was blasted against the [http://string-db.org/newstring_cgi/show_input_page.pl?input_page_type=single_sequence STRING protein database]. This resulted in a number of proteins that are either the correct proteins, or homologs of the introduced proteins.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1" <br />
|'''Biobrick'''<br />
|'''Short name'''<br />
|'''STRING Database ID'''<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398000 BBa_K398000]<br />
|LadA<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=420246.GTNG_3499&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 420246.GTNG_3499]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398001 BBa_K398001]<br />
|AlkB<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=101510.RHA1_ro02534&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 101510.RHA1_ro02534]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398002 BBa_K398002]<br />
|RubA3<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=246196.MSMEG_1840&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 246196.MSMEG_1840]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398003 BBa_K398003]<br />
|RubA4<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=350058.Mvan_1744&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 350058.Mvan_1744]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398004 BBa_K398004]<br />
|Rubredoxin reductase<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=101510.RHA1_ro02537&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 101510.RHA1_ro02537]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398005 BBa_K398005]<br />
|Bt-ADH<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=235909.GK0984&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 235909.GK0984]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398006 BBa_K398006]<br />
|Bt-ALDH<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=235909.GK2772&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 235909.GK2772]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398100 BBa_K398100]<br />
|bbc1<br />
|Dropped: Not in STRING database, and <br />
protein sequence blast only resulted in eukaryotic homologs<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398200 BBa_K398200]<br />
|AlnA<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=62977.ACIAD0697&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 62977.ACIAD0697]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398201 BBa_K398201]<br />
|OprG<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=160488.PP_0504&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 160488.PP_0504]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398300 BBa_K398300]<br />
|AlkS<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=399739.Pmen_0429&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 399739.Pmen_0429]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398400 BBa_K398400]<br />
|Prefoldin-alpha<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=70601.PH0527&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 70601.PH0527]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398401 BBa_K398401]<br />
|Prefoldin-beta<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=70601.PH0532&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 70601.PH0532]<br />
|}<br />
* For each protein, the interactions stored in STRING where gathered. <br />
* For each found interacting protein, ''E. coli'' homologs where gathered.<br />
* This results in a combination of interactions with their ''E. coli'' homologs. The likelyness of this being a true interaction depends on the STRING interaction score (Data from publication text mining, microarrays and other sources) and the BLAST score (The homolog mapping step).<br />
<br />
<br />
===Result===<br />
<br />
==== Prefoldin interaction mapping ====<br />
The network below shows the putative interactions of the [[Team:TU_Delft/Project/tolerance/parts|prefoldin biobricks]], and any interacting proteins that do not have homologs in ''E. coli''.<br />
<br />
<html><br />
<iframe width="800" height="600" src="http://jcnossen.github.com/InteractionHomologMapping/prefoldin-mapping3.htm"><br />
<p>Your browser does not support iframes.</p><br />
</iframe><br />
</html><br />
<br />
* Green: ''E. coli'' homologs of a Pyrococcus protein that is interacting with prefoldin, according to the STRING database.<br />
* Red: Missing homolog.<br />
<br />
<br />
As prefoldin is working in ''E. coli'', one could argue that this is because it is interacting with the green proteins. This could be a hint for more research into the Pyrococcus prefoldin proteins.<br />
<br />
<br />
==== Alkane degradation pathway interaction mapping ====<br />
Additionally, the alkane degradation proteins where also entered into the mapping tool, as shown below. However, we already know how this system works. The STRING interactions probably just show parts of the pathway proteins that are expressed together, hence this analysis is a lot less useful.<br />
<br />
<html><br />
<iframe width="800" height="600" src="http://jcnossen.github.com/InteractionHomologMapping/alkdeg-mapping.htm"><br />
<p>Your browser does not support iframes.</p><br />
</iframe><br />
</html></div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/Software/im-tutorial
Team:TU Delft/Software/im-tutorial
2010-10-27T23:25:02Z
<p>Jcnossen: </p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
== Interaction Mapping Application ==<br />
<br />
The interaction mapping application directs the user through a number of steps to eventually get putative interactions of proteins in a new host organism.<br />
Unfortunately a lot of steps are still required, because the data comes from many sources. Look below for requirements of the application.<br />
<br />
=== Requirements ===<br />
<br />
* Access to running STRING database with the items, network and homology schema's.<br />
* For Cytoscape export, you will need to install the plugin jars from the Interaction Mapping binary package to the Cytoscape plugin directory. <br />
<br />
=== Steps and screenshots ===<br />
Step 1: Enter parts or custom sequences<br />
<html><img src="http://dl.dropbox.com/u/602630/scr/step1-scr.PNG"></html><br />
<br />
Step 2: Use the string-db website to find STRING protein ID for each sequence.<br />
<html><img src="http://dl.dropbox.com/u/602630/scr/step2-scr.PNG"></html><br />
<br />
Step 3: Map interactions to E. coli, or other organisms.<br />
<html><img src="http://dl.dropbox.com/u/602630/scr/step3-scr.PNG"></html><br />
<br />
Step 4: Export to text or Cytoscape<br />
<html><img src="http://dl.dropbox.com/u/602630/scr/step4-scr.PNG"></html><br />
<br />
=== Download ===<br />
Source code is available in a [http://github.com/jcnossen/InteractionHomologMapping github repository], or by directly downloading the [http://github.com/jcnossen/InteractionHomologMapping/zipball/master code in zip format].<br />
<br />
A binary version will be uploaded here:<br />
http://jcnossen.github.com/InteractionHomologMapping/</div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/Software/im-tutorial
Team:TU Delft/Software/im-tutorial
2010-10-27T23:18:39Z
<p>Jcnossen: New page: == Interaction Mapping Application == The interaction mapping application directs the user through a number of steps: Unfortunately a lot of steps are still required, because the data com...</p>
<hr />
<div><br />
== Interaction Mapping Application ==<br />
The interaction mapping application directs the user through a number of steps:<br />
Unfortunately a lot of steps are still required, because the data comes from many sources. Look below for requirements of the application.<br />
<br />
Step 1: Enter parts or custom sequences<br />
<html><img src="http://dl.dropbox.com/u/602630/scr/step1-scr.PNG"></html><br />
<br />
Step 2: Use the string-db website to find STRING protein ID for each sequence.<br />
<html><img src="http://dl.dropbox.com/u/602630/scr/step2-scr.PNG"></html><br />
<br />
Step 3: Map interactions to E. coli, or other organisms.<br />
<html><img src="http://dl.dropbox.com/u/602630/scr/step3-scr.PNG"></html><br />
<br />
Step 4: Export to text or Cytoscape<br />
<html><img src="http://dl.dropbox.com/u/602630/scr/step4-scr.PNG"></html><br />
<br />
=== Download ===<br />
Source code is available in a [http://github.com/jcnossen/InteractionHomologMapping github repository], or by directly downloading the [http://github.com/jcnossen/InteractionHomologMapping/zipball/master code in zip format].<br />
<br />
A binary version will be uploaded here:<br />
http://jcnossen.github.com/InteractionHomologMapping/<br />
<br />
<br />
=== Requirements ===</div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/Modeling/interaction-mapping
Team:TU Delft/Modeling/interaction-mapping
2010-10-27T22:07:39Z
<p>Jcnossen: </p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
==Homolog Interaction Mapping (HIM)==<br />
Part of our project was development of an application that can generate a list of putative interactions for genes in a new host organism. In our project, the clearest example of this is the insertion of a [[Team:TU_Delft/Project/tolerance|prefoldin (phPFDα + phPFDβ)]] coding plasmid into ''E. coli''. Using this application it was possible to get a list of putative interactions with the prefoldin proteins, and therefore give some hints about how these proteins are actually working.<br />
<br />
<br />
===Problems with introducing new genes===<br />
In our project, we are taking genes from various organisms and putting them as a biobrick plasmid into ''E. coli''.<br />
This may cause various unexpected problems in the newly designed biological system:<br />
* The protein in the original organism may need some other functional partners (proteins or RNAs) in order to behave correctly (I.A.W. a part of the imported system is missing)<br />
* The introduced protein can interact with local proteins that are not ment to interact. <br />
<br />
<br />
<br />
===Application===<br />
An application was developed that can run these queries on a PostgreSQL server running the STRING database. This database can be [http://string-db.org/newstring_cgi/show_download_page.pl downloaded] for free for academic use. Unfortunately, the STRING API does not provide enough functionality to do the homolog mapping, so a PostgreSQL database with the STRING data is always needed.<br />
<br />
More detailed use of the application is described in the [[Team:TU_Delft/Software/im-tutorial|homolog interaction mapping tutorial]]. <br />
<br />
<br />
===Searching for interactions===<br />
To get an idea of which interactions may occur, interactions within the original organisms where mapped to their ''E. coli'' homologs:<br />
[[Image:IGEMTUDelft_Interactiongraph.PNG]]<br />
<br />
In more detail:<br />
* The sequence of every introduced protein (See the coding [[Team:TU_Delft/project/biobricks|biobricks]]) was blasted against the [http://string-db.org/newstring_cgi/show_input_page.pl?input_page_type=single_sequence STRING protein database]. This resulted in a number of proteins that are either the correct proteins, or homologs of the introduced proteins.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1" <br />
|'''Biobrick'''<br />
|'''Short name'''<br />
|'''STRING Database ID'''<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398000 BBa_K398000]<br />
|LadA<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=420246.GTNG_3499&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 420246.GTNG_3499]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398001 BBa_K398001]<br />
|AlkB<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=101510.RHA1_ro02534&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 101510.RHA1_ro02534]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398002 BBa_K398002]<br />
|RubA3<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=246196.MSMEG_1840&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 246196.MSMEG_1840]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398003 BBa_K398003]<br />
|RubA4<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=350058.Mvan_1744&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 350058.Mvan_1744]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398004 BBa_K398004]<br />
|Rubredoxin reductase<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=101510.RHA1_ro02537&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 101510.RHA1_ro02537]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398005 BBa_K398005]<br />
|Bt-ADH<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=235909.GK0984&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 235909.GK0984]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398006 BBa_K398006]<br />
|Bt-ALDH<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=235909.GK2772&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 235909.GK2772]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398100 BBa_K398100]<br />
|bbc1<br />
|Dropped: Not in STRING database, and <br />
protein sequence blast only resulted in eukaryotic homologs<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398200 BBa_K398200]<br />
|AlnA<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=62977.ACIAD0697&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 62977.ACIAD0697]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398201 BBa_K398201]<br />
|OprG<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=160488.PP_0504&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 160488.PP_0504]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398300 BBa_K398300]<br />
|AlkS<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=399739.Pmen_0429&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 399739.Pmen_0429]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398400 BBa_K398400]<br />
|Prefoldin-alpha<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=70601.PH0527&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 70601.PH0527]<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K398401 BBa_K398401]<br />
|Prefoldin-beta<br />
|[http://string-db.org/version_8_3/newstring_cgi/show_network_section.pl?identifier=70601.PH0532&all_channels_on=1&interactive=yes&network_flavor=evidence&targetmode=proteins 70601.PH0532]<br />
|}<br />
* For each protein, the interactions stored in STRING where gathered. <br />
* For each found interacting protein, ''E. coli'' homologs where gathered.<br />
* This results in a combination of interactions with their ''E. coli'' homologs. The likelyness of this being a true interaction depends on the STRING interaction score (Data from publication text mining, microarrays and other sources) and the BLAST score (The homolog mapping step).<br />
<br />
<br />
===Result===<br />
<br />
==== Prefoldin interaction mapping ====<br />
The network below shows the putative interactions of the [[Team:TU_Delft/Project/tolerance/parts|prefoldin biobricks]], and any interacting proteins that do not have homologs in ''E. coli''.<br />
<br />
<html><br />
<iframe width="800" height="600" src="http://jcnossen.github.com/InteractionHomologMapping/prefoldin-mapping3.htm"><br />
<p>Your browser does not support iframes.</p><br />
</iframe><br />
</html><br />
<br />
* Green: ''E. coli'' homologs of a Pyrococcus protein that is interacting with prefoldin, according to the STRING database.<br />
* Red: Missing homolog.<br />
<br />
<br />
As prefoldin is working in ''E. coli'', one could argue that this is because it is interacting with the green proteins. This could be a hint for more research into the Pyrococcus prefoldin proteins.<br />
<br />
<br />
==== Alkane degradation pathway interaction mapping ====<br />
Additionally, the alkane degradation proteins where also entered into the mapping tool, as shown below. However, we already know how this system works. The STRING interactions probably just show parts of the pathway proteins that are expressed together, hence this analysis is a lot less useful.<br />
<br />
<html><br />
<iframe width="800" height="600" src="http://jcnossen.github.com/InteractionHomologMapping/alkdeg-mapping.htm"><br />
<p>Your browser does not support iframes.</p><br />
</iframe><br />
</html></div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/Project/rbs-characterization/parts
Team:TU Delft/Project/rbs-characterization/parts
2010-10-27T20:09:25Z
<p>Jcnossen: /* Cloning method */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/parts" target="" /></map></html><br />
<br />
==RBS measurement parts==<br />
<br />
===Experimental setup===<br />
The following RBS sequences from the [http://partsregistry.org/Ribosome_Binding_Sites/Prokaryotic/Constitutive/Anderson Anderson RBS family] were used:<br />
*[http://partsregistry.org/Part:BBa_J61100 BBa_J61100]<br />
*[http://partsregistry.org/Part:BBa_J61101 BBa_J61101]<br />
*[http://partsregistry.org/Part:BBa_J61107 BBa_J61107]<br />
*[http://partsregistry.org/Part:BBa_J61117 BBa_J61117]<br />
*[http://partsregistry.org/Part:BBa_J61127 BBa_J61127]<br />
<br />
For the purpose of comparison and standardization the following RBS was used as a reference for our characterization.<br />
*[http://partsregistry.org/Part:BBa_B0032 BBa_B0032] <br />
<br />
These RBS sequences were positioned upstream of the standard [http://partsregistry.org/Part:BBa_E0040 GFP coding sequence], in order to be able to measure expression.<br />
<br />
===Cloning method===<br />
This is a summary of the cloning methods that succeeded. For more information on the exact methods and all attempts made see what was done under the header 'Characterization of RBS sequences' in our blog posts starting around<br />
[https://2010.igem.org/Team:TU_Delft#page=Notebook/blog&blog=6_July_2010 6 July]<br />
<br />
====The Anderson Constructs====<br />
[http://partsregistry.org/Part:BBa_I13401 BBa_I13401] was PCR amplified by using the universal primers G00100 and G00101. The purified PCR product was back-digested using XbaI and PstI. <br />
The Anderson RBSs are provided in pSB1A2 plasmids. These were digested using SpeI and PstI and ligated with the digested I13401 insert. Transformation into competent TOP10 <i>E.coli</i> cells yielded positives, as determined by UV-light plate analysis.<br />
<br />
====The Reference Construct====<br />
[http://partsregistry.org/Part:BBa_J23100 BBa_J23100], which is contained in the pSB1A2 plasmid was digested using SpeI and PstI. [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] was digested using XbaI and PstI and ligated with the digest product of J23100. Transformation into <i>E.coli</i> TOP10 yielded positives as determined by UV-light.<br />
<br />
====The BioBricks====<br />
The Biobricks generated for the measurements were: [http://partsregistry.org/Part:BBa_K398500 K398500], [http://partsregistry.org/Part:BBa_K398501 K398501], [http://partsregistry.org/Part:BBa_K398502 K398502], [http://partsregistry.org/Part:BBa_K398503 K398503], [http://partsregistry.org/Part:BBa_K398504 K398504] and [http://partsregistry.org/Part:BBa_K173000 K173000]. A general example map of the construction is shown below, where the RBS is displayed in fucsia. Notice the additional XbaI site located between the promoter and RBS sequence. This site is inherent to the Anderson RBS inserts on the distribution plates and was maintained in our constructs in case promoter swapping would be necessary in future.<br />
<br />
[[Image:RBS1.jpg|left|650px]]<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1" <br />
|'''Feature'''<br />
|'''Function'''<br />
|-<br />
|[http://partsregistry.org/Part:BBa_B0032 AmpR]<br />
|Ampicillin resistance<br />
|-<br />
|[http://partsregistry.org/Part:BBa_B0015 B0015]<br />
|Transcriptional (double) terminator<br />
|-<br />
|[http://partsregistry.org/Part:BBa_B0062 B0062]<br />
|Transcriptional terminator<br />
|-<br />
|[http://partsregistry.org/Part:BBa_E0040 E0040]<br />
|GFP<br />
|-<br />
|[http://partsregistry.org/Part:BBa_G00000 G00000]<br />
|Standard prefix<br />
|-<br />
|[http://partsregistry.org/Part:BBa_G00001 G00001]<br />
|Standard suffix<br />
|-<br />
|[http://partsregistry.org/Part:BBa_G00100 G00100]<br />
|VF2 primer binding site<br />
|-<br />
|[http://partsregistry.org/Part:BBa_G00101 G00101]<br />
|VR primer binding site<br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61100 J61100]<br />
|RBS Anderson family<br />
|-<br />
|[http://partsregistry.org/Part:BBa_J23100 J23100]<br />
|Promoter<br />
|}<br />
<br />
<html><div style='clear:both'><br />
</html><br />
<br />
== Parts ==<br />
The constructs used for measurements:<br />
<partinfo>K398500 SpecifiedComponents</partinfo><br />
<br />
<partinfo>K398501 SpecifiedComponents</partinfo><br />
<br />
<partinfo>K398502 SpecifiedComponents</partinfo><br />
<br />
<partinfo>K398503 SpecifiedComponents</partinfo><br />
<br />
<partinfo>K398504 SpecifiedComponents</partinfo><br />
<br />
<partinfo>K173000 SpecifiedComponents</partinfo><br />
<br />
<partinfo>I13401 SpecifiedComponents</partinfo><br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/parts" target="" /></map></html></div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/Project/rbs-characterization/results
Team:TU Delft/Project/rbs-characterization/results
2010-10-27T20:02:26Z
<p>Jcnossen: </p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><br />
<style><br />
#rbs-results table {<br />
margin: 0;<br />
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==The Results==<br />
In the plot below the RBS strengths for the five tested members of the Anderson family are given along with the deviations from the mean. The strengths are given relative to the community RBS BBa_B0034, which has a strength of unity (1). This relationship could be built by our characterization of the community RBS [http://partsregistry.org/Part:BBa_B0032 BBa_B0032] simultaneously, which is known to have a strength (or efficiency) of 0.3 relative to [http://partsregistry.org/Part:BBa_B0032 BBa_B0034]. Therefore in the plot below, the mean value of BBa_B0032 is set to 0.3. The values have been obtained by the experimental setup described [[Team:TU_Delft/Project/rbs-characterization/parts|here]] and using the [[Team:TU_Delft/Modeling/protein-production-model|model]] to correct for growth dilution.<br />
<br />
[[Image:TUDelft_2010_RBS_strength_graph.PNG]]<br />
<br />
<html><div id="rbs-results"></html><br />
<br />
==Conclusions==<br />
We've succeeded in characterizing five members of the Anderson ribosome binding site family by 96 well plate cultivation and simultaneous monitoring of GFP fluorescence and biomass absorbance. Fitting the obtained data to our improved [https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization protein production model] yielded RBS strengths values with negligible standard deviations. To recap the following RBS strengths (efficiencies) were found:<br />
<br />
<br />
<html><!-- Original data<br />
J61100 0.019970|0.005119<br />
J61101 0.119333|0.021401 <br />
J61107 0.077024|0.014804 <br />
J61117 0.012552|0.004484 <br />
J61127 0.065226|0.006604 <br />
B0032 0.300000|0.026904<br />
<br />
Rounded, in percentage<br />
J61100 0.020|0.00512<br />
J61101 0.119|0.02140 <br />
J61107 0.077|0.01480 <br />
J61117 0.013|0.00448 <br />
J61127 0.065|0.00660 <br />
B0032 0.300|0.02690<br />
<br />
--></html><br />
<br />
{|<br />
|<b>Ribosome binding site</b><br />
|<b>Mean relative strength</b><br />
|<b>Standard deviation</b><br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61100 J61100]<br />
|0.020 (2.0%)<br />
|0.00512<br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61101 J61101]<br />
|0.119 (11.9%)<br />
|0.02140 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61107 J61107]<br />
|0.077 (7.7%)<br />
|0.01480 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61117 J61117]<br />
|0.013 (1.3%)<br />
|0.00448 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61127 J61127]<br />
|0.065 (6.5%)<br />
|0.00660 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_B0032 B0032]<br />
|0.300 (Reference, 30%)<br />
|0.02690<br />
|}<br />
<html></div></html><br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/parts" target="" /></map></html></div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/Project/rbs-characterization/characterization
Team:TU Delft/Project/rbs-characterization/characterization
2010-10-27T19:47:25Z
<p>Jcnossen: /* Data normalization method */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
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==Characterization==<br />
<i>By varying only the RBS sequence in a protein generator construct the dependence of the protein expression level on the RBS sequence could be determined by simple fluorescence and biomass curve analysis. The methods are further explained below.</i><br />
<br />
=== What is RBS strength, really ? ===<br />
In the field of synthetic biology the 'strength' of an RBS is defined as the rate at which translation is initiated by the RBS sequence on any given mRNA molecule. Going back a step, we see that translation initiation occurs preferentially at certain mRNA sequences, which show a similarity to the consensus -Shine-Delgarno- sequence. This is due to the optimal binding of the 16S rRNA at these regions. In other words, the RBS 'strength' may loosely be defined as the rate of ribosome binding to any given mRNA molecule. (Click [http://partsregistry.org/Help:Ribosome_Binding_Sites/Mechanism here] for more information.)<br />
<br />
For our project it is not directly the binding equilibria of ribosome to mRNA that is of interest to us, but rather the netto rate of protein production. Thus, in our experimentation we define the RBS strength as the production rate of a given protein downstream of an RBS.<br />
<br />
==The experiment==<br />
The <i>E.coli strains</i> carrying the parts to be tested were cultured for 16 hours on 96-well plates while measuring fluorescence of GFP and biomass absorbance at 10 minute intervals. Click [https://2010.igem.org/Team:TU_Delft/Protocols#RBS_characterization_experiment here] for the more detailed protocol.<br />
<br />
<br />
=== Data normalization method ===<br />
To go from the raw measurements to the RBS strengths, we ran the following steps using a matlab script:<br />
<br />
[[Image:TUDelft2010_Rbs-control-gfp.png|right|thumb|300px]]<br />
the plate reader data consists of 8 rows, each containing multiples of 12:<br />
* 5 strains with Anderson RBSs to be characterized<br />
* B0032 strain to compare to<br />
* I13401 strain as control (GFP-dT without promotor and RBS)<br />
* LB+Amp as blank<br />
<br />
To prepare the data for further calculation, the following was done:<br />
* Subtract the blank GFP and OD values from the data of wells containing E coli. This takes care of any background noise caused by the LB medium.<br />
* The strain containing I13401 produces some nonzero measurements (See right). From this control strain measurements we can calculate how much autofluorescence is reported due to biomass or due to leaky expression.<br />
* For each well, we calculate the GFP that is not a result of biomass or leaky expression:<br />
[[Image:TUDelft_2010_PPM_Autofluorescence.png]]<br />
* These values are curve fitted into our [[Team:TU_Delft/Modeling/protein-production-model|protein production model]], described in the In Silico section. This results in a (exponential phase) protein production rate for each plate reader well. These production rates are then averaged and compared to the B0032 reference RBS to produce our [[Team:TU_Delft/Project/rbs-characterization/results|resulting RBS strengths]].<br />
<br />
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Jcnossen
http://2010.igem.org/Team:TU_Delft/Team/members
Team:TU Delft/Team/members
2010-10-27T16:48:41Z
<p>Jcnossen: </p>
<hr />
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<h2>Team Members</h2><br />
<br />
<p>Why so serious? When it comes to representing Synthetic Biology and iGEM we take things serious. Ofcourse we also had a lot of <a href="https://2010.igem.org/Team:TU_Delft#page=Team/gallery">fun</a>. If you would like to get to know us a bit better, come talk to us at the Jamboree or <a href="https://2010.igem.org/Team:TU_Delft#page=Contact">send us an email</a>!</p><br />
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<td class="name">Main Task:</td><td class="value">Organizational Manager</td><br />
</tr><br />
<tr><br />
<td class="name">Sub Project:</td><td class="value">Alkane Degradation</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Melting Temp:</td><td class="value">87 C</td><br />
</tr><br />
<tr><br />
<td class="name">iJAM:</td><td class="value">Producer</td><br />
</tr><br />
<br />
<tr><br />
<td class="story" colspan=2>"You should buy a Mac"</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<div class="member-container"><br />
<br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:Hugo_87"><img src="https://static.igem.org/mediawiki/2010/8/80/TU_Delft_teamphoto-hugo.jpg" width="120" height="159" /></a><br />
<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:Hugo_87">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:Hugo_87"><h3>Hugo Cueto Rojas (23)</h3></a><br />
<table><br />
<tr><br />
<br />
<td class="name">Education:</td><td class="value">Biochemical Engineering</td><br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">The Characterizor</td><br />
</tr><br />
<br />
<tr><br />
<td class="name">Sub Project:</td><td class="value">Salt & Solvent Tolerance</td><br />
</tr><br />
<tr><br />
<br />
<td class="name">Glucose:</td><td class="value">200 g/L</td><br />
</tr><br />
<tr><br />
<td class="name">iJAM:</td><td class="value">Bass Guitar</td><br />
</tr><br />
<tr><br />
<td class="story" colspan=2>"Fiat voluntas Dei" </td><br />
<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/Team:TU_Delft#page=User:ravandervalk"><img src="https://static.igem.org/mediawiki/2010/9/99/TU_Delft_teamphoto-ramon.jpg" width="120" height="159" /></a><br />
<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:ravandervalk">View Profile</a></p><br />
<br />
</div><br />
<div class="member-description"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:ravandervalk"><h3>Ramon van der Valk (23)</h3></a><br />
<table><br />
<tr><br />
<td class="name">Education:</td><td class="value">Molecular Genetics</td><br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Not-so-safety Manager</td><br />
</tr><br />
<tr><br />
<td class="name">Sub Project:</td><td class="value">Salt & Solvent Tolerance</td><br />
</tr><br />
<tr><br />
<td class="name">Accession #:</td><td class="value">PWN1337</td><br />
</tr><br />
<br />
<tr><br />
<td class="name">iJAM:</td><td class="value">Cheer leader</td><br />
</tr><br />
<tr><br />
<td class="story" colspan=2>"They made me do it! :'("</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/Team:TU_Delft#page=User:mvoges"><img src="https://static.igem.org/mediawiki/2010/8/82/TU_Delft_teamphoto-thias.jpg" width="120" height="159" /></a><br />
<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:mvoges">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:mvoges"><h3>Mathias Voges (22)</h3></a><br />
<table><br />
<tr><br />
<br />
<td class="name">Education:</td><td class="value">Cell Factory</td><br />
</tr><br />
<tr><br />
<td class="name">Main Task:</td><td class="value">Science Manager</td><br />
</tr><br />
<tr><br />
<td class="name">Sub Project:</td><td class="value">Alkane Degradation & RBSs</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Excitation:</td><td class="value">200 nm</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">iJAM:</td><td class="value">Lead Guitar</td><br />
</tr><br />
<tr><br />
<td class="story" colspan=2>"Do not disturb my circles!"</td><br />
</tr><br />
<br />
</table><br />
</div><br />
</div><br />
<br />
<div style="width: 100%; clear: both;"></div><br />
<h2>Instructors</h2><br />
<br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:Aljoscha"><img src="https://static.igem.org/mediawiki/2010/4/4a/TU_Delft_Wahl.jpg" width="120" height="120" /></a><br />
<br />
<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:Aljoscha">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:Aljoscha"><h3>Aljoscha Wahl</h3></a><br />
<table><br />
<tr><br />
<td class="name">Department:</td><td class="value">Bioprocessing Technology</td><br />
<br />
</tr><br />
<tr><br />
<td class="name">Gibbs Free E:</td><td class="value">-300 kJ</td><br />
</tr><br />
<tr><br />
<td class="story" colspan=2>"Nein man! ich will noch nicht gehn ich will noch ein bisschen Tanzen"</td><br />
</tr><br />
<br />
</table><br />
</div><br />
</div><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:Aleabate"><img src="https://static.igem.org/mediawiki/2010/4/41/TU_Delft_Abate.jpg" width="120" height="120" /></a><br />
<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:Aleabate">View Profile</a></p><br />
</div><br />
<br />
<div class="member-description"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:Aleabate"><h3>Alessandro Abate</h3></a><br />
<table><br />
<tr><br />
<td class="name">Department:</td><td class="value">Delft Center for Systems and Control</td><br />
</tr><br />
<tr><br />
<br />
<td class="name">10010011:</td><td class="value">FALSE</td><br />
</tr><br />
<tr><br />
<td class="story" colspan=2>" there is grandeur in this view of life (c. darwin) "</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/Team:TU_Delft#page=User:Esengul"><img src="https://static.igem.org/mediawiki/2010/7/78/TU_Delft_Yildirim.jpg" width="120" height="120" /></a><br />
<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:Esengul">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:Esengul"><h3>Esengul Yildirim</h3></a><br />
<table><br />
<br />
<tr><br />
<td class="name">Department:</td><td class="value">Enzymology</td><br />
</tr><br />
<tr><br />
<td class="name">Conductivity:</td><td class="value">45.2 million S/m</td><br />
</tr><br />
<tr><br />
<br />
<td class="story" colspan=2>"DNA is life, <br />the rest is just translation..."</td><br />
</tr><br />
</table><br />
</div><br />
</div><br />
<div style="width: 100%; clear: both;"></div><br />
<h2>Advisors</h2><br />
<div class="member-container"><br />
<div class="member-picture"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:stefandekok"><img src="https://static.igem.org/mediawiki/2010/b/b2/TU_Delft_Kok.jpg" width="120" height="120" /></a><br />
<p><a href="https://2010.igem.org/Team:TU_Delft#page=User:stefandekok">View Profile</a></p><br />
</div><br />
<br />
<div class="member-description"><br />
<a href="https://2010.igem.org/Team:TU_Delft#page=User:stefandekok"><h3>Stefan de Kok</h3></a><br />
<table><br />
<tr><br />
<td class="name">Department:</td><td class="value">Fermentation Technology</td><br />
</tr><br />
<tr><br />
<br />
<td class="name">Yield:</td><td class="value">15 cmol/cmol</td><br />
</tr><br />
<tr><br />
<td class="story" colspan=2>"I'm the yeasty boy, yeah!"</td><br />
</tr><br />
</table><br />
</div><br />
<br />
</div><br />
<!-- <div class="member-container"><br />
<div class="member-picture"><br />
<a href=""><img src="" width="120" height="120" /></a><br />
<p><a href="">View Profile</a></p><br />
</div><br />
<div class="member-description"><br />
<h3>Domenico Bellomo</h3><br />
<br />
<table><br />
<tr><br />
<td class="name">iGEM Medals:</td><td class="value">2 x gold</td><br />
</tr><br />
<tr><br />
<td class="story" colspan=2>"bla bla bla"</td><br />
</tr><br />
<br />
</table><br />
</div><br />
</div> /--><br />
</div><br />
<!-- Team Overview END /--><br />
</html></div>
Jcnossen
http://2010.igem.org/Team:TU_Delft
Team:TU Delft
2010-10-27T16:40:35Z
<p>Jcnossen: </p>
<hr />
<div>__NOTOC__<br />
<html><br />
<!--<br />
<br />
============================================================================================<br />
*** PLEASE NOTE: ***<br />
<br />
When copying our code, please be so kind to mention so on your wiki.<br />
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You can use our logo https://static.igem.org/mediawiki/2010/7/7b/TU_Delft.jpg to link to our wiki.<br />
<br />
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Pieter van Boheemen, Jelmer Cnossen<br />
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<li><a href="https://2010.igem.org/Team:TU_Delft#page=Collaboration/Rathenau Institute" title="Rathenau Institute">Rathenau Institute</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Collaboration/Science Centre Delft" title="Science Centre Delft">Science Centre Delft</a></li><br />
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<li><a href="https://2010.igem.org/Team:TU_Delft#page=Collaboration/acknowledgements" title="Acknowledgements">Acknowledgements</a></li><br />
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Jcnossen
http://2010.igem.org/Team:TU_Delft/Modeling/protein-production-model
Team:TU Delft/Modeling/protein-production-model
2010-10-27T13:13:46Z
<p>Jcnossen: /* Protein production model */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<br />
==Protein production model==<br />
<br />
This model is an extension of the [https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization RBS-characterization sub-project]<br />
<br />
===Abstract===<br />
Characterizations of RBS sequences (and often promotors as well) require knowing the protein production rate during the exponential growth phase. However, plate reader measurements result in a time series of GFP measurements, reflecting protein concentrations. To convert from protein concentrations to protein production rates we propose fitting the measurement data to a simple model that incorporates protein production, dilution and degradation.<br />
<br />
===Previous models===<br />
We've noticed that [https://2009.igem.org/Team:Groningen/Promoters previous teams] have performed their protein expression rate analysis to only include current protein level (fluorescence measurements) and current biomass concentration (absorption measurements). However, we've found that during measurements, the protein concentration is also influenced by other factors:<br />
* The rapid growth of the bacteria dilutes the protein considerably<br />
* Proteins degrade over time.<br />
<br />
See below for more on how we approached these issues.<br />
<br />
===Growth curve fitting===<br />
From the biomass, or optical density (OD), graph below, it could be concluded that the fastest growth occurs from 1 hour to 3 hours into the experiment. Within this timespan, it is assumed that growth is exponential, and growth rate can be calculated. The graph below shows 72 growth curves. All in all 12 wells were inoculated per strain carrying a different RBS sequence or 'empty' plasmid (I13401).<br />
<br />
[[Image:Tud2010_RBS_OD.png]]<br />
<br />
The exponential section of this graph was thus curve-fitted to an exponential growth model:<br />
<br />
[[Image:TUD2010_RBS_Growth_model.PNG]]<br />
<br />
Which resulted in a growth rate ('mu') which was implemented in the dilution factor in the further calculations.<br />
<br />
===Biomass growth will dilute GFP===<br />
[[Image:TUD2010_Gfpod_all.png|right|400px]]<br />
The conventional method for characterization of RBS or promotor sequences is to assume that the derivative of the GFP fluorescence is equal to the production rate, as shown in the formula from Kelly et al [1].<br />
[[Image:TUDelft2010_RBS_1754-1611-3-4-i9.gif]]<br />
<br />
However, this method would only be representable if the strains are subcultured multiple times from the exponential phase. In that case, a steady state GFP concentration (fluorescence) may be assumed and dilution is ignored.<br />
Due to the decrease in the biomass specific GFP fluorescence at the start of the measurements it was assumed that dilution effects played an important role in our data and thus was taken into account for our model.<br />
<br />
<br style="clear: both" /><br />
<br />
=== Protein production model ===<br />
As discussed earlier, the protein production model will take dilution into account. Furthermore, the GFP protein used used is stable (half life 41 hours), thus the degradation term is negligible compared to dilution. These assumptions call forth the following formulas:<br />
<br />
[[Image:RBS_Expression_model.PNG]]<br />
<br />
where:<br />
* Y is the GFP concentration<br />
* Beta is the production rate (the constant that we're interested in) <br />
* Alpha is the sum of the dilution rate and the degradation rate<br />
<br />
Converting this equation into an explicit form results in:<br />
<br />
[[Image:TU2010_Gfp_explicit.PNG]]<br />
<br />
The 2 unknown parameters (beta and Y1) could now be curve-fitted against the biomass specific GFP data, using the lsqcurvefit command found in MatLab. The beta constants for each of the 72 wells were then determined and compared to the reference RBS BBa-B0032 to yield the relative RBS strengths listed in the [[Team:TU_Delft/Project/rbs-characterization/results|results]] page.<br />
<br />
=== Source code and data ===<br />
https://static.igem.org/mediawiki/2010/8/88/TUDelft_2010_RBS_characterization.zip<br />
<br />
=== References ===<br />
[1] Jason R. Kelly, Adam J. Rubin, Joseph H. Davis, ''et al'' (March 2009). "[http://dx.doi.org/10.1186/1754-1611-3-4 Measuring the activity of BioBrick promoters using an in vivo reference standard]". ''Journal of Biological Engineering'' '''3''': 4</div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/Collaboration/acknowledgements
Team:TU Delft/Collaboration/acknowledgements
2010-10-27T12:31:30Z
<p>Jcnossen: /* Familiy and Friends */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
==Acknowledgements==<br />
<br />
We would like to thank:<br />
<br />
===Our instructors===<br />
For the introduction in the lab and the procedures. We are also very gratefull for the efforts to arrange all our needs. <br />
<br />
*Ing. Esengül Yildirim<br />
<br />
For modelling advice and constructive comments on our progress. <br />
<br />
*Dr. Aljoscha Wahl<br />
*Dr. Alessandro Abate<br />
<br />
Of course, we want to thank all of you for the great times we had during our iGEM project!<br />
<br />
===Enzymology Department===<br />
<br />
For hosting our team by providing lab space.<br />
<br />
*Prof. dr. Wilfred Hagen<br />
*Prof. dr. Simon de Vries<br />
*Ing. Laura Koekoek<br />
*Ing. Marc Strampraad<br />
<br />
===Department of Biotechnology===<br />
<br />
All the people that made it possible to do this project by providing permits.<br />
<br />
*Prof. dr. Han de Winde<br />
*Dr. Lesley Robertson <br />
*Dr. Simon de Goede<br />
*Jos Lispet<br />
<br />
For sacrificing her birthday to check our wiki for spelling errors<br />
<br />
*Angela Paulus <br />
<br />
The technical workshop for providing beamer<br />
<br />
*Arno van den Berg<br />
<br />
For arranging all our equipment in our office<br />
<br />
*Sjaak Lispet<br />
<br />
Arranging extra computers for our office<br />
<br />
*Hans Kemper<br />
<br />
===Advisors===<br />
<br />
The assistance of all advisors is very much appreciated for all tips and help to improve our protocols and experiments. <br />
<br />
*Dr. Domenico Bellomo <br />
*Ir. Amit Deshmukh <br />
*Dr. Emrah Nikerel <br />
*Ir. Stefan de Kok<br />
*Drs. Janine Kiers<br />
<br />
===The Chemical Engineering Department===<br />
<br />
For the use of the Gas Chromotograph<br />
<br />
*Maarten Gorseling <br />
*Remco van Oosten<br />
<br />
===The people involved in our ethical research===<br />
<br />
For reading and evaluating the ethical literature study and also made possible for us to participate the documentary 'Who owns you'<br />
<br />
*Dr. David Koepsell <br />
*Taylor Roesch<br />
<br />
===Communication Department Delft University of Technology===<br />
<br />
For giving us tips dealing with press<br />
<br />
*Michel van Baal <br />
*Nathalie Hugoosgift<br />
<br />
===Nanopodium===<br />
<br />
Giving us the opportunity to making a short movie about our project.<br />
<br />
*Lidy Knol<br />
*Astrid Ventevogel<br />
<br />
===Science Centre and Tinker===<br />
<br />
We are very gratefull for giving us the freedom to make education movies for the Science Museum to our own insight. We are also obliged for the opporunity to collaborate with the decorating the Biotech room. The sponsoring for the development of the serious game was very much appreciated. <br />
<br />
*Michael van der Meer <br />
*Roel Bolhuis <br />
*Stan Boshouwers<br />
*Timo van der Horst<br />
*Rutger Schuit<br />
<br />
===Game developers===<br />
<br />
Supervising the game developers<br />
<br />
*Dr. Rafael Bidarra <br />
<br />
Programming of the game<br />
<br />
*Sverre Rabbelier<br />
*Michel de Ridder<br />
*Thomas Rens <br />
*Martijn Rentmeester <br />
<br />
Making the graphics of the game<br />
*Marin Licina<br />
<br />
===Rathenau institute===<br />
<br />
For the nice collaborating with the workshops<br />
<br />
*Dr. Dirk Stemerding<br />
<br />
===WT & Academie===<br />
<br />
For giving us the opportunaty to give our workshop to teachers<br />
<br />
*Eveline Holla<br />
*Maaike Gorter<br />
<br />
===Wetenschapsdag===<br />
<br />
For giving us the opportunaty to give our workshop<br />
<br />
*Erik van Hellemond<br />
*Liesbeth van Hees<br />
<br />
===For the professional photoshoot===<br />
*Cora van Nieuwkerk <br />
<br />
===For converting our logo to vector images===<br />
*Stan Brinkman<br />
<br />
===Previous iGEM members===<br />
<br />
For sharing their iGEM experiences and giving tips for our presentations<br />
<br />
*Calin Plesa<br />
*Daniel Solis <br />
*Tim Vos<br />
*Tim Weenink<br />
<br />
===The people that attended the brainstorming sessions and BT work discussion at the start of the project===<br />
<br />
===Special thanks to everyone participating in our activities on issues in synthetic biology===<br />
*The people we interviewed during Night of the Nerds<br />
*Prof.ir. K.C.A.M. Luyben<br />
*Prof.dr. P. Osseweijer<br />
<br />
===Family and Friends===<br />
For not letting us down even when we neglected them during our iGEM experience.<br />
<br />
===Fans on our social media===<br />
For all the good support</div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/Parts/characterization
Team:TU Delft/Parts/characterization
2010-10-27T12:28:21Z
<p>Jcnossen: /* Ribosome Binding Site (RBS) sequence characterization */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
==Characterization==<br />
<br />
Our favorite parts were also the ones best characterized. This page is a quick overview of the results. You can find a [[Team:TU_Delft/Project|complete overview of characterization]] on the project pages.<br />
<br />
===Favorite Parts===<br />
====[http://partsregistry.org/Part:BBa_K398029 BBa_K398029] - ALDH====<br />
<br />
[[Image:TUDelftALDH_final.jpg|600px]]<br />
<br />
Our results suggest that the recombinant strain ''E. coli'' 029A functionally express our biobrick [http://partsregistry.org/Part:BBa_K398029 BBa_K398029]. From the statistical analysis that we performed we concluded that the expression of the biobrick [http://partsregistry.org/Part:BBa_K398006 BBa_K398006] under the promoter-rbs combination [http://partsregistry.org/Part:BBa_J13002 BBa_J23100]-[http://partsregistry.org/Part:BBa_J13002 BBa_J61117] increases the dodecanal dehydrogenase activity in ''E. coli'' cell extracts 2-fold. Moreover, the enzymatic activities measured for the construct [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] were equivalent to 33.98% of the ''Pseudomonas putida'' aldehyde dehydrogenase activity. <br />
Read more about it in [https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results our results page]<br />
<br />
View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398331 BBa_K398029 in the '''parts registry''']<br />
<br />
'''Specified Components'''<br />
<partinfo>BBa_K398029 SpecifiedComponents</partinfo><br />
<br />
'''Designer:''' <partinfo>BBa_K398029 Designer</partinfo><br />
<br />
'''Status:''' <partinfo>BBa_K398029 Status</partinfo><br />
<br />
====[http://partsregistry.org/Part:BBa_K398331 BBa_K398331] - pCaif====<br />
<br />
According to our results, the smallest part that we designed it's working!!!<br />
<br />
pCaiF a natural promoter in E. coli that is active at high cAMP levels was characterized attaching E00240 in front of our part. Different GFP production rates were found at the stationary phase showing that GFP production depends on the amount of glucose in the medium. Moreover, growth on a secondary carbon source decreases the GFP production rate.<br />
<br />
[[Image:TU_Delft_pCaiF_final.jpg]]<br />
<br />
Read more about our fascinating results on our [https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results sensing project page]<br />
<br />
View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398331 BBa_K398331 in the '''parts registry''']<br />
<br />
'''Specified Components'''<br />
<partinfo>K398331 SpecifiedComponents</partinfo><br />
<br />
'''Designer:''' <partinfo>K398331 Designer</partinfo><br />
<br />
'''Status:''' <partinfo>K398331 Status</partinfo><br />
<br />
====[http://partsregistry.org/wiki/index.php?title=Part:BBa_K398406 BBa_K398406] - Solvent Tolerance====<br />
The solvent tolerance cluster (BBa_K398406) was expressed on in ''E. coli'' K12. The growth of the cells was tested under different amounts of n-hexane. The results show that this part gives to the cells an growth advantage at higher n-hexane concentrations. The parental strain E. coli K12 grows really slow at 10% (v/v) of n-hexane/M9 mixture.<br />
<br />
[[Image:TU_Delft_Solvent_tolerance.jpg|400px]]<br />
<br />
Read more about these results on our [https://2010.igem.org/Team:TU_Delft#page=Project/tolerance/results survival project page].<br />
<br />
View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398406 BBa_K398406 in the '''parts registry''']<br />
<br />
'''Specified Components'''<br />
<partinfo>K398406 SpecifiedComponents</partinfo><br />
<br />
'''Designer:''' <partinfo>K398406 Designer</partinfo><br />
<br />
'''Status:''' <partinfo>K398406 Status</partinfo><br />
<br />
====[http://partsregistry.org/wiki/index.php?title=Part:BBa_K398108 BBa_K398108] - Salt Tolerance====<br />
The growth of the bbc1 construct (BBa_K398108) was tested as stated in on [[Team:TU_Delft/Project/tolerance/characterization|the salt tolerance characterization page]].<br />
<br />
[[Image:TU_Delft_Salt_tolerance.jpg|500px]]<br />
<br />
Read more about these results on our [https://2010.igem.org/Team:TU_Delft#page=Project/tolerance/results survival project page].<br />
<br />
View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398108 BBa_K398108 in the '''parts registry''']<br />
<br />
'''Specified Components'''<br />
<partinfo>K398108 SpecifiedComponents</partinfo><br />
<br />
'''Designer:''' <partinfo>K398108 Designer</partinfo><br />
<br />
'''Status:''' <partinfo>K398108 Status</partinfo><br />
<br />
====BBa_K398206 - Emulsifier====<br />
[[Image:TU_Delft_Emuls_assay_exp1.jpg|thumb|400px|right|Emulsification of Sudan II by the isolated proteins from the control (BBa_J13002) and AlnA (BBa_K398206) culture. Read more about the [[Team:TU_Delft/Project/solubility/results|BBa_K398206 characteristics]]]]This part contains an IPTG inducible protomor with a protein coding sequence for the production of the emulsifier AlnA. It was the main subject of our [[Team:TU_Delft/Project/solubility|solubility]] subproject.<br />
<br />
View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398206 BBa_K398206 in the '''parts registry''']<br />
<br />
'''Specified Components'''<br />
<partinfo>K398206 SpecifiedComponents</partinfo><br />
<br />
'''Designer:''' <partinfo>K398206 Designer</partinfo><br />
<br />
'''Status:''' <partinfo>K398206 Status</partinfo><br />
<html><span style="clear:both;display:block;width:100%;"></span></html><br />
<br />
====Ribosome Binding Site (RBS) sequence characterization====<br />
Read more about the [[Team:TU_Delft/Project/rbs-characterization|RBS characterization]]. We used the following 5 constructs to perform the characterization.<br />
<partinfo>BBa_K398500 SpecifiedComponents</partinfo><br />
<partinfo>BBa_K398501 SpecifiedComponents</partinfo><br />
<partinfo>BBa_K398502 SpecifiedComponents</partinfo><br />
<partinfo>BBa_K398503 SpecifiedComponents</partinfo><br />
<partinfo>BBa_K398504 SpecifiedComponents</partinfo><br />
<br />
[[Image:TUDelft_2010_RBS_strength_graph.PNG|thumb|400px|right|Characterization of 5 RBS sequences from the Anderson series]]<br />
Resulting RBS strenghts:<br />
{|width="300"<br />
|<b>Ribosome binding site</b><br />
|<b>Mean relative strength</b><br />
|<b>Standard deviation</b><br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61100 J61100]<br />
|0.020 (2.0%)<br />
|0.00512<br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61101 J61101]<br />
|0.119 (11.9%)<br />
|0.02140 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61107 J61107]<br />
|0.077 (7.7%)<br />
|0.01480 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61117 J61117]<br />
|0.013 (1.3%)<br />
|0.00448 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61127 J61127]<br />
|0.065 (6.5%)<br />
|0.00660 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_B0032 B0032]<br />
|0.300 (Reference, 30%)<br />
|0.02690<br />
|}<br />
<html><br style:"clear: both;"/></html></div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/Parts/characterization
Team:TU Delft/Parts/characterization
2010-10-27T12:26:49Z
<p>Jcnossen: /* Ribosome Binding Site (RBS) sequence characterization */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
==Characterization==<br />
<br />
Our favorite parts were also the ones best characterized. This page is a quick overview of the results. You can find a [[Team:TU_Delft/Project|complete overview of characterization]] on the project pages.<br />
<br />
===Favorite Parts===<br />
====[http://partsregistry.org/Part:BBa_K398029 BBa_K398029] - ALDH====<br />
<br />
[[Image:TUDelftALDH_final.jpg|600px]]<br />
<br />
Our results suggest that the recombinant strain ''E. coli'' 029A functionally express our biobrick [http://partsregistry.org/Part:BBa_K398029 BBa_K398029]. From the statistical analysis that we performed we concluded that the expression of the biobrick [http://partsregistry.org/Part:BBa_K398006 BBa_K398006] under the promoter-rbs combination [http://partsregistry.org/Part:BBa_J13002 BBa_J23100]-[http://partsregistry.org/Part:BBa_J13002 BBa_J61117] increases the dodecanal dehydrogenase activity in ''E. coli'' cell extracts 2-fold. Moreover, the enzymatic activities measured for the construct [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] were equivalent to 33.98% of the ''Pseudomonas putida'' aldehyde dehydrogenase activity. <br />
Read more about it in [https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results our results page]<br />
<br />
View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398331 BBa_K398029 in the '''parts registry''']<br />
<br />
'''Specified Components'''<br />
<partinfo>BBa_K398029 SpecifiedComponents</partinfo><br />
<br />
'''Designer:''' <partinfo>BBa_K398029 Designer</partinfo><br />
<br />
'''Status:''' <partinfo>BBa_K398029 Status</partinfo><br />
<br />
====[http://partsregistry.org/Part:BBa_K398331 BBa_K398331] - pCaif====<br />
<br />
According to our results, the smallest part that we designed it's working!!!<br />
<br />
pCaiF a natural promoter in E. coli that is active at high cAMP levels was characterized attaching E00240 in front of our part. Different GFP production rates were found at the stationary phase showing that GFP production depends on the amount of glucose in the medium. Moreover, growth on a secondary carbon source decreases the GFP production rate.<br />
<br />
[[Image:TU_Delft_pCaiF_final.jpg]]<br />
<br />
Read more about our fascinating results on our [https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results sensing project page]<br />
<br />
View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398331 BBa_K398331 in the '''parts registry''']<br />
<br />
'''Specified Components'''<br />
<partinfo>K398331 SpecifiedComponents</partinfo><br />
<br />
'''Designer:''' <partinfo>K398331 Designer</partinfo><br />
<br />
'''Status:''' <partinfo>K398331 Status</partinfo><br />
<br />
====[http://partsregistry.org/wiki/index.php?title=Part:BBa_K398406 BBa_K398406] - Solvent Tolerance====<br />
The solvent tolerance cluster (BBa_K398406) was expressed on in ''E. coli'' K12. The growth of the cells was tested under different amounts of n-hexane. The results show that this part gives to the cells an growth advantage at higher n-hexane concentrations. The parental strain E. coli K12 grows really slow at 10% (v/v) of n-hexane/M9 mixture.<br />
<br />
[[Image:TU_Delft_Solvent_tolerance.jpg|400px]]<br />
<br />
Read more about these results on our [https://2010.igem.org/Team:TU_Delft#page=Project/tolerance/results survival project page].<br />
<br />
View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398406 BBa_K398406 in the '''parts registry''']<br />
<br />
'''Specified Components'''<br />
<partinfo>K398406 SpecifiedComponents</partinfo><br />
<br />
'''Designer:''' <partinfo>K398406 Designer</partinfo><br />
<br />
'''Status:''' <partinfo>K398406 Status</partinfo><br />
<br />
====[http://partsregistry.org/wiki/index.php?title=Part:BBa_K398108 BBa_K398108] - Salt Tolerance====<br />
The growth of the bbc1 construct (BBa_K398108) was tested as stated in on [[Team:TU_Delft/Project/tolerance/characterization|the salt tolerance characterization page]].<br />
<br />
[[Image:TU_Delft_Salt_tolerance.jpg|500px]]<br />
<br />
Read more about these results on our [https://2010.igem.org/Team:TU_Delft#page=Project/tolerance/results survival project page].<br />
<br />
View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398108 BBa_K398108 in the '''parts registry''']<br />
<br />
'''Specified Components'''<br />
<partinfo>K398108 SpecifiedComponents</partinfo><br />
<br />
'''Designer:''' <partinfo>K398108 Designer</partinfo><br />
<br />
'''Status:''' <partinfo>K398108 Status</partinfo><br />
<br />
====BBa_K398206 - Emulsifier====<br />
[[Image:TU_Delft_Emuls_assay_exp1.jpg|thumb|400px|right|Emulsification of Sudan II by the isolated proteins from the control (BBa_J13002) and AlnA (BBa_K398206) culture. Read more about the [[Team:TU_Delft/Project/solubility/results|BBa_K398206 characteristics]]]]This part contains an IPTG inducible protomor with a protein coding sequence for the production of the emulsifier AlnA. It was the main subject of our [[Team:TU_Delft/Project/solubility|solubility]] subproject.<br />
<br />
View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398206 BBa_K398206 in the '''parts registry''']<br />
<br />
'''Specified Components'''<br />
<partinfo>K398206 SpecifiedComponents</partinfo><br />
<br />
'''Designer:''' <partinfo>K398206 Designer</partinfo><br />
<br />
'''Status:''' <partinfo>K398206 Status</partinfo><br />
<html><span style="clear:both;display:block;width:100%;"></span></html><br />
<br />
====Ribosome Binding Site (RBS) sequence characterization====<br />
Read more about the [[Team:TU_Delft/Project/rbs-characterization|RBS characterization]]. We used the following 5 constructs to perform the characterization.<br />
<partinfo>BBa_K398500 SpecifiedComponents</partinfo><br />
<partinfo>BBa_K398501 SpecifiedComponents</partinfo><br />
<partinfo>BBa_K398502 SpecifiedComponents</partinfo><br />
<partinfo>BBa_K398503 SpecifiedComponents</partinfo><br />
<partinfo>BBa_K398504 SpecifiedComponents</partinfo><br />
<br />
Resulting RBS strenghts:<br />
<br />
[[Image:TUDelft_2010_RBS_strength_graph.PNG|thumb|400px|right|Characterization of 5 RBS sequences from the Anderson series]]<br />
{|width="300"<br />
|<b>Ribosome binding site</b><br />
|<b>Mean relative strength</b><br />
|<b>Standard deviation</b><br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61100 J61100]<br />
|0.020 (2.0%)<br />
|0.00512<br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61101 J61101]<br />
|0.119 (11.9%)<br />
|0.02140 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61107 J61107]<br />
|0.077 (7.7%)<br />
|0.01480 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61117 J61117]<br />
|0.013 (1.3%)<br />
|0.00448 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61127 J61127]<br />
|0.065 (6.5%)<br />
|0.00660 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_B0032 B0032]<br />
|0.300 (Reference, 30%)<br />
|0.02690<br />
|}</div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/Parts/characterization
Team:TU Delft/Parts/characterization
2010-10-27T12:25:11Z
<p>Jcnossen: </p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
==Characterization==<br />
<br />
Our favorite parts were also the ones best characterized. This page is a quick overview of the results. You can find a [[Team:TU_Delft/Project|complete overview of characterization]] on the project pages.<br />
<br />
===Favorite Parts===<br />
====[http://partsregistry.org/Part:BBa_K398029 BBa_K398029] - ALDH====<br />
<br />
[[Image:TUDelftALDH_final.jpg|600px]]<br />
<br />
Our results suggest that the recombinant strain ''E. coli'' 029A functionally express our biobrick [http://partsregistry.org/Part:BBa_K398029 BBa_K398029]. From the statistical analysis that we performed we concluded that the expression of the biobrick [http://partsregistry.org/Part:BBa_K398006 BBa_K398006] under the promoter-rbs combination [http://partsregistry.org/Part:BBa_J13002 BBa_J23100]-[http://partsregistry.org/Part:BBa_J13002 BBa_J61117] increases the dodecanal dehydrogenase activity in ''E. coli'' cell extracts 2-fold. Moreover, the enzymatic activities measured for the construct [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] were equivalent to 33.98% of the ''Pseudomonas putida'' aldehyde dehydrogenase activity. <br />
Read more about it in [https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results our results page]<br />
<br />
View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398331 BBa_K398029 in the '''parts registry''']<br />
<br />
'''Specified Components'''<br />
<partinfo>BBa_K398029 SpecifiedComponents</partinfo><br />
<br />
'''Designer:''' <partinfo>BBa_K398029 Designer</partinfo><br />
<br />
'''Status:''' <partinfo>BBa_K398029 Status</partinfo><br />
<br />
====[http://partsregistry.org/Part:BBa_K398331 BBa_K398331] - pCaif====<br />
<br />
According to our results, the smallest part that we designed it's working!!!<br />
<br />
pCaiF a natural promoter in E. coli that is active at high cAMP levels was characterized attaching E00240 in front of our part. Different GFP production rates were found at the stationary phase showing that GFP production depends on the amount of glucose in the medium. Moreover, growth on a secondary carbon source decreases the GFP production rate.<br />
<br />
[[Image:TU_Delft_pCaiF_final.jpg]]<br />
<br />
Read more about our fascinating results on our [https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results sensing project page]<br />
<br />
View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398331 BBa_K398331 in the '''parts registry''']<br />
<br />
'''Specified Components'''<br />
<partinfo>K398331 SpecifiedComponents</partinfo><br />
<br />
'''Designer:''' <partinfo>K398331 Designer</partinfo><br />
<br />
'''Status:''' <partinfo>K398331 Status</partinfo><br />
<br />
====[http://partsregistry.org/wiki/index.php?title=Part:BBa_K398406 BBa_K398406] - Solvent Tolerance====<br />
The solvent tolerance cluster (BBa_K398406) was expressed on in ''E. coli'' K12. The growth of the cells was tested under different amounts of n-hexane. The results show that this part gives to the cells an growth advantage at higher n-hexane concentrations. The parental strain E. coli K12 grows really slow at 10% (v/v) of n-hexane/M9 mixture.<br />
<br />
[[Image:TU_Delft_Solvent_tolerance.jpg|400px]]<br />
<br />
Read more about these results on our [https://2010.igem.org/Team:TU_Delft#page=Project/tolerance/results survival project page].<br />
<br />
View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398406 BBa_K398406 in the '''parts registry''']<br />
<br />
'''Specified Components'''<br />
<partinfo>K398406 SpecifiedComponents</partinfo><br />
<br />
'''Designer:''' <partinfo>K398406 Designer</partinfo><br />
<br />
'''Status:''' <partinfo>K398406 Status</partinfo><br />
<br />
====[http://partsregistry.org/wiki/index.php?title=Part:BBa_K398108 BBa_K398108] - Salt Tolerance====<br />
The growth of the bbc1 construct (BBa_K398108) was tested as stated in on [[Team:TU_Delft/Project/tolerance/characterization|the salt tolerance characterization page]].<br />
<br />
[[Image:TU_Delft_Salt_tolerance.jpg|500px]]<br />
<br />
Read more about these results on our [https://2010.igem.org/Team:TU_Delft#page=Project/tolerance/results survival project page].<br />
<br />
View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398108 BBa_K398108 in the '''parts registry''']<br />
<br />
'''Specified Components'''<br />
<partinfo>K398108 SpecifiedComponents</partinfo><br />
<br />
'''Designer:''' <partinfo>K398108 Designer</partinfo><br />
<br />
'''Status:''' <partinfo>K398108 Status</partinfo><br />
<br />
====BBa_K398206 - Emulsifier====<br />
[[Image:TU_Delft_Emuls_assay_exp1.jpg|thumb|400px|right|Emulsification of Sudan II by the isolated proteins from the control (BBa_J13002) and AlnA (BBa_K398206) culture. Read more about the [[Team:TU_Delft/Project/solubility/results|BBa_K398206 characteristics]]]]This part contains an IPTG inducible protomor with a protein coding sequence for the production of the emulsifier AlnA. It was the main subject of our [[Team:TU_Delft/Project/solubility|solubility]] subproject.<br />
<br />
View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398206 BBa_K398206 in the '''parts registry''']<br />
<br />
'''Specified Components'''<br />
<partinfo>K398206 SpecifiedComponents</partinfo><br />
<br />
'''Designer:''' <partinfo>K398206 Designer</partinfo><br />
<br />
'''Status:''' <partinfo>K398206 Status</partinfo><br />
<html><span style="clear:both;display:block;width:100%;"></span></html><br />
<br />
====Ribosome Binding Site (RBS) sequence characterization====<br />
[[Image:TUDelft_2010_RBS_strength_graph.PNG|thumb|400px|right|Characterization of 5 RBS sequences from the Anderson series]]<br />
Read more about the [[Team:TU_Delft/Project/rbs-characterization|RBS characterization]]. We used the following 5 constructs to perform the characterization.<br />
<partinfo>BBa_K398500 SpecifiedComponents</partinfo><br />
<partinfo>BBa_K398501 SpecifiedComponents</partinfo><br />
<partinfo>BBa_K398502 SpecifiedComponents</partinfo><br />
<partinfo>BBa_K398503 SpecifiedComponents</partinfo><br />
<partinfo>BBa_K398504 SpecifiedComponents</partinfo><br />
<br />
Resulting RBS strenghts:<br />
<br />
{|width="300"<br />
|<b>Ribosome binding site</b><br />
|<b>Mean relative strength</b><br />
|<b>Standard deviation</b><br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61100 J61100]<br />
|0.020 (2.0%)<br />
|0.00512<br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61101 J61101]<br />
|0.119 (11.9%)<br />
|0.02140 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61107 J61107]<br />
|0.077 (7.7%)<br />
|0.01480 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61117 J61117]<br />
|0.013 (1.3%)<br />
|0.00448 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61127 J61127]<br />
|0.065 (6.5%)<br />
|0.00660 <br />
|-<br />
|[http://partsregistry.org/Part:BBa_B0032 B0032]<br />
|0.300 (Reference, 30%)<br />
|0.02690<br />
|}</div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/Project/achievements
Team:TU Delft/Project/achievements
2010-10-27T11:46:05Z
<p>Jcnossen: /* Highlights & Achievements */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
==Highlights & Achievements==<br />
<br />
On this page we would like to elaborate our judgment form a bit more. Of course you can also read our [https://igem.org/Judging_Form.cgi?id=398 official judgement form].<br />
<br />
What did we do? We...<br />
<br />
* Registered our [[Team:TU_Delft/Team/members|team]], had [[Team:TU_Delft/Team/gallery|a great summer]], and plan to have a lot of fun at the Jamboree!<br />
* Came up with our project: [[Team:TU_Delft/Project|The Alkanivore]].<br />
* Build 47 new [[Team:TU_Delft/Parts/biobricks|BioBricks]].<br />
* [[Team:TU_Delft/Parts/characterization|Characterized]] our favourite parts: [http://partsregistry.org/Part:BBa_K398326 BBa_K398326], [http://partsregistry.org/Part:BBa_K398108 BBa_K398108], [http://partsregistry.org/Part:BBa_K398406 BBa_K398406], [http://partsregistry.org/Part:BBa_K398029 BBa_K398029], [http://partsregistry.org/Part:BBa_K398331 BBa_K398331], [http://partsregistry.org/Part:BBa_K398206 BBa_K398206]<br />
* Characterized the [[Team:TU_Delft/Project/rbs-characterization| existing BioBrick Parts]]: [http://partsregistry.org/Part:BBa_J61100 BBa_J61100], [http://partsregistry.org/Part:BBa_J61101 BBa_J61101], [http://partsregistry.org/Part:BBa_J61107 BBa_J61107], [http://partsregistry.org/Part:BBa_J61117 BBa_J61117], [http://partsregistry.org/Part:BBa_J61127 BBa_J61127]<br />
* Passed all the [[Team:TU_Delft/Safety/in-the-lab|biosafety tests]] to work in our VMT lab.<br />
* Simulated the [[Team:TU_Delft/Modeling/HC_regulation|hydrocarbon regulation expression mechanism]].<br />
* Realized an interactive way to show our [[Team:TU_Delft/Modeling/MFA|metabolic flux analysis]].<br />
* Developed a [[Team:TU_Delft/Modeling/interaction-mapping|software tool]] to find putative interactions for proteins in a new host organism.<br />
* Developed a [[Team:TU_Delft/Software/part-search|webservice]] to provide a part query interface to wiki javascript.<br />
* Came up with a new approach to [[Team:TU_Delft/Human-practice|human practice]] - The [[Team:TU_Delft/EPAC|EPAC pyramid]].<br />
* Addressed the impacts of the ongoing advances in synthetic biology and the [[Team:TU_Delft/Safety/ethics|ethical questions]] concerning these impacts.<br />
* Discussed the ongoing debate about [[Team:TU_Delft/Safety/society/who-owns-you|gene patenting]] and open source.<br />
* Investigated the current [[Team:TU_Delft/Safety/survey|perception]] of synthetic biology in general public.<br />
* Made general public [[Team:TU_Delft/Publicity/in-the-news|aware]] of our project.<br />
* Became museum pieces in the [[Team:TU_Delft/Education/science-museum|Science Centre]] in Delft.<br />
* Developed an fun and educational [[Team:TU_Delft/Education/game|iGEM computer game]].<br />
*[[Team:TU_Delft/Collaboration/igem-teams|Helped other iGEM teams]] by creating their [[Team:TU_Delft/Modeling/wiki-tips-tricks|wiki's]].<br />
* Have the [https://2010.igem.org/Special:PopularPages most visited] wiki after [https://2010.igem.org/Main_Page igem main page].<br />
* Made a [[Team:TU_Delft/Tour|tour]] to quickly go through all our highlights and achievements!</div>
Jcnossen
http://2010.igem.org/Team:TU_Delft
Team:TU Delft
2010-10-27T11:40:22Z
<p>Jcnossen: </p>
<hr />
<div>__NOTOC__<br />
<html><br />
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var pos = $('#iGEM_TU_Delft_container > a[name='+anchor+']').offset();<br />
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var showLoadAnim = !!currentPage;<br />
currentPage = page;<br />
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url = wgServer + "/" + page + "?action=render";<br />
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$(window).trigger('page_close');<br />
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$("#TUD-loading-panel").show();<br />
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$("#iGEM_TU_Delft_container").fadeTo(200,0.25);<br />
$('html, body').animate({scrollTop:0}, 'slow');<br />
}<br />
loading_home = false;<br />
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dbgout('processPage: ' + page);<br />
$(window).trigger('page_init');<br />
rewriteLinks($("#iGEM_TU_Delft_container"));<br />
updateContentEditLink();<br />
$("#iGEM_TU_Delft_container").fadeTo(200,1).delay(200).queue(function(n) {<br />
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min-height:100%; /* real browsers */<br />
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<br />
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<a name="top"></a><br />
<br />
<div id="TUD-loading-panel"><br />
<div id="TUD-loading-panel-logo"><br />
<!-- yes im using center --><br />
<center><img width="100" height="120" src="https://static.igem.org/mediawiki/2010/d/d8/TUDelft_2010_AlkanivoreAnimated.gif" /> <h2>Loading...</h2></center><br />
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<div id="TUD-main-wrapper2"><br />
<div id="TUD-main-wrapper"><br />
<br />
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<div id="TUD-header"><br />
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<a class="nohover" href="https://2010.igem.org/Team:TU_Delft"><h1 class="TUD-logo"><span>TU Delft iGEM Team 2010</span></h1></a><br />
<span id="back_to_igem"><a href="https://2010.igem.org/" alt="back to 2010.igem.org" title="back to 2010.igem.org"><img src="https://static.igem.org/mediawiki/2010/3/32/TU_Delft_back_to_igem.png" border="0" /></a></span><br />
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<div id="TUD-menu"><br />
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<ul id="navlist"><br />
<li><a class="menubutton" id="home" href="https://2010.igem.org/Team:TU_Delft/Home" title="Home"><span>Home</span></a></li><br />
<li><a class="menubutton" id="team" href="https://2010.igem.org/Team:TU_Delft/Team" title="Team"><span>Team</span></a><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/members" title="Members">Members</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/organization" title="Organization">Organization</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/university" title="University">University</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/previous-teams" title="Previous Teams">Previous Teams</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/gallery" title="Picture Gallery">Picture Gallery</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/movies" title="Movies">Movies</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/ijam" title="iJAM">iJAM</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="project" href="https://2010.igem.org/Team:TU_Delft#page=Project" title="Project">Project</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/introduction" title="Introduction">Introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation" title="Alkane Degradation">Alkane Degradation</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing" title="Hydrocarbon Sensing">Sensing</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/tolerance" title="Survival">Survival</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility" title="Emulsification">Solubility</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization" title="RBS Characterization">RBS Characterization</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/conclusions" title="General Conclusions">General Conclusions</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/references" title="References">References</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="insilico" href="https://2010.igem.org/Team:TU_Delft#page=Notebook" title="Notebook">Notebook</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/blog" title="Blog">Blog</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/timeline" title="Timeline">Timeline</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/brainstorm" title="Brainstorm">Brainstorm</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/cloning-plan" title="Cloning Plan">Cloning Plan</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols" title="Protocols">Protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/statistics" title="Statistics">Statistics</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="parts" href="https://2010.igem.org/Team:TU_Delft#page=Parts" title="Parts">Parts</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts/biobricks" title="BioBricks">BioBricks</a></li><br />
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Jcnossen
http://2010.igem.org/Team:TU_Delft
Team:TU Delft
2010-10-27T11:39:11Z
<p>Jcnossen: </p>
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<li><a class="menubutton" id="home" href="https://2010.igem.org/Team:TU_Delft/Home" title="Home"><span>Home</span></a></li><br />
<li><a class="menubutton" id="team" href="https://2010.igem.org/Team:TU_Delft/Team" title="Team"><span>Team</span></a><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/members" title="Members">Members</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/organization" title="Organization">Organization</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/university" title="University">University</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/previous-teams" title="Previous Teams">Previous Teams</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/gallery" title="Picture Gallery">Picture Gallery</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/movies" title="Movies">Movies</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/ijam" title="iJAM">iJAM</a></li><br />
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<li><a class="menubutton" id="project" href="https://2010.igem.org/Team:TU_Delft#page=Project" title="Project">Project</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/introduction" title="Introduction">Introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation" title="Alkane Degradation">Alkane Degradation</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing" title="Hydrocarbon Sensing">Sensing</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/tolerance" title="Survival">Survival</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility" title="Emulsification">Solubility</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization" title="RBS Characterization">RBS Characterization</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/conclusions" title="General Conclusions">General Conclusions</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/references" title="References">References</a></li><br />
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<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/blog" title="Blog">Blog</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/timeline" title="Timeline">Timeline</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/brainstorm" title="Brainstorm">Brainstorm</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/cloning-plan" title="Cloning Plan">Cloning Plan</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols" title="Protocols">Protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/statistics" title="Statistics">Statistics</a></li><br />
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<li><a class="menubutton" id="parts" href="https://2010.igem.org/Team:TU_Delft#page=Parts" title="Parts">Parts</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts/biobricks" title="BioBricks">BioBricks</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts/characterization" title="Characterization">Characterization</a></li><br />
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<li><a class="menubutton" id="insilico" href="https://2010.igem.org/Team:TU_Delft#page=Modeling" title="In Silico">In Silico</a><br />
<ul id="insilico_menu"><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/MFA" title="MFA">MFA</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/HC_regulation" title="Hydrocarbon Regulation">Hydrocarbon Regulation</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/protein-production-model" title="Protein Production Model">Protein Production Model</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/pcaif-model" title="Protein Production Model">Protein Production Model</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/interaction-mapping" title="Software Tool">Interaction Mapping</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Software/part-search" title="Part search server & iPhone app">Part search server & iPhone app</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/wiki-tips-tricks" title="Wiki tips & tricks">Wiki Tips &amp; Tricks</a></li><br />
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</li><br />
<li><a class="menubutton" id="safety" href="https://2010.igem.org/Team:TU_Delft#page=Safety" title="Safety">Safety</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Safety" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Safety/in-the-lab" title="Biosafety">Biosafety in the lab</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="humanpractice" href="https://2010.igem.org/Team:TU_Delft#page=Human-practice" title="Human Practice">Human Practice</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Human-practice" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=EPAC" title="EPAC">EPAC</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education" title="Education">Education</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Perception" title="Perception">Perception</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Awareness" title="Awareness">Awareness</a></li><br />
</ul><br />
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Jcnossen
http://2010.igem.org/Team:TU_Delft/Project/conclusions
Team:TU Delft/Project/conclusions
2010-10-27T11:07:56Z
<p>Jcnossen: /* Anderson RBS Characterizations */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
==Conclusions==<br />
Our goal was to tackle the biological conversion of hydrocarbons in an aqueous environment. The basis of the project was generating a "biological chassis", which provides the framework for varying and multiple characteristics needed for the conversion of hydrocarbons, including considerations like conversion ability, hydrocarbon tolerance/solubility and halo (salt) tolerance. This chassis could than be used in for example specifically the biological degradation of oil particles in oil sands tailing water.<br />
<br />
===Alkane Degradation===<br />
<br />
===Survival===<br />
<br />
<br />
====Salt tolerance====<br />
Our biobrick has enabled us to increase the salt tolerance of E.coli by an average of 20%. But due to the range of effects caused by increased salt stress, complete tolerance using a single protein is impossible. As such we hope to have made a first step and that the future iGEM teams will be able to build upon this knowledge.<br />
<br />
===Solubility===<br />
To overcome the mass‐transfer limitation between the water and oil fase, a gene encoding for [[Team:TU_Delft/Project/solubility/alna|AlnA]], a protein with emulsifying properties was expressed. The [[Team:TU_Delft/Project/solubility/results|increased solubility]] of about 20% was determined by [[Team:TU_Delft/Project/solubility/characterization|a new method]]. We suggest that in future research the protein is tagged, so it can be isolated with higher purity.<br />
<br />
===Sensing===<br />
<br />
===Anderson RBS Characterizations===<br />
In order to tune the protein expressions of the alkane degrading genes we've [https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization characterized] 5 members of the Anderson RBS family using an improved protein production model taking dilution into account. The following relative efficiencies were found:<br />
{|style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>RBS</b><br />
|<b>Efficiency</b><br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61100 J61100]<br />
|1.20%<br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61101 J61101]<br />
|11.9%<br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61107 J61107]<br />
|7.70%<br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61117 J61117]<br />
|1.26%<br />
|-<br />
|[http://partsregistry.org/Part:BBa_J61127 J61127]<br />
|6.52%<br />
|-<br />
|[http://partsregistry.org/Part:BBa_B0032 B0032]<br />
|30.0%<br />
|}</div>
Jcnossen
http://2010.igem.org/Team:TU_Delft
Team:TU Delft
2010-10-27T10:35:00Z
<p>Jcnossen: </p>
<hr />
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<li><a class="menubutton" id="team" href="https://2010.igem.org/Team:TU_Delft/Team" title="Team"><span>Team</span></a><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/members" title="Members">Members</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/organization" title="Organization">Organization</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/university" title="University">University</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/previous-teams" title="Previous Teams">Previous Teams</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/gallery" title="Picture Gallery">Picture Gallery</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/movies" title="Movies">Movies</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/ijam" title="iJAM">iJAM</a></li><br />
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<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/introduction" title="Introduction">Introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation" title="Alkane Degradation">Alkane Degradation</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing" title="Hydrocarbon Sensing">Sensing</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/tolerance" title="Survival">Survival</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility" title="Emulsification">Solubility</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization" title="RBS Characterization">RBS Characterization</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/conclusions" title="General Conclusions">General Conclusions</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/references" title="References">References</a></li><br />
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<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/blog" title="Blog">Blog</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/timeline" title="Timeline">Timeline</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/brainstorm" title="Brainstorm">Brainstorm</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/cloning-plan" title="Cloning Plan">Cloning Plan</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols" title="Protocols">Protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/statistics" title="Statistics">Statistics</a></li><br />
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<li><a class="menubutton" id="parts" href="https://2010.igem.org/Team:TU_Delft#page=Parts" title="Parts">Parts</a><br />
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<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts/biobricks" title="BioBricks">BioBricks</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts/characterization" title="Characterization">Characterization</a></li><br />
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<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/MFA" title="MFA">MFA</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/HC_regulation" title="Hydrocarbon Regulation">Hydrocarbon Regulation</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/Protein-production-model" title="Protein Production Model">Protein Production Model</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/interaction-mapping" title="Software Tool">Interaction Mapping</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Software/part-search" title="Part search server & iPhone app">Part search server & iPhone app</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/wiki-tips-tricks" title="Wiki tips & tricks">Wiki Tips &amp; Tricks</a></li><br />
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<li><a class="menubutton" id="safety" href="https://2010.igem.org/Team:TU_Delft#page=Safety" title="Safety">Safety</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Safety" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Safety/in-the-lab" title="Biosafety">Biosafety in the lab</a></li><br />
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<li><a class="menubutton" id="humanpractice" href="https://2010.igem.org/Team:TU_Delft#page=Human-practice" title="Human Practice">Human Practice</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Human-practice" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=EPAC" title="EPAC">EPAC</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education" title="Education">Education</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Perception" title="Perception">Perception</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Awareness" title="Awareness">Awareness</a></li><br />
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<li><a class="menubutton" id="education" href="https://2010.igem.org/Team:TU_Delft#page=Education" title="Education">Education</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education/science-museum" title="Science Museum">Science Museum</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education/game" title="Game">iGEM Game</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education/Workshop_on_synthetic_biology" title="SynBio Workshop">SynBio Workshop</a></li><br />
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Jcnossen
http://2010.igem.org/Team:TU_Delft
Team:TU Delft
2010-10-27T10:15:29Z
<p>Jcnossen: </p>
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<a name="top"></a><br />
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<center><img width="100" height="120" src="https://static.igem.org/mediawiki/2010/d/d8/TUDelft_2010_AlkanivoreAnimated.gif" /> <h2>Loading...</h2></center><br />
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<a class="nohover" href="https://2010.igem.org/Team:TU_Delft"><h1 class="TUD-logo"><span>TU Delft iGEM Team 2010</span></h1></a><br />
<span id="back_to_igem"><a href="https://2010.igem.org/" alt="back to 2010.igem.org" title="back to 2010.igem.org"><img src="https://static.igem.org/mediawiki/2010/3/32/TU_Delft_back_to_igem.png" border="0" /></a></span><br />
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<li><a class="menubutton" id="home" href="https://2010.igem.org/Team:TU_Delft/Home" title="Home"><span>Home</span></a></li><br />
<li><a class="menubutton" id="team" href="https://2010.igem.org/Team:TU_Delft/Team" title="Team"><span>Team</span></a><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/members" title="Members">Members</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/organization" title="Organization">Organization</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/university" title="University">University</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/previous-teams" title="Previous Teams">Previous Teams</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/gallery" title="Picture Gallery">Picture Gallery</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/movies" title="Movies">Movies</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/ijam" title="iJAM">iJAM</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="project" href="https://2010.igem.org/Team:TU_Delft#page=Project" title="Project">Project</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/introduction" title="Introduction">Introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation" title="Alkane Degradation">Alkane Degradation</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing" title="Hydrocarbon Sensing">Sensing</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/tolerance" title="Survival">Survival</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility" title="Emulsification">Solubility</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization" title="RBS Characterization">RBS Characterization</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/conclusions" title="General Conclusions">General Conclusions</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/references" title="References">References</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="insilico" href="https://2010.igem.org/Team:TU_Delft#page=Notebook" title="Notebook">Notebook</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/blog" title="Blog">Blog</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/timeline" title="Timeline">Timeline</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/brainstorm" title="Brainstorm">Brainstorm</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/cloning-plan" title="Cloning Plan">Cloning Plan</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols" title="Protocols">Protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/statistics" title="Statistics">Statistics</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="parts" href="https://2010.igem.org/Team:TU_Delft#page=Parts" title="Parts">Parts</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts/biobricks" title="BioBricks">BioBricks</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts/characterization" title="Characterization">Characterization</a></li><br />
</ul><br />
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<li><a class="menubutton" id="insilico" href="https://2010.igem.org/Team:TU_Delft#page=Modeling" title="In Silico">In Silico</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/MFA" title="MFA">MFA</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/HC_regulation" title="Hydrocarbon Regulation">Hydrocarbon Regulation</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/Protein-production-model" title="Protein Production Model">Protein Production Model</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/interaction-mapping" title="Software Tool">Interaction Mapping</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Software/part-search" title="Part search server & iPhone app">Part search server & iPhone app</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/wiki-tips-tricks" title="Wiki tips & tricks">Wiki Tips &amp; Tricks</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="safety" href="https://2010.igem.org/Team:TU_Delft#page=Safety" title="Safety">Safety</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Safety" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Safety/in-the-lab" title="Biosafety">Biosafety in the lab</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="humanpractice" href="https://2010.igem.org/Team:TU_Delft#page=Human-practice" title="Human Practice">Human Practice</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Human-practice" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=EPAC" title="EPAC">EPAC</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education" title="Education">Education</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Perception" title="Perception">Perception</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Awareness" title="Awareness">Awareness</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="education" href="https://2010.igem.org/Team:TU_Delft#page=Education" title="Education">Education</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education/science-museum" title="Science Museum">Science Museum</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education/game" title="Game">iGEM Game</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education/Workshop_on_synthetic_biology" title="SynBio Workshop">SynBio Workshop</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education/nemo" title="Night of the Nerds">Night of the Nerds</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="publicity" href="https://2010.igem.org/Team:TU_Delft#page=Publicity" title="Publicity">Publicity</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Publicity" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Publicity/communication_plan" title="Communication Plan">Communication Plan</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Publicity/in-the-news" title="In The News">In the Media</a></li><br />
<!-- <li><a href="https://2010.igem.org/Team:TU_Delft#page=Publicity/articles" title="Articles">Articles</a></li> /--><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="collaboration" href="https://2010.igem.org/Team:TU_Delft#page=Collaboration" title="Collaboration">Collaboration</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Collaboration" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Collaboration/sponsors" title="Sponsors">Sponsors</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Collaboration/Rathenau Institute" title="Rathenau Institute">Rathenau Institute</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Collaboration/Science Centre Delft" title="Science Centre Delft">Science Centre Delft</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Collaboration/igem-teams" title="iGEM Teams">iGEM Teams</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Collaboration/acknowledgements" title="Acknowledgements">Acknowledgements</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="collaboration" href="https://2010.igem.org/Team:TU_Delft#page=Contact" title="Contact">Contact</a></li><br />
</ul><br />
</div><br />
<div id="TUD-body-wrapper"><br />
<div id="TUD-body-container"><br />
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Jcnossen
http://2010.igem.org/Team:TU_Delft
Team:TU Delft
2010-10-26T22:15:11Z
<p>Jcnossen: </p>
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Jcnossen
http://2010.igem.org/Team:TU_Delft
Team:TU Delft
2010-10-26T22:14:43Z
<p>Jcnossen: </p>
<hr />
<div>__NOTOC__<br />
<html><br />
<!--<br />
<br />
============================================================================================<br />
*** PLEASE NOTE: ***<br />
<br />
When copying our code, please be so kind to mention so on your wiki.<br />
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You can use our logo https://static.igem.org/mediawiki/2010/7/7b/TU_Delft.jpg to link to our wiki.<br />
<br />
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#TUD-footer-content .socialicon {<br />
margin-top: 4px;<br />
margin-right:10px;<br />
width:32px;<br />
height:32px;<br />
display:block;<br />
float:left;<br />
}<br />
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#TUD-footer-content .socialicon a {<br />
width:100%;<br />
height:100%;<br />
display:block;<br />
}<br />
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margin-top:10px;<br />
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padding-left: 5px;<br />
width: 340px;<br />
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}<br />
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margin: 5px 0 5px 0;<br />
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width: 340px;<br />
}<br />
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color: #0C93BA;<br />
border-bottom-style: solid;<br />
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text-decoration: none;<br />
color: #DDA84E;<br />
}<br />
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#twitter_div ul li a:hover {<br />
text-decoration: none;<br />
color: #D78E42;<br />
}<br />
<br />
#twitter_div p {<br />
text-align: right;<br />
padding-right: 6px;<br />
padding-bottom: 10px;<br />
}<br />
</style><br />
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</div><br />
</div><br />
</div><br />
</div><br />
<script type="text/javascript"><br />
<br />
var _gaq = _gaq || [];<br />
_gaq.push(['_setAccount', 'UA-16783722-1']);<br />
_gaq.push(['_trackPageview']);<br />
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var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true;<br />
ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js';<br />
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Jcnossen
http://2010.igem.org/Team:TU_Delft
Team:TU Delft
2010-10-26T21:21:32Z
<p>Jcnossen: </p>
<hr />
<div>__NOTOC__<br />
<html><br />
<!--<br />
<br />
============================================================================================<br />
*** PLEASE NOTE: ***<br />
<br />
When copying our code, please be so kind to mention so on your wiki.<br />
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You can use our logo https://static.igem.org/mediawiki/2010/7/7b/TU_Delft.jpg to link to our wiki.<br />
<br />
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var page = currentPage ? currentPage : 'Home';<br />
var url = location.pathname + '/' + page + '?action=edit';<br />
$("#edit_content_link").attr('href', url);<br />
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// });<br />
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historyCallback();<br />
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function () { $(this).removeClass("hover"); }<br />
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return kvmap;<br />
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function makeHash(page, kvmap) {<br />
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var i;<br />
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function setHash(page, kvmap) {<br />
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function include_js(file, cb) {<br />
var html_doc = document.getElementsByTagName('head')[0];<br />
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function loadScript(src, callback)<br />
{<br />
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function moveToAnchor(anchor) {<br />
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$(window).trigger('page_close');<br />
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if(showLoadAnim) {<br />
$("#TUD-loading-panel").show();<br />
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$("#iGEM_TU_Delft_container").fadeTo(200,0.25);<br />
$('html, body').animate({scrollTop:0}, 'slow');<br />
}<br />
loading_home = false;<br />
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var processPage = function(next) {<br />
dbgout('processPage: ' + page);<br />
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rewriteLinks($("#iGEM_TU_Delft_container"));<br />
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$("#iGEM_TU_Delft_container").fadeTo(200,1);<br />
next();<br />
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$.get(url, function(data) {<br />
$("#iGEM_TU_Delft_container").html(data).queue(processPage);<br />
buildBreadCrumbTrail(page); <br />
Cufon.replace('h2'); // Works without a selector engine <br />
Cufon.replace('h3'); // Works without a selector engine <br />
Cufon.replace('#sub1'); // Requires a selector engine for IE 6-7, see above <br />
$("tr:nth-child(odd)").addClass("odd");<br />
<br />
$("#TUD-loading-panel").hide();<br />
$("#loading-overlay").hide();<br />
});<br />
}<br />
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hash = hash.substring(1);<br />
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// looks a little clumsy, but js AND operator conflicts with mediawiki markup<br />
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});<br />
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<script type="text/javascript"><br />
var timeout = 500;<br />
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if(constituentFolders[count].substring(0, 5) !== "User:") {<br />
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<style type="text/css"><br />
/* Wiki Hacks - START */<br />
/* Author: Pieter van Boheemen */<br />
/* Team: TU Delft */<br />
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height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
}<br />
#bodyContent { border: none; padding:0; margin:0; width:100%;<br />
height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
}<br />
#top-section { z-index: 2; height: 15px; margin: 0px; margin-left: auto; margin-right: auto; margin-bottom: 0 !important; padding:0; border: none; font-size: 10px;}<br />
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h3#siteSub { display: none;}<br />
#contentSub {display: none;}<br />
p:first-child { display: none;}<br />
h1{border:none; width: 100%; clear: both;}<br />
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<br />
table {<br />
margin: 0;<br />
padding: 0;<br />
font-size: 11px;<br />
border: 1px solid #000;<br />
}<br />
table td {<br />
padding: 5px;<br />
}<br />
table .head {<br />
background-color: #216085 ;<br />
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color: #fff;<br />
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h2 {<br />
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}<br />
<br />
h3 {<br />
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}<br />
<br />
html, body {<br />
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padding: 0;<br />
width: 100%;<br />
height: 100%; <br />
}<br />
<br />
body {<br />
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color: #222222;<br />
min-width: 1050px;<br />
}<br />
<br />
#TUD-main-wrapper {<br />
width: 100%;<br />
height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
text-align: center;<br />
background-image: url('https://static.igem.org/mediawiki/2010/b/b3/TU_Delft_footer_tile.gif');<br />
background-position: bottom left;<br />
background-repeat: repeat-x; <br />
display: block; <br />
font-family: Verdana, Arial;<br />
font-size: 12px;<br />
color: #222222;<br />
}<br />
<br />
#TUD-main-wrapper div {<br />
line-height: 18px;<br />
}<br />
<br />
/* IE ignores this */<br />
html>body #TUD-main-wrapper {<br />
height: auto;<br />
min-height: 100%;<br />
}<br />
<br />
#TUD-main-wrapper2 {<br />
width: 100%;<br />
height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
text-align: center;<br />
background-image: url('https://static.igem.org/mediawiki/2010/9/9d/TU_Delft_bg_tile.gif');<br />
background-repeat: repeat-x;<br />
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color: #222222;<br />
margin-top: -8px;<br />
}<br />
<br />
/* IE ignores this */<br />
html>body #TUD-main-wrapper2 {<br />
height: auto;<br />
min-height: 100%;<br />
}<br />
<br />
#TUD-content-wrapper {<br />
background-color: #fff;<br />
text-align: left;<br />
width: 1024px;<br />
margin: 0 auto;<br />
padding: 0;<br />
height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
}<br />
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width: 1024px;<br />
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padding-top: 1px;/*important*/<br />
margin-top: -1px;/*important*/<br />
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<br />
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/* FireFox collapsing margin fix */<br />
padding-top: 1px;/*important*/<br />
margin-top: -1px;/*important*/<br />
display: block;<br />
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<br />
#TUD-body-content {<br />
background-image: url('https://static.igem.org/mediawiki/2010/5/5f/TU_Delft_body_footer_bg.jpg');<br />
width: 1024px;<br />
background-repeat: no-repeat;<br />
background-position: bottom left;<br />
padding-bottom: 15px<br />
}<br />
<br />
#TUD-main-content {<br />
width: 904px;<br />
margin: 10px 60px 0px 60px;<br />
}<br />
<br />
#iGEM_TU_Delft_container {<br />
min-height: 400px;<br />
}<br />
<br />
#TUD-footer {<br />
width: 100%;<br />
height: 254px;<br />
text-align: center;<br />
}<br />
<br />
h1 {<br />
margin: 0;<br />
padding: 0;<br />
display: block;<br />
float: left;<br />
}<br />
<br />
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background-image: url('https://static.igem.org/mediawiki/2010/0/09/TU_Delft_iGEM_Team_Logo.png');<br />
width: 237px;<br />
height: 95px;<br />
margin-left: 90px;<br />
background-repeat: no-repeat;<br />
border: none;<br />
}<br />
<br />
.TUD-logo span {<br />
visibility: hidden;<br />
}<br />
<br />
<br />
#TUD-loading-panel {<br />
z-index: 100;<br />
position: absolute;<br />
left: 0px;<br />
top: 0px;<br />
width: 100%;<br />
height: 100%;<br />
display:none;<br />
}<br />
<br />
#TUD-loading-panel-logo {<br />
margin: 0 auto;<br />
margin-top: 250px;<br />
}<br />
<br />
#loading-overlay {<br />
z-index: 70;<br />
width: 100%;<br />
height: 100%;<br />
display:none;<br />
opacity:0.4;<br />
filter:alpha(opacity=40);<br />
}<br />
<br />
#back_to_igem {<br />
margin: 15px 0 0 0;<br />
padding:0;<br />
float: right;<br />
}<br />
<br />
/* MENU */<br />
#navlist {<br />
margin: 0 0 0 55px;<br />
padding: 0;<br />
}<br />
<br />
#navlist li {<br />
display: block;<br />
list-style-type: none;<br />
float:left;<br />
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Jcnossen
http://2010.igem.org/Team:TU_Delft/Project/solubility/characterization
Team:TU Delft/Project/solubility/characterization
2010-10-26T21:15:01Z
<p>Jcnossen: /* Our Emulsifier Assay */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility/parts" target="" /></map></html><br />
==Characterization of the Solubility Parts==<br />
<br />
===Protein Production Analysis===<br />
The ''alna'' gene was induced when the culture reached a density of about 10<sup>8</sup> bacteria per mL. Bacterial and medium samples were taken for sodium dodecyl sulfate (SDS)-gel electrophoresis to monitor AlnA production. Because the IPTG inducable protomor is generally regarded as verey strong, we expect that the resulting gel wil show when the expression of AlnA begins and when it reaches its maximum.<br />
<br />
===Standard Emulsifier Assays===<br />
[[Image:TU_Delft_Rajakumari.jpg|thumb|350px|right|''Figure 1'']]Besides proving the production of the protein, we wanted to measure the increase of emulsification as well. The only problem was that emulsification is not easily determined. In literature we found many methods that involve spectrophotometric measurements of turbidity after mixing. Although many articles show nice graphs, we were unable to reproduce them. So, in our opinion measuring turbidity is rather arbitrary and there is a clear need for a new emulsification assay.<br />
<br />
'''Example from literature'''<br />
<br />
Figure 1 on the right shows a calibration graph with a linear increase in absorbance at 660 nm for increasing concentrations of SDS (●). Under the standard detergent assay conditions, various components like 200 mM NaCl (□), 2.0 mM CaCl2 (♦), 10% glycerol (■), 100 μg microsomal membranes (open diamond), 0.2 mM Triton X-100 (▲) and 2.5 mM CHAPS (○) were added and the turbidity was measured. Error bars represent the deviation from five independent experiments and each one performed in duplicate. ''Rajakumari et al'' (2006) Biochemical and Biophysical Methods<br />
<br />
<html><br style="clear:both;" /></html><br />
<br />
===Our Emulsifier Assay===<br />
[[Image:TU_Delft_sudan_ii_balls_stick.png|thumb|250px|right|Ball-and-stick model of the Sudan II molecule. Source [http://commons.wikimedia.org/wiki/File:Sudan-II-3D-balls.png Wikipedia]]]<br />
<br />
For determining the emulsification capacity of a substance we developed our own emulsifier assay. The main difference to the existing assays is that instead of emulsifying a colorless hydrocarbon, we added the orange Sudan II dye. This molecule stains non-polar molecules and hardly dissolves in water. The addition of the dye makes measuring the emulsification much easier, because of it’s nice absorbance peak at 493 nm.<br />
<br />
Read the [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Emulsifier_test Emulsification Assay protocol].<br />
<br />
====Calibration====<br />
The assay was calibrated using SDS, because this detergent is widely used and makes the outcome of the assay comparable to existing assays. Our measurements are shown in the graph on right. It shows that the assay is very sensible at low SDS concentrations.<br />
<html><br style="clear:both;" /></html><br />
[[Image:TU_Delft_Emuls_assay_SDS_calibration.jpg|center|Emulsification Assay calibration curve]]<br />
<br />
====Background====<br />
Next, the influence of the culture media was determined. We used LB medium for the initial growth of the cells, and induced the production of AlnA in M9 medium. The background influence of the media on the assay were measured, as shown below. It shows that LB medium already has some emulsification effect, but M9 shows very little influence.<br />
<br />
[[Image:TU_Delft_Emuls_assay_M9_LB_background.jpg|center|Background measurements of LB and M9 medium]]<br />
<br />
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Jcnossen
http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results
Team:TU Delft/Project/alkane-degradation/results
2010-10-26T21:12:26Z
<p>Jcnossen: /* NADH production in cell extracts */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===Characterization of the alkane hydroxylase system===<br />
====Growth analysis====<br />
We attempted to culture our recombinant AH-carrying ''E.coli'' K12 strains ([http://partsregistry.org/Part:BBa_K398014 BBa_K398014]) on 1% v/v octanol or 1% v/v dodecane. Negligible growth was observed.<br />
<br />
====Resting-cell assays====<br />
The [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Resting-cell_assays_for_E.coli resting cell assays] were performed on our recombinant AH-carrying ''E.coli'' K12 cells ([http://partsregistry.org/Part:BBa_K398014 BBa_K398014]). 100 micromoles of octane was added to 6 mL of growth-stalled cells (1.5 mg cell dry weight total) and incubated at 37 degrees with shaking o/n. The organic phase was [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Ethyl_acetate_extraction_protocol extracted] using EtOAc and analysed by [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=General_gas_chromatography_program_for_alkanes_and_alkanols gas chromatography].<br />
<br />
The following are examples of typical chromatographs obtained:<br />
<br />
<html><span style="display:block;width:100%;clear:both;"> </span></html><br />
[[Image:TUDelft_J13-AH.png|400px|thumb|left|Chromatograph of the negative control for the resting cell assay of the alkane hydroxylase system. The negative control consisted of ''E.coli'' K12 carrying BBa_J13002 in pSB1A2.]]<br />
<br />
[[Image:014 AH.png|400px|left|thumb|Chromatograph of ''E.coli'' K12 strain carrying the AH system.]]<br />
<html><span style="display:block;width:100%;clear:both;"> </span></html><br />
<br />
The peaks shown in the chromatographs belong to the following compounds:<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Retention time [min]</b><br />
|<b>Compound</b><br />
|-<br />
|~2.2<br />
|Ethyl acetate (solvent)<br />
|-<br />
|~5.2<br />
|Octane (substrate)<br />
|-<br />
|~11.8<br />
|Undecane (internal standard, 0.1% v/v of solvent)<br />
|}<br />
<br />
The surface areas of the peaks correspond to the amount of molecules present in the sample. Due to the fact that many factors play a role in establishing an equilibrium between the aqueous and organic phase once the EtOAC is added, it's general procedure to add an internal standard to the EtOAc. In our experiment we decided to use a 0.1% vol/vol of undecane in EtOAc. The <b>ratio</b> between the surface areas of octane and undecane can now give an indication of the amount of octane still present in the sample and be very useful for comparisons between chromatographs.<br />
<br />
The following table contains the surface areas found for the peaks of octane and undecane for each obtained chromatograph:<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Octane surface area</b><br />
|<b>Undecane surface area</b><br />
|<b>Ratio octane/undecane</b><br />
|-<br />
|''E.coli'' K12 negative control<br />
|17903555<br />
|19939270<br />
|0.898<br />
|-<br />
|''E.coli'' K12 014#2<br />
|17426721<br />
|24262069<br />
|0.719<br />
|-<br />
|''E.coli'' K12 014#7<br />
|7824594<br />
|17320861<br />
|0.452<br />
|}<br />
<br />
===LadA===<br />
<br />
===Characterization of the Alcohol DeHydrogenase (ADH) system===<br />
<br />
=====Growth on alcohols as sole carbon source=====<br />
*As we described before, we tried to grow our strain ''Escherichia coli'' 018A ([http://partsregistry.org/Part:BBa_K398018 BBa_K398018] on the plasmid pSB1A2) on the alkanols Octanol-1 and Dodecanol-1 (1% V/V on M9 medium without any other carbon source). No growth was observed after 48 hours. We abandoned this experiment after this result.<br />
<br />
=====Resting cell assays=====<br />
*We grew ''Escherichia coli'' 018A and ''Escherichia coli'' negative control (Biobrick BBa_J13002 on plasmid pSB1A2 ) in 50 mL of M9 medium with glucose and CAS aminoacids.<br />
*The cells were harevested when the O.D. at 600nm was around 0.3; they were spun down at 4000 rpm, for 10 min at 4ºC. And the resting cell assays were prepared according to the [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Resting-cell_assays_for_E.coli standard protocol]<br />
<br />
{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1"; <br />
|'''Strain'''<br />
|'''O.D.'''<br />
|-<br />
|J13002<br />
|0.327<br />
|-<br />
|018A<br />
|0.338<br />
|}<br />
<br />
*After an overnight incubation at 37ºC, the organic phase was extracted using 3 mL of ethyl acetate (dodecane was used as internal standard). We tried to determine production of alkanals or alkanoic acids by Gas Chromatography measurements. The chromatograms are shown below:<br />
<br />
[[Image:TUDelftADH_chrom.jpg|750px|thumb|center|Typical chromatograms obtained after the resting cell assays: (A) Blank, (B) Negative control which is E. coli K12, (C) E. coli 018A (our recombinant strain) and (D) standard of the expected product.]]<br />
<br />
<br />
*According to our results there was no degradation of octanol-1 ='( , after this experiment the protocol was abandoned. We think that the cells lack of transporters for long-chain alcohols, we preferred to check the cell extract in order to know if there was any biological activity in the cytoplasma.<br />
<br />
=====NADH production in cell extracts=====<br />
*'''EXPERIMENT 1'''<br />
Cells were cultured in 50mL of LB medium and harvested when the O.D. 600nm of the culture was around 0.6. Two different cultures of the recominant strain and the negative control were prepared.<br />
{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1"<br />
|Strain<br />
|O.D. 600nm<br />
|-<br />
|J13002 (1)<br />
|0.654<br />
|-<br />
|J13002 (2)<br />
|0.621 <br />
|-<br />
|018A (1)<br />
|0.615<br />
|-<br />
|018A (2)<br />
|0.580<br />
|}<br />
Cytoplasmic proteins were extracted using our standard [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Preparing_cell_lysates_for_enzyme_kinetics_measurements protocol]. And a standard curve for protein quantification was prepared.<br />
[[Image:TUDelftADH_bradford.jpg|600px|thumb|center|Standard curve for total protein quantification.]]<br />
The total protein of each sample was quantified using 20uL of cell extract, the results are shown below.<br />
{| style="color:black; background-color:white;" cellpadding="4" cellspacing="0" border="1"<br />
|Sample<br />
|'''O.D. 562 nm'''<br />
|'''Total ug of protein'''<br />
|'''ug/uL'''<br />
|-<br />
|J13002(1A) <br />
|0.135 <br />
|10.393 <br />
|1.0393<br />
|-<br />
|J13002 (1B) <br />
|0.135 <br />
|10.393 <br />
|1.0393<br />
|-<br />
|J13002 (2A) <br />
|0.128 <br />
|9.7363 <br />
|0.9736<br />
|-<br />
|J13002 (2B) <br />
|0.129 <br />
|9.8302 <br />
|0.9830<br />
|-<br />
|018A (1A) <br />
|0.097 <br />
|6.8275 <br />
|0.6827<br />
|-<br />
|018A (1B) <br />
|0.091 <br />
|6.2645 <br />
|0.6264<br />
|-<br />
|018A (2A) <br />
|0.092 <br />
|6.3583 <br />
|0.6358<br />
|-<br />
|018A (2B) <br />
|0.092 <br />
|6.3583 <br />
|0.6358<br />
|}<br />
The alcohol dehydrogenase activity was measured using the standard [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Alcohol.2FAldehyde_dehydrogenase_activity_assays protocol] and Dodecanol-1 as substrate. You can download our raw data by clicking on the link: [[ Image:TUDelft_ADH_raw.xls]]<br />
After the data treatment, the results that we obtained were the following:<br />
[[Image:TUDelftADH_pH95.jpg|400px]]<br />
[[Image:TUDelftADH_pH8.jpg|400px]]<br />
<br />
*All the enzyme activities were normalized to the TOTAL amount of protein in the cell extract. The results are shown in katal per mg of protein in the cell extract and Enzymatic Units per mg of protein in the cell extract. <br />
<br />
<html><br />
<table><br />
<tr><td></td><td colspan=4 style="text-align: center;"><b>pH=9.5</b></td><td colspan=4 style="text-align: center;"><b>pH=8</b></td></tr> <br />
<tr><td>&nbsp;</td><td colspan=2 style="text-align: center;"><b>Heat</b></td><td colspan=2 style="text-align: center;"><b>Native</b></td><td colspan=2 style="text-align: center;"><b>Heat</b></td><td colspan=2 style="text-align: center;"><b>Native</b></td></tr> <br />
<tr><td>&nbsp;</td><td>J13002</td><td>018A</td><td>J13002</td><td>018A</td><td>J13002</td><td>018A</td><td>J13002</td><td>018A</td></tr> <br />
<tr><td><b>kat/mg</b></td><td>6,156E-12</td><td>1,941E-11</td><td>1,053E-11</td><td>2,806E-11</td><td>3,211E-12</td><td>4,496E-11</td><td>1,302E-11</td><td>2,638E-11</td></tr> <br />
<tr><td><b>U/mg</b></td><td>3,694E-04</td><td>1,091E-03</td><td>6,317E-04</td><td>1,694E-03</td><td>1,927E-04</td><td>2,139E-03</td><td>7,811E-04</td><td>1,608E-03</td></tr> <br />
<tr><td><b>stdev</b></td><td>1,157E-12</td><td>4,579E-12</td><td>1,343E-12</td><td>9,961E-13</td><td>6,395E-12</td><td>2,331E-11</td><td>6,511E-13</td><td>9,961E-13</td></tr> <br />
<tr><td><b>Improvement</b></td><td>-</td><td>215,34%</td><td>-</td><td>166,51%</td><td>-</td><td>1299,87%</td><td>-</td><td>166,51%</td></tr> <br />
<tr><td><b>ttest</b></td><td>-</td><td>0,9963</td><td>-</td><td>1,0000</td><td>-</td><td>0,9988</td><td>-</td><td>1,000</td></tr><br />
</table><br />
</html><br />
<br />
You can download our results file here: [[ Image:TUDelft_ADH_results.xls]]<br />
Maybe you are not familiar with the term "katal", officially the katal is the STANDARD UNIT FOR CATALYTIC ACTIVITY in the International System of Units; the katal is defined as the amount of enzyme that converts 1 mol of substrate each second. [http://www.clinchem.org/cgi/content/full/48/3/586 Click here] to know more about it, and because people are not that familiar with the term, we report the activities in U/mg. One enzyme activity unit is defined as the amount of protein that converts 1 umol of substrate each minute.<br />
T-tests were performed in order to show that there is a difference between E. coli and our recombinant strain; a result for t-test of 1 means that both samples are statistically different; whereas a t-test of nearly 0 means that samples belong to the same group (no difference between both). Our level of significance is 0.025, in two tailed test. That means that if our t-test result is 0.95 we conclude that both samples are statistically different, whereas 0.949 or lower means that both samples are not statistically different.<br />
<br />
<br />
*'''EXPERIMENT 2'''<br />
We ran a second experiment at pH 9.5, with two other strains that seemed to be positive in the colony PCR test. In this test we also added two extra strains of E. coli in order to have more data for the statistical analysis, the strains used were J13002 and 331 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K398331 BBa_K398331] in pSB1A2). We followed the same procedure as in the experiment 1, for this experiment we also included a positive control which was cell extracts from a culture of ''Pseudomonas putida'' growing on octane as sole carbon source, the cells were inoculated the night before and they were harvested when the O.D. at 600 nm was 0.608; the results obtained were the following:<br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th></th><th>kat/mg</th><th>U/mg</th><th>STDEV</th><th>Improvement</th><th>T-test</th><th>Relative to putida</th><th>&nbsp;</th></tr> <tr><td>E. Coli</td><td>8.75E-12</td><td>5.25E-04</td><td>8.18987E-12</td><td>-</td><td>-</td><td>0.90%</td><td>&nbsp;</td></tr> <tr><td>018A-2</td><td>9.30E-12</td><td>5.58E-04</td><td>1.84877E-12</td><td>6.26%</td><td>0.4708</td><td>0.96%</td><td>&nbsp;</td></tr> <tr><td>018A-3</td><td>3.05E-11</td><td>1.83E-03</td><td>6.27802E-12</td><td>249.14%</td><td>0.9808</td><td>3.15%</td><td>&nbsp;</td></tr> <tr><td>018A-4</td><td>8.26E-12</td><td>4.96E-04</td><td>1.8256E-12</td><td>-5.59%</td><td>0.4053</td><td>0.85%</td><td>&nbsp;</td></tr> <tr><td>P. Putida</td><td>9.69E-10</td><td>5.82E-02</td><td>2.09109E-10</td><td>-</td><td>0.9883</td><td>100.00%</td><td></td></tr></table><br />
<br />
According to these results, the other two strains (018A-2 and 018A-4) do not functionally express the part BBa_K398018. However, these results confirm our findings from the first experiment. You can download our result in this link: [[ Image:TUDelft_ADH_results2.xls]] You can also find a summary in this file:[[ Image:TUDelft_ADH_summary.xls]]<br />
<br />
==== Conclusions ====<br />
<br />
[[Image:TUDelftADH_final.jpg|600px]]<br />
<br />
According to our results, a normal ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain 018A has an activity of 2.93e-11 kat/mg (1.76 mU/mg). According to the statistical analysis, the enzymatic activities of both strains are statistically different at confidence level of 0.95 which means that the part BBa_K398018 increases 2 times the alcohol dehydrogenase activity in the cell extract. We can conclude from our data that the part has a biological activity ''in vitro''.<br />
<br />
We still don't know about the ''in vivo'' activity of this part, maybe there is a lack of long-chain alcohol transporter proteins or it is necessary to over-express this part in order to see/measure any biological activity.<br />
<br />
===Characterization of the ALdehyde DeHydrogenase system===<br />
<br />
=====Growth on alcohols as sole carbon source=====<br />
As we described before, we tried to grow our recombinant strains ''Escherichia coli'' 029A ([http://partsregistry.org/Part:BBa_K398029 BBa_K398029] on the plasmid pSB1A2) and ''Escherichia coli'' 030A ([http://partsregistry.org/Part:BBa_K398030 BBa_K398030] on the plasmid pSB1A2) on Octanal and Dodecanal. No growth was observed after 48 hours. We abandoned this experiment after this result.<br />
<br />
=====Resting cell assays=====<br />
*We grew 'Escherichia coli'' 029A, ''Escherichia coli'' 030Aand ''Escherichia coli'' negative control (Biobrick BBa_J13002 on plasmid pSB1A2 ) in 50 mL of M9 medium with glucose and CAS aminoacids.<br />
*The cells were harevested when the O.D. at 600nm was around 0.3; they were spun down at 4000 rpm, for 10 min at 4ºC. And the resting cell assays were prepared according to the [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Resting-cell_assays_for_E.coli standard protocol]<br />
<br />
{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1"; <br />
|'''Strain'''<br />
|'''O.D.'''<br />
|-<br />
|J13002<br />
|0.327<br />
|-<br />
|029A<br />
|0.340<br />
|-<br />
|030A<br />
|0.330<br />
|}<br />
<br />
'''Note''': This experiment was run in parallel with the resting cell assays for 018A, the negative control culture of the strain J13002 used was the same for both experiments.<br />
<br />
*After an overnight incubation at 37ºC with the substrate (octanal), the organic phase was extracted using 3 mL of ethyl acetate (dodecane was used as internal standard). We tried to determine production of alkanoic acids by Gas Chromatography measurements. The chromatograms are shown below:<br />
<br />
[[Image:TUDelftALDH_chrom.jpg|750px|thumb|center| Typical chromatograms obtained after the resting cell assays: (A) Blank, (B) Negative control which is E. coli K12, (C) E. coli 029A (our recombinant strain) and (D) E. coli 029A (our recombinant strain).]]<br />
<br />
<br />
*According to our results there was a peak reduction for octanal, however we were worried because of the fact that we didn't see the peak of the product (dodecanoic acid) appearing. It could be that the cells are just storing the product inside the cell. The experiment required to much time in order to get more results: the extractions, preparation of triplicates, cultures, cell suspensions... etc. it was a work that would require an effort of all the team for a couple of weeks. We decided to go for something easier and faster to measure; since we had some experience with the NADH determinations that was our choice.<br />
<br />
==== NADH production in cell extracts====<br />
Cells were cultured in 50mL of LB medium and harvested when the O.D. 600nm of the culture was between 0.5-0.8. Two different cultures of each recominant strain and the negative control were prepared.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1"<br />
|Strain<br />
|O.D. 600nm<br />
|-<br />
|J13002 (1)<br />
|0.825<br />
|-<br />
|J13002 (2)<br />
|0.774 <br />
|-<br />
|029A (1)<br />
|0.746<br />
|-<br />
|029A (2)<br />
|0.753<br />
|-<br />
|030A (1)<br />
|0.532<br />
|-<br />
|030A (2)<br />
|0.532<br />
|}<br />
Cytoplasmic proteins were extracted using our standard [https://2010.igem.org/Team:TU_Delft#Preparing_cell_lysates_for_enzyme_kinetics_measurements protocol]. And a standard curve for protein quantification was prepared.<br />
<br />
[[Image:TUDelftALDH_bradford.jpg|600px|thumb|center|Standard curve for total protein quantification.]]<br />
<br />
The total protein of each sample was quantified using 20uL of cell extract, the results are shown below.<br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th></th><th>Total protein [ug]</th><th>ug/uL</th></tr> <tr><td>J1300</td><td>31.108</td><td>1.555</td></tr> <tr><td>029A (1)</td><td>19.974</td><td>0.999</td></tr> <tr><td>030A (1)</td><td>12.610</td><td>0.630</td></tr> <tr><td>029A (2)</td><td>12.854</td><td>0.782</td></tr> <tr><td>030 (2)</td><td>18.578</td><td>0.929</td></tr> <tr><td>P. Puitda</td><td>9.783</td><td>0.489</td></tr></table><br />
<br />
<br />
The aldehyde dehydrogenase activity was measured using the standard [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Alcohol.2FAldehyde_dehydrogenase_activity_assays protocol] and Dodecanal as substrate.<br />
<br />
After the data treatment, the results that we obtained were the following:<br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th></th><th>&nbsp;</th><th>J13002</th><th>029A (1)</th><th>029A (2)</th><th>030A (1)</th><th>030A (2)</th><th>P. putida</th></tr> <tr><td>Specific activity</td><td>kat/mg</td><td>8.604E-11</td><td>1.470E-10</td><td>2.853E-10</td><td>2.433E-10</td><td>2.912E-10</td><td>6.362E-10</td></tr> <tr><td>&nbsp;</td><td>U/mg</td><td>5.162E-03</td><td>8.820E-03</td><td>1.712E-02</td><td>1.460E-02</td><td>1.747E-02</td><td>3.817E-02</td></tr> <tr><td>Standard deviation</td><td>&nbsp;</td><td>1.778E-12</td><td>6.187E-12</td><td>4.026E-11</td><td>3.539E-11</td><td>4.319E-11</td><td>3.361E-11</td></tr> <tr><td>Relative to (J13002)</td><td>&nbsp;</td><td>100.00%</td><td>170.86%</td><td>331.59%</td><td>282.79%</td><td>338.46%</td><td>739.41%</td></tr> <tr><td>Relative activity (P. putida)</td><td>&nbsp;</td><td>13.52%</td><td>23.11%</td><td>44.84%</td><td>38.25%</td><td>45.78%</td><td>100.00%</td></tr> <tr><td>T-test vs J13002</td><td>&nbsp;</td><td>-</td><td>0.9827</td><td>0.9599</td><td>0.9764</td><td>0.9976</td><td>0.9976</td></tr> <tr><td>T-test vs P. putida</td><td>&nbsp;</td><td>0.9999</td><td>1.0000</td><td>0.9998</td><td>0.9974</td><td>1.0000</td><td>-</td></tr> <tr><td>Average activity [kat/mg]</td><td>&nbsp;</td><td>8.604E-11</td><td>2.161E-10</td><td>&nbsp;</td><td>2.673E-10</td><td>&nbsp;</td><td>6.362E-10</td></tr></table><br />
<br />
You can download a file with our raw data, results anda summary here: [[ Image:TUDelft_ALDH_results.xls]]<br />
<br />
==== Conclusion====<br />
<br />
Our results suggest that the recombinant strains ''E. coli'' 029A and ''E. coli'' 030A functionally express our biobricks, we couldn't prove ''in vivo'' activity. However, it is clear from the statistical analysis performed that the expression of ... under the promoter-rbs combination increases the dodecanal dehydrogenase activity in E. coli cell extracts 2-fold; whereas the expression of the same protein using the part J13002 increases the same activity 3-fold. Moreover, the enzymatic activities measured are equivalent to % and % of the Pseudomonas putida aldehyde dehydrogenase activity. <br />
<br />
<br />
[[Image:TUDelftALDH_final.jpg|600px]]<br />
<br />
<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results
Team:TU Delft/Project/alkane-degradation/results
2010-10-26T21:10:11Z
<p>Jcnossen: /* Resting cell assays */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===Characterization of the alkane hydroxylase system===<br />
====Growth analysis====<br />
We attempted to culture our recombinant AH-carrying ''E.coli'' K12 strains ([http://partsregistry.org/Part:BBa_K398014 BBa_K398014]) on 1% v/v octanol or 1% v/v dodecane. Negligible growth was observed.<br />
<br />
====Resting-cell assays====<br />
The [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Resting-cell_assays_for_E.coli resting cell assays] were performed on our recombinant AH-carrying ''E.coli'' K12 cells ([http://partsregistry.org/Part:BBa_K398014 BBa_K398014]). 100 micromoles of octane was added to 6 mL of growth-stalled cells (1.5 mg cell dry weight total) and incubated at 37 degrees with shaking o/n. The organic phase was [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Ethyl_acetate_extraction_protocol extracted] using EtOAc and analysed by [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=General_gas_chromatography_program_for_alkanes_and_alkanols gas chromatography].<br />
<br />
The following are examples of typical chromatographs obtained:<br />
<br />
<html><span style="display:block;width:100%;clear:both;"> </span></html><br />
[[Image:TUDelft_J13-AH.png|400px|thumb|left|Chromatograph of the negative control for the resting cell assay of the alkane hydroxylase system. The negative control consisted of ''E.coli'' K12 carrying BBa_J13002 in pSB1A2.]]<br />
<br />
[[Image:014 AH.png|400px|left|thumb|Chromatograph of ''E.coli'' K12 strain carrying the AH system.]]<br />
<html><span style="display:block;width:100%;clear:both;"> </span></html><br />
<br />
The peaks shown in the chromatographs belong to the following compounds:<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Retention time [min]</b><br />
|<b>Compound</b><br />
|-<br />
|~2.2<br />
|Ethyl acetate (solvent)<br />
|-<br />
|~5.2<br />
|Octane (substrate)<br />
|-<br />
|~11.8<br />
|Undecane (internal standard, 0.1% v/v of solvent)<br />
|}<br />
<br />
The surface areas of the peaks correspond to the amount of molecules present in the sample. Due to the fact that many factors play a role in establishing an equilibrium between the aqueous and organic phase once the EtOAC is added, it's general procedure to add an internal standard to the EtOAc. In our experiment we decided to use a 0.1% vol/vol of undecane in EtOAc. The <b>ratio</b> between the surface areas of octane and undecane can now give an indication of the amount of octane still present in the sample and be very useful for comparisons between chromatographs.<br />
<br />
The following table contains the surface areas found for the peaks of octane and undecane for each obtained chromatograph:<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Octane surface area</b><br />
|<b>Undecane surface area</b><br />
|<b>Ratio octane/undecane</b><br />
|-<br />
|''E.coli'' K12 negative control<br />
|17903555<br />
|19939270<br />
|0.898<br />
|-<br />
|''E.coli'' K12 014#2<br />
|17426721<br />
|24262069<br />
|0.719<br />
|-<br />
|''E.coli'' K12 014#7<br />
|7824594<br />
|17320861<br />
|0.452<br />
|}<br />
<br />
===LadA===<br />
<br />
===Characterization of the Alcohol DeHydrogenase (ADH) system===<br />
<br />
=====Growth on alcohols as sole carbon source=====<br />
*As we described before, we tried to grow our strain ''Escherichia coli'' 018A ([http://partsregistry.org/Part:BBa_K398018 BBa_K398018] on the plasmid pSB1A2) on the alkanols Octanol-1 and Dodecanol-1 (1% V/V on M9 medium without any other carbon source). No growth was observed after 48 hours. We abandoned this experiment after this result.<br />
<br />
=====Resting cell assays=====<br />
*We grew ''Escherichia coli'' 018A and ''Escherichia coli'' negative control (Biobrick BBa_J13002 on plasmid pSB1A2 ) in 50 mL of M9 medium with glucose and CAS aminoacids.<br />
*The cells were harevested when the O.D. at 600nm was around 0.3; they were spun down at 4000 rpm, for 10 min at 4ºC. And the resting cell assays were prepared according to the [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Resting-cell_assays_for_E.coli standard protocol]<br />
<br />
{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1"; <br />
|'''Strain'''<br />
|'''O.D.'''<br />
|-<br />
|J13002<br />
|0.327<br />
|-<br />
|018A<br />
|0.338<br />
|}<br />
<br />
*After an overnight incubation at 37ºC, the organic phase was extracted using 3 mL of ethyl acetate (dodecane was used as internal standard). We tried to determine production of alkanals or alkanoic acids by Gas Chromatography measurements. The chromatograms are shown below:<br />
<br />
[[Image:TUDelftADH_chrom.jpg|750px|thumb|center|Typical chromatograms obtained after the resting cell assays: (A) Blank, (B) Negative control which is E. coli K12, (C) E. coli 018A (our recombinant strain) and (D) standard of the expected product.]]<br />
<br />
<br />
*According to our results there was no degradation of octanol-1 ='( , after this experiment the protocol was abandoned. We think that the cells lack of transporters for long-chain alcohols, we preferred to check the cell extract in order to know if there was any biological activity in the cytoplasma.<br />
<br />
=====NADH production in cell extracts=====<br />
*'''EXPERIMENT 1'''<br />
Cells were cultured in 50mL of LB medium and harvested when the O.D. 600nm of the culture was around 0.6. Two different cultures of the recominant strain and the negative control were prepared.<br />
{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1"<br />
|Strain<br />
|O.D. 600nm<br />
|-<br />
|J13002 (1)<br />
|0.654<br />
|-<br />
|J13002 (2)<br />
|0.621 <br />
|-<br />
|018A (1)<br />
|0.615<br />
|-<br />
|018A (2)<br />
|0.580<br />
|}<br />
Cytoplasmic proteins were extracted using our standard [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Preparing_cell_lysates_for_enzyme_kinetics_measurements protocol]. And a standard curve for protein quantification was prepared.<br />
[[Image:TUDelftADH_bradford.jpg|600px|thumb|center|Standard curve for total protein quantification.]]<br />
The total protein of each sample was quantified using 20uL of cell extract, the results are shown below.<br />
{| style="color:black; background-color:white;" cellpadding="4" cellspacing="0" border="1"<br />
|Sample<br />
|'''O.D. 562 nm'''<br />
|'''Total ug of protein'''<br />
|'''ug/uL'''<br />
|-<br />
|J13002(1A) <br />
|0.135 <br />
|10.393 <br />
|1.0393<br />
|-<br />
|J13002 (1B) <br />
|0.135 <br />
|10.393 <br />
|1.0393<br />
|-<br />
|J13002 (2A) <br />
|0.128 <br />
|9.7363 <br />
|0.9736<br />
|-<br />
|J13002 (2B) <br />
|0.129 <br />
|9.8302 <br />
|0.9830<br />
|-<br />
|018A (1A) <br />
|0.097 <br />
|6.8275 <br />
|0.6827<br />
|-<br />
|018A (1B) <br />
|0.091 <br />
|6.2645 <br />
|0.6264<br />
|-<br />
|018A (2A) <br />
|0.092 <br />
|6.3583 <br />
|0.6358<br />
|-<br />
|018A (2B) <br />
|0.092 <br />
|6.3583 <br />
|0.6358<br />
|}<br />
The alcohol dehydrogenase activity was measured using the standard [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Alcohol.2FAldehyde_dehydrogenase_activity_assays protocol] and Dodecanol-1 as substrate. You can download our raw data by clicking on the link: [[ Image:TUDelft_ADH_raw.xls]]<br />
After the data treatment, the results that we obtained were the following:<br />
[[Image:TUDelftADH_pH95.jpg|400px]]<br />
[[Image:TUDelftADH_pH8.jpg|400px]]<br />
<br />
*All the enzyme activities were normalized to the TOTAL amount of protein in the cell extract. The results are shown in katal per mg of protein in the cell extract and Enzymatic Units per mg of protein in the cell extract. <br />
<br />
<html><br />
<table><br />
<tr><td></td><td colspan=4 style="text-align: center;"><b>pH=9.5</b></td><td colspan=4 style="text-align: center;"><b>pH=8</b></td></tr> <br />
<tr><td>&nbsp;</td><td colspan=2 style="text-align: center;"><b>Heat</b></td><td colspan=2 style="text-align: center;"><b>Native</b></td><td colspan=2 style="text-align: center;"><b>Heat</b></td><td colspan=2 style="text-align: center;"><b>Native</b></td></tr> <br />
<tr><td>&nbsp;</td><td>J13002</td><td>018A</td><td>J13002</td><td>018A</td><td>J13002</td><td>018A</td><td>J13002</td><td>018A</td></tr> <br />
<tr><td><b>kat/mg</b></td><td>6,156E-12</td><td>1,941E-11</td><td>1,053E-11</td><td>2,806E-11</td><td>3,211E-12</td><td>4,496E-11</td><td>1,302E-11</td><td>2,638E-11</td></tr> <br />
<tr><td><b>U/mg</b></td><td>3,694E-04</td><td>1,091E-03</td><td>6,317E-04</td><td>1,694E-03</td><td>1,927E-04</td><td>2,139E-03</td><td>7,811E-04</td><td>1,608E-03</td></tr> <br />
<tr><td><b>stdev</b></td><td>1,157E-12</td><td>4,579E-12</td><td>1,343E-12</td><td>9,961E-13</td><td>6,395E-12</td><td>2,331E-11</td><td>6,511E-13</td><td>9,961E-13</td></tr> <br />
<tr><td><b>Improvement</b></td><td>-</td><td>215,34%</td><td>-</td><td>166,51%</td><td>-</td><td>1299,87%</td><td>-</td><td>166,51%</td></tr> <br />
<tr><td><b>ttest</b></td><td>-</td><td>0,9963</td><td>-</td><td>1,0000</td><td>-</td><td>0,9988</td><td>-</td><td>1,000</td></tr><br />
</table><br />
</html><br />
<br />
You can download our results file here: [[ Image:TUDelft_ADH_results.xls]]<br />
Maybe you are not familiar with the term "katal", officially the katal is the STANDARD UNIT FOR CATALYTIC ACTIVITY in the International System of Units; the katal is defined as the amount of enzyme that converts 1 mol of substrate each second. [http://www.clinchem.org/cgi/content/full/48/3/586 Click here] to know more about it, and because people are not that familiar with the term, we report the activities in U/mg. One enzyme activity unit is defined as the amount of protein that converts 1 umol of substrate each minute.<br />
T-tests were performed in order to show that there is a difference between E. coli and our recombinant strain; a result for t-test of 1 means that both samples are statistically different; whereas a t-test of nearly 0 means that samples belong to the same group (no difference between both). Our level of significance is 0.025, in two tailed test. That means that if our t-test result is 0.95 we conclude that both samples are statistically different, whereas 0.949 or lower means that both samples are not statistically different.<br />
<br />
<br />
*'''EXPERIMENT 2'''<br />
We ran a second experiment at pH 9.5, with two other strains that seemed to be positive in the colony PCR test. In this test we also added two extra strains of E. coli in order to have more data for the statistical analysis, the strains used were J13002 and 331 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K398331 BBa_K398331] in pSB1A2). We followed the same procedure as in the experiment 1, the results obtained were the following:<br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th></th><th>kat/mg</th><th>U/mg</th><th>STDEV</th><th>Improvement</th><th>T-test</th><th>Relative to putida</th><th>&nbsp;</th></tr> <tr><td>E. Coli</td><td>8.75E-12</td><td>5.25E-04</td><td>8.18987E-12</td><td>-</td><td>-</td><td>0.90%</td><td>&nbsp;</td></tr> <tr><td>018A-2</td><td>9.30E-12</td><td>5.58E-04</td><td>1.84877E-12</td><td>6.26%</td><td>0.4708</td><td>0.96%</td><td>&nbsp;</td></tr> <tr><td>018A-3</td><td>3.05E-11</td><td>1.83E-03</td><td>6.27802E-12</td><td>249.14%</td><td>0.9808</td><td>3.15%</td><td>&nbsp;</td></tr> <tr><td>018A-4</td><td>8.26E-12</td><td>4.96E-04</td><td>1.8256E-12</td><td>-5.59%</td><td>0.4053</td><td>0.85%</td><td>&nbsp;</td></tr> <tr><td>P. Putida</td><td>9.69E-10</td><td>5.82E-02</td><td>2.09109E-10</td><td>-</td><td>0.9883</td><td>100.00%</td><td></td></tr></table><br />
<br />
According to these results, the other two strains (018A-2 and 018A-4) do not functionally express the part BBa_K398018. However, these results confirm our findings from the first experiment. You can download our result in this link: [[ Image:TUDelft_ADH_results2.xls]] You can also find a summary in this file:[[ Image:TUDelft_ADH_summary.xls]]<br />
<br />
==== Conclusions ====<br />
<br />
[[Image:TUDelftADH_final.jpg|600px]]<br />
<br />
According to our results, a normal ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain 018A has an activity of 2.93e-11 kat/mg (1.76 mU/mg). According to the statistical analysis, the enzymatic activities of both strains are statistically different at confidence level of 0.95 which means that the part BBa_K398018 increases 2 times the alcohol dehydrogenase activity in the cell extract. We can conclude from our data that the part has a biological activity ''in vitro''.<br />
<br />
We still don't know about the ''in vivo'' activity of this part, maybe there is a lack of long-chain alcohol transporter proteins or it is necessary to over-express this part in order to see/measure any biological activity.<br />
<br />
===Characterization of the ALdehyde DeHydrogenase system===<br />
<br />
=====Growth on alcohols as sole carbon source=====<br />
As we described before, we tried to grow our recombinant strains ''Escherichia coli'' 029A ([http://partsregistry.org/Part:BBa_K398029 BBa_K398029] on the plasmid pSB1A2) and ''Escherichia coli'' 030A ([http://partsregistry.org/Part:BBa_K398030 BBa_K398030] on the plasmid pSB1A2) on Octanal and Dodecanal. No growth was observed after 48 hours. We abandoned this experiment after this result.<br />
<br />
=====Resting cell assays=====<br />
*We grew 'Escherichia coli'' 029A, ''Escherichia coli'' 030Aand ''Escherichia coli'' negative control (Biobrick BBa_J13002 on plasmid pSB1A2 ) in 50 mL of M9 medium with glucose and CAS aminoacids.<br />
*The cells were harevested when the O.D. at 600nm was around 0.3; they were spun down at 4000 rpm, for 10 min at 4ºC. And the resting cell assays were prepared according to the [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Resting-cell_assays_for_E.coli standard protocol]<br />
<br />
{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1"; <br />
|'''Strain'''<br />
|'''O.D.'''<br />
|-<br />
|J13002<br />
|0.327<br />
|-<br />
|029A<br />
|0.340<br />
|-<br />
|030A<br />
|0.330<br />
|}<br />
<br />
'''Note''': This experiment was run in parallel with the resting cell assays for 018A, the negative control culture of the strain J13002 used was the same for both experiments.<br />
<br />
*After an overnight incubation at 37ºC with the substrate (octanal), the organic phase was extracted using 3 mL of ethyl acetate (dodecane was used as internal standard). We tried to determine production of alkanoic acids by Gas Chromatography measurements. The chromatograms are shown below:<br />
<br />
[[Image:TUDelftALDH_chrom.jpg|750px|thumb|center| Typical chromatograms obtained after the resting cell assays: (A) Blank, (B) Negative control which is E. coli K12, (C) E. coli 029A (our recombinant strain) and (D) E. coli 029A (our recombinant strain).]]<br />
<br />
<br />
*According to our results there was a peak reduction for octanal, however we were worried because of the fact that we didn't see the peak of the product (dodecanoic acid) appearing. It could be that the cells are just storing the product inside the cell. The experiment required to much time in order to get more results: the extractions, preparation of triplicates, cultures, cell suspensions... etc. it was a work that would require an effort of all the team for a couple of weeks. We decided to go for something easier and faster to measure; since we had some experience with the NADH determinations that was our choice.<br />
<br />
==== NADH production in cell extracts====<br />
Cells were cultured in 50mL of LB medium and harvested when the O.D. 600nm of the culture was between 0.5-0.8. Two different cultures of each recominant strain and the negative control were prepared.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1"<br />
|Strain<br />
|O.D. 600nm<br />
|-<br />
|J13002 (1)<br />
|0.825<br />
|-<br />
|J13002 (2)<br />
|0.774 <br />
|-<br />
|029A (1)<br />
|0.746<br />
|-<br />
|029A (2)<br />
|0.753<br />
|-<br />
|030A (1)<br />
|0.532<br />
|-<br />
|030A (2)<br />
|0.532<br />
|}<br />
Cytoplasmic proteins were extracted using our standard [https://2010.igem.org/Team:TU_Delft#Preparing_cell_lysates_for_enzyme_kinetics_measurements protocol]. And a standard curve for protein quantification was prepared.<br />
<br />
[[Image:TUDelftALDH_bradford.jpg|600px|thumb|center|Standard curve for total protein quantification.]]<br />
<br />
The total protein of each sample was quantified using 20uL of cell extract, the results are shown below.<br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th></th><th>Total protein [ug]</th><th>ug/uL</th></tr> <tr><td>J1300</td><td>31.108</td><td>1.555</td></tr> <tr><td>029A (1)</td><td>19.974</td><td>0.999</td></tr> <tr><td>030A (1)</td><td>12.610</td><td>0.630</td></tr> <tr><td>029A (2)</td><td>12.854</td><td>0.782</td></tr> <tr><td>030 (2)</td><td>18.578</td><td>0.929</td></tr> <tr><td>P. Puitda</td><td>9.783</td><td>0.489</td></tr></table><br />
<br />
<br />
The aldehyde dehydrogenase activity was measured using the standard [https://2010.igem.org/Team:TU_Delft#Alcohol.2FAldehyde_dehydrogenase_activity_assays protocol] and Dodecanal as substrate.<br />
<br />
After the data treatment, the results that we obtained were the following:<br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th></th><th>&nbsp;</th><th>J13002</th><th>029A (1)</th><th>029A (2)</th><th>030A (1)</th><th>030A (2)</th><th>P. putida</th></tr> <tr><td>Specific activity</td><td>kat/mg</td><td>8.604E-11</td><td>1.470E-10</td><td>2.853E-10</td><td>2.433E-10</td><td>2.912E-10</td><td>6.362E-10</td></tr> <tr><td>&nbsp;</td><td>U/mg</td><td>5.162E-03</td><td>8.820E-03</td><td>1.712E-02</td><td>1.460E-02</td><td>1.747E-02</td><td>3.817E-02</td></tr> <tr><td>Standard deviation</td><td>&nbsp;</td><td>1.778E-12</td><td>6.187E-12</td><td>4.026E-11</td><td>3.539E-11</td><td>4.319E-11</td><td>3.361E-11</td></tr> <tr><td>Relative to (J13002)</td><td>&nbsp;</td><td>100.00%</td><td>170.86%</td><td>331.59%</td><td>282.79%</td><td>338.46%</td><td>739.41%</td></tr> <tr><td>Relative activity (P. putida)</td><td>&nbsp;</td><td>13.52%</td><td>23.11%</td><td>44.84%</td><td>38.25%</td><td>45.78%</td><td>100.00%</td></tr> <tr><td>T-test vs J13002</td><td>&nbsp;</td><td>-</td><td>0.9827</td><td>0.9599</td><td>0.9764</td><td>0.9976</td><td>0.9976</td></tr> <tr><td>T-test vs P. putida</td><td>&nbsp;</td><td>0.9999</td><td>1.0000</td><td>0.9998</td><td>0.9974</td><td>1.0000</td><td>-</td></tr> <tr><td>Average activity [kat/mg]</td><td>&nbsp;</td><td>8.604E-11</td><td>2.161E-10</td><td>&nbsp;</td><td>2.673E-10</td><td>&nbsp;</td><td>6.362E-10</td></tr></table><br />
<br />
You can download a file with our raw data, results anda summary here: [[ Image:TUDelft_ALDH_results.xls]]<br />
<br />
==== Conclusion====<br />
<br />
Our results suggest that the recombinant strains ''E. coli'' 029A and ''E. coli'' 030A functionally express our biobricks, we couldn't prove ''in vivo'' activity. However, it is clear from the statistical analysis performed that the expression of ... under the promoter-rbs combination increases the dodecanal dehydrogenase activity in E. coli cell extracts 2-fold; whereas the expression of the same protein using the part J13002 increases the same activity 3-fold. Moreover, the enzymatic activities measured are equivalent to % and % of the Pseudomonas putida aldehyde dehydrogenase activity. <br />
<br />
<br />
[[Image:TUDelftALDH_final.jpg|600px]]<br />
<br />
<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results
Team:TU Delft/Project/alkane-degradation/results
2010-10-26T21:08:42Z
<p>Jcnossen: /* NADH production in cell extracts */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===Characterization of the alkane hydroxylase system===<br />
====Growth analysis====<br />
We attempted to culture our recombinant AH-carrying ''E.coli'' K12 strains ([http://partsregistry.org/Part:BBa_K398014 BBa_K398014]) on 1% v/v octanol or 1% v/v dodecane. Negligible growth was observed.<br />
<br />
====Resting-cell assays====<br />
The [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Resting-cell_assays_for_E.coli resting cell assays] were performed on our recombinant AH-carrying ''E.coli'' K12 cells ([http://partsregistry.org/Part:BBa_K398014 BBa_K398014]). 100 micromoles of octane was added to 6 mL of growth-stalled cells (1.5 mg cell dry weight total) and incubated at 37 degrees with shaking o/n. The organic phase was [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Ethyl_acetate_extraction_protocol extracted] using EtOAc and analysed by [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=General_gas_chromatography_program_for_alkanes_and_alkanols gas chromatography].<br />
<br />
The following are examples of typical chromatographs obtained:<br />
<br />
<html><span style="display:block;width:100%;clear:both;"> </span></html><br />
[[Image:TUDelft_J13-AH.png|400px|thumb|left|Chromatograph of the negative control for the resting cell assay of the alkane hydroxylase system. The negative control consisted of ''E.coli'' K12 carrying BBa_J13002 in pSB1A2.]]<br />
<br />
[[Image:014 AH.png|400px|left|thumb|Chromatograph of ''E.coli'' K12 strain carrying the AH system.]]<br />
<html><span style="display:block;width:100%;clear:both;"> </span></html><br />
<br />
The peaks shown in the chromatographs belong to the following compounds:<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Retention time [min]</b><br />
|<b>Compound</b><br />
|-<br />
|~2.2<br />
|Ethyl acetate (solvent)<br />
|-<br />
|~5.2<br />
|Octane (substrate)<br />
|-<br />
|~11.8<br />
|Undecane (internal standard, 0.1% v/v of solvent)<br />
|}<br />
<br />
The surface areas of the peaks correspond to the amount of molecules present in the sample. Due to the fact that many factors play a role in establishing an equilibrium between the aqueous and organic phase once the EtOAC is added, it's general procedure to add an internal standard to the EtOAc. In our experiment we decided to use a 0.1% vol/vol of undecane in EtOAc. The <b>ratio</b> between the surface areas of octane and undecane can now give an indication of the amount of octane still present in the sample and be very useful for comparisons between chromatographs.<br />
<br />
The following table contains the surface areas found for the peaks of octane and undecane for each obtained chromatograph:<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Octane surface area</b><br />
|<b>Undecane surface area</b><br />
|<b>Ratio octane/undecane</b><br />
|-<br />
|''E.coli'' K12 negative control<br />
|17903555<br />
|19939270<br />
|0.898<br />
|-<br />
|''E.coli'' K12 014#2<br />
|17426721<br />
|24262069<br />
|0.719<br />
|-<br />
|''E.coli'' K12 014#7<br />
|7824594<br />
|17320861<br />
|0.452<br />
|}<br />
<br />
===LadA===<br />
<br />
===Characterization of the Alcohol DeHydrogenase (ADH) system===<br />
<br />
=====Growth on alcohols as sole carbon source=====<br />
*As we described before, we tried to grow our strain ''Escherichia coli'' 018A ([http://partsregistry.org/Part:BBa_K398018 BBa_K398018] on the plasmid pSB1A2) on the alkanols Octanol-1 and Dodecanol-1 (1% V/V on M9 medium without any other carbon source). No growth was observed after 48 hours. We abandoned this experiment after this result.<br />
<br />
=====Resting cell assays=====<br />
*We grew ''Escherichia coli'' 018A and ''Escherichia coli'' negative control (Biobrick BBa_J13002 on plasmid pSB1A2 ) in 50 mL of M9 medium with glucose and CAS aminoacids.<br />
*The cells were harevested when the O.D. at 600nm was around 0.3; they were spun down at 4000 rpm, for 10 min at 4ºC. And the resting cell assays were prepared according to the [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Resting-cell_assays_for_E.coli standard protocol]<br />
<br />
{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1"; <br />
|'''Strain'''<br />
|'''O.D.'''<br />
|-<br />
|J13002<br />
|0.327<br />
|-<br />
|018A<br />
|0.338<br />
|}<br />
<br />
*After an overnight incubation at 37ºC, the organic phase was extracted using 3 mL of ethyl acetate (dodecane was used as internal standard). We tried to determine production of alkanals or alkanoic acids by Gas Chromatography measurements. The chromatograms are shown below:<br />
<br />
[[Image:TUDelftADH_chrom.jpg|750px|thumb|center|Typical chromatograms obtained after the resting cell assays: (A) Blank, (B) Negative control which is E. coli K12, (C) E. coli 018A (our recombinant strain) and (D) standard of the expected product.]]<br />
<br />
<br />
*According to our results there was no degradation of octanol-1 ='( , after this experiment the protocol was abandoned. We think that the cells lack of transporters for long-chain alcohols, we preferred to check the cell extract in order to know if there was any biological activity in the cytoplasma.<br />
<br />
=====NADH production in cell extracts=====<br />
*'''EXPERIMENT 1'''<br />
Cells were cultured in 50mL of LB medium and harvested when the O.D. 600nm of the culture was around 0.6. Two different cultures of the recominant strain and the negative control were prepared.<br />
{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1"<br />
|Strain<br />
|O.D. 600nm<br />
|-<br />
|J13002 (1)<br />
|0.654<br />
|-<br />
|J13002 (2)<br />
|0.621 <br />
|-<br />
|018A (1)<br />
|0.615<br />
|-<br />
|018A (2)<br />
|0.580<br />
|}<br />
Cytoplasmic proteins were extracted using our standard [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Preparing_cell_lysates_for_enzyme_kinetics_measurements protocol]. And a standard curve for protein quantification was prepared.<br />
[[Image:TUDelftADH_bradford.jpg|600px|thumb|center|Standard curve for total protein quantification.]]<br />
The total protein of each sample was quantified using 20uL of cell extract, the results are shown below.<br />
{| style="color:black; background-color:white;" cellpadding="4" cellspacing="0" border="1"<br />
|Sample<br />
|'''O.D. 562 nm'''<br />
|'''Total ug of protein'''<br />
|'''ug/uL'''<br />
|-<br />
|J13002(1A) <br />
|0.135 <br />
|10.393 <br />
|1.0393<br />
|-<br />
|J13002 (1B) <br />
|0.135 <br />
|10.393 <br />
|1.0393<br />
|-<br />
|J13002 (2A) <br />
|0.128 <br />
|9.7363 <br />
|0.9736<br />
|-<br />
|J13002 (2B) <br />
|0.129 <br />
|9.8302 <br />
|0.9830<br />
|-<br />
|018A (1A) <br />
|0.097 <br />
|6.8275 <br />
|0.6827<br />
|-<br />
|018A (1B) <br />
|0.091 <br />
|6.2645 <br />
|0.6264<br />
|-<br />
|018A (2A) <br />
|0.092 <br />
|6.3583 <br />
|0.6358<br />
|-<br />
|018A (2B) <br />
|0.092 <br />
|6.3583 <br />
|0.6358<br />
|}<br />
The alcohol dehydrogenase activity was measured using the standard [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Alcohol.2FAldehyde_dehydrogenase_activity_assays protocol] and Dodecanol-1 as substrate. You can download our raw data by clicking on the link: [[ Image:TUDelft_ADH_raw.xls]]<br />
After the data treatment, the results that we obtained were the following:<br />
[[Image:TUDelftADH_pH95.jpg|400px]]<br />
[[Image:TUDelftADH_pH8.jpg|400px]]<br />
<br />
*All the enzyme activities were normalized to the TOTAL amount of protein in the cell extract. The results are shown in katal per mg of protein in the cell extract and Enzymatic Units per mg of protein in the cell extract. <br />
<br />
<html><br />
<table><br />
<tr><td></td><td colspan=4 style="text-align: center;"><b>pH=9.5</b></td><td colspan=4 style="text-align: center;"><b>pH=8</b></td></tr> <br />
<tr><td>&nbsp;</td><td colspan=2 style="text-align: center;"><b>Heat</b></td><td colspan=2 style="text-align: center;"><b>Native</b></td><td colspan=2 style="text-align: center;"><b>Heat</b></td><td colspan=2 style="text-align: center;"><b>Native</b></td></tr> <br />
<tr><td>&nbsp;</td><td>J13002</td><td>018A</td><td>J13002</td><td>018A</td><td>J13002</td><td>018A</td><td>J13002</td><td>018A</td></tr> <br />
<tr><td><b>kat/mg</b></td><td>6,156E-12</td><td>1,941E-11</td><td>1,053E-11</td><td>2,806E-11</td><td>3,211E-12</td><td>4,496E-11</td><td>1,302E-11</td><td>2,638E-11</td></tr> <br />
<tr><td><b>U/mg</b></td><td>3,694E-04</td><td>1,091E-03</td><td>6,317E-04</td><td>1,694E-03</td><td>1,927E-04</td><td>2,139E-03</td><td>7,811E-04</td><td>1,608E-03</td></tr> <br />
<tr><td><b>stdev</b></td><td>1,157E-12</td><td>4,579E-12</td><td>1,343E-12</td><td>9,961E-13</td><td>6,395E-12</td><td>2,331E-11</td><td>6,511E-13</td><td>9,961E-13</td></tr> <br />
<tr><td><b>Improvement</b></td><td>-</td><td>215,34%</td><td>-</td><td>166,51%</td><td>-</td><td>1299,87%</td><td>-</td><td>166,51%</td></tr> <br />
<tr><td><b>ttest</b></td><td>-</td><td>0,9963</td><td>-</td><td>1,0000</td><td>-</td><td>0,9988</td><td>-</td><td>1,000</td></tr><br />
</table><br />
</html><br />
<br />
You can download our results file here: [[ Image:TUDelft_ADH_results.xls]]<br />
Maybe you are not familiar with the term "katal", officially the katal is the STANDARD UNIT FOR CATALYTIC ACTIVITY in the International System of Units; the katal is defined as the amount of enzyme that converts 1 mol of substrate each second. [http://www.clinchem.org/cgi/content/full/48/3/586 Click here] to know more about it, and because people are not that familiar with the term, we report the activities in U/mg. One enzyme activity unit is defined as the amount of protein that converts 1 umol of substrate each minute.<br />
T-tests were performed in order to show that there is a difference between E. coli and our recombinant strain; a result for t-test of 1 means that both samples are statistically different; whereas a t-test of nearly 0 means that samples belong to the same group (no difference between both). Our level of significance is 0.025, in two tailed test. That means that if our t-test result is 0.95 we conclude that both samples are statistically different, whereas 0.949 or lower means that both samples are not statistically different.<br />
<br />
<br />
*'''EXPERIMENT 2'''<br />
We ran a second experiment at pH 9.5, with two other strains that seemed to be positive in the colony PCR test. In this test we also added two extra strains of E. coli in order to have more data for the statistical analysis, the strains used were J13002 and 331 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K398331 BBa_K398331] in pSB1A2). We followed the same procedure as in the experiment 1, the results obtained were the following:<br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th></th><th>kat/mg</th><th>U/mg</th><th>STDEV</th><th>Improvement</th><th>T-test</th><th>Relative to putida</th><th>&nbsp;</th></tr> <tr><td>E. Coli</td><td>8.75E-12</td><td>5.25E-04</td><td>8.18987E-12</td><td>-</td><td>-</td><td>0.90%</td><td>&nbsp;</td></tr> <tr><td>018A-2</td><td>9.30E-12</td><td>5.58E-04</td><td>1.84877E-12</td><td>6.26%</td><td>0.4708</td><td>0.96%</td><td>&nbsp;</td></tr> <tr><td>018A-3</td><td>3.05E-11</td><td>1.83E-03</td><td>6.27802E-12</td><td>249.14%</td><td>0.9808</td><td>3.15%</td><td>&nbsp;</td></tr> <tr><td>018A-4</td><td>8.26E-12</td><td>4.96E-04</td><td>1.8256E-12</td><td>-5.59%</td><td>0.4053</td><td>0.85%</td><td>&nbsp;</td></tr> <tr><td>P. Putida</td><td>9.69E-10</td><td>5.82E-02</td><td>2.09109E-10</td><td>-</td><td>0.9883</td><td>100.00%</td><td></td></tr></table><br />
<br />
According to these results, the other two strains (018A-2 and 018A-4) do not functionally express the part BBa_K398018. However, these results confirm our findings from the first experiment. You can download our result in this link: [[ Image:TUDelft_ADH_results2.xls]] You can also find a summary in this file:[[ Image:TUDelft_ADH_summary.xls]]<br />
<br />
==== Conclusions ====<br />
<br />
[[Image:TUDelftADH_final.jpg|600px]]<br />
<br />
According to our results, a normal ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain 018A has an activity of 2.93e-11 kat/mg (1.76 mU/mg). According to the statistical analysis, the enzymatic activities of both strains are statistically different at confidence level of 0.95 which means that the part BBa_K398018 increases 2 times the alcohol dehydrogenase activity in the cell extract. We can conclude from our data that the part has a biological activity ''in vitro''.<br />
<br />
We still don't know about the ''in vivo'' activity of this part, maybe there is a lack of long-chain alcohol transporter proteins or it is necessary to over-express this part in order to see/measure any biological activity.<br />
<br />
===Characterization of the ALdehyde DeHydrogenase system===<br />
<br />
=====Growth on alcohols as sole carbon source=====<br />
As we described before, we tried to grow our recombinant strains ''Escherichia coli'' 029A ([http://partsregistry.org/Part:BBa_K398029 BBa_K398029] on the plasmid pSB1A2) and ''Escherichia coli'' 030A ([http://partsregistry.org/Part:BBa_K398030 BBa_K398030] on the plasmid pSB1A2) on Octanal and Dodecanal. No growth was observed after 48 hours. We abandoned this experiment after this result.<br />
<br />
=====Resting cell assays=====<br />
*We grew 'Escherichia coli'' 029A, ''Escherichia coli'' 030Aand ''Escherichia coli'' negative control (Biobrick BBa_J13002 on plasmid pSB1A2 ) in 50 mL of M9 medium with glucose and CAS aminoacids.<br />
*The cells were harevested when the O.D. at 600nm was around 0.3; they were spun down at 4000 rpm, for 10 min at 4ºC. And the resting cell assays were prepared according to the [https://2010.igem.org/Team:TU_Delft#Resting-cell_assays_for_E.coli standard protocol]<br />
<br />
{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1"; <br />
|'''Strain'''<br />
|'''O.D.'''<br />
|-<br />
|J13002<br />
|0.327<br />
|-<br />
|029A<br />
|0.340<br />
|-<br />
|030A<br />
|0.330<br />
|}<br />
<br />
'''Note''': This experiment was run in parallel with the resting cell assays for 018A, the negative control culture of the strain J13002 used was the same for both experiments.<br />
<br />
*After an overnight incubation at 37ºC with the substrate (octanal), the organic phase was extracted using 3 mL of ethyl acetate (dodecane was used as internal standard). We tried to determine production of alkanoic acids by Gas Chromatography measurements. The chromatograms are shown below:<br />
<br />
[[Image:TUDelftALDH_chrom.jpg|750px|thumb|center| Typical chromatograms obtained after the resting cell assays: (A) Blank, (B) Negative control which is E. coli K12, (C) E. coli 029A (our recombinant strain) and (D) E. coli 029A (our recombinant strain).]]<br />
<br />
<br />
*According to our results there was a peak reduction for octanal, however we were worried because of the fact that we didn't see the peak of the product (dodecanoic acid) appearing. It could be that the cells are just storing the product inside the cell. The experiment required to much time in order to get more results: the extractions, preparation of triplicates, cultures, cell suspensions... etc. it was a work that would require an effort of all the team for a couple of weeks. We decided to go for something easier and faster to measure; since we had some experience with the NADH determinations that was our choice.<br />
<br />
==== NADH production in cell extracts====<br />
Cells were cultured in 50mL of LB medium and harvested when the O.D. 600nm of the culture was between 0.5-0.8. Two different cultures of each recominant strain and the negative control were prepared.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1"<br />
|Strain<br />
|O.D. 600nm<br />
|-<br />
|J13002 (1)<br />
|0.825<br />
|-<br />
|J13002 (2)<br />
|0.774 <br />
|-<br />
|029A (1)<br />
|0.746<br />
|-<br />
|029A (2)<br />
|0.753<br />
|-<br />
|030A (1)<br />
|0.532<br />
|-<br />
|030A (2)<br />
|0.532<br />
|}<br />
Cytoplasmic proteins were extracted using our standard [https://2010.igem.org/Team:TU_Delft#Preparing_cell_lysates_for_enzyme_kinetics_measurements protocol]. And a standard curve for protein quantification was prepared.<br />
<br />
[[Image:TUDelftALDH_bradford.jpg|600px|thumb|center|Standard curve for total protein quantification.]]<br />
<br />
The total protein of each sample was quantified using 20uL of cell extract, the results are shown below.<br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th></th><th>Total protein [ug]</th><th>ug/uL</th></tr> <tr><td>J1300</td><td>31.108</td><td>1.555</td></tr> <tr><td>029A (1)</td><td>19.974</td><td>0.999</td></tr> <tr><td>030A (1)</td><td>12.610</td><td>0.630</td></tr> <tr><td>029A (2)</td><td>12.854</td><td>0.782</td></tr> <tr><td>030 (2)</td><td>18.578</td><td>0.929</td></tr> <tr><td>P. Puitda</td><td>9.783</td><td>0.489</td></tr></table><br />
<br />
<br />
The aldehyde dehydrogenase activity was measured using the standard [https://2010.igem.org/Team:TU_Delft#Alcohol.2FAldehyde_dehydrogenase_activity_assays protocol] and Dodecanal as substrate.<br />
<br />
After the data treatment, the results that we obtained were the following:<br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th></th><th>&nbsp;</th><th>J13002</th><th>029A (1)</th><th>029A (2)</th><th>030A (1)</th><th>030A (2)</th><th>P. putida</th></tr> <tr><td>Specific activity</td><td>kat/mg</td><td>8.604E-11</td><td>1.470E-10</td><td>2.853E-10</td><td>2.433E-10</td><td>2.912E-10</td><td>6.362E-10</td></tr> <tr><td>&nbsp;</td><td>U/mg</td><td>5.162E-03</td><td>8.820E-03</td><td>1.712E-02</td><td>1.460E-02</td><td>1.747E-02</td><td>3.817E-02</td></tr> <tr><td>Standard deviation</td><td>&nbsp;</td><td>1.778E-12</td><td>6.187E-12</td><td>4.026E-11</td><td>3.539E-11</td><td>4.319E-11</td><td>3.361E-11</td></tr> <tr><td>Relative to (J13002)</td><td>&nbsp;</td><td>100.00%</td><td>170.86%</td><td>331.59%</td><td>282.79%</td><td>338.46%</td><td>739.41%</td></tr> <tr><td>Relative activity (P. putida)</td><td>&nbsp;</td><td>13.52%</td><td>23.11%</td><td>44.84%</td><td>38.25%</td><td>45.78%</td><td>100.00%</td></tr> <tr><td>T-test vs J13002</td><td>&nbsp;</td><td>-</td><td>0.9827</td><td>0.9599</td><td>0.9764</td><td>0.9976</td><td>0.9976</td></tr> <tr><td>T-test vs P. putida</td><td>&nbsp;</td><td>0.9999</td><td>1.0000</td><td>0.9998</td><td>0.9974</td><td>1.0000</td><td>-</td></tr> <tr><td>Average activity [kat/mg]</td><td>&nbsp;</td><td>8.604E-11</td><td>2.161E-10</td><td>&nbsp;</td><td>2.673E-10</td><td>&nbsp;</td><td>6.362E-10</td></tr></table><br />
<br />
You can download a file with our raw data, results anda summary here: [[ Image:TUDelft_ALDH_results.xls]]<br />
<br />
==== Conclusion====<br />
<br />
Our results suggest that the recombinant strains ''E. coli'' 029A and ''E. coli'' 030A functionally express our biobricks, we couldn't prove ''in vivo'' activity. However, it is clear from the statistical analysis performed that the expression of ... under the promoter-rbs combination increases the dodecanal dehydrogenase activity in E. coli cell extracts 2-fold; whereas the expression of the same protein using the part J13002 increases the same activity 3-fold. Moreover, the enzymatic activities measured are equivalent to % and % of the Pseudomonas putida aldehyde dehydrogenase activity. <br />
<br />
<br />
[[Image:TUDelftALDH_final.jpg|600px]]<br />
<br />
<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results
Team:TU Delft/Project/alkane-degradation/results
2010-10-26T21:07:28Z
<p>Jcnossen: /* Resting cell assays */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===Characterization of the alkane hydroxylase system===<br />
====Growth analysis====<br />
We attempted to culture our recombinant AH-carrying ''E.coli'' K12 strains ([http://partsregistry.org/Part:BBa_K398014 BBa_K398014]) on 1% v/v octanol or 1% v/v dodecane. Negligible growth was observed.<br />
<br />
====Resting-cell assays====<br />
The [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Resting-cell_assays_for_E.coli resting cell assays] were performed on our recombinant AH-carrying ''E.coli'' K12 cells ([http://partsregistry.org/Part:BBa_K398014 BBa_K398014]). 100 micromoles of octane was added to 6 mL of growth-stalled cells (1.5 mg cell dry weight total) and incubated at 37 degrees with shaking o/n. The organic phase was [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Ethyl_acetate_extraction_protocol extracted] using EtOAc and analysed by [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=General_gas_chromatography_program_for_alkanes_and_alkanols gas chromatography].<br />
<br />
The following are examples of typical chromatographs obtained:<br />
<br />
<html><span style="display:block;width:100%;clear:both;"> </span></html><br />
[[Image:TUDelft_J13-AH.png|400px|thumb|left|Chromatograph of the negative control for the resting cell assay of the alkane hydroxylase system. The negative control consisted of ''E.coli'' K12 carrying BBa_J13002 in pSB1A2.]]<br />
<br />
[[Image:014 AH.png|400px|left|thumb|Chromatograph of ''E.coli'' K12 strain carrying the AH system.]]<br />
<html><span style="display:block;width:100%;clear:both;"> </span></html><br />
<br />
The peaks shown in the chromatographs belong to the following compounds:<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Retention time [min]</b><br />
|<b>Compound</b><br />
|-<br />
|~2.2<br />
|Ethyl acetate (solvent)<br />
|-<br />
|~5.2<br />
|Octane (substrate)<br />
|-<br />
|~11.8<br />
|Undecane (internal standard, 0.1% v/v of solvent)<br />
|}<br />
<br />
The surface areas of the peaks correspond to the amount of molecules present in the sample. Due to the fact that many factors play a role in establishing an equilibrium between the aqueous and organic phase once the EtOAC is added, it's general procedure to add an internal standard to the EtOAc. In our experiment we decided to use a 0.1% vol/vol of undecane in EtOAc. The <b>ratio</b> between the surface areas of octane and undecane can now give an indication of the amount of octane still present in the sample and be very useful for comparisons between chromatographs.<br />
<br />
The following table contains the surface areas found for the peaks of octane and undecane for each obtained chromatograph:<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Octane surface area</b><br />
|<b>Undecane surface area</b><br />
|<b>Ratio octane/undecane</b><br />
|-<br />
|''E.coli'' K12 negative control<br />
|17903555<br />
|19939270<br />
|0.898<br />
|-<br />
|''E.coli'' K12 014#2<br />
|17426721<br />
|24262069<br />
|0.719<br />
|-<br />
|''E.coli'' K12 014#7<br />
|7824594<br />
|17320861<br />
|0.452<br />
|}<br />
<br />
===LadA===<br />
<br />
===Characterization of the Alcohol DeHydrogenase (ADH) system===<br />
<br />
=====Growth on alcohols as sole carbon source=====<br />
*As we described before, we tried to grow our strain ''Escherichia coli'' 018A ([http://partsregistry.org/Part:BBa_K398018 BBa_K398018] on the plasmid pSB1A2) on the alkanols Octanol-1 and Dodecanol-1 (1% V/V on M9 medium without any other carbon source). No growth was observed after 48 hours. We abandoned this experiment after this result.<br />
<br />
=====Resting cell assays=====<br />
*We grew ''Escherichia coli'' 018A and ''Escherichia coli'' negative control (Biobrick BBa_J13002 on plasmid pSB1A2 ) in 50 mL of M9 medium with glucose and CAS aminoacids.<br />
*The cells were harevested when the O.D. at 600nm was around 0.3; they were spun down at 4000 rpm, for 10 min at 4ºC. And the resting cell assays were prepared according to the [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Resting-cell_assays_for_E.coli standard protocol]<br />
<br />
{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1"; <br />
|'''Strain'''<br />
|'''O.D.'''<br />
|-<br />
|J13002<br />
|0.327<br />
|-<br />
|018A<br />
|0.338<br />
|}<br />
<br />
*After an overnight incubation at 37ºC, the organic phase was extracted using 3 mL of ethyl acetate (dodecane was used as internal standard). We tried to determine production of alkanals or alkanoic acids by Gas Chromatography measurements. The chromatograms are shown below:<br />
<br />
[[Image:TUDelftADH_chrom.jpg|750px|thumb|center|Typical chromatograms obtained after the resting cell assays: (A) Blank, (B) Negative control which is E. coli K12, (C) E. coli 018A (our recombinant strain) and (D) standard of the expected product.]]<br />
<br />
<br />
*According to our results there was no degradation of octanol-1 ='( , after this experiment the protocol was abandoned. We think that the cells lack of transporters for long-chain alcohols, we preferred to check the cell extract in order to know if there was any biological activity in the cytoplasma.<br />
<br />
=====NADH production in cell extracts=====<br />
*'''EXPERIMENT 1'''<br />
Cells were cultured in 50mL of LB medium and harvested when the O.D. 600nm of the culture was around 0.6. Two different cultures of the recominant strain and the negative control were prepared.<br />
{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1"<br />
|Strain<br />
|O.D. 600nm<br />
|-<br />
|J13002 (1)<br />
|0.654<br />
|-<br />
|J13002 (2)<br />
|0.621 <br />
|-<br />
|018A (1)<br />
|0.615<br />
|-<br />
|018A (2)<br />
|0.580<br />
|}<br />
Cytoplasmic proteins were extracted using our standard [https://2010.igem.org/Team:TU_Delft#Preparing_cell_lysates_for_enzyme_kinetics_measurements protocol]. And a standard curve for protein quantification was prepared.<br />
[[Image:TUDelftADH_bradford.jpg|600px|thumb|center|Standard curve for total protein quantification.]]<br />
The total protein of each sample was quantified using 20uL of cell extract, the results are shown below.<br />
{| style="color:black; background-color:white;" cellpadding="4" cellspacing="0" border="1"<br />
|Sample<br />
|'''O.D. 562 nm'''<br />
|'''Total ug of protein'''<br />
|'''ug/uL'''<br />
|-<br />
|J13002(1A) <br />
|0.135 <br />
|10.393 <br />
|1.0393<br />
|-<br />
|J13002 (1B) <br />
|0.135 <br />
|10.393 <br />
|1.0393<br />
|-<br />
|J13002 (2A) <br />
|0.128 <br />
|9.7363 <br />
|0.9736<br />
|-<br />
|J13002 (2B) <br />
|0.129 <br />
|9.8302 <br />
|0.9830<br />
|-<br />
|018A (1A) <br />
|0.097 <br />
|6.8275 <br />
|0.6827<br />
|-<br />
|018A (1B) <br />
|0.091 <br />
|6.2645 <br />
|0.6264<br />
|-<br />
|018A (2A) <br />
|0.092 <br />
|6.3583 <br />
|0.6358<br />
|-<br />
|018A (2B) <br />
|0.092 <br />
|6.3583 <br />
|0.6358<br />
|}<br />
The alcohol dehydrogenase activity was measured using the standard [https://2010.igem.org/Team:TU_Delft#Alcohol.2FAldehyde_dehydrogenase_activity_assays protocol] and Dodecanol-1 as substrate. You can download our raw data by clicking on the link: [[ Image:TUDelft_ADH_raw.xls]]<br />
After the data treatment, the results that we obtained were the following:<br />
[[Image:TUDelftADH_pH95.jpg|400px]]<br />
[[Image:TUDelftADH_pH8.jpg|400px]]<br />
<br />
*All the enzyme activities were normalized to the TOTAL amount of protein in the cell extract. The results are shown in katal per mg of protein in the cell extract and Enzymatic Units per mg of protein in the cell extract. <br />
<br />
<html><br />
<table><br />
<tr><td></td><td colspan=4 style="text-align: center;"><b>pH=9.5</b></td><td colspan=4 style="text-align: center;"><b>pH=8</b></td></tr> <br />
<tr><td>&nbsp;</td><td colspan=2 style="text-align: center;"><b>Heat</b></td><td colspan=2 style="text-align: center;"><b>Native</b></td><td colspan=2 style="text-align: center;"><b>Heat</b></td><td colspan=2 style="text-align: center;"><b>Native</b></td></tr> <br />
<tr><td>&nbsp;</td><td>J13002</td><td>018A</td><td>J13002</td><td>018A</td><td>J13002</td><td>018A</td><td>J13002</td><td>018A</td></tr> <br />
<tr><td><b>kat/mg</b></td><td>6,156E-12</td><td>1,941E-11</td><td>1,053E-11</td><td>2,806E-11</td><td>3,211E-12</td><td>4,496E-11</td><td>1,302E-11</td><td>2,638E-11</td></tr> <br />
<tr><td><b>U/mg</b></td><td>3,694E-04</td><td>1,091E-03</td><td>6,317E-04</td><td>1,694E-03</td><td>1,927E-04</td><td>2,139E-03</td><td>7,811E-04</td><td>1,608E-03</td></tr> <br />
<tr><td><b>stdev</b></td><td>1,157E-12</td><td>4,579E-12</td><td>1,343E-12</td><td>9,961E-13</td><td>6,395E-12</td><td>2,331E-11</td><td>6,511E-13</td><td>9,961E-13</td></tr> <br />
<tr><td><b>Improvement</b></td><td>-</td><td>215,34%</td><td>-</td><td>166,51%</td><td>-</td><td>1299,87%</td><td>-</td><td>166,51%</td></tr> <br />
<tr><td><b>ttest</b></td><td>-</td><td>0,9963</td><td>-</td><td>1,0000</td><td>-</td><td>0,9988</td><td>-</td><td>1,000</td></tr><br />
</table><br />
</html><br />
<br />
You can download our results file here: [[ Image:TUDelft_ADH_results.xls]]<br />
Maybe you are not familiar with the term "katal", officially the katal is the STANDARD UNIT FOR CATALYTIC ACTIVITY in the International System of Units; the katal is defined as the amount of enzyme that converts 1 mol of substrate each second. [http://www.clinchem.org/cgi/content/full/48/3/586 Click here] to know more about it, and because people are not that familiar with the term, we report the activities in U/mg. One enzyme activity unit is defined as the amount of protein that converts 1 umol of substrate each minute.<br />
T-tests were performed in order to show that there is a difference between E. coli and our recombinant strain; a result for t-test of 1 means that both samples are statistically different; whereas a t-test of nearly 0 means that samples belong to the same group (no difference between both). Our level of significance is 0.025, in two tailed test. That means that if our t-test result is 0.95 we conclude that both samples are statistically different, whereas 0.949 or lower means that both samples are not statistically different.<br />
<br />
<br />
*'''EXPERIMENT 2'''<br />
We ran a second experiment at pH 9.5, with two other strains that seemed to be positive in the colony PCR test. In this test we also added two extra strains of E. coli in order to have more data for the statistical analysis, the strains used were J13002 and 331 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K398331 BBa_K398331] in pSB1A2). We followed the same procedure as in the experiment 1, the results obtained were the following:<br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th></th><th>kat/mg</th><th>U/mg</th><th>STDEV</th><th>Improvement</th><th>T-test</th><th>Relative to putida</th><th>&nbsp;</th></tr> <tr><td>E. Coli</td><td>8.75E-12</td><td>5.25E-04</td><td>8.18987E-12</td><td>-</td><td>-</td><td>0.90%</td><td>&nbsp;</td></tr> <tr><td>018A-2</td><td>9.30E-12</td><td>5.58E-04</td><td>1.84877E-12</td><td>6.26%</td><td>0.4708</td><td>0.96%</td><td>&nbsp;</td></tr> <tr><td>018A-3</td><td>3.05E-11</td><td>1.83E-03</td><td>6.27802E-12</td><td>249.14%</td><td>0.9808</td><td>3.15%</td><td>&nbsp;</td></tr> <tr><td>018A-4</td><td>8.26E-12</td><td>4.96E-04</td><td>1.8256E-12</td><td>-5.59%</td><td>0.4053</td><td>0.85%</td><td>&nbsp;</td></tr> <tr><td>P. Putida</td><td>9.69E-10</td><td>5.82E-02</td><td>2.09109E-10</td><td>-</td><td>0.9883</td><td>100.00%</td><td></td></tr></table><br />
<br />
According to these results, the other two strains (018A-2 and 018A-4) do not functionally express the part BBa_K398018. However, these results confirm our findings from the first experiment. You can download our result in this link: [[ Image:TUDelft_ADH_results2.xls]] You can also find a summary in this file:[[ Image:TUDelft_ADH_summary.xls]]<br />
<br />
==== Conclusions ====<br />
<br />
[[Image:TUDelftADH_final.jpg|600px]]<br />
<br />
According to our results, a normal ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain 018A has an activity of 2.93e-11 kat/mg (1.76 mU/mg). According to the statistical analysis, the enzymatic activities of both strains are statistically different at confidence level of 0.95 which means that the part BBa_K398018 increases 2 times the alcohol dehydrogenase activity in the cell extract. We can conclude from our data that the part has a biological activity ''in vitro''.<br />
<br />
We still don't know about the ''in vivo'' activity of this part, maybe there is a lack of long-chain alcohol transporter proteins or it is necessary to over-express this part in order to see/measure any biological activity.<br />
<br />
===Characterization of the ALdehyde DeHydrogenase system===<br />
<br />
=====Growth on alcohols as sole carbon source=====<br />
As we described before, we tried to grow our recombinant strains ''Escherichia coli'' 029A ([http://partsregistry.org/Part:BBa_K398029 BBa_K398029] on the plasmid pSB1A2) and ''Escherichia coli'' 030A ([http://partsregistry.org/Part:BBa_K398030 BBa_K398030] on the plasmid pSB1A2) on Octanal and Dodecanal. No growth was observed after 48 hours. We abandoned this experiment after this result.<br />
<br />
=====Resting cell assays=====<br />
*We grew 'Escherichia coli'' 029A, ''Escherichia coli'' 030Aand ''Escherichia coli'' negative control (Biobrick BBa_J13002 on plasmid pSB1A2 ) in 50 mL of M9 medium with glucose and CAS aminoacids.<br />
*The cells were harevested when the O.D. at 600nm was around 0.3; they were spun down at 4000 rpm, for 10 min at 4ºC. And the resting cell assays were prepared according to the [https://2010.igem.org/Team:TU_Delft#Resting-cell_assays_for_E.coli standard protocol]<br />
<br />
{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1"; <br />
|'''Strain'''<br />
|'''O.D.'''<br />
|-<br />
|J13002<br />
|0.327<br />
|-<br />
|029A<br />
|0.340<br />
|-<br />
|030A<br />
|0.330<br />
|}<br />
<br />
'''Note''': This experiment was run in parallel with the resting cell assays for 018A, the negative control culture of the strain J13002 used was the same for both experiments.<br />
<br />
*After an overnight incubation at 37ºC with the substrate (octanal), the organic phase was extracted using 3 mL of ethyl acetate (dodecane was used as internal standard). We tried to determine production of alkanoic acids by Gas Chromatography measurements. The chromatograms are shown below:<br />
<br />
[[Image:TUDelftALDH_chrom.jpg|750px|thumb|center| Typical chromatograms obtained after the resting cell assays: (A) Blank, (B) Negative control which is E. coli K12, (C) E. coli 029A (our recombinant strain) and (D) E. coli 029A (our recombinant strain).]]<br />
<br />
<br />
*According to our results there was a peak reduction for octanal, however we were worried because of the fact that we didn't see the peak of the product (dodecanoic acid) appearing. It could be that the cells are just storing the product inside the cell. The experiment required to much time in order to get more results: the extractions, preparation of triplicates, cultures, cell suspensions... etc. it was a work that would require an effort of all the team for a couple of weeks. We decided to go for something easier and faster to measure; since we had some experience with the NADH determinations that was our choice.<br />
<br />
==== NADH production in cell extracts====<br />
Cells were cultured in 50mL of LB medium and harvested when the O.D. 600nm of the culture was between 0.5-0.8. Two different cultures of each recominant strain and the negative control were prepared.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1"<br />
|Strain<br />
|O.D. 600nm<br />
|-<br />
|J13002 (1)<br />
|0.825<br />
|-<br />
|J13002 (2)<br />
|0.774 <br />
|-<br />
|029A (1)<br />
|0.746<br />
|-<br />
|029A (2)<br />
|0.753<br />
|-<br />
|030A (1)<br />
|0.532<br />
|-<br />
|030A (2)<br />
|0.532<br />
|}<br />
Cytoplasmic proteins were extracted using our standard [https://2010.igem.org/Team:TU_Delft#Preparing_cell_lysates_for_enzyme_kinetics_measurements protocol]. And a standard curve for protein quantification was prepared.<br />
<br />
[[Image:TUDelftALDH_bradford.jpg|600px|thumb|center|Standard curve for total protein quantification.]]<br />
<br />
The total protein of each sample was quantified using 20uL of cell extract, the results are shown below.<br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th></th><th>Total protein [ug]</th><th>ug/uL</th></tr> <tr><td>J1300</td><td>31.108</td><td>1.555</td></tr> <tr><td>029A (1)</td><td>19.974</td><td>0.999</td></tr> <tr><td>030A (1)</td><td>12.610</td><td>0.630</td></tr> <tr><td>029A (2)</td><td>12.854</td><td>0.782</td></tr> <tr><td>030 (2)</td><td>18.578</td><td>0.929</td></tr> <tr><td>P. Puitda</td><td>9.783</td><td>0.489</td></tr></table><br />
<br />
<br />
The aldehyde dehydrogenase activity was measured using the standard [https://2010.igem.org/Team:TU_Delft#Alcohol.2FAldehyde_dehydrogenase_activity_assays protocol] and Dodecanal as substrate.<br />
<br />
After the data treatment, the results that we obtained were the following:<br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th></th><th>&nbsp;</th><th>J13002</th><th>029A (1)</th><th>029A (2)</th><th>030A (1)</th><th>030A (2)</th><th>P. putida</th></tr> <tr><td>Specific activity</td><td>kat/mg</td><td>8.604E-11</td><td>1.470E-10</td><td>2.853E-10</td><td>2.433E-10</td><td>2.912E-10</td><td>6.362E-10</td></tr> <tr><td>&nbsp;</td><td>U/mg</td><td>5.162E-03</td><td>8.820E-03</td><td>1.712E-02</td><td>1.460E-02</td><td>1.747E-02</td><td>3.817E-02</td></tr> <tr><td>Standard deviation</td><td>&nbsp;</td><td>1.778E-12</td><td>6.187E-12</td><td>4.026E-11</td><td>3.539E-11</td><td>4.319E-11</td><td>3.361E-11</td></tr> <tr><td>Relative to (J13002)</td><td>&nbsp;</td><td>100.00%</td><td>170.86%</td><td>331.59%</td><td>282.79%</td><td>338.46%</td><td>739.41%</td></tr> <tr><td>Relative activity (P. putida)</td><td>&nbsp;</td><td>13.52%</td><td>23.11%</td><td>44.84%</td><td>38.25%</td><td>45.78%</td><td>100.00%</td></tr> <tr><td>T-test vs J13002</td><td>&nbsp;</td><td>-</td><td>0.9827</td><td>0.9599</td><td>0.9764</td><td>0.9976</td><td>0.9976</td></tr> <tr><td>T-test vs P. putida</td><td>&nbsp;</td><td>0.9999</td><td>1.0000</td><td>0.9998</td><td>0.9974</td><td>1.0000</td><td>-</td></tr> <tr><td>Average activity [kat/mg]</td><td>&nbsp;</td><td>8.604E-11</td><td>2.161E-10</td><td>&nbsp;</td><td>2.673E-10</td><td>&nbsp;</td><td>6.362E-10</td></tr></table><br />
<br />
You can download a file with our raw data, results anda summary here: [[ Image:TUDelft_ALDH_results.xls]]<br />
<br />
==== Conclusion====<br />
<br />
[[Image:TUDelftALDH_final.jpg|600px]]<br />
<br />
<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/results
Team:TU Delft/Project/alkane-degradation/results
2010-10-26T21:06:53Z
<p>Jcnossen: /* Resting-cell assays */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Results & Conclusions==<br />
[[Image:TUDelft_Alkane_degradation_route.png|600px|thumb|center|'''Figure 1''' – Schematic description of the alkane degradation pathway with the corresponding genes.]]<br />
<br />
===Characterization of the alkane hydroxylase system===<br />
====Growth analysis====<br />
We attempted to culture our recombinant AH-carrying ''E.coli'' K12 strains ([http://partsregistry.org/Part:BBa_K398014 BBa_K398014]) on 1% v/v octanol or 1% v/v dodecane. Negligible growth was observed.<br />
<br />
====Resting-cell assays====<br />
The [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Resting-cell_assays_for_E.coli resting cell assays] were performed on our recombinant AH-carrying ''E.coli'' K12 cells ([http://partsregistry.org/Part:BBa_K398014 BBa_K398014]). 100 micromoles of octane was added to 6 mL of growth-stalled cells (1.5 mg cell dry weight total) and incubated at 37 degrees with shaking o/n. The organic phase was [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Ethyl_acetate_extraction_protocol extracted] using EtOAc and analysed by [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=General_gas_chromatography_program_for_alkanes_and_alkanols gas chromatography].<br />
<br />
The following are examples of typical chromatographs obtained:<br />
<br />
<html><span style="display:block;width:100%;clear:both;"> </span></html><br />
[[Image:TUDelft_J13-AH.png|400px|thumb|left|Chromatograph of the negative control for the resting cell assay of the alkane hydroxylase system. The negative control consisted of ''E.coli'' K12 carrying BBa_J13002 in pSB1A2.]]<br />
<br />
[[Image:014 AH.png|400px|left|thumb|Chromatograph of ''E.coli'' K12 strain carrying the AH system.]]<br />
<html><span style="display:block;width:100%;clear:both;"> </span></html><br />
<br />
The peaks shown in the chromatographs belong to the following compounds:<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Retention time [min]</b><br />
|<b>Compound</b><br />
|-<br />
|~2.2<br />
|Ethyl acetate (solvent)<br />
|-<br />
|~5.2<br />
|Octane (substrate)<br />
|-<br />
|~11.8<br />
|Undecane (internal standard, 0.1% v/v of solvent)<br />
|}<br />
<br />
The surface areas of the peaks correspond to the amount of molecules present in the sample. Due to the fact that many factors play a role in establishing an equilibrium between the aqueous and organic phase once the EtOAC is added, it's general procedure to add an internal standard to the EtOAc. In our experiment we decided to use a 0.1% vol/vol of undecane in EtOAc. The <b>ratio</b> between the surface areas of octane and undecane can now give an indication of the amount of octane still present in the sample and be very useful for comparisons between chromatographs.<br />
<br />
The following table contains the surface areas found for the peaks of octane and undecane for each obtained chromatograph:<br />
<br />
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"<br />
|<b>Strain</b><br />
|<b>Octane surface area</b><br />
|<b>Undecane surface area</b><br />
|<b>Ratio octane/undecane</b><br />
|-<br />
|''E.coli'' K12 negative control<br />
|17903555<br />
|19939270<br />
|0.898<br />
|-<br />
|''E.coli'' K12 014#2<br />
|17426721<br />
|24262069<br />
|0.719<br />
|-<br />
|''E.coli'' K12 014#7<br />
|7824594<br />
|17320861<br />
|0.452<br />
|}<br />
<br />
===LadA===<br />
<br />
===Characterization of the Alcohol DeHydrogenase (ADH) system===<br />
<br />
=====Growth on alcohols as sole carbon source=====<br />
*As we described before, we tried to grow our strain ''Escherichia coli'' 018A ([http://partsregistry.org/Part:BBa_K398018 BBa_K398018] on the plasmid pSB1A2) on the alkanols Octanol-1 and Dodecanol-1 (1% V/V on M9 medium without any other carbon source). No growth was observed after 48 hours. We abandoned this experiment after this result.<br />
<br />
=====Resting cell assays=====<br />
*We grew ''Escherichia coli'' 018A and ''Escherichia coli'' negative control (Biobrick BBa_J13002 on plasmid pSB1A2 ) in 50 mL of M9 medium with glucose and CAS aminoacids.<br />
*The cells were harevested when the O.D. at 600nm was around 0.3; they were spun down at 4000 rpm, for 10 min at 4ºC. And the resting cell assays were prepared according to the [https://2010.igem.org/Team:TU_Delft#Resting-cell_assays_for_E.coli standard protocol]<br />
<br />
{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1"; <br />
|'''Strain'''<br />
|'''O.D.'''<br />
|-<br />
|J13002<br />
|0.327<br />
|-<br />
|018A<br />
|0.338<br />
|}<br />
<br />
*After an overnight incubation at 37ºC, the organic phase was extracted using 3 mL of ethyl acetate (dodecane was used as internal standard). We tried to determine production of alkanals or alkanoic acids by Gas Chromatography measurements. The chromatograms are shown below:<br />
<br />
[[Image:TUDelftADH_chrom.jpg|750px|thumb|center|Typical chromatograms obtained after the resting cell assays: (A) Blank, (B) Negative control which is E. coli K12, (C) E. coli 018A (our recombinant strain) and (D) standard of the expected product.]]<br />
<br />
<br />
*According to our results there was no degradation of octanol-1 ='( , after this experiment the protocol was abandoned. We think that the cells lack of transporters for long-chain alcohols, we preferred to check the cell extract in order to know if there was any biological activity in the cytoplasma.<br />
<br />
=====NADH production in cell extracts=====<br />
*'''EXPERIMENT 1'''<br />
Cells were cultured in 50mL of LB medium and harvested when the O.D. 600nm of the culture was around 0.6. Two different cultures of the recominant strain and the negative control were prepared.<br />
{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1"<br />
|Strain<br />
|O.D. 600nm<br />
|-<br />
|J13002 (1)<br />
|0.654<br />
|-<br />
|J13002 (2)<br />
|0.621 <br />
|-<br />
|018A (1)<br />
|0.615<br />
|-<br />
|018A (2)<br />
|0.580<br />
|}<br />
Cytoplasmic proteins were extracted using our standard [https://2010.igem.org/Team:TU_Delft#Preparing_cell_lysates_for_enzyme_kinetics_measurements protocol]. And a standard curve for protein quantification was prepared.<br />
[[Image:TUDelftADH_bradford.jpg|600px|thumb|center|Standard curve for total protein quantification.]]<br />
The total protein of each sample was quantified using 20uL of cell extract, the results are shown below.<br />
{| style="color:black; background-color:white;" cellpadding="4" cellspacing="0" border="1"<br />
|Sample<br />
|'''O.D. 562 nm'''<br />
|'''Total ug of protein'''<br />
|'''ug/uL'''<br />
|-<br />
|J13002(1A) <br />
|0.135 <br />
|10.393 <br />
|1.0393<br />
|-<br />
|J13002 (1B) <br />
|0.135 <br />
|10.393 <br />
|1.0393<br />
|-<br />
|J13002 (2A) <br />
|0.128 <br />
|9.7363 <br />
|0.9736<br />
|-<br />
|J13002 (2B) <br />
|0.129 <br />
|9.8302 <br />
|0.9830<br />
|-<br />
|018A (1A) <br />
|0.097 <br />
|6.8275 <br />
|0.6827<br />
|-<br />
|018A (1B) <br />
|0.091 <br />
|6.2645 <br />
|0.6264<br />
|-<br />
|018A (2A) <br />
|0.092 <br />
|6.3583 <br />
|0.6358<br />
|-<br />
|018A (2B) <br />
|0.092 <br />
|6.3583 <br />
|0.6358<br />
|}<br />
The alcohol dehydrogenase activity was measured using the standard [https://2010.igem.org/Team:TU_Delft#Alcohol.2FAldehyde_dehydrogenase_activity_assays protocol] and Dodecanol-1 as substrate. You can download our raw data by clicking on the link: [[ Image:TUDelft_ADH_raw.xls]]<br />
After the data treatment, the results that we obtained were the following:<br />
[[Image:TUDelftADH_pH95.jpg|400px]]<br />
[[Image:TUDelftADH_pH8.jpg|400px]]<br />
<br />
*All the enzyme activities were normalized to the TOTAL amount of protein in the cell extract. The results are shown in katal per mg of protein in the cell extract and Enzymatic Units per mg of protein in the cell extract. <br />
<br />
<html><br />
<table><br />
<tr><td></td><td colspan=4 style="text-align: center;"><b>pH=9.5</b></td><td colspan=4 style="text-align: center;"><b>pH=8</b></td></tr> <br />
<tr><td>&nbsp;</td><td colspan=2 style="text-align: center;"><b>Heat</b></td><td colspan=2 style="text-align: center;"><b>Native</b></td><td colspan=2 style="text-align: center;"><b>Heat</b></td><td colspan=2 style="text-align: center;"><b>Native</b></td></tr> <br />
<tr><td>&nbsp;</td><td>J13002</td><td>018A</td><td>J13002</td><td>018A</td><td>J13002</td><td>018A</td><td>J13002</td><td>018A</td></tr> <br />
<tr><td><b>kat/mg</b></td><td>6,156E-12</td><td>1,941E-11</td><td>1,053E-11</td><td>2,806E-11</td><td>3,211E-12</td><td>4,496E-11</td><td>1,302E-11</td><td>2,638E-11</td></tr> <br />
<tr><td><b>U/mg</b></td><td>3,694E-04</td><td>1,091E-03</td><td>6,317E-04</td><td>1,694E-03</td><td>1,927E-04</td><td>2,139E-03</td><td>7,811E-04</td><td>1,608E-03</td></tr> <br />
<tr><td><b>stdev</b></td><td>1,157E-12</td><td>4,579E-12</td><td>1,343E-12</td><td>9,961E-13</td><td>6,395E-12</td><td>2,331E-11</td><td>6,511E-13</td><td>9,961E-13</td></tr> <br />
<tr><td><b>Improvement</b></td><td>-</td><td>215,34%</td><td>-</td><td>166,51%</td><td>-</td><td>1299,87%</td><td>-</td><td>166,51%</td></tr> <br />
<tr><td><b>ttest</b></td><td>-</td><td>0,9963</td><td>-</td><td>1,0000</td><td>-</td><td>0,9988</td><td>-</td><td>1,000</td></tr><br />
</table><br />
</html><br />
<br />
You can download our results file here: [[ Image:TUDelft_ADH_results.xls]]<br />
Maybe you are not familiar with the term "katal", officially the katal is the STANDARD UNIT FOR CATALYTIC ACTIVITY in the International System of Units; the katal is defined as the amount of enzyme that converts 1 mol of substrate each second. [http://www.clinchem.org/cgi/content/full/48/3/586 Click here] to know more about it, and because people are not that familiar with the term, we report the activities in U/mg. One enzyme activity unit is defined as the amount of protein that converts 1 umol of substrate each minute.<br />
T-tests were performed in order to show that there is a difference between E. coli and our recombinant strain; a result for t-test of 1 means that both samples are statistically different; whereas a t-test of nearly 0 means that samples belong to the same group (no difference between both). Our level of significance is 0.025, in two tailed test. That means that if our t-test result is 0.95 we conclude that both samples are statistically different, whereas 0.949 or lower means that both samples are not statistically different.<br />
<br />
<br />
*'''EXPERIMENT 2'''<br />
We ran a second experiment at pH 9.5, with two other strains that seemed to be positive in the colony PCR test. In this test we also added two extra strains of E. coli in order to have more data for the statistical analysis, the strains used were J13002 and 331 ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K398331 BBa_K398331] in pSB1A2). We followed the same procedure as in the experiment 1, the results obtained were the following:<br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th></th><th>kat/mg</th><th>U/mg</th><th>STDEV</th><th>Improvement</th><th>T-test</th><th>Relative to putida</th><th>&nbsp;</th></tr> <tr><td>E. Coli</td><td>8.75E-12</td><td>5.25E-04</td><td>8.18987E-12</td><td>-</td><td>-</td><td>0.90%</td><td>&nbsp;</td></tr> <tr><td>018A-2</td><td>9.30E-12</td><td>5.58E-04</td><td>1.84877E-12</td><td>6.26%</td><td>0.4708</td><td>0.96%</td><td>&nbsp;</td></tr> <tr><td>018A-3</td><td>3.05E-11</td><td>1.83E-03</td><td>6.27802E-12</td><td>249.14%</td><td>0.9808</td><td>3.15%</td><td>&nbsp;</td></tr> <tr><td>018A-4</td><td>8.26E-12</td><td>4.96E-04</td><td>1.8256E-12</td><td>-5.59%</td><td>0.4053</td><td>0.85%</td><td>&nbsp;</td></tr> <tr><td>P. Putida</td><td>9.69E-10</td><td>5.82E-02</td><td>2.09109E-10</td><td>-</td><td>0.9883</td><td>100.00%</td><td></td></tr></table><br />
<br />
According to these results, the other two strains (018A-2 and 018A-4) do not functionally express the part BBa_K398018. However, these results confirm our findings from the first experiment. You can download our result in this link: [[ Image:TUDelft_ADH_results2.xls]] You can also find a summary in this file:[[ Image:TUDelft_ADH_summary.xls]]<br />
<br />
==== Conclusions ====<br />
<br />
[[Image:TUDelftADH_final.jpg|600px]]<br />
<br />
According to our results, a normal ''E. coli'' cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain 018A has an activity of 2.93e-11 kat/mg (1.76 mU/mg). According to the statistical analysis, the enzymatic activities of both strains are statistically different at confidence level of 0.95 which means that the part BBa_K398018 increases 2 times the alcohol dehydrogenase activity in the cell extract. We can conclude from our data that the part has a biological activity ''in vitro''.<br />
<br />
We still don't know about the ''in vivo'' activity of this part, maybe there is a lack of long-chain alcohol transporter proteins or it is necessary to over-express this part in order to see/measure any biological activity.<br />
<br />
===Characterization of the ALdehyde DeHydrogenase system===<br />
<br />
=====Growth on alcohols as sole carbon source=====<br />
As we described before, we tried to grow our recombinant strains ''Escherichia coli'' 029A ([http://partsregistry.org/Part:BBa_K398029 BBa_K398029] on the plasmid pSB1A2) and ''Escherichia coli'' 030A ([http://partsregistry.org/Part:BBa_K398030 BBa_K398030] on the plasmid pSB1A2) on Octanal and Dodecanal. No growth was observed after 48 hours. We abandoned this experiment after this result.<br />
<br />
=====Resting cell assays=====<br />
*We grew 'Escherichia coli'' 029A, ''Escherichia coli'' 030Aand ''Escherichia coli'' negative control (Biobrick BBa_J13002 on plasmid pSB1A2 ) in 50 mL of M9 medium with glucose and CAS aminoacids.<br />
*The cells were harevested when the O.D. at 600nm was around 0.3; they were spun down at 4000 rpm, for 10 min at 4ºC. And the resting cell assays were prepared according to the [https://2010.igem.org/Team:TU_Delft#Resting-cell_assays_for_E.coli standard protocol]<br />
<br />
{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1"; <br />
|'''Strain'''<br />
|'''O.D.'''<br />
|-<br />
|J13002<br />
|0.327<br />
|-<br />
|029A<br />
|0.340<br />
|-<br />
|030A<br />
|0.330<br />
|}<br />
<br />
'''Note''': This experiment was run in parallel with the resting cell assays for 018A, the negative control culture of the strain J13002 used was the same for both experiments.<br />
<br />
*After an overnight incubation at 37ºC with the substrate (octanal), the organic phase was extracted using 3 mL of ethyl acetate (dodecane was used as internal standard). We tried to determine production of alkanoic acids by Gas Chromatography measurements. The chromatograms are shown below:<br />
<br />
[[Image:TUDelftALDH_chrom.jpg|750px|thumb|center| Typical chromatograms obtained after the resting cell assays: (A) Blank, (B) Negative control which is E. coli K12, (C) E. coli 029A (our recombinant strain) and (D) E. coli 029A (our recombinant strain).]]<br />
<br />
<br />
*According to our results there was a peak reduction for octanal, however we were worried because of the fact that we didn't see the peak of the product (dodecanoic acid) appearing. It could be that the cells are just storing the product inside the cell. The experiment required to much time in order to get more results: the extractions, preparation of triplicates, cultures, cell suspensions... etc. it was a work that would require an effort of all the team for a couple of weeks. We decided to go for something easier and faster to measure; since we had some experience with the NADH determinations that was our choice.<br />
<br />
==== NADH production in cell extracts====<br />
Cells were cultured in 50mL of LB medium and harvested when the O.D. 600nm of the culture was between 0.5-0.8. Two different cultures of each recominant strain and the negative control were prepared.<br />
<br />
{| style="color:black; background-color:white;" cellpadding="2" cellspacing="0" border="1"<br />
|Strain<br />
|O.D. 600nm<br />
|-<br />
|J13002 (1)<br />
|0.825<br />
|-<br />
|J13002 (2)<br />
|0.774 <br />
|-<br />
|029A (1)<br />
|0.746<br />
|-<br />
|029A (2)<br />
|0.753<br />
|-<br />
|030A (1)<br />
|0.532<br />
|-<br />
|030A (2)<br />
|0.532<br />
|}<br />
Cytoplasmic proteins were extracted using our standard [https://2010.igem.org/Team:TU_Delft#Preparing_cell_lysates_for_enzyme_kinetics_measurements protocol]. And a standard curve for protein quantification was prepared.<br />
<br />
[[Image:TUDelftALDH_bradford.jpg|600px|thumb|center|Standard curve for total protein quantification.]]<br />
<br />
The total protein of each sample was quantified using 20uL of cell extract, the results are shown below.<br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th></th><th>Total protein [ug]</th><th>ug/uL</th></tr> <tr><td>J1300</td><td>31.108</td><td>1.555</td></tr> <tr><td>029A (1)</td><td>19.974</td><td>0.999</td></tr> <tr><td>030A (1)</td><td>12.610</td><td>0.630</td></tr> <tr><td>029A (2)</td><td>12.854</td><td>0.782</td></tr> <tr><td>030 (2)</td><td>18.578</td><td>0.929</td></tr> <tr><td>P. Puitda</td><td>9.783</td><td>0.489</td></tr></table><br />
<br />
<br />
The aldehyde dehydrogenase activity was measured using the standard [https://2010.igem.org/Team:TU_Delft#Alcohol.2FAldehyde_dehydrogenase_activity_assays protocol] and Dodecanal as substrate.<br />
<br />
After the data treatment, the results that we obtained were the following:<br />
<br />
<table class="tableizer-table"><br />
<tr class="tableizer-firstrow"><th></th><th>&nbsp;</th><th>J13002</th><th>029A (1)</th><th>029A (2)</th><th>030A (1)</th><th>030A (2)</th><th>P. putida</th></tr> <tr><td>Specific activity</td><td>kat/mg</td><td>8.604E-11</td><td>1.470E-10</td><td>2.853E-10</td><td>2.433E-10</td><td>2.912E-10</td><td>6.362E-10</td></tr> <tr><td>&nbsp;</td><td>U/mg</td><td>5.162E-03</td><td>8.820E-03</td><td>1.712E-02</td><td>1.460E-02</td><td>1.747E-02</td><td>3.817E-02</td></tr> <tr><td>Standard deviation</td><td>&nbsp;</td><td>1.778E-12</td><td>6.187E-12</td><td>4.026E-11</td><td>3.539E-11</td><td>4.319E-11</td><td>3.361E-11</td></tr> <tr><td>Relative to (J13002)</td><td>&nbsp;</td><td>100.00%</td><td>170.86%</td><td>331.59%</td><td>282.79%</td><td>338.46%</td><td>739.41%</td></tr> <tr><td>Relative activity (P. putida)</td><td>&nbsp;</td><td>13.52%</td><td>23.11%</td><td>44.84%</td><td>38.25%</td><td>45.78%</td><td>100.00%</td></tr> <tr><td>T-test vs J13002</td><td>&nbsp;</td><td>-</td><td>0.9827</td><td>0.9599</td><td>0.9764</td><td>0.9976</td><td>0.9976</td></tr> <tr><td>T-test vs P. putida</td><td>&nbsp;</td><td>0.9999</td><td>1.0000</td><td>0.9998</td><td>0.9974</td><td>1.0000</td><td>-</td></tr> <tr><td>Average activity [kat/mg]</td><td>&nbsp;</td><td>8.604E-11</td><td>2.161E-10</td><td>&nbsp;</td><td>2.673E-10</td><td>&nbsp;</td><td>6.362E-10</td></tr></table><br />
<br />
You can download a file with our raw data, results anda summary here: [[ Image:TUDelft_ALDH_results.xls]]<br />
<br />
==== Conclusion====<br />
<br />
[[Image:TUDelftALDH_final.jpg|600px]]<br />
<br />
<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/characterization
Team:TU Delft/Project/alkane-degradation/characterization
2010-10-26T21:03:42Z
<p>Jcnossen: /* Characterization of the long-chain alkane monooxygenase LadA */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Characterization==<br />
<br />
===Introduction===<br />
In summary, the following strains were characterized:<br />
*''E.coli'' K12 carrying [http://partsregistry.org/Part:BBa_K398014 BBa_K398014] in pSB1A2 (AH system)<br />
*''E.coli'' K12 carrying [http://partsregistry.org/Part:BBa_K398017 BBa_K398017] in pSB1A2 (LadA)<br />
*''E.coli'' TOP10 carrying [http://partsregistry.org/Part:BBa_K398027 BBa_K398027] in pSB1A2 (LadA)<br />
*''E.coli'' K12 carrying [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] in pSB1A2 (ADH)<br />
*''E.coli'' K12 carrying [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] in pSB1A2 (ALDH)<br />
*''E.coli'' K12 carrying [http://partsregistry.org/Part:BBa_K398030 BBa_K398030] in pSB1A2 (ALDH)<br />
<br />
The characterization will generally be executed along with an 'empty' plasmid carrying strain:<br />
*''E.coli'' K12 carrying [http://partsregistry.org/Part:BBa_J13002 BBa_J13002] in pSB1A2 ('empty' plasmid)<br />
<br />
===Characterization of the alkane hydroxylase system===<br />
==== Growth analysis====<br />
Of course, one of the first characterization experiments was to test growth of the ''E.coli'' strains carrying BBa_K398014 on alkanes. The alkanes octane and dodecane were tested as possible substrates in M9 minimal medium. The protocol can be found found [https://2010.igem.org/Team:TU_Delft/Protocols#Protocol_for_growth_of_E.coli_K12_on_alkanes here]. The idea behind this is that ''E.coli'' might inherently contain an ADH and ALDH that, while it might be at an extremely low activity, can be able to degrade larger chain alkanes thus releasing energy for growth.<br />
<br />
====Resting-cell assays====<br />
As explained earlier the catalytic component of the alkane hydroxylase system is an integral membrane protein. Characterization must thus be done using an intact-membrane setup. An option which has been explored in literature [1] is the resting-cell assay a.k.a. biotransformation assay. These assays will indicate the presence or absence of the desired enzymes, regardless of the alkane’s utilization for growth. The logic behind this is to stall the growth of a large volume of cells by using nitrogen-deficient medium to test their alkane conversion capabilities at near-zero growth.<br />
Extraction hydrocarbons from the medium using an apolar solvent (such as ethyl acetate) after the reaction and subsequent analysis by gas chromatography would indicate the presence of the corresponding alkanol and/or the decrease of alkane. For more on the experimental setup see the following pages:<br />
*[https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Resting-cell_assays_for_E.coli Resting-cell assays] <br />
*[https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Ethyl_acetate_extraction_protocol EtOAc extraction]<br />
*[https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=General_gas_chromatography_program_for_alkanes_and_alkanols Gas chromatography program]<br />
<br />
[1] Liu Li, Xueqian Liu, Wen Yang, Feng Xu, Wei Wang, Lu Feng, Mark Bartlam, Lei Wang and Zihe Rao. Crystal Structure of Long-Chain Alkane Monooxygenase (LadA) in Complex with Coenzyme FMN: Unveiling the Long-Chain Alkane Hydroxylase. ''Journal of molecular biology'', 376: 453–465 ('''2008''')<br />
<br />
===Characterization of the long-chain alkane monooxygenase LadA===<br />
====Enzyme activity assay based on GC-analysis====<br />
The most favorable way to analyse the activity of the LadA enzyme is by creating an environment in which it can properly function in-vitro. As explained earlier the enzyme required NADH and flavin mononucleotide as cofactors. Furthermore the optimal activity was found at a temperature of 60 degrees and a pH of 7.5. In accordance with these properties a [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Enzyme_activity_assay_for_LadA_by_GC protocol] was set up for the conversion of hexadecane using the lysates of the ''E.coli'' cells carrying the ladA constructs. After the reaction the hydrocarbons are extracted with an apolar solvent and analysed by gas chromatography (click [https://2010.igem.org/Team:TU_Delft/Protocols#General_gas_chromatography_program_for_alkanes_and_alkanols here] for the GC program and the obtained GC calibration graph).<br />
<br />
====Enzyme activity assay based on NADH absorbance====<br />
A less favored, yet very well accepted method for enzyme activity determination is by following the consumption of NADH at an absorption wavelength of 340 nm. By using a buffer at the appropriate pH, containing FMN and proper amounts of NADH the kinetics could easily be monitored by a 96-well plate reader. The protocol is described in detail [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Enzyme_activity_assay_for_LadA_by_NADH_absorption here].<br />
<br />
===Characterization of the Alcohol DeHydrogenase (ADH) and ALdehyde DeHydrogenase (ALDH) systems===<br />
<br />
==== Growth on alcohols as sole carbon source====<br />
As in the case of alkane monooxygenases, our first attempt was to grow our recombinant strains on long-chain alcohols and aldehydes; hoping that E. coli will do the further degradation steps. For that purpose, we used octanol-1 and dodecanol-1 for the ADH system; whereas we used octanal and dodecanal for the ALDH system. If everything goes well, we should see microbial growth on minimal medium with these compounds as sole carbon source. <br />
<br />
==== Resting cell assays====<br />
Our second attempt was to grow cells, until the exponential phase is reached; then the cells were collected and the spent medium was changed for a buffer with glucose that lacked of N-source, this was done in order to avoid more biomass production. Additionally we added alcohols and aldehydes to the mixture, so that if our part is working they will be converted in other molecules: alcohols to aldehydes and aldehydes to alkanoic acids.<br />
<br />
With this approach, we are hoping that the enzymes produced during the growth phase will remain inside the cells. The glucose added will serve for maintenance purposes and co-factor regeneration. If everything works well, there will be some nice peaks appearing on our GC analysis. <br />
<br />
==== NADH production in cell extracts====<br />
We called this assay: THE LAST RESORT =s <br />
<br />
Basically, we grew cells until an O.D. (600nm) between 0.5-1.0. We needed chubby healthy happy exponential-growth cells because most of our constructions are meant for protein production in this growth phase. <br />
<br />
Once, we got a lot of cells... we disrupted them by sonication and we analyze the NADH production by their guts using a simple spectrophotometrical analysis. If our parts are working, we should see a higher NADH production when the substrate (dodecanol-1 or dodecanal) to the buffer with microbial guts and NAD buffer. We can use this method because NAD is the natural co-factor of our proteins. Fortunately for us, NAD reduction could be easily quantified by absorbance measurements at 340 nm.<br />
<br />
We called this protocol "the last resort" because the others didn't work. Which means that alcohols and aldehydes do not cross the cell membrane, thus in order to grow cell on these compounds as sole carbon sources... WE NEEDED TO ADD TRANSPORTERS =(<br />
<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/Project/alkane-degradation/characterization
Team:TU Delft/Project/alkane-degradation/characterization
2010-10-26T21:02:39Z
<p>Jcnossen: /* Resting-cell assays */</p>
<hr />
<div>{{Team:TU_Delft/frame_check}}<br />
__NOTOC__<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html><br />
==Alkane Degradation Characterization==<br />
<br />
===Introduction===<br />
In summary, the following strains were characterized:<br />
*''E.coli'' K12 carrying [http://partsregistry.org/Part:BBa_K398014 BBa_K398014] in pSB1A2 (AH system)<br />
*''E.coli'' K12 carrying [http://partsregistry.org/Part:BBa_K398017 BBa_K398017] in pSB1A2 (LadA)<br />
*''E.coli'' TOP10 carrying [http://partsregistry.org/Part:BBa_K398027 BBa_K398027] in pSB1A2 (LadA)<br />
*''E.coli'' K12 carrying [http://partsregistry.org/Part:BBa_K398018 BBa_K398018] in pSB1A2 (ADH)<br />
*''E.coli'' K12 carrying [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] in pSB1A2 (ALDH)<br />
*''E.coli'' K12 carrying [http://partsregistry.org/Part:BBa_K398030 BBa_K398030] in pSB1A2 (ALDH)<br />
<br />
The characterization will generally be executed along with an 'empty' plasmid carrying strain:<br />
*''E.coli'' K12 carrying [http://partsregistry.org/Part:BBa_J13002 BBa_J13002] in pSB1A2 ('empty' plasmid)<br />
<br />
===Characterization of the alkane hydroxylase system===<br />
==== Growth analysis====<br />
Of course, one of the first characterization experiments was to test growth of the ''E.coli'' strains carrying BBa_K398014 on alkanes. The alkanes octane and dodecane were tested as possible substrates in M9 minimal medium. The protocol can be found found [https://2010.igem.org/Team:TU_Delft/Protocols#Protocol_for_growth_of_E.coli_K12_on_alkanes here]. The idea behind this is that ''E.coli'' might inherently contain an ADH and ALDH that, while it might be at an extremely low activity, can be able to degrade larger chain alkanes thus releasing energy for growth.<br />
<br />
====Resting-cell assays====<br />
As explained earlier the catalytic component of the alkane hydroxylase system is an integral membrane protein. Characterization must thus be done using an intact-membrane setup. An option which has been explored in literature [1] is the resting-cell assay a.k.a. biotransformation assay. These assays will indicate the presence or absence of the desired enzymes, regardless of the alkane’s utilization for growth. The logic behind this is to stall the growth of a large volume of cells by using nitrogen-deficient medium to test their alkane conversion capabilities at near-zero growth.<br />
Extraction hydrocarbons from the medium using an apolar solvent (such as ethyl acetate) after the reaction and subsequent analysis by gas chromatography would indicate the presence of the corresponding alkanol and/or the decrease of alkane. For more on the experimental setup see the following pages:<br />
*[https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Resting-cell_assays_for_E.coli Resting-cell assays] <br />
*[https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Ethyl_acetate_extraction_protocol EtOAc extraction]<br />
*[https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=General_gas_chromatography_program_for_alkanes_and_alkanols Gas chromatography program]<br />
<br />
[1] Liu Li, Xueqian Liu, Wen Yang, Feng Xu, Wei Wang, Lu Feng, Mark Bartlam, Lei Wang and Zihe Rao. Crystal Structure of Long-Chain Alkane Monooxygenase (LadA) in Complex with Coenzyme FMN: Unveiling the Long-Chain Alkane Hydroxylase. ''Journal of molecular biology'', 376: 453–465 ('''2008''')<br />
<br />
===Characterization of the long-chain alkane monooxygenase LadA===<br />
====Enzyme activity assay based on GC-analysis====<br />
The most favorable way to analyse the activity of the LadA enzyme is by creating an environment in which it can properly function in-vitro. As explained earlier the enzyme required NADH and flavin mononucleotide as cofactors. Furthermore the optimal activity was found at a temperature of 60 degrees and a pH of 7.5. In accordance with these properties a [https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols protocol] was set up for the conversion of hexadecane using the lysates of the ''E.coli'' cells carrying the ladA constructs. After the reaction the hydrocarbons are extracted with an apolar solvent and analysed by gas chromatography (click [https://2010.igem.org/Team:TU_Delft/Protocols#General_gas_chromatography_program_for_alkanes_and_alkanols here] for the GC program and the obtained GC calibration graph).<br />
<br />
====Enzyme activity assay based on NADH absorbance====<br />
A less favored, yet very well accepted method for enzyme activity determination is by following the consumption of NADH at an absorption wavelength of 340 nm. By using a buffer at the appropriate pH, containing FMN and proper amounts of NADH the kinetics could easily be monitored by a 96-well plate reader. The protocol is described in detail [https://2010.igem.org/Team:TU_Delft/Protocols#Enzyme_activity_assay_for_LadA_by_NADH_absorption here].<br />
<br />
<br />
===Characterization of the Alcohol DeHydrogenase (ADH) and ALdehyde DeHydrogenase (ALDH) systems===<br />
<br />
==== Growth on alcohols as sole carbon source====<br />
As in the case of alkane monooxygenases, our first attempt was to grow our recombinant strains on long-chain alcohols and aldehydes; hoping that E. coli will do the further degradation steps. For that purpose, we used octanol-1 and dodecanol-1 for the ADH system; whereas we used octanal and dodecanal for the ALDH system. If everything goes well, we should see microbial growth on minimal medium with these compounds as sole carbon source. <br />
<br />
==== Resting cell assays====<br />
Our second attempt was to grow cells, until the exponential phase is reached; then the cells were collected and the spent medium was changed for a buffer with glucose that lacked of N-source, this was done in order to avoid more biomass production. Additionally we added alcohols and aldehydes to the mixture, so that if our part is working they will be converted in other molecules: alcohols to aldehydes and aldehydes to alkanoic acids.<br />
<br />
With this approach, we are hoping that the enzymes produced during the growth phase will remain inside the cells. The glucose added will serve for maintenance purposes and co-factor regeneration. If everything works well, there will be some nice peaks appearing on our GC analysis. <br />
<br />
==== NADH production in cell extracts====<br />
We called this assay: THE LAST RESORT =s <br />
<br />
Basically, we grew cells until an O.D. (600nm) between 0.5-1.0. We needed chubby healthy happy exponential-growth cells because most of our constructions are meant for protein production in this growth phase. <br />
<br />
Once, we got a lot of cells... we disrupted them by sonication and we analyze the NADH production by their guts using a simple spectrophotometrical analysis. If our parts are working, we should see a higher NADH production when the substrate (dodecanol-1 or dodecanal) to the buffer with microbial guts and NAD buffer. We can use this method because NAD is the natural co-factor of our proteins. Fortunately for us, NAD reduction could be easily quantified by absorbance measurements at 340 nm.<br />
<br />
We called this protocol "the last resort" because the others didn't work. Which means that alcohols and aldehydes do not cross the cell membrane, thus in order to grow cell on these compounds as sole carbon sources... WE NEEDED TO ADD TRANSPORTERS =(<br />
<br />
<br />
<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/parts" target="" /></map></html></div>
Jcnossen
http://2010.igem.org/Team:TU_Delft
Team:TU Delft
2010-10-26T20:57:48Z
<p>Jcnossen: </p>
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$("#TUD-loading-panel").show();<br />
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<a name="top"></a><br />
<br />
<div id="TUD-loading-panel"><br />
<div id="TUD-loading-panel-logo"><br />
<!-- yes im using center --><br />
<center><img width="100" height="120" src="https://static.igem.org/mediawiki/2010/d/d8/TUDelft_2010_AlkanivoreAnimated.gif" /> <h2>Loading...</h2></center><br />
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<a class="nohover" href="https://2010.igem.org/Team:TU_Delft"><h1 class="TUD-logo"><span>TU Delft iGEM Team 2010</span></h1></a><br />
<span id="back_to_igem"><a href="https://2010.igem.org/" alt="back to 2010.igem.org" title="back to 2010.igem.org"><img src="https://static.igem.org/mediawiki/2010/3/32/TU_Delft_back_to_igem.png" border="0" /></a></span><br />
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<div id="TUD-menu"><br />
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<li><a class="menubutton" id="home" href="https://2010.igem.org/Team:TU_Delft/Home" title="Home"><span>Home</span></a></li><br />
<li><a class="menubutton" id="team" href="https://2010.igem.org/Team:TU_Delft/Team" title="Team"><span>Team</span></a><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/members" title="Members">Members</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/organization" title="Organization">Organization</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/university" title="University">University</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/previous-teams" title="Previous Teams">Previous Teams</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/gallery" title="Picture Gallery">Picture Gallery</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/movies" title="Movies">Movies</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/ijam" title="iJAM">iJAM</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="project" href="https://2010.igem.org/Team:TU_Delft#page=Project" title="Project">Project</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/introduction" title="Introduction">Introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation" title="Alkane Degradation">Alkane Degradation</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing" title="Hydrocarbon Sensing">Sensing</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/tolerance" title="Survival">Survival</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility" title="Emulsification">Solubility</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization" title="RBS Characterization">RBS Characterization</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/conclusions" title="General Conclusions">General Conclusions</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/references" title="References">References</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="insilico" href="https://2010.igem.org/Team:TU_Delft#page=Notebook" title="Notebook">Notebook</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/blog" title="Blog">Blog</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/timeline" title="Timeline">Timeline</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/brainstorm" title="Brainstorm">Brainstorm</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/cloning-plan" title="Cloning Plan">Cloning Plan</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols" title="Protocols">Protocols</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="parts" href="https://2010.igem.org/Team:TU_Delft#page=Parts" title="Parts">Parts</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts/biobricks" title="BioBricks">BioBricks</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts/characterization" title="Characterization">Characterization</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="insilico" href="https://2010.igem.org/Team:TU_Delft#page=Modeling" title="In Silico">In Silico</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/MFA" title="MFA">MFA</a></li><br />
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Jcnossen
http://2010.igem.org/Team:TU_Delft/test
Team:TU Delft/test
2010-10-26T20:56:38Z
<p>Jcnossen: </p>
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<a name="top"></a><br />
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<center><img width="100" height="120" src="https://static.igem.org/mediawiki/2010/d/d8/TUDelft_2010_AlkanivoreAnimated.gif" /> <h2>Loading...</h2></center><br />
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<a class="nohover" href="https://2010.igem.org/Team:TU_Delft"><h1 class="TUD-logo"><span>TU Delft iGEM Team 2010</span></h1></a><br />
<span id="back_to_igem"><a href="https://2010.igem.org/" alt="back to 2010.igem.org" title="back to 2010.igem.org"><img src="https://static.igem.org/mediawiki/2010/3/32/TU_Delft_back_to_igem.png" border="0" /></a></span><br />
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<li><a class="menubutton" id="home" href="https://2010.igem.org/Team:TU_Delft/Home" title="Home"><span>Home</span></a></li><br />
<li><a class="menubutton" id="team" href="https://2010.igem.org/Team:TU_Delft/Team" title="Team"><span>Team</span></a><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/members" title="Members">Members</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/organization" title="Organization">Organization</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/university" title="University">University</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/previous-teams" title="Previous Teams">Previous Teams</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/gallery" title="Picture Gallery">Picture Gallery</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/movies" title="Movies">Movies</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/ijam" title="iJAM">iJAM</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="project" href="https://2010.igem.org/Team:TU_Delft#page=Project" title="Project">Project</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/introduction" title="Introduction">Introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation" title="Alkane Degradation">Alkane Degradation</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing" title="Hydrocarbon Sensing">Sensing</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/tolerance" title="Survival">Survival</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility" title="Emulsification">Solubility</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization" title="RBS Characterization">RBS Characterization</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/conclusions" title="General Conclusions">General Conclusions</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/references" title="References">References</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="insilico" href="https://2010.igem.org/Team:TU_Delft#page=Notebook" title="Notebook">Notebook</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/blog" title="Blog">Blog</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/timeline" title="Timeline">Timeline</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/brainstorm" title="Brainstorm">Brainstorm</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/cloning-plan" title="Cloning Plan">Cloning Plan</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols" title="Protocols">Protocols</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="parts" href="https://2010.igem.org/Team:TU_Delft#page=Parts" title="Parts">Parts</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts/biobricks" title="BioBricks">BioBricks</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts/characterization" title="Characterization">Characterization</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="insilico" href="https://2010.igem.org/Team:TU_Delft#page=Modeling" title="In Silico">In Silico</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/MFA" title="MFA">MFA</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/HC_regulation" title="Hydrocarbon Regulation">Hydrocarbon Regulation</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/Protein-production-model" title="Protein Production Model">Protein Production Model</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/interaction-mapping" title="Software Tool">Interaction Mapping</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Software/part-search" title="Part search server & iPhone app">Part search server & iPhone app</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/wiki-tips-tricks" title="Wiki tips & tricks">Wiki Tips &amp; Tricks</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="safety" href="https://2010.igem.org/Team:TU_Delft#page=Safety" title="Safety">Safety</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Safety" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Safety/in-the-lab" title="Biosafety">Biosafety in the lab</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="humanpractice" href="https://2010.igem.org/Team:TU_Delft#page=Human-practice" title="Human Practice">Human Practice</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Human-practice" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=EPAC" title="EPAC">EPAC</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education" title="Education">Education</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Perception" title="Perception">Perception</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Awareness" title="Awareness">Awareness</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="education" href="https://2010.igem.org/Team:TU_Delft#page=Education" title="Education">Education</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education/science-museum" title="Science Museum">Science Museum</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education/game" title="Game">iGEM Game</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education/Workshop_on_synthetic_biology" title="SynBio Workshop">SynBio Workshop</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education/nemo" title="Night of the Nerds">Night of the Nerds</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="publicity" href="https://2010.igem.org/Team:TU_Delft#page=Publicity" title="Publicity">Publicity</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Publicity" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Publicity/communication_plan" title="Communication Plan">Communication Plan</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Publicity/in-the-news" title="In The News">In the Media</a></li><br />
<!-- <li><a href="https://2010.igem.org/Team:TU_Delft#page=Publicity/articles" title="Articles">Articles</a></li> /--><br />
</ul><br />
</li><br />
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Jcnossen
http://2010.igem.org/Team:TU_Delft/test
Team:TU Delft/test
2010-10-26T20:50:09Z
<p>Jcnossen: </p>
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<li><a href="/Team:TU_Delft#page=Education">Education</a></li><br />
<li><a href="/Team:TU_Delft#page=Publicity">Publicity</a></li><br />
<li><a href="/Team:TU_Delft#page=Collaboration">Collaboration</a></li><br />
<li><a href="/Team:TU_Delft#page=Contact">Contact</a></li><br />
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<div class="socialicon" style="background-image:url(https://static.igem.org/mediawiki/2010/2/22/Linkedin-32x32.png);"><a href="http://www.linkedin.com/groups?gid=3070941" title="TU Delft iGEM Team LinkedIn"></a></div><br />
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_gaq.push(['_setAccount', 'UA-16783722-1']);<br />
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Jcnossen
http://2010.igem.org/Team:TU_Delft/test
Team:TU Delft/test
2010-10-26T20:46:36Z
<p>Jcnossen: </p>
<hr />
<div>__NOTOC__<br />
<html><br />
<!--<br />
<br />
============================================================================================<br />
*** PLEASE NOTE: ***<br />
<br />
When copying our code, please be so kind to mention so on your wiki.<br />
<br />
You can use our logo https://static.igem.org/mediawiki/2010/7/7b/TU_Delft.jpg to link to our wiki.<br />
<br />
For questions about this, contact us at info@igemtudelft.nl.<br />
<br />
Pieter van Boheemen, Jelmer Cnossen<br />
<br />
============================================================================================<br />
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/--><br />
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<link rel="stylesheet" type="text/css" href="http://yui.yahooapis.com/2.8.1/build/base/base-min.css"><br />
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var page = currentPage ? currentPage : 'Home';<br />
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$("#edit_content_link").attr('href', url);<br />
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<br />
<!-- Main CSS - START /--><br />
<style type="text/css"><br />
/* Wiki Hacks - START */<br />
/* Author: Pieter van Boheemen */<br />
/* Team: TU Delft */<br />
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min-height:100%; /* real browsers */<br />
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table td {<br />
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height: 14px;<br />
display: block;<br />
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background-color: #266c96;<br />
padding: 0 5px;<br />
}<br />
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#TUD-footer-navlist ul li a {<br />
text-decoration: none;<br />
}<br />
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#TUD-footer-navlist ul li a:hover {<br />
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}<br />
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#TUD-footer-content .socialicon {<br />
margin-top: 4px;<br />
margin-right:10px;<br />
width:32px;<br />
height:32px;<br />
display:block;<br />
float:left;<br />
}<br />
<br />
#TUD-footer-content .socialicon a {<br />
width:100%;<br />
height:100%;<br />
display:block;<br />
}<br />
<br />
<br />
#twitter_div {<br />
background-image: url('https://static.igem.org/mediawiki/2010/e/ec/TU_Delft_twitterBG.gif');<br />
background-repeat: no-repeat;<br />
border-bottom-style: solid;<br />
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border-bottom-color: #5AA5BC;<br />
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font-size: 0.9em;<br />
margin-top:10px;<br />
padding-top: 33px;<br />
padding-right: 5px;<br />
padding-left: 5px;<br />
width: 340px;<br />
overflow: hidden;<br />
}<br />
<br />
#twitter_div ul {<br />
list-style-type: none;<br />
margin: 5px 0 5px 0;<br />
padding: 0;<br />
width: 340px;<br />
}<br />
<br />
#twitter_div ul li {<br />
color: #0C93BA;<br />
border-bottom-style: solid;<br />
border-bottom-width: 1px;<br />
border-bottom-color: #A1E8F7;<br />
margin: 0 0 3px 0;<br />
}<br />
<br />
#twitter_div ul li a {<br />
text-decoration: none;<br />
color: #DDA84E;<br />
}<br />
<br />
#twitter_div ul li a:hover {<br />
text-decoration: none;<br />
color: #D78E42;<br />
}<br />
<br />
#twitter_div p {<br />
text-align: right;<br />
padding-right: 6px;<br />
padding-bottom: 10px;<br />
}<br />
</style><br />
<br />
</div><br />
</div><br />
</div><br />
</div><br />
<script type="text/javascript"><br />
<br />
var _gaq = _gaq || [];<br />
_gaq.push(['_setAccount', 'UA-16783722-1']);<br />
_gaq.push(['_trackPageview']);<br />
<br />
(function() {<br />
var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true;<br />
ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js';<br />
var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s);<br />
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</script><br />
</html></div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/test
Team:TU Delft/test
2010-10-26T20:40:22Z
<p>Jcnossen: </p>
<hr />
<div>__NOTOC__<br />
<html><br />
<!--<br />
<br />
============================================================================================<br />
*** PLEASE NOTE: ***<br />
<br />
When copying our code, please be so kind to mention so on your wiki.<br />
<br />
You can use our logo https://static.igem.org/mediawiki/2010/7/7b/TU_Delft.jpg to link to our wiki.<br />
<br />
For questions about this, contact us at info@igemtudelft.nl.<br />
<br />
Pieter van Boheemen, Jelmer Cnossen<br />
<br />
============================================================================================<br />
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<br />
<!-- Reset CSS - START <br />
<link rel="stylesheet" type="text/css" href="http://yui.yahooapis.com/2.8.1/build/base/base-min.css"><br />
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<script type="text/javascript" src="http://github.com/cowboy/jquery-hashchange/raw/v1.3/jquery.ba-hashchange.js"></script><br />
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<script src="https://2010.igem.org/Team:TU_Delft/files/Mido_500.font.js?action=raw&ctype=text/javascript" type="text/javascript"></script><br />
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if(is_chrome) console.log(msg);<br />
}<br />
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function updateContentEditLink() {<br />
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return;<br />
}<br />
<br />
var page = currentPage ? currentPage : 'Home';<br />
var url = location.pathname + '/' + page + '?action=edit';<br />
$("#edit_content_link").attr('href', url);<br />
// $("#edit_content_link").click(function() {<br />
// $("#iGEM_TU_Delft_container").html('<iframe src="'+url+'" width="100%" height="100%"></iframe>');<br />
// });<br />
<br />
}<br />
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// Set document ready callback<br />
$(function() {<br />
if (wgUserName) {<br />
// Create content edit link<br />
$(".left-menu ul > *:last").after('<li><a id="edit_content_link">edit content</a></li>');<br />
updateContentEditLink();<br />
}<br />
<br />
// Initialize history plugin.<br />
$(window).hashchange(historyCallback);<br />
rewriteLinks($("#navlist"));<br />
<br />
historyCallback();<br />
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// setup hovering<br />
$(".menu_button").hover(<br />
function () { $(this).addClass("hover"); },<br />
function () { $(this).removeClass("hover"); }<br />
);<br />
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function isDefined(variable)<br />
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kvmap[s[0]] = s[1];<br />
}<br />
return kvmap;<br />
}<br />
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function makeHash(page, kvmap) {<br />
var str = '#page=' + page;<br />
var i;<br />
if (kvmap) {<br />
for(i in kvmap) {<br />
str += ampersandStr + i + '=' + kvmap[i];<br />
}<br />
}<br />
return str; <br />
}<br />
<br />
function setHash(page, kvmap) {<br />
location.hash = makeHash(page,kvmap);<br />
}<br />
<br />
<br />
function include_js(file, cb) {<br />
var html_doc = document.getElementsByTagName('head')[0];<br />
var js = document.createElement('script');<br />
js.setAttribute('type', 'text/javascript');<br />
js.setAttribute('src', file);<br />
html_doc.appendChild(js);<br />
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<br />
function loadScript(src, callback)<br />
{<br />
include_js(src, callback);<br />
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function moveToAnchor(anchor) {<br />
var pos = $('#load > a[name='+anchor+']').offset();<br />
dbgout('anchor: '+anchor+'; pos=' + pos.left + ','+pos.top );<br />
window.scroll(pos.left, pos.top);<br />
}<br />
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function loadPage(page, anchor)<br />
{<br />
var showLoadAnim = !!currentPage;<br />
currentPage = page;<br />
var url = wgServer + "/Team:TU_Delft/" + page + "?action=render";<br />
if(page.substring(0, 5) == "User:") {<br />
url = wgServer + "/" + page + "?action=render";<br />
}<br />
$(window).trigger('page_close');<br />
<br />
if(showLoadAnim) {<br />
$("#TUD-loading-panel").show();<br />
//$("#loading-overlay").show();<br />
$("#iGEM_TU_Delft_container").fadeTo(200,0.25);<br />
$('html, body').animate({scrollTop:0}, 'slow');<br />
}<br />
loading_home = false;<br />
<br />
var processPage = function(next) {<br />
dbgout('processPage: ' + page);<br />
$(window).trigger('page_init');<br />
rewriteLinks($("#iGEM_TU_Delft_container"));<br />
if (anchor) moveToAnchor(anchor);<br />
updateContentEditLink();<br />
$("#iGEM_TU_Delft_container").fadeTo(200,1);<br />
next();<br />
}<br />
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$.get(url, function(data) {<br />
$("#iGEM_TU_Delft_container").html(data).queue(processPage);<br />
buildBreadCrumbTrail(page); <br />
Cufon.replace('h2'); // Works without a selector engine <br />
Cufon.replace('h3'); // Works without a selector engine <br />
Cufon.replace('#sub1'); // Requires a selector engine for IE 6-7, see above <br />
$("tr:nth-child(odd)").addClass("odd");<br />
<br />
$("#TUD-loading-panel").hide();<br />
$("#loading-overlay").hide();<br />
});<br />
}<br />
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function historyCallback() {<br />
var hash = location.hash;<br />
if(hash) {<br />
hash = hash.substring(1);<br />
if(hash.split('=').length > 1) {<br />
var kvmap = splitHash(hash);<br />
var changepage;<br />
<br />
// looks a little clumsy, but js AND operator conflicts with mediawiki markup<br />
if (kvmap.page) if(kvmap.page != currentPage) changepage = kvmap.page;<br />
<br />
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$(window).trigger('hashupdate');<br />
}<br />
}<br />
} else {<br />
setHash('Home');<br />
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<br />
function rewriteLinks(elem) {<br />
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var url = this.href;<br />
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if (anchor.substring(0,5)!='=page')<br />
this.href = '#page=' + currentPage + ampersandStr + 'anchor=' + anchor;<br />
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} else if(txt != "edit")<br />
this.href = this.href.replace("https://2010.igem.org/Team:TU_Delft/", "#page=");<br />
<br />
dbgout('rewriting ' + url + ' to ' + this.href);<br />
});<br />
}<br />
<br />
<br />
</script><br />
<script type="text/javascript"><br />
var timeout = 500;<br />
var closetimer = 0;<br />
var ddmenuitem = 0;<br />
<br />
function jsddm_open() {<br />
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jsddm_close();<br />
ddmenuitem = $(this).find('ul').css('display', 'block');<br />
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}<br />
<br />
function jsddm_close() {<br />
if(ddmenuitem) ddmenuitem.css('display', 'none');<br />
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}<br />
<br />
function jsddm_timer() {<br />
closetimer = window.setTimeout(jsddm_close, timeout);<br />
}<br />
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}<br />
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$(function() {<br />
$('#navlist > li').bind('mouseover', jsddm_open);<br />
$('#navlist > li').bind('mouseout', jsddm_timer);<br />
// $("#navlist li ul li:even").addClass("alt");<br />
});<br />
<br />
document.onclick = jsddm_close;<br />
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<script language="javascript" type="text/javascript"><br />
function capitaliseFirstLetter(string)<br />
{<br />
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function buildDepth(array,count)<br />
{<br />
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{<br />
depthStr=depthStr + array[i] + "/" ;<br />
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}<br />
function buildBreadCrumbTrail(page)<br />
{<br />
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constituentFolders=currentURL.split("/");<br />
var outputStr="<a href='https://2010.igem.org/Team:TU_Delft#page=Home'>Home</a>";<br />
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{<br />
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}<br />
if(constituentFolders[count].substring(0, 5) !== "User:") {<br />
outputStr=outputStr + " <span class='doubleright'>&raquo;</span> <a href='https://2010.igem.org/Team:TU_Delft#page=" + buildDepth(constituentFolders,count) + constituentFolders[count] + "'>" + capitaliseFirstLetter(constituentFolders[count]).replace(/-/gi," ") + "</a>";<br />
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}<br />
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$("#breadcrumbs").html(outputStr);<br />
}<br />
</script><br />
<!-- Scripting END /--><br />
<br />
<!-- Main CSS - START /--><br />
<style type="text/css"><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%;<br />
height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
}<br />
#content { z-index: 1; background-color: transparent; border: none; padding: 0; margin: 0; width: 100%; overflow: hidden; margin-top: -15px !important; margin-top: 0px;<br />
height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
}<br />
#bodyContent { border: none; padding:0; margin:0; width:100%;<br />
height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
}<br />
#top-section { z-index: 2; 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 { background-color: #216085; border: none; width: 100%; margin: -10px auto 0 auto; padding: 20px 0;}<br />
.visualClear { display: none; }<br />
#footer { border: none; width: 965px; margin: 0 auto; padding: 0;}<br />
.firstHeading { display: none;}<br />
#f-list a { color: #333; font-size: 10px;}<br />
#f-list a:hover { color: #666;}<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 />
#search-controls { display:none; }<br />
h3#siteSub { display: none;}<br />
#contentSub {display: none;}<br />
p:first-child { display: none;}<br />
h1{border:none; width: 100%; clear: both;}<br />
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<br />
table {<br />
margin: 0;<br />
padding: 0;<br />
font-size: 11px;<br />
border: 1px solid #000;<br />
}<br />
table td {<br />
padding: 5px;<br />
}<br />
table .head {<br />
background-color: #216085 ;<br />
font-weight: bold;<br />
color: #fff;<br />
}<br />
table .odd {<br />
background-color: #d0f1fa;<br />
}<br />
<br />
h2 {<br />
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border: none;<br />
}<br />
<br />
h3 {<br />
font-size: 20px;<br />
border: none;<br />
}<br />
<br />
html, body {<br />
margin: 0;<br />
padding: 0;<br />
width: 100%;<br />
height: 100%; <br />
}<br />
<br />
body {<br />
background-color: #d0f1fa;<br />
font-family: Verdana, Arial;<br />
font-size: 12px;<br />
color: #222222;<br />
min-width: 1050px;<br />
}<br />
<br />
#TUD-main-wrapper {<br />
width: 100%;<br />
height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
text-align: center;<br />
background-image: url('https://static.igem.org/mediawiki/2010/b/b3/TU_Delft_footer_tile.gif');<br />
background-position: bottom left;<br />
background-repeat: repeat-x; <br />
display: block; <br />
font-family: Verdana, Arial;<br />
font-size: 12px;<br />
color: #222222;<br />
}<br />
<br />
#TUD-main-wrapper div {<br />
line-height: 18px;<br />
}<br />
<br />
/* IE ignores this */<br />
html>body #TUD-main-wrapper {<br />
height: auto;<br />
min-height: 100%;<br />
}<br />
<br />
#TUD-main-wrapper2 {<br />
width: 100%;<br />
height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
text-align: center;<br />
background-image: url('https://static.igem.org/mediawiki/2010/9/9d/TU_Delft_bg_tile.gif');<br />
background-repeat: repeat-x;<br />
background-repeat: repeat-x; <br />
display: block; <br />
font-family: Verdana, Arial;<br />
font-size: 12px;<br />
color: #222222;<br />
margin-top: -8px;<br />
}<br />
<br />
/* IE ignores this */<br />
html>body #TUD-main-wrapper2 {<br />
height: auto;<br />
min-height: 100%;<br />
}<br />
<br />
#TUD-content-wrapper {<br />
background-color: #fff;<br />
text-align: left;<br />
width: 1024px;<br />
margin: 0 auto;<br />
padding: 0;<br />
height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
}<br />
<br />
#TUD-header {<br />
width: 1024px;<br />
background-image: url('https://static.igem.org/mediawiki/2010/5/58/TU_Delft_header_bg.gif');<br />
height: 120px;<br />
}<br />
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background-image: url('https://static.igem.org/mediawiki/2010/c/c5/TU_Delft_menu_bg.gif');<br />
height: 38px;<br />
}<br />
<br />
<br />
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padding-top: 1px;/*important*/<br />
margin-top: -1px;/*important*/<br />
}<br />
<br />
#TUD-body-container {<br />
width: 100%;<br />
background-image: url('https://static.igem.org/mediawiki/2010/5/5d/TU_Delft_body_top_bg.jpg');<br />
background-repeat: no-repeat;<br />
/* FireFox collapsing margin fix */<br />
padding-top: 1px;/*important*/<br />
margin-top: -1px;/*important*/<br />
display: block;<br />
}<br />
<br />
#TUD-body-content {<br />
background-image: url('https://static.igem.org/mediawiki/2010/5/5f/TU_Delft_body_footer_bg.jpg');<br />
width: 1024px;<br />
background-repeat: no-repeat;<br />
background-position: bottom left;<br />
padding-bottom: 15px<br />
}<br />
<br />
#TUD-main-content {<br />
width: 904px;<br />
margin: 10px 60px 0px 60px;<br />
}<br />
<br />
#iGEM_TU_Delft_container {<br />
min-height: 400px;<br />
}<br />
<br />
#TUD-footer {<br />
width: 100%;<br />
height: 254px;<br />
text-align: center;<br />
}<br />
<br />
h1 {<br />
margin: 0;<br />
padding: 0;<br />
display: block;<br />
float: left;<br />
}<br />
<br />
.TUD-logo {<br />
background-image: url('https://static.igem.org/mediawiki/2010/0/09/TU_Delft_iGEM_Team_Logo.png');<br />
width: 237px;<br />
height: 95px;<br />
margin-left: 90px;<br />
background-repeat: no-repeat;<br />
border: none;<br />
}<br />
<br />
.TUD-logo span {<br />
visibility: hidden;<br />
}<br />
<br />
<br />
#TUD-loading-panel {<br />
z-index: 100;<br />
position: absolute;<br />
left: 0px;<br />
top: 0px;<br />
width: 100%;<br />
height: 100%;<br />
display:none;<br />
}<br />
<br />
#TUD-loading-panel-logo {<br />
margin: 0 auto;<br />
margin-top: 250px;<br />
}<br />
<br />
#loading-overlay {<br />
z-index: 70;<br />
width: 100%;<br />
height: 100%;<br />
display:none;<br />
opacity:0.4;<br />
filter:alpha(opacity=40);<br />
}<br />
<br />
#back_to_igem {<br />
margin: 15px 0 0 0;<br />
padding:0;<br />
float: right;<br />
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Jcnossen
http://2010.igem.org/Team:TU_Delft/test
Team:TU Delft/test
2010-10-26T20:38:43Z
<p>Jcnossen: </p>
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/* Wiki Hacks - START */<br />
/* Author: Pieter van Boheemen */<br />
/* Team: TU Delft */<br />
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width: 100%;<br />
height: 254px;<br />
text-align: center;<br />
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h1 {<br />
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margin-left: 90px;<br />
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z-index: 100;<br />
position: absolute;<br />
left: 0px;<br />
top: 0px;<br />
width: 100%;<br />
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display:none;<br />
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margin-top: 250px;<br />
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width: 100%;<br />
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padding:0;<br />
float: right;<br />
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/* MENU */<br />
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padding: 0;<br />
}<br />
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display: block;<br />
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#navlist li ul li a:hover {<br />
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text-decoration: underline;<br />
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display:block;<br />
margin: 13px 10px 0px 10px;<br />
}<br />
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width: 904px;<br />
margin: 0 auto;<br />
text-align: left;<br />
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/*TABLE .odd {<br />
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<br />
</style><br />
<!-- Main CSS - END /--><br />
<a name="top"></a><br />
<br />
<div id="TUD-loading-panel"><br />
<div id="TUD-loading-panel-logo"><br />
<!-- yes im using center --><br />
<center><img width="100" height="120" src="https://static.igem.org/mediawiki/2010/d/d8/TUDelft_2010_AlkanivoreAnimated.gif" /> <h2>Loading...</h2></center><br />
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</div><br />
<br />
<div id="TUD-main-wrapper2"><br />
<div id="TUD-main-wrapper"><br />
<br />
<div id="TUD-content-wrapper"><br />
<div id="TUD-header"><br />
<style><br />
a.nohover:hover {<br />
text-decoration: none;<br />
}<br />
</style><br />
<a class="nohover" href="https://2010.igem.org/Team:TU_Delft"><h1 class="TUD-logo"><span>TU Delft iGEM Team 2010</span></h1></a><br />
<span id="back_to_igem"><a href="https://2010.igem.org/" alt="back to 2010.igem.org" title="back to 2010.igem.org"><img src="https://static.igem.org/mediawiki/2010/3/32/TU_Delft_back_to_igem.png" border="0" /></a></span><br />
</div><br />
<div id="TUD-menu"><br />
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<ul id="navlist"><br />
<li><a class="menubutton" id="home" href="https://2010.igem.org/Team:TU_Delft/Home" title="Home"><span>Home</span></a></li><br />
<li><a class="menubutton" id="team" href="https://2010.igem.org/Team:TU_Delft/Team" title="Team"><span>Team</span></a><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/members" title="Members">Members</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/organization" title="Organization">Organization</a></li><br />
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Jcnossen
http://2010.igem.org/Team:TU_Delft/test
Team:TU Delft/test
2010-10-26T20:36:20Z
<p>Jcnossen: </p>
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<a name="top"></a><br />
<br />
<div id="TUD-loading-panel"><br />
<div id="TUD-loading-panel-logo"><br />
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<center><img width="100" height="120" src="https://static.igem.org/mediawiki/2010/d/d8/TUDelft_2010_AlkanivoreAnimated.gif" /> <h2>Loading...</h2></center><br />
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<span id="back_to_igem"><a href="https://2010.igem.org/" alt="back to 2010.igem.org" title="back to 2010.igem.org"><img src="https://static.igem.org/mediawiki/2010/3/32/TU_Delft_back_to_igem.png" border="0" /></a></span><br />
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<li><a class="menubutton" id="home" href="https://2010.igem.org/Team:TU_Delft/Home" title="Home"><span>Home</span></a></li><br />
<li><a class="menubutton" id="team" href="https://2010.igem.org/Team:TU_Delft/Team" title="Team"><span>Team</span></a><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/members" title="Members">Members</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/organization" title="Organization">Organization</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/university" title="University">University</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/previous-teams" title="Previous Teams">Previous Teams</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/gallery" title="Picture Gallery">Picture Gallery</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/movies" title="Movies">Movies</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/ijam" title="iJAM">iJAM</a></li><br />
</ul><br />
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<li><a class="menubutton" id="project" href="https://2010.igem.org/Team:TU_Delft#page=Project" title="Project">Project</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/introduction" title="Introduction">Introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation" title="Alkane Degradation">Alkane Degradation</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing" title="Hydrocarbon Sensing">Sensing</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/tolerance" title="Survival">Survival</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility" title="Emulsification">Solubility</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization" title="RBS Characterization">RBS Characterization</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/conclusions" title="General Conclusions">General Conclusions</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/references" title="References">References</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="insilico" href="https://2010.igem.org/Team:TU_Delft#page=Notebook" title="Notebook">Notebook</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/blog" title="Blog">Blog</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/timeline" title="Timeline">Timeline</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/brainstorm" title="Brainstorm">Brainstorm</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/cloning-plan" title="Cloning Plan">Cloning Plan</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols" title="Protocols">Protocols</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="parts" href="https://2010.igem.org/Team:TU_Delft#page=Parts" title="Parts">Parts</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts/biobricks" title="BioBricks">BioBricks</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts/characterization" title="Characterization">Characterization</a></li><br />
</ul><br />
</li><br />
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http://2010.igem.org/Team:TU_Delft/test
Team:TU Delft/test
2010-10-26T20:30:46Z
<p>Jcnossen: </p>
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<center><img width="100" height="120" src="https://static.igem.org/mediawiki/2010/d/d8/TUDelft_2010_AlkanivoreAnimated.gif" /> <h2>Loading...</h2></center><br />
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<span id="back_to_igem"><a href="https://2010.igem.org/" alt="back to 2010.igem.org" title="back to 2010.igem.org"><img src="https://static.igem.org/mediawiki/2010/3/32/TU_Delft_back_to_igem.png" border="0" /></a></span><br />
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<li><a class="menubutton" id="home" href="https://2010.igem.org/Team:TU_Delft/Home" title="Home"><span>Home</span></a></li><br />
<li><a class="menubutton" id="team" href="https://2010.igem.org/Team:TU_Delft/Team" title="Team"><span>Team</span></a><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/members" title="Members">Members</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/organization" title="Organization">Organization</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/university" title="University">University</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/previous-teams" title="Previous Teams">Previous Teams</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/gallery" title="Picture Gallery">Picture Gallery</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/movies" title="Movies">Movies</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/ijam" title="iJAM">iJAM</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="project" href="https://2010.igem.org/Team:TU_Delft#page=Project" title="Project">Project</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/introduction" title="Introduction">Introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation" title="Alkane Degradation">Alkane Degradation</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing" title="Hydrocarbon Sensing">Sensing</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/tolerance" title="Survival">Survival</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility" title="Emulsification">Solubility</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization" title="RBS Characterization">RBS Characterization</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/conclusions" title="General Conclusions">General Conclusions</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/references" title="References">References</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="insilico" href="https://2010.igem.org/Team:TU_Delft#page=Notebook" title="Notebook">Notebook</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/blog" title="Blog">Blog</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/timeline" title="Timeline">Timeline</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/brainstorm" title="Brainstorm">Brainstorm</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/cloning-plan" title="Cloning Plan">Cloning Plan</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols" title="Protocols">Protocols</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="parts" href="https://2010.igem.org/Team:TU_Delft#page=Parts" title="Parts">Parts</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts/biobricks" title="BioBricks">BioBricks</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts/characterization" title="Characterization">Characterization</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="insilico" href="https://2010.igem.org/Team:TU_Delft#page=Modeling" title="In Silico">In Silico</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/MFA" title="MFA">MFA</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/HC_regulation" title="Hydrocarbon Regulation">Hydrocarbon Regulation</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/Protein-production-model" title="Protein Production Model">Protein Production Model</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/interaction-mapping" title="Software Tool">Interaction Mapping</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Software/part-search" title="Part search server & iPhone app">Part search server & iPhone app</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Modeling/wiki-tips-tricks" title="Wiki tips & tricks">Wiki Tips &amp; Tricks</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="safety" href="https://2010.igem.org/Team:TU_Delft#page=Safety" title="Safety">Safety</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Safety" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Safety/in-the-lab" title="Biosafety">Biosafety in the lab</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="humanpractice" href="https://2010.igem.org/Team:TU_Delft#page=Human-practice" title="Human Practice">Human Practice</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Human-practice" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=EPAC" title="EPAC">EPAC</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education" title="Education">Education</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Perception" title="Perception">Perception</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Awareness" title="Awareness">Awareness</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="education" href="https://2010.igem.org/Team:TU_Delft#page=Education" title="Education">Education</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education/science-museum" title="Science Museum">Science Museum</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education/game" title="Game">iGEM Game</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education/Workshop_on_synthetic_biology" title="SynBio Workshop">SynBio Workshop</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Education/nemo" title="Night of the Nerds">Night of the Nerds</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="publicity" href="https://2010.igem.org/Team:TU_Delft#page=Publicity" title="Publicity">Publicity</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Publicity" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Publicity/communication_plan" title="Communication Plan">Communication Plan</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Publicity/in-the-news" title="In The News">In the Media</a></li><br />
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Jcnossen
http://2010.igem.org/Team:TU_Delft/test
Team:TU Delft/test
2010-10-26T20:29:36Z
<p>Jcnossen: </p>
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Jcnossen
http://2010.igem.org/Team:TU_Delft/test
Team:TU Delft/test
2010-10-26T20:28:32Z
<p>Jcnossen: </p>
<hr />
<div>__NOTOC__<br />
<html><br />
<!--<br />
<br />
============================================================================================<br />
*** PLEASE NOTE: ***<br />
<br />
When copying our code, please be so kind to mention so on your wiki.<br />
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You can use our logo https://static.igem.org/mediawiki/2010/7/7b/TU_Delft.jpg to link to our wiki.<br />
<br />
For questions about this, contact us at info@igemtudelft.nl.<br />
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Pieter van Boheemen, Jelmer Cnossen<br />
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margin-right:10px;<br />
width:32px;<br />
height:32px;<br />
display:block;<br />
float:left;<br />
}<br />
<br />
#TUD-footer-content .socialicon a {<br />
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display:block;<br />
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#twitter_div {<br />
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font-size: 0.9em;<br />
margin-top:10px;<br />
padding-top: 33px;<br />
padding-right: 5px;<br />
padding-left: 5px;<br />
width: 340px;<br />
overflow: hidden;<br />
}<br />
<br />
#twitter_div ul {<br />
list-style-type: none;<br />
margin: 5px 0 5px 0;<br />
padding: 0;<br />
width: 340px;<br />
}<br />
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color: #0C93BA;<br />
border-bottom-style: solid;<br />
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border-bottom-color: #A1E8F7;<br />
margin: 0 0 3px 0;<br />
}<br />
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#twitter_div ul li a {<br />
text-decoration: none;<br />
color: #DDA84E;<br />
}<br />
<br />
#twitter_div ul li a:hover {<br />
text-decoration: none;<br />
color: #D78E42;<br />
}<br />
<br />
#twitter_div p {<br />
text-align: right;<br />
padding-right: 6px;<br />
padding-bottom: 10px;<br />
}<br />
</style><br />
<br />
</div><br />
</div><br />
</div><br />
</div><br />
<script type="text/javascript"><br />
<br />
var _gaq = _gaq || [];<br />
_gaq.push(['_setAccount', 'UA-16783722-1']);<br />
_gaq.push(['_trackPageview']);<br />
<br />
(function() {<br />
var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true;<br />
ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js';<br />
var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s);<br />
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</html></div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/test
Team:TU Delft/test
2010-10-26T20:26:52Z
<p>Jcnossen: </p>
<hr />
<div>__NOTOC__<br />
<html><br />
<!--<br />
<br />
============================================================================================<br />
*** PLEASE NOTE: ***<br />
<br />
When copying our code, please be so kind to mention so on your wiki.<br />
<br />
You can use our logo https://static.igem.org/mediawiki/2010/7/7b/TU_Delft.jpg to link to our wiki.<br />
<br />
For questions about this, contact us at info@igemtudelft.nl.<br />
<br />
Pieter van Boheemen, Jelmer Cnossen<br />
<br />
============================================================================================<br />
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/--><br />
<br />
<!-- Reset CSS - START <br />
<link rel="stylesheet" type="text/css" href="http://yui.yahooapis.com/2.8.1/build/base/base-min.css"><br />
Reset CSS - END /--><br />
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<script type="text/javascript" src="http://github.com/cowboy/jquery-hashchange/raw/v1.3/jquery.ba-hashchange.js"></script><br />
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<script src="https://2010.igem.org/Team:TU_Delft/files/Mido_500.font.js?action=raw&ctype=text/javascript" type="text/javascript"></script><br />
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if(is_chrome) console.log(msg);<br />
}<br />
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return;<br />
}<br />
<br />
var page = currentPage ? currentPage : 'Home';<br />
var url = location.pathname + '/' + page + '?action=edit';<br />
$("#edit_content_link").attr('href', url);<br />
// $("#edit_content_link").click(function() {<br />
// $("#iGEM_TU_Delft_container").html('<iframe src="'+url+'" width="100%" height="100%"></iframe>');<br />
// });<br />
<br />
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if (wgUserName) {<br />
// Create content edit link<br />
$(".left-menu ul > *:last").after('<li><a id="edit_content_link">edit content</a></li>');<br />
updateContentEditLink();<br />
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// Initialize history plugin.<br />
$(window).hashchange(historyCallback);<br />
rewriteLinks($("#navlist"));<br />
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historyCallback();<br />
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$(".menu_button").hover(<br />
function () { $(this).addClass("hover"); },<br />
function () { $(this).removeClass("hover"); }<br />
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}<br />
return kvmap;<br />
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function makeHash(page, kvmap) {<br />
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var i;<br />
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for(i in kvmap) {<br />
str += ampersandStr + i + '=' + kvmap[i];<br />
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}<br />
return str; <br />
}<br />
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function setHash(page, kvmap) {<br />
location.hash = makeHash(page,kvmap);<br />
}<br />
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<br />
function include_js(file, cb) {<br />
var html_doc = document.getElementsByTagName('head')[0];<br />
var js = document.createElement('script');<br />
js.setAttribute('type', 'text/javascript');<br />
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}<br />
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}<br />
$(window).trigger('page_close');<br />
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if(showLoadAnim) {<br />
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$("#iGEM_TU_Delft_container").fadeTo(200,0.25);<br />
$('html, body').animate({scrollTop:0}, 'slow');<br />
}<br />
loading_home = false;<br />
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var processPage = function(next) {<br />
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$(window).trigger('page_init');<br />
rewriteLinks($("#iGEM_TU_Delft_container"));<br />
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$.get(url, function(data) {<br />
$("#iGEM_TU_Delft_container").html(data).queue(processPage);<br />
buildBreadCrumbTrail(page); <br />
Cufon.replace('h2'); // Works without a selector engine <br />
Cufon.replace('h3'); // Works without a selector engine <br />
Cufon.replace('#sub1'); // Requires a selector engine for IE 6-7, see above <br />
$("tr:nth-child(odd)").addClass("odd");<br />
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hash = hash.substring(1);<br />
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var kvmap = splitHash(hash);<br />
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this.href = this.href.replace("https://2010.igem.org/Team:TU_Delft/", "#page=");<br />
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dbgout('rewriting ' + url + ' to ' + this.href);<br />
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</script><br />
<script type="text/javascript"><br />
var timeout = 500;<br />
var closetimer = 0;<br />
var ddmenuitem = 0;<br />
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ddmenuitem = $(this).find('ul').css('display', 'block');<br />
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}<br />
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function jsddm_close() {<br />
if(ddmenuitem) ddmenuitem.css('display', 'none');<br />
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}<br />
<br />
function jsddm_timer() {<br />
closetimer = window.setTimeout(jsddm_close, timeout);<br />
}<br />
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$('#navlist > li').bind('mouseover', jsddm_open);<br />
$('#navlist > li').bind('mouseout', jsddm_timer);<br />
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});<br />
<br />
document.onclick = jsddm_close;<br />
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<script language="javascript" type="text/javascript"><br />
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{<br />
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{<br />
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function buildBreadCrumbTrail(page)<br />
{<br />
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constituentFolders=currentURL.split("/");<br />
var outputStr="<a href='https://2010.igem.org/Team:TU_Delft#page=Home'>Home</a>";<br />
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{<br />
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}<br />
if(constituentFolders[count].substring(0, 5) !== "User:") {<br />
outputStr=outputStr + " <span class='doubleright'>&raquo;</span> <a href='https://2010.igem.org/Team:TU_Delft#page=" + buildDepth(constituentFolders,count) + constituentFolders[count] + "'>" + capitaliseFirstLetter(constituentFolders[count]).replace(/-/gi," ") + "</a>";<br />
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$("#breadcrumbs").html(outputStr);<br />
}<br />
</script><br />
<!-- Scripting END /--><br />
<br />
<!-- Main CSS - START /--><br />
<style type="text/css"><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%;<br />
height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
}<br />
#content { z-index: 1; background-color: transparent; border: none; padding: 0; margin: 0; width: 100%; overflow: hidden; margin-top: -15px !important; margin-top: 0px;<br />
height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
}<br />
#bodyContent { border: none; padding:0; margin:0; width:100%;<br />
height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
}<br />
#top-section { z-index: 2; height: 15px; margin: 0px; margin-left: auto; margin-right: auto; margin-bottom: 0 !important; padding:0; border: none; font-size: 10px;}<br />
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.left-menu { width: 500px !important; display:block; margin-top:-80px; border: none; text-align: right;}<br />
.left-menu ul { border: none; }<br />
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.right-menu ul { border: none; width: 300px;}<br />
#footer-box { background-color: #216085; border: none; width: 100%; margin: -10px auto 0 auto; padding: 20px 0;}<br />
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#footer { border: none; width: 965px; margin: 0 auto; padding: 0;}<br />
.firstHeading { display: none;}<br />
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#footer ul { margin: 0; padding: 0;}<br />
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h3#siteSub { display: none;}<br />
#contentSub {display: none;}<br />
p:first-child { display: none;}<br />
h1{border:none; width: 100%; clear: both;}<br />
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table {<br />
margin: 0;<br />
padding: 0;<br />
font-size: 11px;<br />
border: 1px solid #000;<br />
}<br />
table td {<br />
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}<br />
table .head {<br />
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color: #fff;<br />
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table .odd {<br />
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<br />
h2 {<br />
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}<br />
<br />
h3 {<br />
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border: none;<br />
}<br />
<br />
html, body {<br />
margin: 0;<br />
padding: 0;<br />
width: 100%;<br />
height: 100%; <br />
}<br />
<br />
body {<br />
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<br />
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height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
text-align: center;<br />
background-image: url('https://static.igem.org/mediawiki/2010/b/b3/TU_Delft_footer_tile.gif');<br />
background-position: bottom left;<br />
background-repeat: repeat-x; <br />
display: block; <br />
font-family: Verdana, Arial;<br />
font-size: 12px;<br />
color: #222222;<br />
}<br />
<br />
#TUD-main-wrapper div {<br />
line-height: 18px;<br />
}<br />
<br />
/* IE ignores this */<br />
html>body #TUD-main-wrapper {<br />
height: auto;<br />
min-height: 100%;<br />
}<br />
<br />
#TUD-main-wrapper2 {<br />
width: 100%;<br />
height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
text-align: center;<br />
background-image: url('https://static.igem.org/mediawiki/2010/9/9d/TU_Delft_bg_tile.gif');<br />
background-repeat: repeat-x;<br />
background-repeat: repeat-x; <br />
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font-size: 12px;<br />
color: #222222;<br />
margin-top: -8px;<br />
}<br />
<br />
/* IE ignores this */<br />
html>body #TUD-main-wrapper2 {<br />
height: auto;<br />
min-height: 100%;<br />
}<br />
<br />
#TUD-content-wrapper {<br />
background-color: #fff;<br />
text-align: left;<br />
width: 1024px;<br />
margin: 0 auto;<br />
padding: 0;<br />
height:auto !important; /* real browsers */<br />
height:100%; /* IE6: treaded as min-height*/<br />
min-height:100%; /* real browsers */<br />
}<br />
<br />
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width: 1024px;<br />
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padding-top: 1px;/*important*/<br />
margin-top: -1px;/*important*/<br />
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<br />
#TUD-body-container {<br />
width: 100%;<br />
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background-repeat: no-repeat;<br />
/* FireFox collapsing margin fix */<br />
padding-top: 1px;/*important*/<br />
margin-top: -1px;/*important*/<br />
display: block;<br />
}<br />
<br />
#TUD-body-content {<br />
background-image: url('https://static.igem.org/mediawiki/2010/5/5f/TU_Delft_body_footer_bg.jpg');<br />
width: 1024px;<br />
background-repeat: no-repeat;<br />
background-position: bottom left;<br />
padding-bottom: 15px<br />
}<br />
<br />
#TUD-main-content {<br />
width: 904px;<br />
margin: 10px 60px 0px 60px;<br />
}<br />
<br />
#iGEM_TU_Delft_container {<br />
min-height: 400px;<br />
}<br />
<br />
#TUD-footer {<br />
width: 100%;<br />
height: 254px;<br />
text-align: center;<br />
}<br />
<br />
h1 {<br />
margin: 0;<br />
padding: 0;<br />
display: block;<br />
float: left;<br />
}<br />
<br />
.TUD-logo {<br />
background-image: url('https://static.igem.org/mediawiki/2010/0/09/TU_Delft_iGEM_Team_Logo.png');<br />
width: 237px;<br />
height: 95px;<br />
margin-left: 90px;<br />
background-repeat: no-repeat;<br />
border: none;<br />
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<br />
.TUD-logo span {<br />
visibility: hidden;<br />
}<br />
<br />
<br />
#TUD-loading-panel {<br />
z-index: 100;<br />
position: absolute;<br />
left: 0px;<br />
top: 0px;<br />
width: 100%;<br />
height: 100%;<br />
display:none;<br />
}<br />
<br />
#TUD-loading-panel-logo {<br />
margin: 0 auto;<br />
margin-top: 250px;<br />
}<br />
<br />
#loading-overlay {<br />
z-index: 70;<br />
width: 100%;<br />
height: 100%;<br />
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Jcnossen
http://2010.igem.org/Team:TU_Delft/test
Team:TU Delft/test
2010-10-26T20:19:43Z
<p>Jcnossen: </p>
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<!-- Main CSS - END /--><br />
<a name="top"></a><br />
<br />
<div id="TUD-loading-panel"><br />
<div id="TUD-loading-panel-logo"><br />
<!-- yes im using center --><br />
<center><img width="100" height="120" src="https://static.igem.org/mediawiki/2010/d/d8/TUDelft_2010_AlkanivoreAnimated.gif" /> <h2>Loading...</h2></center><br />
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</div><br />
<br />
<div id="TUD-main-wrapper2"><br />
<div id="TUD-main-wrapper"><br />
<br />
<div id="TUD-content-wrapper"><br />
<div id="TUD-header"><br />
<style><br />
a.nohover:hover {<br />
text-decoration: none;<br />
}<br />
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<a class="nohover" href="https://2010.igem.org/Team:TU_Delft"><h1 class="TUD-logo"><span>TU Delft iGEM Team 2010</span></h1></a><br />
<span id="back_to_igem"><a href="https://2010.igem.org/" alt="back to 2010.igem.org" title="back to 2010.igem.org"><img src="https://static.igem.org/mediawiki/2010/3/32/TU_Delft_back_to_igem.png" border="0" /></a></span><br />
</div><br />
<div id="TUD-menu"><br />
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<ul id="navlist"><br />
<li><a class="menubutton" id="home" href="https://2010.igem.org/Team:TU_Delft#page=Home" title="Home"><span>Home</span></a></li><br />
<li><a class="menubutton" id="team" href="https://2010.igem.org/Team:TU_Delft#page=Team" title="Team"><span>Team</span></a><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/members" title="Members">Members</a></li><br />
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Jcnossen
http://2010.igem.org/Team:TU_Delft/test
Team:TU Delft/test
2010-10-26T20:17:09Z
<p>Jcnossen: </p>
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<a name="top"></a><br />
<br />
<div id="TUD-loading-panel"><br />
<div id="TUD-loading-panel-logo"><br />
<!-- yes im using center --><br />
<center><img width="100" height="120" src="https://static.igem.org/mediawiki/2010/d/d8/TUDelft_2010_AlkanivoreAnimated.gif" /> <h2>Loading...</h2></center><br />
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<div id="TUD-main-wrapper2"><br />
<div id="TUD-main-wrapper"><br />
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<span id="back_to_igem"><a href="https://2010.igem.org/" alt="back to 2010.igem.org" title="back to 2010.igem.org"><img src="https://static.igem.org/mediawiki/2010/3/32/TU_Delft_back_to_igem.png" border="0" /></a></span><br />
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<div id="TUD-menu"><br />
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<ul id="navlist"><br />
<li><a class="menubutton" id="home" href="https://2010.igem.org/Team:TU_Delft#page=Home" title="Home"><span>Home</span></a></li><br />
<li><a class="menubutton" id="team" href="https://2010.igem.org/Team:TU_Delft#page=Team" title="Team"><span>Team</span></a><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/members" title="Members">Members</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/organization" title="Organization">Organization</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/university" title="University">University</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/previous-teams" title="Previous Teams">Previous Teams</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/gallery" title="Picture Gallery">Picture Gallery</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/movies" title="Movies">Movies</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Team/ijam" title="iJAM">iJAM</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="project" href="https://2010.igem.org/Team:TU_Delft#page=Project" title="Project">Project</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/introduction" title="Introduction">Introduction</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation" title="Alkane Degradation">Alkane Degradation</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/sensing" title="Hydrocarbon Sensing">Sensing</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/tolerance" title="Survival">Survival</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/solubility" title="Emulsification">Solubility</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization" title="RBS Characterization">RBS Characterization</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/conclusions" title="General Conclusions">General Conclusions</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Project/references" title="References">References</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="insilico" href="https://2010.igem.org/Team:TU_Delft#page=Notebook" title="Notebook">Notebook</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/blog" title="Blog">Blog</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/timeline" title="Timeline">Timeline</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/brainstorm" title="Brainstorm">Brainstorm</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/cloning-plan" title="Cloning Plan">Cloning Plan</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Notebook/protocols" title="Protocols">Protocols</a></li><br />
</ul><br />
</li><br />
<li><a class="menubutton" id="parts" href="https://2010.igem.org/Team:TU_Delft#page=Parts" title="Parts">Parts</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts" title="Overview">Overview</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts/biobricks" title="BioBricks">BioBricks</a></li><br />
<li><a href="https://2010.igem.org/Team:TU_Delft#page=Parts/characterization" title="Characterization">Characterization</a></li><br />
</ul><br />
</li><br />
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Jcnossen
http://2010.igem.org/Team:TU_Delft/PartViewExample
Team:TU Delft/PartViewExample
2010-10-26T20:02:41Z
<p>Jcnossen: </p>
<hr />
<div><html><br />
<style><br />
.partview_label { <br />
font-weight: bold; <br />
width: 200px;<br />
}<br />
.partview_data {<br />
margin: 10px;<br />
font-family: courier;<br />
width: 400px;<br />
overflow: auto;<br />
}<br />
</style><br />
<div id="partview"><br />
<div class="partview_label">Part Name:</div><div class="partview_name partview_data"></div><br />
<div class="partview_label">Part Description:</div><div class="partview_desc partview_data"></div><br />
<div class="partview_label">Part Author:</div><div class="partview_author partview_data"></div><br />
<div class="partview_label">Part Sequence Length:</div><div class="partview_sequence_len partview_data"></div><br />
<div class="partview_label">Part Sequence:</div><div class="partview_sequence partview_data"></div><br />
</div><br />
<br />
<script type="text/javascript"><br />
<br />
function PartInfoCallback(parts) {<br />
var part = parts[0];<br />
$(".partview_name").text(part.name);<br />
$(".partview_desc").text(part.desc);<br />
$(".partview_author").text(part.author);<br />
$(".partview_sequence").text(part.sequence);<br />
$(".partview_sequence_len").text(part.sequence.length);<br />
}<br />
</script><br />
<br />
</html><br />
<br />
{{Team:TU_Delft/PartView|part=BBa_K398005|callback=PartInfoCallback}}<br />
<br />
Made by using template [[Template:Team:TU_Delft/PartView|PartView]]<br />
<html><br />
<pre><br />
{{Team:TU_Delft/PartView|part=BBa_K398005|callback=PartInfoCallback}}<br />
</pre></html></div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/PartViewExample
Team:TU Delft/PartViewExample
2010-10-26T20:00:48Z
<p>Jcnossen: </p>
<hr />
<div><html><br />
<style><br />
.partview_label { <br />
font-weight: bold; <br />
width: 200px;<br />
}<br />
.partview_data {<br />
margin: 10px;<br />
font-family: courier;<br />
width: 400px;<br />
overflow: auto;<br />
}<br />
</style><br />
<div id="partview"><br />
<div class="partview_label">Part Name:</div><div class="partview_name partview_data"></div><br />
<div class="partview_label">Part Description:</div><div class="partview_desc partview_data"></div><br />
<div class="partview_label">Part Author:</div><div class="partview_author partview_data"></div><br />
<div class="partview_label">Part Sequence Length:</div><div class="partview_sequence_len partview_data"></div><br />
<div class="partview_label">Part Sequence:</div><div class="partview_sequence partview_data"></div><br />
</div><br />
<br />
<script type="text/javascript"><br />
<br />
function PartInfoCallback(parts) {<br />
var part = parts[0];<br />
$(".partview_name").text(part.name);<br />
$(".partview_desc").text(part.desc);<br />
$(".partview_author").text(part.author);<br />
$(".partview_sequence").text(part.sequence);<br />
$(".partview_sequence_len").text(part.sequence.length);<br />
}<br />
</script><br />
<br />
</html><br />
<br />
{{Team:TU_Delft/PartView|part=BBa_K398005|callback=PartInfoCallback}}<br />
<br />
Made by using template:<br />
<html><br />
<pre><br />
{{Team:TU_Delft/PartView|part=BBa_K398005|callback=PartInfoCallback}}<br />
</pre></html></div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/PartViewExample
Team:TU Delft/PartViewExample
2010-10-26T19:59:20Z
<p>Jcnossen: </p>
<hr />
<div><html><br />
<style><br />
.partview_label { <br />
font-weight: bold; <br />
width: 200px;<br />
}<br />
.partview_data {<br />
margin: 10px;<br />
font-family: courier;<br />
width: 400px;<br />
overflow: auto;<br />
}<br />
</style><br />
<div id="partview"><br />
<div class="partview_label">Part Name:</div><div class="partview_name partview_data"></div><br />
<div class="partview_label">Part Description:</div><div class="partview_desc partview_data"></div><br />
<div class="partview_label">Part Author:</div><div class="partview_author partview_data"></div><br />
<div class="partview_label">Part Sequence Length:</div><div class="partview_sequence_len partview_data"></div><br />
<div class="partview_label">Part Sequence:</div><div class="partview_sequence partview_data"></div><br />
</div><br />
<br />
<script type="text/javascript"><br />
<br />
function PartInfoCallback(parts) {<br />
var part = parts[0];<br />
$(".partview_name").text(part.name);<br />
$(".partview_desc").text(part.desc);<br />
$(".partview_author").text(part.author);<br />
$(".partview_sequence").text(part.sequence);<br />
$(".partview_sequence_len").text(part.sequence.length);<br />
}<br />
</script><br />
<br />
</html><br />
<br />
{{Team:TU_Delft/PartView|part=BBa_K398005|callback=PartInfoCallback}}<br />
<br />
Made by using template:<br />
{{Team:TU_Delft/PartView|part=BBa_K398005|callback=PartInfoCallback}}</div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/Software/part-search
Team:TU Delft/Software/part-search
2010-10-26T19:58:06Z
<p>Jcnossen: /* Why make another webservice? */</p>
<hr />
<div>__NOTOC__<br />
{{Team:TU_Delft/frame_check}}<br />
<html><h2><a href="http://igempartview.appspot.com/">Part search server</a></h2></html><br />
Because the partsregistry has limited capabilities, a part information service was created, running at: <b>[http://igempartview.appspot.com igempartview.appspot.com]</b>. <br />
You can use this server to browse parts by category, as specified by each part in the partsregistry editor.<br />
It also supports a range of other webservices that can be used by javascript or other applications, described [http://igempartview.appspot.com/api.jsp here]. <br />
<br />
===Why make another webservice?===<br />
The current partsregistry [http://partsregistry.org/Registry_API web interfaces] are pretty limited. <br />
* There is no interface to search for parts.<br />
* The XML interface cannot be used by scripts that are not on the partsregistry wiki, due to [http://en.wikipedia.org/wiki/Same_origin_policy Same-Origin-Policy]<br />
<br />
This service supports a webservice mechanism called [http://en.wikipedia.org/wiki/JSON#JSONP JSONP], which allows team wiki javascript to query part information (properties, categories, or DNA sequence) in a dynamic way, something that is not possible with the current [http://partsregistry.org/Registry_API registry API]. A small example of using this interface can be found [[Team:TU_Delft/PartViewExample|here]], but next year teams could make more advanced use of this (Protein sequence for example)<br />
<br />
===Implementation===<br />
The server uses the [http://partsregistry.org/DAS_-_Distributed_Annotation_System DAS server] to look for new parts, and the [http://partsregistry.org/Registry_API#XML_Part_Information XML Part information] to read part information. It refreshes it's content once a week (Look [http://igempartview.appspot.com/content_update.jsp here] for accurate update information).<br />
<br />
The service runs on [http://code.google.com/intl/nl-NL/appengine/ Google App Engine], so uptime is pretty ok. There are no server maintainance costs, because it will be able to run within the [http://code.google.com/intl/nl-NL/appengine/docs/quotas.html free quota's] of Google App Engine.<br />
<br />
<html><h2><a href="http://igempartview.appspot.com/iphone.jsp">iPhone part browser app</a></h2></html><br />
[[Image:TU_Delft_2010_Iphone.jpg|right]]<br />
The TU Delft team is working on an iPhone app to allow quick lookup of part properties, and browse parts by category. The new webservice above is being used for this. There used to be a more extensive application on the App Store known as "[http://www.macworld.com/appguide/app.html?id=107869&expand=false Biobrick Studio]" but it seems to have disappeared.<br />
<br />
iPhone apps have to be approved by Apple before they end up in the App Store. Once this is done for our app, details will be put on this page:<br />
http://igempartview.appspot.com/iphone.jsp<br />
<br />
<br />
<html><br style="clear:both" /></html></div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/PartViewExample
Team:TU Delft/PartViewExample
2010-10-26T19:55:24Z
<p>Jcnossen: </p>
<hr />
<div><html><br />
<style><br />
.partview_label { <br />
font-weight: bold; <br />
width: 200px;<br />
}<br />
.partview_data {<br />
margin: 10px;<br />
font-family: courier;<br />
width: 400px;<br />
overflow: auto;<br />
}<br />
</style><br />
<div id="partview"><br />
<div class="partview_label">Part Name:</div><div class="partview_name partview_data"></div><br />
<div class="partview_label">Part Description:</div><div class="partview_desc partview_data"></div><br />
<div class="partview_label">Part Author:</div><div class="partview_author partview_data"></div><br />
<div class="partview_label">Part Sequence Length:</div><div class="partview_sequence_len partview_data"></div><br />
<div class="partview_label">Part Sequence:</div><div class="partview_sequence partview_data"></div><br />
</div><br />
<br />
<script type="text/javascript"><br />
<br />
function PartInfoCallback(parts) {<br />
var part = parts[0];<br />
$(".partview_name").text(part.name);<br />
$(".partview_desc").text(part.desc);<br />
$(".partview_author").text(part.author);<br />
$(".partview_sequence").text(part.sequence);<br />
$(".partview_sequence_len").text(part.sequence.length);<br />
}<br />
</script><br />
<br />
</html><br />
<br />
{{Team:TU_Delft/PartView|part=BBa_K398005|callback=PartInfoCallback}}</div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/PartViewExample
Team:TU Delft/PartViewExample
2010-10-26T19:54:59Z
<p>Jcnossen: </p>
<hr />
<div><html><br />
<style><br />
.partview_label { <br />
font-weight: bold; <br />
width: 200px;<br />
}<br />
.partview_data {<br />
margin: 10px;<br />
font-family: courier;<br />
width: 400px;<br />
overflow: scroll;<br />
}<br />
</style><br />
<div id="partview"><br />
<div class="partview_label">Part Name:</div><div class="partview_name partview_data"></div><br />
<div class="partview_label">Part Description:</div><div class="partview_desc partview_data"></div><br />
<div class="partview_label">Part Author:</div><div class="partview_author partview_data"></div><br />
<div class="partview_label">Part Sequence Length:</div><div class="partview_sequence_len partview_data"></div><br />
<div class="partview_label">Part Sequence:</div><div class="partview_sequence partview_data"></div><br />
</div><br />
<br />
<script type="text/javascript"><br />
<br />
function PartInfoCallback(parts) {<br />
var part = parts[0];<br />
$(".partview_name").text(part.name);<br />
$(".partview_desc").text(part.desc);<br />
$(".partview_author").text(part.author);<br />
$(".partview_sequence").text(part.sequence);<br />
$(".partview_sequence_len").text(part.sequence.length);<br />
}<br />
</script><br />
<br />
</html><br />
<br />
{{Team:TU_Delft/PartView|part=BBa_K398005|callback=PartInfoCallback}}</div>
Jcnossen
http://2010.igem.org/Team:TU_Delft/PartViewExample
Team:TU Delft/PartViewExample
2010-10-26T19:54:05Z
<p>Jcnossen: </p>
<hr />
<div><html><br />
<style><br />
.partview_label { <br />
font-weight: bold; <br />
width: 200px;<br />
}<br />
.partview_data div {<br />
margin: 10px;<br />
font-family: courier;<br />
width: 400px;<br />
overflow: scroll;<br />
}<br />
</style><br />
<div id="partview"><br />
<div class="partview_label">Part Name:</div><div class="partview_name partview_data"></div><br />
<div class="partview_label">Part Description:</div><div class="partview_desc partview_data"></div><br />
<div class="partview_label">Part Author:</div><div class="partview_author partview_data"></div><br />
<div class="partview_label">Part Sequence Length:</div><div class="partview_sequence_len partview_data"></div><br />
<div class="partview_label">Part Sequence:</div><div class="partview_sequence partview_data"></div><br />
</div><br />
<br />
<script type="text/javascript"><br />
<br />
function PartInfoCallback(parts) {<br />
var part = parts[0];<br />
$(".partview_name").text(part.name);<br />
$(".partview_desc").text(part.desc);<br />
$(".partview_author").text(part.author);<br />
$(".partview_sequence").text(part.sequence);<br />
$(".partview_sequence_len").text(part.sequence.length);<br />
}<br />
</script><br />
<br />
</html><br />
<br />
{{Team:TU_Delft/PartView|part=BBa_K398005|callback=PartInfoCallback}}</div>
Jcnossen