http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=500&target=MaartenR2012.igem.org - User contributions [en]2024-03-28T14:16:59ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/File:Amsterdam_exp_fig_14.pngFile:Amsterdam exp fig 14.png2012-09-30T12:59:28Z<p>MaartenR: uploaded a new version of &quot;File:Amsterdam exp fig 14.png&quot;</p>
<hr />
<div></div>MaartenRhttp://2012.igem.org/Team:Amsterdam/project/molecular_designTeam:Amsterdam/project/molecular design2012-09-27T04:00:47Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
__NOTOC__<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<center>'''Click on one of the buttons to get the information on our modules!'''</center><br />
<html><br />
<br />
<script type='text/javascript' src='https://2012.igem.org/Template:Team:Amsterdam/scripts/mapper.js?action=raw'></script><br />
<script type='text/javascript'><br />
function sensorText(){<br />
$('#moldesign-content').empty()<br />
$('#moldesign-content').append("<h1>Sensor</h1><h4>What does a sensor mean to the Cellular Logbook?</h4><p>A logbook aims to store the occurrence, encounter or presence of an external signal. Whether this signal comes from a metabolite, a chemical or a toxin, compound or substrate, does not matter! What matters for our Cellular Logbook is if the signal can be taken up by the cell and is able to activate a promoter that will induce the transcription of our writer module. Therefore any well-characterized promoter in the parts registry can be used in the sensor module.</p><p>For our proof-of-concept we chose to implement two different promoters as sensors:</p><h4>Lac-hybrid promoter:</h4><p>The lac promoter and its different protein components have been studied for decades and is widely used as one of the common systems for recombinant protein production in <i>E. coli</i>.</p><p>In its repressive state, the LacI repressor, an allosteric protein constitutively expressed by <i>E. Coli</i>, binds the promoter with high affinity, thereby preventing transcription. Once bound by an inducer such as lactose or IPTG, the repressor is released from the promoter and the RNA polymerase complex can be formed to enable synthesis.</p><p>Our first sensor uses the lac-hybrid promoter (BBa_R0011) together with a medium ribosomal binding site (BBa_B0032). Derived from the original Lac Operon (BBa_R0010), this adaptation does not rely on the presence of glucose, but only on lactose.</p><h4>Arabinose promoter:</h4><p>This promoter is derived from wild-type E. coli and has a modified <i>AraI1</i> site, which causes this promoter to be less responsive to low concentrations of induction and therefore exhibits a lower maximum response.</p><p>pBAD is very specifically activated by L-Arabinose. In the absence of arabinose, the repressor protein AraC binds to <i>AraI1 AraO2</i>, blocking transcription. In the presence of arabinose, <i>AraC</i> binds to it and changes its conformation such that it interacts with the <i>AraI1</i> and <i>AraI2</i> operator sites, allowing transcription.</p><p>For the characterization of our system, we intentionally chose a weak version of the promoter, the pBAD-weak (BBa_K206001), from the parts registry.</p>")<br />
}<br />
<br />
function writerText(){<br />
$('#moldesign-content').empty()<br />
$('#moldesign-content').append("<h1>Writer</h1><p>To efficiently write information in our Cellular Logbook, we need a good pen! Methylation of DNA is one of the main epigenetic marks used in eukaryotes to store epigenetic information and stably alter the gene expression pattern in cells over cell division. In our Cellular Logbook design we aim to take advantage of this natural epigenetic memory system to build the writer module.</p><h4>A methyltransferase to write</h4><p>The M.ScaI protein is a type II methyltransferase expressed in Streptomyces caespitosus that recognizes specifically the sequence 5’- AGTACT- ‘3 and leaves an N4-methylcytosine (m4) on the 5th cytosine of this sequence. m4 methylation is natively absent from <i>E. coli</i> and the site that M.ScaI recognises is not methylated by any of <i>E. coli</i>'s native methylation systems (Dam, Dcm). We designed our writer module by utilizing the M.ScaI’s ability to methylate a specific site, thereby creating a writer module. The M.ScaI sequence was taken from Streptomyces caespitosus (REBASE), with some silent point mutations included to avoid forbidden sites and illegal sites specified by the parts registry. The modified M.ScaI methyltransferase sequence (BBa_K874000) constitutes the foundation of the writer module present in the Cellular Logbook.</p><h4>A polydactyl Zinc Finger (PZF) for the site</h4><p>In an attempt to achieve high specificity of the M.ScaI methyltransferase, we fused it to a Polydactyl Zinc Finger (BBa_K874001) consisting of 6 Zinc-fingers using a myc-linker (BBa_K874021). BBa_K874001 recognises the 18 bp E2C transcription factor motif 5’- GGGGCCGGAGCCGCAGTG- 3’. Assuming that PZFs have a higher binding affinity compared to methyltransferases, M.ScaI can be used for multiple sensors, which establishes a vital part of the expandability of the Cellular Logbook to log multiple signals via different sensors.</p><p>Altogether, the M.ScaI methyltransferase and the PZF create the writer module of the Cellular Logbook where the PZF provides the specificity or structure (the pen) for the M.ScaI to register events (the ink) in the Cellular Logbook.</p>")<br />
}<br />
<br />
function readerText(){<br />
$('#moldesign-content').empty()<br />
$('#moldesign-content').append("<h1>Reader</h1><p>After storing a signal through our writer module, the occurrence of DNA methylation still needs to be detected. We call this process the reader.</p><p>In prokaryotes it is understood that DNA methylation is used to protect endogenous DNA from restriction by endogenous restriction enzymes (RE). The M.ScaI methyltransferase is able to methylate a specific DNA sequence which can be recognized by the ScaI restriction enzyme. Addition of a methyl group to the 5th cytosine of this sequence inhibits the binding of ScaI restriction enzyme and cutting of the DNA at this specific recognition site.</p><p>To make a complete reader module an interface for the signal to be stored and retrieved was required. For this purpose the reader module (BBa_K874040) was created comprising of the consensus ScaI restriction site surrounded by two Polydactyl Zinc Finger binding sites from the writer module.</p><p>The information stored in the Cellular Logbook and can be retrieved as described in the “Experimental Results”. Following plasmid isolation and restriction digestion using the ScaI restriction enzyme, the methylation status of the Cellular Logbook can be inferred according to the restriction profile visualized on gel electrophoresis.</p>")<br />
}<br />
</script><br />
<div><br />
<a href="https://2012.igem.org/Team:Amsterdam/software/logbook_designer/webtool><img src="https://static.igem.org/mediawiki/2012/d/d7/Fred.png" width="100px" \></a><br />
<img src="https://static.igem.org/mediawiki/2012/e/ee/Amsterdam_moldesign.png" alt="" usemap="#MolDesign" style="border-style:none; width:auto; border=0" class="mapper" /><br />
</div><br />
<div><br />
<map id="MolDesign" name="MolDesign"><br />
<area shape="poly" alt="" coords="177,50,287,50,287,51,289,51,289,52,291,52,291,53,293,53,293,54,294,54,294,55,295,55,295,56,296,56,296,57,297,57,297,58,298,58,298,60,299,60,299,62,300,62,300,65,301,65,301,101,301,103,300,103,300,106,299,106,299,108,298,108,298,110,297,110,297,111,296,111,296,112,295,112,295,113,294,113,294,114,293,114,293,115,291,115,291,116,289,116,289,117,289,117,286,117,286,118,174,118,174,117,171,117,171,116,169,116,169,115,167,115,167,114,166,114,166,113,165,113,165,112,164,112,164,111,163,111,163,110,162,110,162,108,161,108,161,106,160,106,160,103,159,103,159,65,160,65,160,62,161,62,161,60,162,60,162,58,163,58,163,57,164,57,164,56,165,56,165,55,166,55,166,54,167,54,167,53,169,53,169,52,171,52,171,51,175,51,175,50,178,50,181,50,178,50" nohref title="" onClick="sensorText()" /><br />
<area shape="poly" alt="" coords="482,49,593,49,593,50,596,50,596,51,598,51,598,52,600,52,600,53,601,53,601,54,602,54,602,55,603,55,603,56,604,56,604,57,605,57,605,58,606,59,606,60,607,61,607,62,607,63,608,64,608,101,608,105,607,107,606,109,605,110,604,112,602,114,599,116,597,117,596,118,595,118,594,119,478,119,476,118,474,117,472,115,470,114,466,109,465,106,464,104,464,63,469,55,478,49" nohref title="" onClick="readerText()" /><br />
<area shape="poly" alt="" coords="330,48,435,48,437,49,441,49,441,50,443,50,443,51,445,51,449,55,453,59,453,61,454,63,455,66,455,99,455,104,450,113,444,117,437,119,325,119,319,115,314,110,312,107,311,104,311,63,317,54,322,51,327,48" nohref title="" onClick="writerText()" /><br />
<area shape="default" nohref="nohref" alt="" /><br />
</map><br />
</div><br />
</html><br />
<center>[https://2012.igem.org/Team:Amsterdam/software/logbook_designer/webtool '''Design your own logbook!''']</center><br\><br\><br />
<div id='moldesign-content'><br />
<h1>Sensor</h1><h4>What does a sensor mean to the Cellular Logbook?</h4><p>A logbook aims to store the occurrence, encounter or presence of an external signal. Whether this signal comes from a metabolite, a chemical or a toxin, compound or substrate, does not matter! What matters for our Cellular Logbook is if the signal can be taken up by the cell and is able to activate a promoter that will induce the transcription of our writer module. Therefore any well-characterized promoter in the parts registry can be used in the sensor module.</p><p>For our proof-of-concept we chose to implement two different promoters as sensors:</p><h4>Lac-hybrid promoter:</h4><p>The lac promoter and its different protein components have been studied for decades and is widely used as one of the common systems for recombinant protein production in <i>E. coli</i>.</p><p>In its repressive state, the LacI repressor, an allosteric protein constitutively expressed by <i>E. Coli</i>, binds the promoter with high affinity, thereby preventing transcription. Once bound by an inducer such as lactose or IPTG, the repressor is released from the promoter and the RNA polymerase complex can be formed to enable synthesis.</p><p>Our first sensor uses the lac-hybrid promoter (BBa_R0011) together with a medium ribosomal binding site (BBa_B0032). Derived from the original Lac Operon (BBa_R0010), this adaptation does not rely on the presence of glucose, but only on lactose.</p><h4>Arabinose promoter:</h4><p>This promoter is derived from wild-type <i>E. coli</i> and has a modified <i>AraI1</i> site, which causes this promoter to be less responsive to low concentrations of induction and therefore exhibits a lower maximum response.</p><p>pBAD is very specifically activated by L-Arabinose. In the absence of arabinose, the repressor protein AraC binds to AraI1 AraO2, blocking transcription. In the presence of arabinose, <i>AraC</i> binds to it and changes its conformation such that it interacts with the <i>AraI1</i> and <i>AraI2</i> operator sites, allowing transcription.</p><p>For the characterization of our system, we intentionally chose a weak version of the promoter, the pBAD-weak (BBa_K206001), from the parts registry.</p><br />
</div><br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/project/molecular_designTeam:Amsterdam/project/molecular design2012-09-27T04:00:27Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
__NOTOC__<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<center>'''Click on one of the buttons to get the information on our modules! Click the Logbook Designer to design your own logbook!'''</center><br />
<html><br />
<br />
<script type='text/javascript' src='https://2012.igem.org/Template:Team:Amsterdam/scripts/mapper.js?action=raw'></script><br />
<script type='text/javascript'><br />
function sensorText(){<br />
$('#moldesign-content').empty()<br />
$('#moldesign-content').append("<h1>Sensor</h1><h4>What does a sensor mean to the Cellular Logbook?</h4><p>A logbook aims to store the occurrence, encounter or presence of an external signal. Whether this signal comes from a metabolite, a chemical or a toxin, compound or substrate, does not matter! What matters for our Cellular Logbook is if the signal can be taken up by the cell and is able to activate a promoter that will induce the transcription of our writer module. Therefore any well-characterized promoter in the parts registry can be used in the sensor module.</p><p>For our proof-of-concept we chose to implement two different promoters as sensors:</p><h4>Lac-hybrid promoter:</h4><p>The lac promoter and its different protein components have been studied for decades and is widely used as one of the common systems for recombinant protein production in <i>E. coli</i>.</p><p>In its repressive state, the LacI repressor, an allosteric protein constitutively expressed by <i>E. Coli</i>, binds the promoter with high affinity, thereby preventing transcription. Once bound by an inducer such as lactose or IPTG, the repressor is released from the promoter and the RNA polymerase complex can be formed to enable synthesis.</p><p>Our first sensor uses the lac-hybrid promoter (BBa_R0011) together with a medium ribosomal binding site (BBa_B0032). Derived from the original Lac Operon (BBa_R0010), this adaptation does not rely on the presence of glucose, but only on lactose.</p><h4>Arabinose promoter:</h4><p>This promoter is derived from wild-type E. coli and has a modified <i>AraI1</i> site, which causes this promoter to be less responsive to low concentrations of induction and therefore exhibits a lower maximum response.</p><p>pBAD is very specifically activated by L-Arabinose. In the absence of arabinose, the repressor protein AraC binds to <i>AraI1 AraO2</i>, blocking transcription. In the presence of arabinose, <i>AraC</i> binds to it and changes its conformation such that it interacts with the <i>AraI1</i> and <i>AraI2</i> operator sites, allowing transcription.</p><p>For the characterization of our system, we intentionally chose a weak version of the promoter, the pBAD-weak (BBa_K206001), from the parts registry.</p>")<br />
}<br />
<br />
function writerText(){<br />
$('#moldesign-content').empty()<br />
$('#moldesign-content').append("<h1>Writer</h1><p>To efficiently write information in our Cellular Logbook, we need a good pen! Methylation of DNA is one of the main epigenetic marks used in eukaryotes to store epigenetic information and stably alter the gene expression pattern in cells over cell division. In our Cellular Logbook design we aim to take advantage of this natural epigenetic memory system to build the writer module.</p><h4>A methyltransferase to write</h4><p>The M.ScaI protein is a type II methyltransferase expressed in Streptomyces caespitosus that recognizes specifically the sequence 5’- AGTACT- ‘3 and leaves an N4-methylcytosine (m4) on the 5th cytosine of this sequence. m4 methylation is natively absent from <i>E. coli</i> and the site that M.ScaI recognises is not methylated by any of <i>E. coli</i>'s native methylation systems (Dam, Dcm). We designed our writer module by utilizing the M.ScaI’s ability to methylate a specific site, thereby creating a writer module. The M.ScaI sequence was taken from Streptomyces caespitosus (REBASE), with some silent point mutations included to avoid forbidden sites and illegal sites specified by the parts registry. The modified M.ScaI methyltransferase sequence (BBa_K874000) constitutes the foundation of the writer module present in the Cellular Logbook.</p><h4>A polydactyl Zinc Finger (PZF) for the site</h4><p>In an attempt to achieve high specificity of the M.ScaI methyltransferase, we fused it to a Polydactyl Zinc Finger (BBa_K874001) consisting of 6 Zinc-fingers using a myc-linker (BBa_K874021). BBa_K874001 recognises the 18 bp E2C transcription factor motif 5’- GGGGCCGGAGCCGCAGTG- 3’. Assuming that PZFs have a higher binding affinity compared to methyltransferases, M.ScaI can be used for multiple sensors, which establishes a vital part of the expandability of the Cellular Logbook to log multiple signals via different sensors.</p><p>Altogether, the M.ScaI methyltransferase and the PZF create the writer module of the Cellular Logbook where the PZF provides the specificity or structure (the pen) for the M.ScaI to register events (the ink) in the Cellular Logbook.</p>")<br />
}<br />
<br />
function readerText(){<br />
$('#moldesign-content').empty()<br />
$('#moldesign-content').append("<h1>Reader</h1><p>After storing a signal through our writer module, the occurrence of DNA methylation still needs to be detected. We call this process the reader.</p><p>In prokaryotes it is understood that DNA methylation is used to protect endogenous DNA from restriction by endogenous restriction enzymes (RE). The M.ScaI methyltransferase is able to methylate a specific DNA sequence which can be recognized by the ScaI restriction enzyme. Addition of a methyl group to the 5th cytosine of this sequence inhibits the binding of ScaI restriction enzyme and cutting of the DNA at this specific recognition site.</p><p>To make a complete reader module an interface for the signal to be stored and retrieved was required. For this purpose the reader module (BBa_K874040) was created comprising of the consensus ScaI restriction site surrounded by two Polydactyl Zinc Finger binding sites from the writer module.</p><p>The information stored in the Cellular Logbook and can be retrieved as described in the “Experimental Results”. Following plasmid isolation and restriction digestion using the ScaI restriction enzyme, the methylation status of the Cellular Logbook can be inferred according to the restriction profile visualized on gel electrophoresis.</p>")<br />
}<br />
</script><br />
<div><br />
<a href="https://2012.igem.org/Team:Amsterdam/software/logbook_designer/webtool><img src="https://static.igem.org/mediawiki/2012/d/d7/Fred.png" width="100px" \></a><br />
<img src="https://static.igem.org/mediawiki/2012/e/ee/Amsterdam_moldesign.png" alt="" usemap="#MolDesign" style="border-style:none; width:auto; border=0" class="mapper" /><br />
</div><br />
<div><br />
<map id="MolDesign" name="MolDesign"><br />
<area shape="poly" alt="" coords="177,50,287,50,287,51,289,51,289,52,291,52,291,53,293,53,293,54,294,54,294,55,295,55,295,56,296,56,296,57,297,57,297,58,298,58,298,60,299,60,299,62,300,62,300,65,301,65,301,101,301,103,300,103,300,106,299,106,299,108,298,108,298,110,297,110,297,111,296,111,296,112,295,112,295,113,294,113,294,114,293,114,293,115,291,115,291,116,289,116,289,117,289,117,286,117,286,118,174,118,174,117,171,117,171,116,169,116,169,115,167,115,167,114,166,114,166,113,165,113,165,112,164,112,164,111,163,111,163,110,162,110,162,108,161,108,161,106,160,106,160,103,159,103,159,65,160,65,160,62,161,62,161,60,162,60,162,58,163,58,163,57,164,57,164,56,165,56,165,55,166,55,166,54,167,54,167,53,169,53,169,52,171,52,171,51,175,51,175,50,178,50,181,50,178,50" nohref title="" onClick="sensorText()" /><br />
<area shape="poly" alt="" coords="482,49,593,49,593,50,596,50,596,51,598,51,598,52,600,52,600,53,601,53,601,54,602,54,602,55,603,55,603,56,604,56,604,57,605,57,605,58,606,59,606,60,607,61,607,62,607,63,608,64,608,101,608,105,607,107,606,109,605,110,604,112,602,114,599,116,597,117,596,118,595,118,594,119,478,119,476,118,474,117,472,115,470,114,466,109,465,106,464,104,464,63,469,55,478,49" nohref title="" onClick="readerText()" /><br />
<area shape="poly" alt="" coords="330,48,435,48,437,49,441,49,441,50,443,50,443,51,445,51,449,55,453,59,453,61,454,63,455,66,455,99,455,104,450,113,444,117,437,119,325,119,319,115,314,110,312,107,311,104,311,63,317,54,322,51,327,48" nohref title="" onClick="writerText()" /><br />
<area shape="default" nohref="nohref" alt="" /><br />
</map><br />
</div><br />
</html><br />
<center>[https://2012.igem.org/Team:Amsterdam/software/logbook_designer/webtool '''Design your own logbook!''']</center><br\><br\><br />
<div id='moldesign-content'><br />
<h1>Sensor</h1><h4>What does a sensor mean to the Cellular Logbook?</h4><p>A logbook aims to store the occurrence, encounter or presence of an external signal. Whether this signal comes from a metabolite, a chemical or a toxin, compound or substrate, does not matter! What matters for our Cellular Logbook is if the signal can be taken up by the cell and is able to activate a promoter that will induce the transcription of our writer module. Therefore any well-characterized promoter in the parts registry can be used in the sensor module.</p><p>For our proof-of-concept we chose to implement two different promoters as sensors:</p><h4>Lac-hybrid promoter:</h4><p>The lac promoter and its different protein components have been studied for decades and is widely used as one of the common systems for recombinant protein production in <i>E. coli</i>.</p><p>In its repressive state, the LacI repressor, an allosteric protein constitutively expressed by <i>E. Coli</i>, binds the promoter with high affinity, thereby preventing transcription. Once bound by an inducer such as lactose or IPTG, the repressor is released from the promoter and the RNA polymerase complex can be formed to enable synthesis.</p><p>Our first sensor uses the lac-hybrid promoter (BBa_R0011) together with a medium ribosomal binding site (BBa_B0032). Derived from the original Lac Operon (BBa_R0010), this adaptation does not rely on the presence of glucose, but only on lactose.</p><h4>Arabinose promoter:</h4><p>This promoter is derived from wild-type <i>E. coli</i> and has a modified <i>AraI1</i> site, which causes this promoter to be less responsive to low concentrations of induction and therefore exhibits a lower maximum response.</p><p>pBAD is very specifically activated by L-Arabinose. In the absence of arabinose, the repressor protein AraC binds to AraI1 AraO2, blocking transcription. In the presence of arabinose, <i>AraC</i> binds to it and changes its conformation such that it interacts with the <i>AraI1</i> and <i>AraI2</i> operator sites, allowing transcription.</p><p>For the characterization of our system, we intentionally chose a weak version of the promoter, the pBAD-weak (BBa_K206001), from the parts registry.</p><br />
</div><br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/project/molecular_designTeam:Amsterdam/project/molecular design2012-09-27T03:59:52Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
__NOTOC__<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<center>'''Click on one of the buttons to get the information on our modules! Click the Logbook Designer to design your own logbook!'''</center><br />
<html><br />
<br />
<script type='text/javascript' src='https://2012.igem.org/Template:Team:Amsterdam/scripts/mapper.js?action=raw'></script><br />
<script type='text/javascript'><br />
function sensorText(){<br />
$('#moldesign-content').empty()<br />
$('#moldesign-content').append("<h1>Sensor</h1><h4>What does a sensor mean to the Cellular Logbook?</h4><p>A logbook aims to store the occurrence, encounter or presence of an external signal. Whether this signal comes from a metabolite, a chemical or a toxin, compound or substrate, does not matter! What matters for our Cellular Logbook is if the signal can be taken up by the cell and is able to activate a promoter that will induce the transcription of our writer module. Therefore any well-characterized promoter in the parts registry can be used in the sensor module.</p><p>For our proof-of-concept we chose to implement two different promoters as sensors:</p><h4>Lac-hybrid promoter:</h4><p>The lac promoter and its different protein components have been studied for decades and is widely used as one of the common systems for recombinant protein production in <i>E. coli</i>.</p><p>In its repressive state, the LacI repressor, an allosteric protein constitutively expressed by <i>E. Coli</i>, binds the promoter with high affinity, thereby preventing transcription. Once bound by an inducer such as lactose or IPTG, the repressor is released from the promoter and the RNA polymerase complex can be formed to enable synthesis.</p><p>Our first sensor uses the lac-hybrid promoter (BBa_R0011) together with a medium ribosomal binding site (BBa_B0032). Derived from the original Lac Operon (BBa_R0010), this adaptation does not rely on the presence of glucose, but only on lactose.</p><h4>Arabinose promoter:</h4><p>This promoter is derived from wild-type E. coli and has a modified <i>AraI1</i> site, which causes this promoter to be less responsive to low concentrations of induction and therefore exhibits a lower maximum response.</p><p>pBAD is very specifically activated by L-Arabinose. In the absence of arabinose, the repressor protein AraC binds to <i>AraI1 AraO2</i>, blocking transcription. In the presence of arabinose, <i>AraC</i> binds to it and changes its conformation such that it interacts with the <i>AraI1</i> and <i>AraI2</i> operator sites, allowing transcription.</p><p>For the characterization of our system, we intentionally chose a weak version of the promoter, the pBAD-weak (BBa_K206001), from the parts registry.</p>")<br />
}<br />
<br />
function writerText(){<br />
$('#moldesign-content').empty()<br />
$('#moldesign-content').append("<h1>Writer</h1><p>To efficiently write information in our Cellular Logbook, we need a good pen! Methylation of DNA is one of the main epigenetic marks used in eukaryotes to store epigenetic information and stably alter the gene expression pattern in cells over cell division. In our Cellular Logbook design we aim to take advantage of this natural epigenetic memory system to build the writer module.</p><h4>A methyltransferase to write</h4><p>The M.ScaI protein is a type II methyltransferase expressed in Streptomyces caespitosus that recognizes specifically the sequence 5’- AGTACT- ‘3 and leaves an N4-methylcytosine (m4) on the 5th cytosine of this sequence. m4 methylation is natively absent from <i>E. coli</i> and the site that M.ScaI recognises is not methylated by any of <i>E. coli</i>'s native methylation systems (Dam, Dcm). We designed our writer module by utilizing the M.ScaI’s ability to methylate a specific site, thereby creating a writer module. The M.ScaI sequence was taken from Streptomyces caespitosus (REBASE), with some silent point mutations included to avoid forbidden sites and illegal sites specified by the parts registry. The modified M.ScaI methyltransferase sequence (BBa_K874000) constitutes the foundation of the writer module present in the Cellular Logbook.</p><h4>A polydactyl Zinc Finger (PZF) for the site</h4><p>In an attempt to achieve high specificity of the M.ScaI methyltransferase, we fused it to a Polydactyl Zinc Finger (BBa_K874001) consisting of 6 Zinc-fingers using a myc-linker (BBa_K874021). BBa_K874001 recognises the 18 bp E2C transcription factor motif 5’- GGGGCCGGAGCCGCAGTG- 3’. Assuming that PZFs have a higher binding affinity compared to methyltransferases, M.ScaI can be used for multiple sensors, which establishes a vital part of the expandability of the Cellular Logbook to log multiple signals via different sensors.</p><p>Altogether, the M.ScaI methyltransferase and the PZF create the writer module of the Cellular Logbook where the PZF provides the specificity or structure (the pen) for the M.ScaI to register events (the ink) in the Cellular Logbook.</p>")<br />
}<br />
<br />
function readerText(){<br />
$('#moldesign-content').empty()<br />
$('#moldesign-content').append("<h1>Reader</h1><p>After storing a signal through our writer module, the occurrence of DNA methylation still needs to be detected. We call this process the reader.</p><p>In prokaryotes it is understood that DNA methylation is used to protect endogenous DNA from restriction by endogenous restriction enzymes (RE). The M.ScaI methyltransferase is able to methylate a specific DNA sequence which can be recognized by the ScaI restriction enzyme. Addition of a methyl group to the 5th cytosine of this sequence inhibits the binding of ScaI restriction enzyme and cutting of the DNA at this specific recognition site.</p><p>To make a complete reader module an interface for the signal to be stored and retrieved was required. For this purpose the reader module (BBa_K874040) was created comprising of the consensus ScaI restriction site surrounded by two Polydactyl Zinc Finger binding sites from the writer module.</p><p>The information stored in the Cellular Logbook and can be retrieved as described in the “Experimental Results”. Following plasmid isolation and restriction digestion using the ScaI restriction enzyme, the methylation status of the Cellular Logbook can be inferred according to the restriction profile visualized on gel electrophoresis.</p>")<br />
}<br />
</script><br />
<div><br />
<a href="https://2012.igem.org/Team:Amsterdam/software/logbook_designer/webtool><img src="https://static.igem.org/mediawiki/2012/d/d7/Fred.png" width="100px" \></a><br />
<img src="https://static.igem.org/mediawiki/2012/e/ee/Amsterdam_moldesign.png" alt="" usemap="#MolDesign" style="border-style:none; width:auto; border=0" class="mapper" /><br />
</div><br />
<div><br />
<map id="MolDesign" name="MolDesign"><br />
<area shape="poly" alt="" coords="177,50,287,50,287,51,289,51,289,52,291,52,291,53,293,53,293,54,294,54,294,55,295,55,295,56,296,56,296,57,297,57,297,58,298,58,298,60,299,60,299,62,300,62,300,65,301,65,301,101,301,103,300,103,300,106,299,106,299,108,298,108,298,110,297,110,297,111,296,111,296,112,295,112,295,113,294,113,294,114,293,114,293,115,291,115,291,116,289,116,289,117,289,117,286,117,286,118,174,118,174,117,171,117,171,116,169,116,169,115,167,115,167,114,166,114,166,113,165,113,165,112,164,112,164,111,163,111,163,110,162,110,162,108,161,108,161,106,160,106,160,103,159,103,159,65,160,65,160,62,161,62,161,60,162,60,162,58,163,58,163,57,164,57,164,56,165,56,165,55,166,55,166,54,167,54,167,53,169,53,169,52,171,52,171,51,175,51,175,50,178,50,181,50,178,50" nohref title="" onClick="sensorText()" /><br />
<area shape="poly" alt="" coords="482,49,593,49,593,50,596,50,596,51,598,51,598,52,600,52,600,53,601,53,601,54,602,54,602,55,603,55,603,56,604,56,604,57,605,57,605,58,606,59,606,60,607,61,607,62,607,63,608,64,608,101,608,105,607,107,606,109,605,110,604,112,602,114,599,116,597,117,596,118,595,118,594,119,478,119,476,118,474,117,472,115,470,114,466,109,465,106,464,104,464,63,469,55,478,49" nohref title="" onClick="readerText()" /><br />
<area shape="poly" alt="" coords="330,48,435,48,437,49,441,49,441,50,443,50,443,51,445,51,449,55,453,59,453,61,454,63,455,66,455,99,455,104,450,113,444,117,437,119,325,119,319,115,314,110,312,107,311,104,311,63,317,54,322,51,327,48" nohref title="" onClick="writerText()" /><br />
<area shape="default" nohref="nohref" alt="" /><br />
</map><br />
</div><br />
</html><br />
[https://2012.igem.org/Team:Amsterdam/software/logbook_designer/webtool Design your own logbook!]<br\><br />
<div id='moldesign-content'><br />
<h1>Sensor</h1><h4>What does a sensor mean to the Cellular Logbook?</h4><p>A logbook aims to store the occurrence, encounter or presence of an external signal. Whether this signal comes from a metabolite, a chemical or a toxin, compound or substrate, does not matter! What matters for our Cellular Logbook is if the signal can be taken up by the cell and is able to activate a promoter that will induce the transcription of our writer module. Therefore any well-characterized promoter in the parts registry can be used in the sensor module.</p><p>For our proof-of-concept we chose to implement two different promoters as sensors:</p><h4>Lac-hybrid promoter:</h4><p>The lac promoter and its different protein components have been studied for decades and is widely used as one of the common systems for recombinant protein production in <i>E. coli</i>.</p><p>In its repressive state, the LacI repressor, an allosteric protein constitutively expressed by <i>E. Coli</i>, binds the promoter with high affinity, thereby preventing transcription. Once bound by an inducer such as lactose or IPTG, the repressor is released from the promoter and the RNA polymerase complex can be formed to enable synthesis.</p><p>Our first sensor uses the lac-hybrid promoter (BBa_R0011) together with a medium ribosomal binding site (BBa_B0032). Derived from the original Lac Operon (BBa_R0010), this adaptation does not rely on the presence of glucose, but only on lactose.</p><h4>Arabinose promoter:</h4><p>This promoter is derived from wild-type <i>E. coli</i> and has a modified <i>AraI1</i> site, which causes this promoter to be less responsive to low concentrations of induction and therefore exhibits a lower maximum response.</p><p>pBAD is very specifically activated by L-Arabinose. In the absence of arabinose, the repressor protein AraC binds to AraI1 AraO2, blocking transcription. In the presence of arabinose, <i>AraC</i> binds to it and changes its conformation such that it interacts with the <i>AraI1</i> and <i>AraI2</i> operator sites, allowing transcription.</p><p>For the characterization of our system, we intentionally chose a weak version of the promoter, the pBAD-weak (BBa_K206001), from the parts registry.</p><br />
</div><br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/project/molecular_designTeam:Amsterdam/project/molecular design2012-09-27T03:59:03Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
__NOTOC__<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<center>'''Click on one of the buttons to get the information on our modules! Click the Logbook Designer to design your own logbook!'''</center><br />
<html><br />
<br />
<script type='text/javascript' src='https://2012.igem.org/Template:Team:Amsterdam/scripts/mapper.js?action=raw'></script><br />
<script type='text/javascript'><br />
function sensorText(){<br />
$('#moldesign-content').empty()<br />
$('#moldesign-content').append("<h1>Sensor</h1><h4>What does a sensor mean to the Cellular Logbook?</h4><p>A logbook aims to store the occurrence, encounter or presence of an external signal. Whether this signal comes from a metabolite, a chemical or a toxin, compound or substrate, does not matter! What matters for our Cellular Logbook is if the signal can be taken up by the cell and is able to activate a promoter that will induce the transcription of our writer module. Therefore any well-characterized promoter in the parts registry can be used in the sensor module.</p><p>For our proof-of-concept we chose to implement two different promoters as sensors:</p><h4>Lac-hybrid promoter:</h4><p>The lac promoter and its different protein components have been studied for decades and is widely used as one of the common systems for recombinant protein production in <i>E. coli</i>.</p><p>In its repressive state, the LacI repressor, an allosteric protein constitutively expressed by <i>E. Coli</i>, binds the promoter with high affinity, thereby preventing transcription. Once bound by an inducer such as lactose or IPTG, the repressor is released from the promoter and the RNA polymerase complex can be formed to enable synthesis.</p><p>Our first sensor uses the lac-hybrid promoter (BBa_R0011) together with a medium ribosomal binding site (BBa_B0032). Derived from the original Lac Operon (BBa_R0010), this adaptation does not rely on the presence of glucose, but only on lactose.</p><h4>Arabinose promoter:</h4><p>This promoter is derived from wild-type E. coli and has a modified <i>AraI1</i> site, which causes this promoter to be less responsive to low concentrations of induction and therefore exhibits a lower maximum response.</p><p>pBAD is very specifically activated by L-Arabinose. In the absence of arabinose, the repressor protein AraC binds to <i>AraI1 AraO2</i>, blocking transcription. In the presence of arabinose, <i>AraC</i> binds to it and changes its conformation such that it interacts with the <i>AraI1</i> and <i>AraI2</i> operator sites, allowing transcription.</p><p>For the characterization of our system, we intentionally chose a weak version of the promoter, the pBAD-weak (BBa_K206001), from the parts registry.</p>")<br />
}<br />
<br />
function writerText(){<br />
$('#moldesign-content').empty()<br />
$('#moldesign-content').append("<h1>Writer</h1><p>To efficiently write information in our Cellular Logbook, we need a good pen! Methylation of DNA is one of the main epigenetic marks used in eukaryotes to store epigenetic information and stably alter the gene expression pattern in cells over cell division. In our Cellular Logbook design we aim to take advantage of this natural epigenetic memory system to build the writer module.</p><h4>A methyltransferase to write</h4><p>The M.ScaI protein is a type II methyltransferase expressed in Streptomyces caespitosus that recognizes specifically the sequence 5’- AGTACT- ‘3 and leaves an N4-methylcytosine (m4) on the 5th cytosine of this sequence. m4 methylation is natively absent from <i>E. coli</i> and the site that M.ScaI recognises is not methylated by any of <i>E. coli</i>'s native methylation systems (Dam, Dcm). We designed our writer module by utilizing the M.ScaI’s ability to methylate a specific site, thereby creating a writer module. The M.ScaI sequence was taken from Streptomyces caespitosus (REBASE), with some silent point mutations included to avoid forbidden sites and illegal sites specified by the parts registry. The modified M.ScaI methyltransferase sequence (BBa_K874000) constitutes the foundation of the writer module present in the Cellular Logbook.</p><h4>A polydactyl Zinc Finger (PZF) for the site</h4><p>In an attempt to achieve high specificity of the M.ScaI methyltransferase, we fused it to a Polydactyl Zinc Finger (BBa_K874001) consisting of 6 Zinc-fingers using a myc-linker (BBa_K874021). BBa_K874001 recognises the 18 bp E2C transcription factor motif 5’- GGGGCCGGAGCCGCAGTG- 3’. Assuming that PZFs have a higher binding affinity compared to methyltransferases, M.ScaI can be used for multiple sensors, which establishes a vital part of the expandability of the Cellular Logbook to log multiple signals via different sensors.</p><p>Altogether, the M.ScaI methyltransferase and the PZF create the writer module of the Cellular Logbook where the PZF provides the specificity or structure (the pen) for the M.ScaI to register events (the ink) in the Cellular Logbook.</p>")<br />
}<br />
<br />
function readerText(){<br />
$('#moldesign-content').empty()<br />
$('#moldesign-content').append("<h1>Reader</h1><p>After storing a signal through our writer module, the occurrence of DNA methylation still needs to be detected. We call this process the reader.</p><p>In prokaryotes it is understood that DNA methylation is used to protect endogenous DNA from restriction by endogenous restriction enzymes (RE). The M.ScaI methyltransferase is able to methylate a specific DNA sequence which can be recognized by the ScaI restriction enzyme. Addition of a methyl group to the 5th cytosine of this sequence inhibits the binding of ScaI restriction enzyme and cutting of the DNA at this specific recognition site.</p><p>To make a complete reader module an interface for the signal to be stored and retrieved was required. For this purpose the reader module (BBa_K874040) was created comprising of the consensus ScaI restriction site surrounded by two Polydactyl Zinc Finger binding sites from the writer module.</p><p>The information stored in the Cellular Logbook and can be retrieved as described in the “Experimental Results”. Following plasmid isolation and restriction digestion using the ScaI restriction enzyme, the methylation status of the Cellular Logbook can be inferred according to the restriction profile visualized on gel electrophoresis.</p>")<br />
}<br />
</script><br />
<div><br />
<a href="https://2012.igem.org/Team:Amsterdam/software/logbook_designer/webtool><img src="https://static.igem.org/mediawiki/2012/d/d7/Fred.png" width="100px" \></a><br />
<img src="https://static.igem.org/mediawiki/2012/e/ee/Amsterdam_moldesign.png" alt="" usemap="#MolDesign" style="border-style:none; width:auto; border=0" class="mapper" /><br />
</div><br />
<div><br />
<map id="MolDesign" name="MolDesign"><br />
<area shape="poly" alt="" coords="177,50,287,50,287,51,289,51,289,52,291,52,291,53,293,53,293,54,294,54,294,55,295,55,295,56,296,56,296,57,297,57,297,58,298,58,298,60,299,60,299,62,300,62,300,65,301,65,301,101,301,103,300,103,300,106,299,106,299,108,298,108,298,110,297,110,297,111,296,111,296,112,295,112,295,113,294,113,294,114,293,114,293,115,291,115,291,116,289,116,289,117,289,117,286,117,286,118,174,118,174,117,171,117,171,116,169,116,169,115,167,115,167,114,166,114,166,113,165,113,165,112,164,112,164,111,163,111,163,110,162,110,162,108,161,108,161,106,160,106,160,103,159,103,159,65,160,65,160,62,161,62,161,60,162,60,162,58,163,58,163,57,164,57,164,56,165,56,165,55,166,55,166,54,167,54,167,53,169,53,169,52,171,52,171,51,175,51,175,50,178,50,181,50,178,50" nohref title="" onClick="sensorText()" /><br />
<area shape="poly" alt="" coords="482,49,593,49,593,50,596,50,596,51,598,51,598,52,600,52,600,53,601,53,601,54,602,54,602,55,603,55,603,56,604,56,604,57,605,57,605,58,606,59,606,60,607,61,607,62,607,63,608,64,608,101,608,105,607,107,606,109,605,110,604,112,602,114,599,116,597,117,596,118,595,118,594,119,478,119,476,118,474,117,472,115,470,114,466,109,465,106,464,104,464,63,469,55,478,49" nohref title="" onClick="readerText()" /><br />
<area shape="poly" alt="" coords="330,48,435,48,437,49,441,49,441,50,443,50,443,51,445,51,449,55,453,59,453,61,454,63,455,66,455,99,455,104,450,113,444,117,437,119,325,119,319,115,314,110,312,107,311,104,311,63,317,54,322,51,327,48" nohref title="" onClick="writerText()" /><br />
<area shape="default" nohref="nohref" alt="" /><br />
</map><br />
</div><br />
</html><br />
<div id='moldesign-content'><br />
<h1>Sensor</h1><h4>What does a sensor mean to the Cellular Logbook?</h4><p>A logbook aims to store the occurrence, encounter or presence of an external signal. Whether this signal comes from a metabolite, a chemical or a toxin, compound or substrate, does not matter! What matters for our Cellular Logbook is if the signal can be taken up by the cell and is able to activate a promoter that will induce the transcription of our writer module. Therefore any well-characterized promoter in the parts registry can be used in the sensor module.</p><p>For our proof-of-concept we chose to implement two different promoters as sensors:</p><h4>Lac-hybrid promoter:</h4><p>The lac promoter and its different protein components have been studied for decades and is widely used as one of the common systems for recombinant protein production in <i>E. coli</i>.</p><p>In its repressive state, the LacI repressor, an allosteric protein constitutively expressed by <i>E. Coli</i>, binds the promoter with high affinity, thereby preventing transcription. Once bound by an inducer such as lactose or IPTG, the repressor is released from the promoter and the RNA polymerase complex can be formed to enable synthesis.</p><p>Our first sensor uses the lac-hybrid promoter (BBa_R0011) together with a medium ribosomal binding site (BBa_B0032). Derived from the original Lac Operon (BBa_R0010), this adaptation does not rely on the presence of glucose, but only on lactose.</p><h4>Arabinose promoter:</h4><p>This promoter is derived from wild-type <i>E. coli</i> and has a modified <i>AraI1</i> site, which causes this promoter to be less responsive to low concentrations of induction and therefore exhibits a lower maximum response.</p><p>pBAD is very specifically activated by L-Arabinose. In the absence of arabinose, the repressor protein AraC binds to AraI1 AraO2, blocking transcription. In the presence of arabinose, <i>AraC</i> binds to it and changes its conformation such that it interacts with the <i>AraI1</i> and <i>AraI2</i> operator sites, allowing transcription.</p><p>For the characterization of our system, we intentionally chose a weak version of the promoter, the pBAD-weak (BBa_K206001), from the parts registry.</p><br />
</div><br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/project/molecular_designTeam:Amsterdam/project/molecular design2012-09-27T03:58:45Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
__NOTOC__<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<center>'''Click on one of the buttons to get the information on our modules! Click the Logbook Designer to design your own logbook!'''</center><br />
<html><br />
<a href="https://2012.igem.org/Team:Amsterdam/software/logbook_designer/webtool><img src="https://static.igem.org/mediawiki/2012/d/d7/Fred.png" width="100px" \></a><br />
<script type='text/javascript' src='https://2012.igem.org/Template:Team:Amsterdam/scripts/mapper.js?action=raw'></script><br />
<script type='text/javascript'><br />
function sensorText(){<br />
$('#moldesign-content').empty()<br />
$('#moldesign-content').append("<h1>Sensor</h1><h4>What does a sensor mean to the Cellular Logbook?</h4><p>A logbook aims to store the occurrence, encounter or presence of an external signal. Whether this signal comes from a metabolite, a chemical or a toxin, compound or substrate, does not matter! What matters for our Cellular Logbook is if the signal can be taken up by the cell and is able to activate a promoter that will induce the transcription of our writer module. Therefore any well-characterized promoter in the parts registry can be used in the sensor module.</p><p>For our proof-of-concept we chose to implement two different promoters as sensors:</p><h4>Lac-hybrid promoter:</h4><p>The lac promoter and its different protein components have been studied for decades and is widely used as one of the common systems for recombinant protein production in <i>E. coli</i>.</p><p>In its repressive state, the LacI repressor, an allosteric protein constitutively expressed by <i>E. Coli</i>, binds the promoter with high affinity, thereby preventing transcription. Once bound by an inducer such as lactose or IPTG, the repressor is released from the promoter and the RNA polymerase complex can be formed to enable synthesis.</p><p>Our first sensor uses the lac-hybrid promoter (BBa_R0011) together with a medium ribosomal binding site (BBa_B0032). Derived from the original Lac Operon (BBa_R0010), this adaptation does not rely on the presence of glucose, but only on lactose.</p><h4>Arabinose promoter:</h4><p>This promoter is derived from wild-type E. coli and has a modified <i>AraI1</i> site, which causes this promoter to be less responsive to low concentrations of induction and therefore exhibits a lower maximum response.</p><p>pBAD is very specifically activated by L-Arabinose. In the absence of arabinose, the repressor protein AraC binds to <i>AraI1 AraO2</i>, blocking transcription. In the presence of arabinose, <i>AraC</i> binds to it and changes its conformation such that it interacts with the <i>AraI1</i> and <i>AraI2</i> operator sites, allowing transcription.</p><p>For the characterization of our system, we intentionally chose a weak version of the promoter, the pBAD-weak (BBa_K206001), from the parts registry.</p>")<br />
}<br />
<br />
function writerText(){<br />
$('#moldesign-content').empty()<br />
$('#moldesign-content').append("<h1>Writer</h1><p>To efficiently write information in our Cellular Logbook, we need a good pen! Methylation of DNA is one of the main epigenetic marks used in eukaryotes to store epigenetic information and stably alter the gene expression pattern in cells over cell division. In our Cellular Logbook design we aim to take advantage of this natural epigenetic memory system to build the writer module.</p><h4>A methyltransferase to write</h4><p>The M.ScaI protein is a type II methyltransferase expressed in Streptomyces caespitosus that recognizes specifically the sequence 5’- AGTACT- ‘3 and leaves an N4-methylcytosine (m4) on the 5th cytosine of this sequence. m4 methylation is natively absent from <i>E. coli</i> and the site that M.ScaI recognises is not methylated by any of <i>E. coli</i>'s native methylation systems (Dam, Dcm). We designed our writer module by utilizing the M.ScaI’s ability to methylate a specific site, thereby creating a writer module. The M.ScaI sequence was taken from Streptomyces caespitosus (REBASE), with some silent point mutations included to avoid forbidden sites and illegal sites specified by the parts registry. The modified M.ScaI methyltransferase sequence (BBa_K874000) constitutes the foundation of the writer module present in the Cellular Logbook.</p><h4>A polydactyl Zinc Finger (PZF) for the site</h4><p>In an attempt to achieve high specificity of the M.ScaI methyltransferase, we fused it to a Polydactyl Zinc Finger (BBa_K874001) consisting of 6 Zinc-fingers using a myc-linker (BBa_K874021). BBa_K874001 recognises the 18 bp E2C transcription factor motif 5’- GGGGCCGGAGCCGCAGTG- 3’. Assuming that PZFs have a higher binding affinity compared to methyltransferases, M.ScaI can be used for multiple sensors, which establishes a vital part of the expandability of the Cellular Logbook to log multiple signals via different sensors.</p><p>Altogether, the M.ScaI methyltransferase and the PZF create the writer module of the Cellular Logbook where the PZF provides the specificity or structure (the pen) for the M.ScaI to register events (the ink) in the Cellular Logbook.</p>")<br />
}<br />
<br />
function readerText(){<br />
$('#moldesign-content').empty()<br />
$('#moldesign-content').append("<h1>Reader</h1><p>After storing a signal through our writer module, the occurrence of DNA methylation still needs to be detected. We call this process the reader.</p><p>In prokaryotes it is understood that DNA methylation is used to protect endogenous DNA from restriction by endogenous restriction enzymes (RE). The M.ScaI methyltransferase is able to methylate a specific DNA sequence which can be recognized by the ScaI restriction enzyme. Addition of a methyl group to the 5th cytosine of this sequence inhibits the binding of ScaI restriction enzyme and cutting of the DNA at this specific recognition site.</p><p>To make a complete reader module an interface for the signal to be stored and retrieved was required. For this purpose the reader module (BBa_K874040) was created comprising of the consensus ScaI restriction site surrounded by two Polydactyl Zinc Finger binding sites from the writer module.</p><p>The information stored in the Cellular Logbook and can be retrieved as described in the “Experimental Results”. Following plasmid isolation and restriction digestion using the ScaI restriction enzyme, the methylation status of the Cellular Logbook can be inferred according to the restriction profile visualized on gel electrophoresis.</p>")<br />
}<br />
</script><br />
<div><br />
<img src="https://static.igem.org/mediawiki/2012/e/ee/Amsterdam_moldesign.png" alt="" usemap="#MolDesign" style="border-style:none; width:auto; border=0" class="mapper" /><br />
</div><br />
<div><br />
<map id="MolDesign" name="MolDesign"><br />
<area shape="poly" alt="" coords="177,50,287,50,287,51,289,51,289,52,291,52,291,53,293,53,293,54,294,54,294,55,295,55,295,56,296,56,296,57,297,57,297,58,298,58,298,60,299,60,299,62,300,62,300,65,301,65,301,101,301,103,300,103,300,106,299,106,299,108,298,108,298,110,297,110,297,111,296,111,296,112,295,112,295,113,294,113,294,114,293,114,293,115,291,115,291,116,289,116,289,117,289,117,286,117,286,118,174,118,174,117,171,117,171,116,169,116,169,115,167,115,167,114,166,114,166,113,165,113,165,112,164,112,164,111,163,111,163,110,162,110,162,108,161,108,161,106,160,106,160,103,159,103,159,65,160,65,160,62,161,62,161,60,162,60,162,58,163,58,163,57,164,57,164,56,165,56,165,55,166,55,166,54,167,54,167,53,169,53,169,52,171,52,171,51,175,51,175,50,178,50,181,50,178,50" nohref title="" onClick="sensorText()" /><br />
<area shape="poly" alt="" coords="482,49,593,49,593,50,596,50,596,51,598,51,598,52,600,52,600,53,601,53,601,54,602,54,602,55,603,55,603,56,604,56,604,57,605,57,605,58,606,59,606,60,607,61,607,62,607,63,608,64,608,101,608,105,607,107,606,109,605,110,604,112,602,114,599,116,597,117,596,118,595,118,594,119,478,119,476,118,474,117,472,115,470,114,466,109,465,106,464,104,464,63,469,55,478,49" nohref title="" onClick="readerText()" /><br />
<area shape="poly" alt="" coords="330,48,435,48,437,49,441,49,441,50,443,50,443,51,445,51,449,55,453,59,453,61,454,63,455,66,455,99,455,104,450,113,444,117,437,119,325,119,319,115,314,110,312,107,311,104,311,63,317,54,322,51,327,48" nohref title="" onClick="writerText()" /><br />
<area shape="default" nohref="nohref" alt="" /><br />
</map><br />
</div><br />
</html><br />
<div id='moldesign-content'><br />
<h1>Sensor</h1><h4>What does a sensor mean to the Cellular Logbook?</h4><p>A logbook aims to store the occurrence, encounter or presence of an external signal. Whether this signal comes from a metabolite, a chemical or a toxin, compound or substrate, does not matter! What matters for our Cellular Logbook is if the signal can be taken up by the cell and is able to activate a promoter that will induce the transcription of our writer module. Therefore any well-characterized promoter in the parts registry can be used in the sensor module.</p><p>For our proof-of-concept we chose to implement two different promoters as sensors:</p><h4>Lac-hybrid promoter:</h4><p>The lac promoter and its different protein components have been studied for decades and is widely used as one of the common systems for recombinant protein production in <i>E. coli</i>.</p><p>In its repressive state, the LacI repressor, an allosteric protein constitutively expressed by <i>E. Coli</i>, binds the promoter with high affinity, thereby preventing transcription. Once bound by an inducer such as lactose or IPTG, the repressor is released from the promoter and the RNA polymerase complex can be formed to enable synthesis.</p><p>Our first sensor uses the lac-hybrid promoter (BBa_R0011) together with a medium ribosomal binding site (BBa_B0032). Derived from the original Lac Operon (BBa_R0010), this adaptation does not rely on the presence of glucose, but only on lactose.</p><h4>Arabinose promoter:</h4><p>This promoter is derived from wild-type <i>E. coli</i> and has a modified <i>AraI1</i> site, which causes this promoter to be less responsive to low concentrations of induction and therefore exhibits a lower maximum response.</p><p>pBAD is very specifically activated by L-Arabinose. In the absence of arabinose, the repressor protein AraC binds to AraI1 AraO2, blocking transcription. In the presence of arabinose, <i>AraC</i> binds to it and changes its conformation such that it interacts with the <i>AraI1</i> and <i>AraI2</i> operator sites, allowing transcription.</p><p>For the characterization of our system, we intentionally chose a weak version of the promoter, the pBAD-weak (BBa_K206001), from the parts registry.</p><br />
</div><br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/File:Fred.pngFile:Fred.png2012-09-27T03:57:16Z<p>MaartenR: </p>
<hr />
<div></div>MaartenRhttp://2012.igem.org/Team:Amsterdam/extra/softwareTeam:Amsterdam/extra/software2012-09-27T03:56:14Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<h1>Software</h1><br />
<br />
__NOTOC__<br />
The complexity of biology calls for the aid of computers in a synthetic biology design process.<br />
Over the summer we have created a range of useful applications to make the life of the synthetic biologist easier.<br />
<div class='clear'></div><br />
<br />
<br />
<h2>Local MediaWiki Editor 0.1</h2><br />
Also tired of using the MediaWiki standard interface for editing? Check out the script we made to make a local copy of the MediaWiki directory structure! It contains features such as uploading/downloading newer files and it works with any editor. <b>Use at your own risk though!</b> since its all automated a settup mistake can result in loss or pollution of data on your wiki.<br><br />
[https://github.com/mreijnders/Local-MediaWiki You can check it out here at git-hub]<br />
<br />
<h2>DNA Template Compiler</h2><br />
Creating custom sequences of DNA is a none trivial task. It requires checks for example RFC-10 forbidden sites, <i>E. coli</i> native restiction and GC contents optimization. This is why we create a script compiles a specially formatted DNA template file into a custom sequences that has been checked and optimized for these parameters.<br><br />
[https://github.com/mreijnders/DNA-Template-Compiler Click here to check the DNA Template Compiler out at github!]<br />
<br />
<h2>MDL2LaTeX</h2><br />
[[File:MDL2LaTeXlogo.png|frameless|center|400px]]<br />
<br />
[http://www.stompy.sourceforge.net StochPy] is a versatile stochastic simulation package implemented in Python for the simulation of biochemical networks. Its inherits the easy to learn model description language [http://www.stompy.sourceforge.net/html/inputfile_doc.html MDL] from its parent package [http://pysces.sourceforge.net/PySCeS PySCeS], which is a modelling environment with deterministic capabilities. By defining models in MDL and executing them through high-level commands in the Python or IPython shell, an easy-going experience is presented to the modeller in which one (almost) doesn&rsquo;t need to worry about implementation details.<br />
<br />
[https://github.com/slagtermaarten/MDL2Latex MDL2LaTeX] is another step towards making a modeller&rsquo;s life as carefree as possible. Exactly as its name implies: it converts MDL files to clean LaTeX representations, so the models can be easily integrated into reports. No more time wasted on tedious equation copying into LaTeX!<br />
<br />
'''Installation'''<br />
<br />
MLD2LaTeX has been tested in both Linux and Unix (Mac OS). For easy installation, first install [http://www.github.com git].<br />
<br />
Then run:<br />
<br />
<pre>git clone https://github.com/slagtermaarten/MDL2Latex.git</pre><br />
If you can&rsquo;t access git, simply go to this [https://github.com/slagtermaarten/MDL2Latex MDL2LaTeX github page] and download and extract the .zip.<br />
<br />
'''Usage'''<br />
<br />
Edit the<br />
<br />
<pre>Makefile</pre><br />
inside of this folder and change the pscfile variable to filepath of the the .psc (MDL model) file you want to convert. Save and run<br />
<br />
<pre>make</pre><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/extra/faqTeam:Amsterdam/extra/faq2012-09-27T03:56:00Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<h1>Frequently Asked Questions</h1><br />
__NOTOC__<br />
<div class='clear'></div><br />
<br />
<h4>Why not use a fluorescent protein?</h4><br />
Using a fluorescent protein has its advantages compared with the Cellular Loogbook on the other hand the Cellular Logbook also has advantages compared using a fluorescent protein(FP). The main advantage of the Cellular Logbook is that there is no limit to expandability. Spectral characteristics of FPs limit their expandibility.. <br />
<br />
<h4>Won’t endogenous methylation cause interference?</h4><br />
E. coli posses several endogenous methyltransferases which are used for epigenetic purposes of E. coli’s own genome. Our scan of these endogenous methyltransferases does not indicate that any of them posses the ability to bind to our M.ScaI-recognition site and are thus they are not able to methylate the detection site.<br />
<br />
<h4>Will the fusion-protein fold correctly or be processed by E. coli’s native systems?</h4><br />
At the moment there is no indication that this will be the case. Separately the parts of the protein have been translated and expressed in E. coli without complications. <br />
<br />
<h4>Will the introduced methyltranferase cause problems inside the microorganism?</h4><br />
Right now all our experiments are conducted in the DH5alpha strain of E. coli which does not posses the M.ScaI methyltransferase we use. Actually the M.ScaI is a type 4 methyltransferase, that does not occur naturally in E. coli. After BLAST searching the E. coli genome we also did not find any potential binding site for our M.ScaI.<br />
<br />
<h4>Won’t the memory plasmid be cut or processed by the endogenous restriction enzymes?</h4><br />
The chosen restriction sites in our plasmid are not recognized by E. coli’s endogenous restriction enzymes. The plasmid backbones and vectors that are used have all been previously used or expressed in E. coli without any complications.<br />
<br />
<h4>If there is leaky expression of the protein, would this cause interference?</h4><br />
Replication creates a progeny without a logged signal, eventually you would lose the logged signals. This perspective differs in system specific applications. If something is being sensed that continues to activate the sensors linked to the system this is not a problem. If the sensed compound is not degradable this is also not a problem. If a time-indication based sensing is done the loss of logged signal to the progeny is actually a desired phenomenon. The choice of high- or a low-copy plasmid can influence the amount of progeny methylation. In the case of using a high-copy plasmid there will be enough of the fusion protein divided during replication to keep the progeny methylated.<br />
<br />
<h4>How much specificity is created by the Zinc-finger?</h4><br />
The Zinc-finger uses an unique 18 bp sequence which is not found anywhere else on the plasmid or in the genome of E. coli. The Zinc-finger multiplies the overall probability of binding (PoB) to a much higher specificity when being attached to the M.ScaI which has its own PoB. <br />
<br />
<h4>Would constitutive active promoters cause random or unspecific methylation?</h4><br />
This is fine-tuned and/or dependent on the conditions that the experiment is being done in. Factors contributing in this are; the degradation time of the fusion protein, high or low copy plasmid and the amount of different sites present. If the degradation time is really fast then a constitutive active promoter is desirable. If the degradaton time is slow then this is unwanted. When using a low copy plasmid it could be desirable to use a constitutively active promotor, when using a high copy plasmid t this could be unwanted.<br />
<br />
<h4>When is choosing between a high or low copy plasmid the best approach?</h4><br />
If the intention is to store the occurrence of something for a longer period the best approach would be to choose for the high-copy plasmid. <br />
<br />
In the event of a time-concentration storage a low-copy plasmid would be preferable since there is less chance of the progeny receiving any Mtase during replication.<br />
<br />
<h4>What is the degredation time of the fusion protein?</h4><br />
The time of fusion protein degradation depends on the three parts of which the fusion protein is composed of. First of all the degradation of the methyltransferase is the main part which will cause loss of function when degraded. The issue of methyltransferase degradation is the most critical to answer this question regarding fusion protein degradation.<br />
The Zn-finger would, when degraded, only lower the specificity of the fusion protein and enable the M.ScaI part to bind in random places.<br />
<br />
The Myc-linker part would also create an unfortunate circumstance when degraded by releasing the Mtase into the cytoplasm for free binding at random sites.<br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/extra/protocolsTeam:Amsterdam/extra/protocols2012-09-27T03:55:42Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<h1>Protocols</h1><br/><br />
<br />
__NOTOC__<br />
<div class='clear'></div><br />
<br />
<h2>Transformation Protocol in DH5a (Invitrogen)</h2><br />
*Thaw an aliquot of competent bacteria on ice.<br />
*Add 50 µl of DH5a competent cells gently in a sterile 15 ml polypropylene tube.<br />
*Add 1 µl of ligation mixture or Gibson Assembly reaction (1 – 10 ng of DNA) to the polypropylene tube<br />
*Incubate on ice for 30 minutes.<br />
*Heat shock for 45 seconds in a water bath at 42°C, then quickly back on ice for 2 minutes.<br />
*Add 450 ml of room temperature SOC medium (Work sterile!!).<br />
*Incubate 1 hour at 37°C for antibiotic resistance expression, while shaking (225 rpm).<br />
*Spread 1/10 and 9/10 on LB plates (containing the right antibiotic).<br/><br />
<br />
<br />
Notes:<br />
*Do not shake the polypropylene tubes during the heat shock!<br />
*To increase the yield, centrifuge gently (at low speed), remove the excess of medium and then spread on the plates.<br />
*To determine transformation efficiency<br/><br/><br />
<br />
</div><br />
<div id="sub-menu" class="content-block"><br />
<br />
<h2>Gel Electrophoresis</h2><br />
*Dissolve 2g of Agarose in 200 ml TAE or TBE buffer. Heat until the solution is clear. Do not boil.<br />
*Allow to cool down and add 2µl of ethidium bromide.<br />
*Transfer to the casts + combs and leave at room temperature. Store at 4°C for later use.<br />
*Input:<br />
**5 µl DNA ladder<br />
**5 µl sample + 5 µl H2O + 1 µl loading buffer<br/><br/><br />
<br />
</div><br />
<div id="sub-menu" class="content-block"><br />
<br />
<h2>Preparation of LB Medium and Agar Plates</h2><br />
*Luria-Bertani (LB) medium is a nutritionally rich medium that can be used for the preparation of plasmid DNA and recombinant proteins. It is one of the most common media used for maintaining and cultivating recombinant strains of Escherichia coli.<br />
<br />
*To 950 ml of deionised water, add:<br />
**Bacto Tryptone – 10 grams<br />
**NaCl – 10 grams <br />
**Yeast extract – 5 grams<br />
<br />
*Shake until all the solutes have completely dissolved. Adjust the pH to 7.0 with 5N NaOH (˜ 0.2 ml). Adjust the total volume to one litre with deionised water. For LB plates, add 15g/litre of Bacto agar to the medium before autoclaving.<br />
Sterilise by autoclaving (standard autoclaving liquid cycle protocol).<br />
<br />
*Allow to cool down and add the right amount of antibiotic.<br />
<br />
*Add about 15 – 20 ml of solution to plates in a sterile environment. Allow to settle and store at 4°C.<br/><br/><br />
<br />
</div><br />
<div id="sub-menu" class="content-block"><br />
<br />
<h2>Gibson Assembly</h2><br />
<h4>Preparation of reagents</h4><br />
<br />
*Preparation of 6ml of 5X isothermal reaction buffer by combining (This buffer can be aliquoted and stored at –20°C):<br />
**3 ml of 1M Tris-HCl pH 7.5<br />
**150 ml of 2M MgCl2<br />
**60 ml of 100mM dNTP<br />
**300 ml of 1M DTT<br />
**1,5 g PEG-8000<br />
**300 ml of 100mM NAD<br/><br/><br />
<br />
<h4>One-step isothermal DNA assembly protocol (Gibson Reaction)</h4><br />
*For one reaction containing 40 µl:<br />
**8 µl 5X isothermal buffer<br />
**0.8 µl of 0.2 U.µl –1 or 1.0 U.µl –1 T5 exonuclease<br />
**4 µl of 40 U.µl –1 Taq DNA ligase<br />
**0.5 µl of 2 U.µl –1 Phusion DNA polymerase.<br />
**5 µl of DNA<br />
**Water up to 40 µl<br/><br />
<br />
*50 ºC ----- 1 hour<br/><br />
<br />
[[File:Amsterdam_Gibson_pic.png|700px]]<br\><br />
Adapted from:<br\><br />
http://www.syntheticgenomics.com <br\><br />
http://eu.idtdna.com<br />
<br />
<br />
Notes:<br />
*All isothermal assembly components can be stored at –20°C in a single mixture at 1.33X concentration for more than one year. The enzymes are still active after more than ten freeze-thaw cycles. The aliquots should be kept on ice until ready to use.<br />
*The exonuclease amount is ideal for the assembly of DNA molecules with 20– 150 bp overlaps.<br />
*Between 10 and 100 ng of each ?6 kb DNA fragment was added.<br />
*For larger DNA segments, proportional amounts of DNA should be added (for example, 250 ng of each 150 kb DNA segment).<br/><br />
<br />
</div><br />
<div id="sub-menu" class="content-block"><br />
<br />
<h2>DNA Precipitation</h2><br />
*Add 1/10 of sodium acetate (3M, pH 5.2) to the total volume of DNA to be precipitated. Vortex to make sure that the solution is well mixed.<br />
*Add 2.5 X total volume in eppendorf of 100% ethanol. Vortex and keep the mixture at -20°C for 30 minutes.<br />
*Centrifuge at maximum speed for 20 minutes.<br />
*Discard the supernatant without disturbing the pellet and add about 500 µl of 70% ethanol.<br />
*Centrifuge for one minute at maximum speed. Discard the supernatant.<br />
*Add about 500 µl of 70% ethanol again. Centrifuge for one minute at maximum speed. Discard the supernatant.<br />
*Remove as much ethanol as possible using a glass Pasteur pipette. Allow to air dry.<br />
*Resuspend the DNA in 20 µl of deionised sterile water.<br/><br />
<br />
<br />
Notes:<br />
*Keep the 100% ethanol at -20°C. The ethanol must always be cold prior to use.<br />
*Centrifugation at 4°C is preferable.<br/><br />
<br />
</div><br />
<div id="sub-menu" class="content-block"><br />
<br />
<h2>Preparation of Competent E. coli</h2><br />
<br />
<h4>Preparation of Reagents</h4><br />
<br />
*Buffer 1:<br />
**30 mM KOAc, 100 mM RbC12, 10 mM CaCl2, 50 mM MnC12, 15% glycerol, pH 5.8<br />
**For 500 ml: 1.47 g KOAc (MW 98.14)<br />
**6.04 g RbC12 (MW 120.92)<br />
**0.74 g CaC12 (MW 147.02)<br />
**4.94 g MnC12-4H20 (MW 197.9)<br />
**75 ml glycerol<br />
**pH to 5.8 with dilute acetic acid.<br />
*Filter sterilize<br />
*Buffer 2:<br />
**10 mM MOPS/KOH pH 6.5, 75 mM CaC12, 10 mM RbC12, 15% glycerol<br />
**For 100 ml: 0.21 g MOPS (MW 209.26)<br />
**1.10 g CaC12 (MW 147.02)<br />
**0.12 g RbC12 (MW 120.92)<br />
**15 ml glycerol<br />
**pH to 6.5 with 1 N KOH.<br />
*Filter sterilize.<br/><br />
<br />
<h4>Protocol</h4><br />
<br />
*Streak desired E. coli strain on fresh LB plate. Grow overnight at 37°C.<br />
*Inoculate a single colony into 5 ml LB. Grow overnight, shaking at 37° C.<br />
*Inoculate about 1 ml into 200 ml LB in a 2 1 flask. Shake at 37°C until OD550 = 0.5.<br />
*Chill flask in ice-water 5 minutes.<br />
*Spin 5 minutes at 6,000 rpm in GS3 rotor.<br />
*Resuspend pellet in 80 ml ice cold Buffer 1.<br />
*Chill in ice-water 5 minutes.<br />
*Spin 5 minutes at 6,000 rpm in GS3 rotor.<br />
*Resuspend pellet in 8 ml ice cold Buffer 2.<br />
*Chill in ice-water 15 minutes.<br />
*Make 50, 100 and 200 ml aliquots in 1.5 ml Eppendorf tubes.<br />
*Flash-freeze in liquid nitrogen.<br />
*Store at -80 degrees.<br/><br />
<br />
<br />
Notes:<br />
*Keep buffers, tips, tubes, rotors, etc. ice cold<br/><br/><br />
<br />
<h2>Long time exposure protocol</h2><br />
'''Day 1'''<br />
<br />
*1. End of the day: Prepare and incubate (37C) 6x 3ml pSB1AT3+LacH containing bacterial cultures (with appropriate antibiotic)<br />
*2. End of the day: Prepare and incubate (37C) 6x 3ml pSB1AT3+pBAD containing bacterial cultures (with appropriate antibiotic)<br />
<br />
'''Day 2'''<br />
<br />
*1. Start of the day: To 3 cultures from Day 1, Step 1 add to each 15 ul of 1M IPTG solution<br />
*2. Start of the day: To 3 cultures from Day 1, Step 2 add to each 120 ul of 100% (1g/ml) Arabinose solution<br />
*3. Keep all cultures incubated at 37C<br />
<br />
'''Day 3'''<br />
<br />
*1. Start of day: From 1 of the cultures from Day 2, Step 1 take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (IPTG+ 1 day exposure)<br />
*2. Start of day: From 1 of the cultures from Day 2, Step 2 take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (IPTG- 1 day negative control)<br />
*3. Start of day: From 1 of the cultures from Day 1, Step 1 (and not used in Day 2, Step 1) take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (ARA+ 1 day exposure)<br />
*4. Start of day: From 1 of the cultures from Day 1, Step 2 (and not used in Day 2, Step 2) take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (ARA- 1 day negative control)<br />
*5. Start of day: To the 2 remaining cultures of Day 2, Step 2 each add 120 ul of 100% (1g/ml) Arabinose solution<br />
*6. Keep all (remaining) cultures incubated at 37C<br />
<br />
'''Day 4'''<br />
<br />
*1. Start of day: From 1 of the (remaining) cultures from Day 2, Step 1 take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (IPTG+ 2days exposure)<br />
*2. Start of day: From 1 of the (remaining) cultures from Day 2, Step 2 take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (IPTG- 2 days negative control)<br />
*3. Start of day: From 1 of the (remaining) cultures from Day 1, Step 1 (and not used in Day 2, Step 1) take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (ARA+ 2 days exposure)<br />
*4. Start of day: From 1 of the (remaining) cultures from Day 1, Step 2 (and not used in Day 2, Step 2) take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (ARA- 2 days negative control)<br />
*5. Start of day: To the 1 remaining culture of Day 2, Step 2 add 120 ul of 100% (1g/ml) Arabinose solution<br />
*6. Keep all (remaining) cultures incubated at 37C<br />
<br />
'''Day 5'''<br />
<br />
*1. Start of day: From the 1 remaining culture from Day 2, Step 1 take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (IPTG+ 3 days exposure)<br />
*2. Start of day: From the 1 remaining culture from Day 2, Step 2 take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (IPTG- 3 days negative control)<br />
*3. Start of day: From the 1 remaining culture from Day 1, Step 1 (and not used in Day 2, Step 1) take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (ARA+ 3 days exposure)<br />
*4. Start of day: From the 1 remaining culture from Day 1, Step 2 (and not used in Day 2, Step 2) take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (ARA- 3 days negative control)<br />
<br />
<br\><br\><br />
<br />
<h2>Exposure Window Experiment</h2><br />
'''Day 1'''<br\><br />
*Set in 2 o/n cultures of 10 ml with the psb1at3-lac/bad-mtase (mutated) with antibiotics<br />
<br />
'''Day 2'''<br\><br />
*Set each o/n culture through in 4 different tubes. Take 2 ml and fill up to 3 ml with LB and antibiotics.<br />
<br />
'''Day 3'''<br\><br />
*Give 15 ul of 1M IPTG and 1200 ul of 10% Arabinose creating;<br />
**LacH; 1;normal (-), 2;normal(-), 3;signal(+), 4;signal(+)<br />
**pBAD;1;normal (-), 2;normal(-), 3;signal(+), 4;signal(+)<br />
<br />
'''Day 4'''<br\><br />
*Take 1 ml sample checking day 3 created settings<br />
*Give again same IPTG and Arabinose creating;<br />
**LacH; 1;normal (--), 2;signal(-+), 3;normal(+-), 4;signal(++)<br />
**pBAD;1; normal (--), 2;signal(-+), 3;normal(+-), 4;signal(++)<br />
<br />
'''Day 5'''<br\><br />
*Take 1 ml sample checking day 4 created settings<br />
*Mini-prep all taken samples and digest with ScaI<br />
<br />
<br\><br\><br />
<br />
<h2>Growth curve experiment 5 september</h2><br />
<br />
'''4 samples'''<br\><br />
*LacH promoter + Mtase in psb1at3 [with and without IPTG]<br />
*pBAD promoter + Mtase in psb1at3 [with and without Arabinose]<br />
<br />
'''The starting samples'''<br\><br />
*100 (95*) ml LB broth<br />
*5 ml of O/N culture (one of two constructs creating a 5% state)<br />
*antibiotic: Ampicilin ( 1% of total will make for 1 ml / Erlenmeyer )<br />
<br />
One of each construct will be kept as Blanco and one of each construct will receive IPTG or Arabinose<br />
<br />
IPTG (10mM)<br />
v<br />
Arabinose (~1% is generally used) [Meaning you will need 1 ml of a 10% stock]<br />
<br />
'''Growth curve time points'''<br\><br />
<br />
This contained a time laps of 30min for the first period of time (lets say first 3 hours) and after that time lapses of a hour would be better.<br />
<br />
'''Growth curve samples'''<br\><br />
*1 ml is taken to measure OD<br />
*1 ml is taken for WB analysis<br />
*1 ml is taken for digest analysis<br />
<br />
<br\><br\><br />
<br />
<h2>Degradation of Signal at varying exposure times and time points</h2><br />
'''Day 1 (13-9-2012)'''<br\><br />
Setup:<br />
*Prepare 2x 10ml pSB1AT3 + LacH cultures (in normal LB and with appropriate antibiotic added)<br />
*Prepare 2x 10ml pSB1AT3 + pBAD cultures (in normal LB and with appropriate antibiotic added)<br />
*Incubate at approximately 37 C for 20 hours <br />
<br />
'''Day 2 '''<br\><br />
Setup:<br />
*Stop incubation of cultures after 20 hours of growth , cultures should now be well into the stationary faze.<br />
*Prepare 8x a 500ml flasks containing 49.5ml of LB each<br />
*For 4 flasks add appropriate antibiotic for culture 1 (pSB1AT3 + LacH containing bacteria from day 1 - step 1)<br />
*For 4 flasks add appropriate antibiotic for culture 2 (pSB1AT3 + pBAD containing bacteria from day 1 - step 2)<br />
*To the 4 flasks from step 3 add 0.5ml of culture 1<br />
*To the 4 flasks from step 4 add 0.5ml of culture 2<br />
*Incubate new cultures (step 5 & 6) at 37 degrees for 18 hours <br />
<br />
'''Day 3 '''<br\><br />
Experiment:<br />
*Stop incubation of cultures after 18 hours of growth (at 12:00), cultures should now be well into the stationary faze.<br />
*From all 8 cultures take 1.5 ml sample and put on ice (-20 C) these are negative control reference points (IPTG- and ARA-)<br />
*To 3 of 4 cultures) (day 2 – step 5) each add 100mM IPTG, these will become the IPTGex30, IPTGex60, IPTGex120 cultures<br />
*To 3 of 4 cultures (day 2 – step 6) each add excessive amount of Arabinose, these will become the ARAex30, ARAex60, ARAex120 cultures<br />
*From all 8 cultures (IPTG-, IPTGex30, IPTGex60, IPTGex120, ARA-, ARAex30, ARAex60, ARAex120) take 1.5 ml sample and put on ice (-20 C) (IPTG t0 & ARA t0)<br />
*At 30 min take for all 8 cultures (IPTG-, IPTGex30, IPTGex60, IPTGex120, ARA-, ARAex30, ARAex60, ARAex120) a 1.5 ml sample and put on ice (-20 C) (IPTG t30 & ARA t30)<br />
*Afterwards wash 1 (IPTGex30) of the 3 cultures from step 3 and also from 1 (ARAex30) of 3 of the cultures from step 4 according to the following washing procedure (approx. 30 min):<br />
**Centrifuge at 4000 RMP for 10 min (minimalize cell death at low RPM)<br />
**Remove supernatant<br />
**Resuspendent pellet in 45.5ml LB<br />
**Repeat steps a through c 4 times.<br />
*Put washed and resuspended IPTGex30 and ARAex30 cultures from step 7 into 2 clean (don’t think they have to be sterile) 500ml flasks.<br />
*At 60 min take for all 8 cultures (IPTG-, IPTGex30, IPTGex60, IPTGex120, ARA-, ARAex30, ARAex60, ARAex120) a 1.5 ml sample and put on ice (-20 C) (IPTG t60 & ARA t60)<br />
*Immediately after step 9, repeat steps 7&8 for IPTGex60 & ARAex60 cultures (approx 30 min). Note that the resuspenention during the washing (step 7) should be done in 44ml of LB instead of 45.5ml!<br />
*At 90 min take for all 8 cultures (IPTG-, IPTGex30, IPTGex60, IPTGex120, ARA-, ARAex30, ARAex60, ARAex120) a 1.5 ml sample and put on ice (-20 C) (IPTG t90 & ARA t90)<br />
*At 120 min take for all 8 cultures (IPTG-, IPTGex30, IPTGex60, IPTGex120, ARA-, ARAex30, ARAex60, ARAex120) a 1.5 ml sample and put on ice (-20 C) (IPTG t120 & ARA t120)<br />
*Immediately after step 12, repeat washing procedure of step 7&8 for IPTGex120 & ARAex120 cultures (approx 30 min). Note that the resuspenention during the washing (step 7) should be done in 41ml of LB instead of 45.5ml!<br />
*At 150 min take for all 8 cultures (IPTG-, IPTGex30, IPTGex60, IPTGex120, ARA-, ARAex30, ARAex60, ARAex120) a 1.5 ml sample and put on ice (-20 C) (IPTG t150 & ARA t150)<br />
*Repeat step 14 for every next 30 min until out of time (t180, t210, t240, t270, t300, enz)<br\><br\><br />
<br />
<h2>Long time exposure protocol</h2><br />
<br />
'''Day 1'''<br\><br />
<br />
*End of the day: Prepare and incubate (37C) 6x 3ml pSB1AT3+LacH containing bacterial cultures (with appropriate antibiotic)<br />
*End of the day: Prepare and incubate (37C) 6x 3ml pSB1AT3+pBAD containing bacterial cultures (with appropriate antibiotic)<br />
<br />
'''Day 2'''<br\><br />
<br />
*Start of the day: To 3 cultures from Day 1, Step 1 add to each 15 ul of 1M IPTG solution<br />
*Start of the day: To 3 cultures from Day 1, Step 2 add to each 120 ul of 100% (1g/ml) Arabinose solution<br />
*Keep all cultures incubated at 37C<br />
<br />
'''Day 3'''<br\><br />
<br />
*Start of day: From 1 of the cultures from Day 2, Step 1 take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (IPTG+ 1 day exposure)<br />
*Start of day: From 1 of the cultures from Day 2, Step 2 take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (IPTG- 1 day negative control)<br />
*Start of day: From 1 of the cultures from Day 1, Step 1 (and not used in Day 2, Step 1) take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (ARA+ 1 day<br />
exposure)<br />
*Start of day: From 1 of the cultures from Day 1, Step 2 (and not used in Day 2, Step 2) take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (ARA- 1 day<br />
negative control)<br />
*Start of day: To the 2 remaining cultures of Day 2, Step 2 each add 120 ul of 100% (1g/ml) Arabinose solution<br />
*Keep all (remaining) cultures incubated at 37C<br />
<br />
'''Day 4'''<br\><br />
<br />
*Start of day: From 1 of the (remaining) cultures from Day 2, Step 1 take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (IPTG+ 2days exposure)<br />
*Start of day: From 1 of the (remaining) cultures from Day 2, Step 2 take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (IPTG- 2 days negative control)<br />
*Start of day: From 1 of the (remaining) cultures from Day 1, Step 1 (and not used in Day 2, Step 1) take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (ARA+ 2 days exposure)<br />
*Start of day: From 1 of the (remaining) cultures from Day 1, Step 2 (and not used in Day 2, Step 2) take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (ARA- 2 days negative control)<br />
*Start of day: To the 1 remaining culture of Day 2, Step 2 add 120 ul of 100% (1g/ml) Arabinose solution<br />
*Keep all (remaining) cultures incubated at 37C<br />
<br />
'''Day 5'''<br />
<br />
*Start of day: From the 1 remaining culture from Day 2, Step 1 take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (IPTG+ 3 days exposure)<br />
*Start of day: From the 1 remaining culture from Day 2, Step 2 take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (IPTG- 3 days negative control)<br />
*Start of day: From the 1 remaining culture from Day 1, Step 1 (and not used in Day 2, Step 1) take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (ARA+ 3 days exposure)<br />
*Start of day: From the 1 remaining culture from Day 1, Step 2 (and not used in Day 2, Step 2) take 2x a 1.65ml sample (1 for miniprep 1 for western blot), discard rest of culture (ARA- 3 days negative control)<br />
<br />
<br\><br\><br />
<br />
<h2>Experimental setup for consistent testing of IPTG+/- pSB1AT3+MTase containing bacteria in different growth phases (Log and Stationary)</h2><br />
<br />
<h4>Step 1 – Growth Curve Measurement</h4><br />
Growthcuves should be measured in for the pSB1AT3 + Mtase construct containing bacteria. This is critical because all tests should be performed on plasmids extracted from cultures that are either in the log (exponential) phase or fully grown cultures. Bought conditions should be used for bought IPTG+ and IPTG- cultures.<br />
<br />
'''Setup'''<br\><br />
'''Starting media:'''<br\><br />
*100ml LB<br />
*100ml LB containing 100milliM IPTG<br />
<br />
'''Starting bacteria:'''<br\><br />
*2x 10ul pSB1AT3 + MTase containing bacteria form overnight (stationary phase) culture<br />
<br />
'''Other requirements:'''<br\><br />
*1ml cuvette accepting spectrophotometer<br />
*44x 1ml cuvette<br />
*1x 1ml Demi or MilliQ containing cuvette (for calibration)<br />
<br />
'''Experiment'''<br\><br />
*1.Add 10ul pSB1AT3 + MTase containing bacteria from overnight (stationary phase) culture to 100ml LB and to a 100ml LB containing 100milliM IPTG media<br />
*2.Incubate at 37 C in shaker<br />
*3.Measure OD of both cultures every 30 min for 10 hours<br />
*4.After 10h leave cultures in 37C shaker<br />
*5.Next day measure OD again for bought cultures, and 30 min later (to confirm that the culture is actually stationary at this time)<br />
<br />
'''Expectation'''<br\><br />
Literature suggests that (if there is enough medium available) the culture will be in the log fase after 1h till about 6h.<br />
<br />
Experience suggests that 10ml cultures will be in stationary phase after 16h of 37C growth (overnight cultures)<br />
<br />
<h4>Step 2 – Stationary phase Positive (IPTG+) and Negative (IPTG-) controls</h4><br />
These experiments will tell us if the leakiness of the promoter is enough to cause all sites to be methylated. And will (in conjunction with the results from step 3) later tell us something the influence of growth rate on the equilibrium between methylation loss (due to plasmid replication and death) and gain (due to leakiness of the promoter).<br />
<br />
'''Setup'''<br\><br />
<br />
'''Starting media:'''<br\><br />
*10ml LB<br />
*10ml LB containing 100milliM IPTG<br />
<br />
'''Starting bacteria:'''<br\><br />
*2x 10ul pSB1AT3 + MTase containing bacteria form overnight (stationary fase) culture<br />
<br />
'''Other requirements:'''<br\><br />
*Miniprep kit<br />
*Nanodrop machine<br />
*ScaI restriction enzyme<br />
*EcoRI restriction enzyme<br />
*Gels<br />
*Standard ladder<br />
<br />
'''Experiment'''<br\><br />
*1.Add 10ul pSB1AT3 + MTase containing bacteria from overnight (stationary phase) culture to 10ml LB and to a 10ml LB containing 100milliM IPTG media<br />
*2.Put cultures in shaker and incubate at 37C<br />
*3.Wait till cultures are in stationary phase, exact time is determined in step 1 (experience suggests that this is after 16h which means an overnight culture in practice)<br />
*4.Do a plasmid extraction from bought cultures using miniprep, note however that a little bit of both cultures (10ul) should be preserved for step 3!<br />
*5.Measure DNA yields from both extractions using nanodrop<br />
*6.Dilute or concentrate until DNA concentration for both extractions is the same (aim for 1000ug/ul *Guess*)<br />
*7.Do standard 1h digestions of both DNA extracts in the following variations ScaI, ScaI + EcoRI, EcoRI, None.<br />
*8.Run DNA from both cultures for all variations on gel, with the ladder on both sides and in the middle. Exact layout: **Ladder, IPTG- DNA digested by ScaI, IPTG- DNA digested by ScaI + EcoRI, IPTG- DNA digested by EcoRI, IPTG- DNA digested by None, Ladder, IPTG+ DNA digested by ScaI, IPTG+ DNA digested by ScaI + EcoRI, IPTG+ DNA digested by EcoRI, IPTG+ DNA digested by None, Ladder.<br />
<br />
'''Expectation'''<br\><br />
Ideal results would be that the Positive control (IPTG+) will result in intensity shift to the uncut/1cut plasmid band in the ScaI digestion and a shift to the top band (plasmid only cut by EcoRI) in the ScaI + EcoRI digestion. Whereas for the Negative control (IPTG-) this shift would be the opposite. Pilot experiments already suggest that this shift of the Negative control is not as severe as would be desired.<br />
<br />
Might we find that the Positive and Negative control of this experiment are similar and this result is more like what we would like to see from the positive control it would indicate that in the case of a static culture leaky expression is enough to methylate all the sites.<br />
<br />
Seeing similar results but no clear shift (similar to the pilot experiment) will indicate that all sites are possibly methylated but that ScaI have a small chance of cutting methylated sites.<br />
<br />
<h4>Step 3 – Log phase Positive (IPTG+) and Negative (IPTG-) controls</h4><br />
This experiment is done in order to determine the effect that bacterial growth has on the loss and gain of methylated sites and the equilibrium it might establish.<br />
<br />
'''Setup'''<br\><br />
Starting media (much more than in step 3 because less DNA will be extracted per ml due to lower growth time):<br />
*100ml LB<br />
*100ml LB containing 100milliM IPTG<br />
<br />
'''Starting bacteria:'''<br\><br />
*10ul pSB1AT3 + MTase containing bacteria form step 2.4 (stationary phase) IPTG+ culture<br />
*10ul pSB1AT3 + MTase containing bacteria form step 2.4 (stationary phase) IPTG- culture<br />
<br />
'''Other requirements:'''<br\><br />
*Miniprep kit<br />
*Nanodrop machine<br />
*ScaI restriction enzyme<br />
*EcoRI restriction enzyme<br />
*Gels<br />
*Standard ladder<br />
<br />
'''Experiment'''<br\><br />
*1.Add 10ul pSB1AT3 + MTase containing bacteria from step 2.4 (stationary phase) IPTG+ culture to the 100ml LB containing 100milliM IPTG media<br />
*2.Add 10ul pSB1AT3 + MTase containing bacteria from step 2.4 (stationary phase) IPTG- culture to the 100ml LB media<br />
*3.Put cultures in shaker and incubate at 37C<br />
*4.Wait till cultures are in log phase, and let them grow till near the end of the log phase, exact time is determined in step 1 (literature suggests that this is after 6h which means that this can be on the same day as you do step 2.4)<br />
*5.Do a plasmid extraction from bought cultures using miniprep<br />
*6.Measure DNA yields from both extractions using nanodrop<br />
*7.Dilute or concentrate until DNA concentration for both extractions is the same (aim for 1000ug/ul *Guess*). To achieve the desired DNA concentration step 5 and 6 may need to be repeated, this is also the reason why the cultures in step 3 needed to be bigger.<br />
*8.Do standard 1h digestions of both DNA extracts in the following variations ScaI, ScaI + EcoRI, EcoRI, None.<br />
*9.Run DNA from both cultures for all variations on gel, with the ladder on both sides and in the middle. Exact layout: Ladder, IPTG- DNA digested by ScaI, IPTG- DNA digested by ScaI + EcoRI, IPTG- DNA digested by EcoRI, IPTG- DNA digested by None, Ladder, IPTG+ DNA digested by ScaI, IPTG+ DNA digested by ScaI + EcoRI, IPTG+ DNA digested by EcoRI, IPTG+ DNA digested by None, Ladder.<br />
<br />
'''Expectation'''<br\><br />
In the ideal case the Positive result (IPTG+) would only show the uncut/1 cut plasmid band in the ScaI digestion and only the top band (uncut plasmid) in the ScaI + EcoRI digestion. And the opposite results in the negative control (IPTG-)<br />
If similar results to the expected shift in step 2 are observed than the growth rate of the culture doesn’t have any impact on the methylation gain and methylation loss equilibrium.<br />
<br />
If similar results to step 2 but no shift is observer this means that ScaI is able to cut a certain methylated sites in some with a low affinity and that all sites are methylated.<br />
<br />
<h4>Possible Step – Testing under different concentrations of IPTG</h4><br />
Repeat Step 2 and 3 using a different concentrations of IPTG. If step 1 suggest that IPTG influences the growth rate of the bacteria significantly also redo step 1 with the different concentrations of IPTG.<br />
<br />
<h4>Possible Step – Testing different concentrations or exposure times of/to ScaI</h4><br />
If ScaI is found to be able to sporadically cut methylated sites its useful to find out how sporadic this occurs. This can be done by repeating steps 2 or/and 3 using lower concentrations of ScaI or varying the exposure time (normally 1h) to the restriction enzyme. Note however when varying the exposure times to ScaI you need to perform the EcoRI digestion first and separately.<br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/safety/questionsTeam:Amsterdam/safety/questions2012-09-27T03:55:24Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<br />
<h1>Safety Questions</h1><br />
__NOTOC__<br />
<h4>Would any of your project ideas raise safety issues in terms of: researcher safety, public safety or environmental safety</h4><br />
<br />
Researchers work with safety guidelines that must be upheld by any lab to ensure the safety necessary to work with bacteria. The Cellular Logbook does not raise any immediate suspicion of potential hazards, since non-pathogenic bacteria or bacterial product was used. iGEM Amsterdam is aware that introducing non-endogenous genes to E. coli might give rise to unexpected metabolic toxic products that could threaten the safety of the researcher and/or public. The general ML-I and ML-II regulations have been respected at all times. No additional laboratory safety rules were considered necessary for the purpose of this project.<br />
<br />
The greatest concern arises in cases where the Cellular Logbook would be used for environmental measurements. These cases would involve the use of a semi-permeable biofilm that would contain the bacteria and prevent release in the environment. Another safety measure thought of involves the reduction of the bacterial growth rate, hence excluding potential invasion of the natural biosphere. However, none of the immediate project plans involve release of the genetically modified bacteria into the environment. In this view, the project ideas do not pose any threat to both the public and the environmental safety. Each application that will be brought forward involving interaction with the natural environment will remain theoretical for the duration of the project.<br />
<br />
<h4>Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, did you document these issues in the Registry? how did you manage to handle the safety issue? How could other teams learn from your experience?</h4><br />
<br />
The main components used for our construct assembly involved biobricks provided by iGEM headquarters, a methyltransferase synthesised by a company and thus has been subjected to vigorous testing, and finally a polydactyl Zinc-Finger obtained from a research group at Leids Universitair Medisch Centrum (LUMC), The Netherlands.[1] The above-mentioned research group did not address any particular safety issues. Xu et al introduced M.ScaI into E. coli in one of their previous studies.[2] Based on this knowledge, it is very unlikely that the M.ScaI would render E. coli pathogenic or would pose a threat of any kind to the researcher. The methyltransferase M.ScaI comes from Streptomyces caespitosus which is known as a non-pathogenic bacteria. <br />
<br />
<h4>Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?</h4><br />
<br />
Dr. Pernette J. Verschure, one of the iGEM team advisors, is tasked with overseeing biosafety at the lab where we have perfomed all our experiments, i.e. the Systems and Synthetic Biology/Nuclear Organization Group at the Swammerdam Institute for Life Sciences (SILS), University of Amsterdam. Dr. Verschure takes care of the GMO database, safety, and official registration of GMOs (i.e. GGO 01-045, 01-052 and 02-241). Dr. Verschure is closely involved with the the Cellular Logbook project, attends our meetings, and keeps an eye to ensure we maintain a safe working environment. Furthermore we have to abide to the biosafety regulation policy of the Dutch government. This is monitored/organized by the RIVM and regulated via permits and GGO’s.[3]<br />
<br />
<h4>Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?</h4><br />
<br />
When working with GMOs or planning to create GMOs there should be easy access to any work already done on the matter. A central database in which all bio-engineering safety data would be stored and catalogued could greatly help in this respect. Alternatively, an active discussion panel where project ideas can be discussed and addressed regarding their expected and perhaps unexpected safety issues.<br />
<br />
<h4>Reference List</h4><br\><br />
1. de,P.S., Neuteboom,L.W., Pinas,J.E., Hooykaas,P.J., & van der Zaal,B.J. ZFN-induced mutagenesis and gene-targeting in Arabidopsis through Agrobacterium-mediated floral dip transformation. Plant Biotechnol. J. 7, 821-835 (2009).<br />
<br />
2. Xu,S.Y. et al. Cloning and expression of the ApaLI, NspI, NspHI, SacI, ScaI, and SapI restriction-modification systems in Escherichia coli. Mol. Gen. Genet. 260, 226-231 (1998).<br />
<br />
3. http://www.biosafety-europe.eu/d20public_300309.pdf<br />
<br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/practices/resultsTeam:Amsterdam/practices/results2012-09-27T03:55:04Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<h1>Human Practices: Results</h1><br />
__NOTOC__<br />
<br />
<h4>Social scientist</h4><br />
We started working on our human practice through a ‘crash course’ on SB and iGEM organized by the supervisors of the Amsterdam and Delft 2012 iGEM teams. One of our advisors, [https://2012.igem.org/Team:Amsterdam/team/advisors#Wieke_Betten ''Wieke Betten''], gave a lecture about the importance of ‘good’ incorporation of the societal context in the development of SB and embedding of SB in society. The key word from this presentation was [https://2012.igem.org/Team:Amsterdam/practices/methods#Key_Concepts ''valorization'']. The only way this can be achieved, she stated, was by early involvement of stakeholders and public in the design of a new scientific project. This lecture inspired us to outline a generic approach in human practice that could be applied not only to our present iGEM topics, the Cellular Logbook, but also to all future iGEM teams. Such a generic approach in human practice would reflect on the ethical, societal, safety and security issues concerning each specific project. Our Interactive iGEM research approach was born.<br />
<br />
<h4>Experts in biotechnology</h4><br />
[[File:Amsterdam_practices_1.jpg|300px|right|thumb|Presenting our project in an early stage to the experts at the UvA]]<br />
In the first month of our project we organized presentations at both universities (UvA and VU) and invited scientists from the departments of [http://www.falw.vu.nl/nl/onderzoek/molecular-cell-biology/ ''Molecular Cell Biology''] and the [http://sils.uva.nl/ ''Swammerdam Institute for Life Sciences (SILS)'']. During these presentations we explained how we wanted to realize our project, the Cellular Logbook. We proposed our designed [https://2012.igem.org/Team:Amsterdam/project/molecular_design ''molecular mechanisms''] of the Cellular Logbook to these experts. Although most feedback confirmed the neatness of our project design, we received some relevant feedback:<br />
<br />
* Low concentrations of IPTG may (1-100 µM) not enter the cell, because of the phenomenon called ‘inducer exclusion’. In this case, glucose transport leads to phosphorylation of a compound of the phosphotransferase system (PTS), which in turn blocks the lacY transporter of IPTG. During our experiments we prevented this from happening by using higher concentrations of IPTG.<br />
<br />
* The basal activity of the envisioned LacH promotor might result in background noise because of leaky expression of the methyltransferase. We tried to solve this problem by doing experiments with the [https://2012.igem.org/Team:Amsterdam/data/experimental#Reducing_basal_activity_of_the_LacH_promoter_using_LacIQ_E._Coli_strain: ''LacIQ strain of E.coli and by using the pBAD promotor.'']<br />
<br />
<h4>Innovation manager & Business developer</h4><br />
Early in the stage of our project we realized that the Cellular Logbook had the potential to become a [https://2012.igem.org/Team:Amsterdam/practices/methods#Key_Concepts ''platform technology''] for synthetic biology. Although we could think of numerous different applications for our system in the future, none of these applications were really evident at the moment; our main focus was to get the proof of concept that our multi-signal sensing system would work. We did realize though, that we needed to have a clear story on how to present our project to all kinds of audiences.<br />
<br />
In order to get some advice on this, we went to talk to Willem Fokkema, innovation manager and business developer at the UvA Technology Transfer Office. Willem Fokkema is specialized in the applicability of knowledge and aiding life scientists to find commercial partners. We started of with an explanation of our project, after which he gave us some tips:<br />
<br />
* Work on your elevator pitch. You should be able to explain your project in one minute.<br />
<br />
* Don’t use ‘cheap or low costs’ as an argument in favor of your technology. Focus on what your technology can do and help companies see how this can help them.<br />
<br />
* Concerning legal issues: he stated that we shouldn’t think about patenting until we would have a clear ‘product and application’ in mind. He proposed he would give us advice regarding patenting at a later stage of our project.<br />
<br />
* In thinking of applications for your project, consider going through the list of [http://www.millennium-project.org/millennium/challeng.html ''Global Grand Challenges''].<br />
<br />
We went through the list of 15 Global Grand Challenges and figured out for which points our Cellular Logbook system could provide a solution. Click [https://2012.igem.org/Team:Amsterdam/project/features_and_applications#Global_Challenges ''here''] to see the results.<br />
<br />
<h4>Stakeholders in potential fields</h4><br />
<br />
<h5>BioDetection Systems B.V (Amsterdam, The Netherlands)</h5><br />
<br />
We tried to further investigate the future potential of our system as a multi-sensor that could register different signals in the environment. In the light of this, we consulted [http://www.biodetectionsystems.com/ ''BioDetection Systems b.v. (BDS)'']. We spoke with dr. Bart van der Burg, Chief Scientific Officer at BDS b.v.<br />
<br />
'''Bioassays'''<br\><br />
BDS uses human and animal cells to detect compounds in samples, as a cheaper alternative to existing chemical screening methods. This is done by measuring the acute toxic effects of the compound on the cell instead of directly measuring the presence of the compound. Measured substances are: (I) aromatic carbon-hydroxides (dioxins) and (II) hormones. Dioxins are very stable and not metabolized by the cells that measure their toxic effects, hence easily measurable using this technique. Hormones are sometimes metabolized by the chassis cells used, which affects the outcome of the measurement. European and international legislation concerning analytical chemistry is mostly directed at chemical measurements of dioxins, not the biological measurement.<br />
<br />
'''Cons to bioassays'''<br\><br />
Mass spectrometry methods are much more widely used in the field and more familiar to companies looking for detection systems. This technique allows to measure various chemicals simultaneously, this technique is very expensive. Mass spectrometry methods are also not completely unambiguous either. For our product to be a viable and interesting measurement method, it has to be much easier and cheaper than already known chemical analytical methods.<br />
<br />
'''View on applications'''<br\><br />
We summarized the molecular mechanism of the Cellular Logbook for Bart van der Burg and asked him if he could see advantages of a memory module in a multi-sensor system. Applications he could think of on the spot:<br />
<br />
* Forensics: Intrigued by the facet of [https://2012.igem.org/Team:Amsterdam/project/features_and_applications#Time_Indicator ''time indication''] that is inherent to the Cellular Logbook, he thought of bacteria that would solve a crime, by inferring the time on which the criminal was at the crime scene. One major bottleneck would be that the crime scene would have to be supplied with bacteria containing the Cellular Logbook beforehand.<br />
<br />
* Control of compound emission at industrial sites (e.g. factory): he envisioned to place the bacteria at multiple locations around a factory site to get an idea where and when chemicals were released.<br />
<br />
'''Problems envisioned'''<br\><br />
* Release of GMOs into the environment<br />
<br />
* Survivability of the cell in the environment<br />
<br />
* Robustness of the test<br />
<br />
* Specificity for a particular compound - lots of validation steps are required.<br />
<br />
'''Points of action'''<br\><br />
Bart van der Burg then encouraged us to have a talk with the Dutch Water company Waternet, and specifically to their toxicologist Ron van der Oost.<br />
<br />
<h5>Waternet (Amsterdam, the Netherlands)</h5><br />
Ron van der Oost is a toxicologist at a Dutch water company, named [https://www.waternet.nl/ ''Waternet'']. He is specialized in research on risks, effects and behavior of emerging substances in the water cycle. Waternet is the only company in the Netherlands that focuses on the whole water cycle. Waternet is responsible for cleaning wastewater, making water drinkable and monitor and clean surface water.<br />
[[File:Amsterdam_practices_2.jpg|300px|right|thumbs|Ron van Oost from waternet discussing possible project applications]]<br />
Ron van der Oost was really interested in our multi-sensor idea. Right now they use 20 different sensors for different groups of compounds. These bioassays are not optimal. They are really expensive and it is hard to normalize the data (at what point does a certain concentration become toxic?). Therefore it is often necessary to get a toxicologist to look at the data from bioassays, what makes it even more expensive. He pointed out some crucial issues regarding the potential use of our Cellular Logbook:<br />
<br />
* The output of our system would have to be easy to interpret.<br />
<br />
* He also put emphasis on the possibility to register concentrations of compounds, using our system. What [https://2012.igem.org/Team:Amsterdam/project/features_and_applications#Concentration_Indicator ''concentrations''] can we measure?<br />
<br />
* The sensitivity of the multi-sensor has to be good; you don’t want the system to register the presence of a toxic compound only when the concentration of this toxin is already high.<br />
<br />
* The sensor should be able to monitor it’s environment for quit some time; the system needs to be ‘up and running’; a lot of systems they use now can be in the water for weeks/months. The time-indicator is an interesting feature -> let the system be in the water for 4 weeks and register when a certain compound was registered.<br />
<br />
* What groups of compounds can we measure?<br />
<br />
* Maybe most important: how do you want to put the GMO’s in the water? By using some sort of filter, where the bacteria can’t escape from, but can sense their environment. He mentioned passive sampling.<br />
<br />
'''Points of action'''<br />
* Check which (20) compounds we could detect<br />
<br />
* Develop the concentration and time indicator<br />
<br />
<h4>Biosafety Officer</h4><br />
<br />
Some safety and security questions were already addressed during our talks with scientist and companies. But for a more extensive and fundamental way of addressing these kinds of issues, we contacted dr. Cécile van der Vlugt, from the [http://www.rivm.nl/ ''National Institute of Public Health and the Environment (RIVM)'']. She is specialized in the risk assessment of Genetically Modified Organisms (GMOs). We were interested in what she would think about a possible application and thereby controlled release of our Cellular Logbook into the environment.<br />
<br />
'''Biosafety'''<br\><br />
Dr. van der Vlugt stated that although the positive intention of synthetic biology (SB) is clear, the developments in SB raise new questions concerning biosafety. “You may never exclude unintentional risks, but these risks should not impede progression”, she continued. On the question whether SB required a new legislative framework, she answered that a new framework would probably only hinder scientists and companies, without improving the current risk assessment. According to dr. van der Vlugt, each GMO should be assessed by a specific analysis on the genes introduced in that GMO. This way the risk assessment is built on contemporary knowledge, without having to replace the current biosafety framework.<br />
<br />
'''Controlled environmental release of GMOs'''<br\><br />
GMOs could threaten the environment if they release hazardous gene products. Therefore these GMOs should be analyzed for so called potentially hazardous gene products (PHGPs). Examples of PHGPs are: protein toxins, gene products and sequences that are involved in genome rearrangements, gene products involved in apoptosis or activated proto-oncogenes. GMOs that carry a construct that may encode for a PHGP are handled with extreme precaution, until the risks of the specific construct have been assessed. [[#reference1|[1]]]<br />
<br />
'''Points of action'''<br\><br />
Since our Cellular Logbook system contains genes of which there products have a role in genome rearrangements (the M.ScaI methyltranserase and a Zinc Finger), we included a little [https://2012.igem.org/Team:Amsterdam/safety/questions ''risk assessment''] in the design process of our Cellular Logbook.<br />
<br />
<h4>Human Outreach</h4><br />
<br />
We had two main goals concerning public engagement and human outreach:<br />
<br />
'''1. Introducing SB to the general public and engaging in a dialogue.'''<br\><br />
We approached a broad audience by:<br />
<br />
* Featuring in a [https://2012.igem.org/Team:Amsterdam/practices/results#Documentary ''documentary''] that will be broadcasted next year in Germany, Belgium and the Netherlands.<br />
<br />
* Participating on a national art, music and science festival, named [https://2012.igem.org/Team:Amsterdam/practices/results#Discovery_Festival ''Discovery Festival''].<br />
<br />
'''2. Creating awareness amongst students of the two universities of Amsterdam (UvA and VU).'''<br\><br />
Young, bright minded scientist earn to get acquainted with the exciting and promising field of SB. Especially, since we have noticed that only a little group of students in Amsterdam ever heard about SB and iGEM, we felt called upon to change this. We decided to do this in two ways:<br />
<br />
* Giving a short interactive presentation to first year life science students, during the introduction week of the universities.<br />
<br />
* By press releases in the online and print version of the universities magazines.<br />
<br />
<h5>Discovery Festival</h5><br />
The Discovery Festival is all about experiencing new things in the form of art, music and science. It will be held on the 28th of September in 3 major Dutch cities: Rotterdam, Eindhoven and Amsterdam. The general theme of the Discovery Festival 2012 is Do It Yourself (DIY). This gave us a nice opportunity of introducing SB to the public and giving the visitors the opportunity to experience what SB is about.<br />
<br />
<h5>National iGEM Collaboration</h5><br />
[[File:Amsterdam_practices_3.jpg|300px|right|thumb|Meeting with the other Dutch teams to discuss the program of the Discovery Festival]]<br />
We collaborated with the iGEM teams from Eindhoven, Wageningen and Groningen to make iGEM and SB feature in the program of Discovery Festival. During the summer we had several meetings with representatives from each collaborating iGEM team. During these sessions we discussed, together with the organization of the event, on how to incorporate SB into the program. All teams took their responsibility by providing and preparing part of the program. In addition, we divided all participating teams over the three locations, so that iGEM would be represented on all locations. Because of the excellent teamwork and effort, we were able to realize the following program:<br />
<br />
'''The program'''<br />
[[File:Amsterdam_practices_4.jpg|300px|thumb|Flyer for the Discovery Festival]]<br />
'''1. The SB-experience'''<br\><br />
As main part of the program, people will experience the process of genetically modifying bacteria by following a 7-step workshop in an improvised laboratorial setting: From ordering DNA online, to implementing the perceivable characteristic into bacteria and showing the resulting bacteria on agar plate. Safety is of course a priority: no actual GMOs or E.coli will be used.<br />
<br />
'''2. The posters'''<br\><br />
We have made a wall full of posters, on which several fictional and non-fictional iGEM-projects are shown. The goal of this is to give the people an idea of the potential of SB, but also to inspire them and see how they will react on this.<br />
<br />
'''3. What would you do with your own iGEM project?'''<br\><br />
After the experience, people are asked what they would do if they could start their own iGEM project. All these ideas are recorded on a video guestbook.<br />
<br />
'''4. Tuur van Balen'''<br\><br />
We invited [http://www.tuurvanbalen.com/ ''Tuur van Balen''], an artist who got heavily inspired by SB, to the Discovery Festival in Amsterdam. His work will prove to be a stimulus in introducing SB to the public.<br />
<br />
Because the event will take place after the wiki-freeze we are unable to show the results. Click [http://www.facebook.com/IgemAmsterdam2012 ''here''] to see a report of the festival!<br />
<br />
<h5>Documentary</h5><br />
[[File:Amsterdam_practices_5.jpg|300px|right|thumb|Frank Theys, the documentary maker]]<br />
Together with the iGEM team from Delft, the Netherlands Institute for Neuroscience (NIN) and the Advanced Nano Characterization Center (ANCC) from Japan, we will feature in a documentary called [http://www.savagefilm.be/documentary/LAB-LIFE ''Lab Life''], by Belgian documentary maker Frank Theys. The documentary will show a portrait of the daily life around innovative scientific research projects. Moreover, the documentary will focus on responsible innovation by showing the projects through the perspective of a social scientist that follows these projects. By highlighting the aspects of CTA and MM, this documentary is the perfect extension of our Interactive iGEM research approach. Lab Life will be broadcasted next year on German, Belgian and Dutch television.<br />
<br />
<h5>Press releases</h5><br />
'''Press releases in magazines from UvA and VU'''<br />
<br />
VU: [http://www.advalvas.vu.nl/nieuws/op-naar-het-wk-synthetische-biologie ''Ad Valvas'']<br />
<br />
UvA: Folia. Unfortunately not published yet.<br />
<br />
<h1>References</h1><br />
<p><br />
<span id="reference1"><br />
<sup><br />
[1]Hans Bergmans, Colin Logie, Kees Van Maanen, Harm Hermsen, Michelle Meredyth and Cécile Van Der Vlugt (2008). Identification of potentially hazardous human gene products in GMO risk assessment. Environmental Biosafety Research, 7, pp 1-9 doi:10.1051/ebr:2008001<br />
</sup><br />
</span><br />
</p><br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/software/logbook_designer/future_perspectiveTeam:Amsterdam/software/logbook designer/future perspective2012-09-27T03:54:32Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<h1>Logbook Designer: Future Perspective</h1><br />
<br />
__NOTOC__<br />
The Logbook Designer is not yet a product that is able to fully push the boundaries of the Cellular Logbook such as, for example, Blast is in its field. This page provides some insight on what is needed to add and adjust to this webtool if the Cellular Logbook is to be a massive platform for new technology.<br />
<div class='clear'></div><br />
<br />
<h2>Optimization</h2><br />
The Logbook Designer presents the user with a construct that does not break any rules when plasmid design is taken into account. However, it does not attempt to create the optimal Cellular Logbook plasmid. A sub-optimal plasmid is more than good enough when creating a proof of concept, which is what we are doing with our iGEM project. But when we think about industrializing the Cellular Logbook, it is necessary that every plasmid is optimized. If the Logbook Designer wants to achieve this, the algorithm does not only have to take into account the optimal distances between features, optimal features and optimal combination of features. The algorithm also has to think ahead when it comes to primer design and cloning steps required to create the plasmid. In order to have a fully functional and industrialized Logbook Designer, the optimization of the algorithm is an absolute necessity.<br />
<br />
<h2>Database</h2><br />
[[File:Amsterdam_db_future.png|right|300px|thumb|Figure 1: An abstract view of the user interaction with a database, allowing the user to communicate with the database by uploading, downloading and editting plasmids]]<br />
In order to make the Cellular Logbook the standard biosensor system not only is quick and efficient plasmid design a necessity, but also the easy to retrieve, edit and distribute already existing plasmids. The easiest way to accomplish all this is by designing and maintaining a Cellular Logbook database on a dedicated server. This database should allow for easy to use uploading, downloading and editing of Cellular Logbook plasmids.<br />
<br />
<h2>Literature</h2><br />
Scientific research stands and falls with literature and experiments backing up your hypothesis.<br />
If the Cellular Logbook plasmid constructed by the tool is used directly in research and as reference in papers, it is very plausible that there are notable flaws in the design and reviewers won't take your research seriously. To prevent this, cross-checking of every feature is required. Text mining of literature for each component is an easy way to present the user with extra information about the plasmid and its components. This way all results can be double checked by the tool and the given literature.<br />
<br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/software/logbook_designer/setupTeam:Amsterdam/software/logbook designer/setup2012-09-27T03:54:11Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<h1>Logbook Designer: Setup</h1><br />
<br />
__NOTOC__<br />
We have designed an online tool where a user can create their Cellular Logbook in silico by giving a backbone sequence and selecting one or more sensors manually or predefined from the parts registry database. An algorithm then computes one or more plasmids, taking into account the rules we use for our real life Cellular Logbook, and outputs the appropriate genbank files as well as the visualized version of the plasmid. The tool will also generate all possible permutations of gel bands which can be used as a read-out sheet for the gel electrophoresis results. By using this tool we are able to quickly design a plasmid with multiple sensors.<br />
<br />
<h2>Genbank Output</h2><br />
[[File:Amsterdam_tool_genbank.png|right|300px|thumb|Figure 1: Genbank output given by the Logbook Designer]]<br />
Standard formats for biological data are essential to communicate and pass along biological findings. The Genbank format is the most widely used format for genome and sequence annotation. As such the Plasmid Designer is able to produce Genbank files for each plasmid with the click of a button. This way the user can use the designed construct in other biological tools such as the freeware plasmid editor ApE[http://biologylabs.utah.edu/jorgensen/wayned/ape/].<br\><br\><br\><br\><br />
<br />
<h2>Plasmid Visualization</h2><br />
[[File:Amsterdam tool visualization1.png|left|300px|thumb|Figure 2: Graphical display of a plasmid using the Logbook Designer]]<br />
In order to grant the user a quick and dynamic overview of the plasmids that were just designed, a visualization of each plasmid is available with just a click on a button. Each of these visualized plasmids shows the specific features of that plasmid by color code. Hovering over each feature gives additional information, and clicking on a feature gives its DNA sequence.<br />
<br />
</div><br />
<div id="sub-menu" class="content-block"><br />
<br />
<h2>Gel Readout</h2><br />
[[File:Amsterdam_tool_readout.png|right|330px|thumb|Figure 3: Sensor belonging to the given band sizes]]<br />
The amount of gel bands increases exponentially as more sensors are engineered into the construct. Therefore having to figure out which signals have come to expression and which have not can prove to be an impossible task. We have included an algorithm inside the tool that computes all possible band combinations, and selects the bands that belong to each band position given by the user.<br />
<br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/data/background_activityTeam:Amsterdam/data/background activity2012-09-27T03:53:55Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<br />
__NOTOC__<br />
<h1>Background Activity</h1><br />
Here we will propose a solution to the the background noise observed in the experiments, and conclude what rates of leakiness are acceptable to the system.<br />
In this, we model the system both using both ordinary differential equations and stochastically using the Gillespie SSA algorithm.<br />
<br />
We will focus on the effects of two rate constants in particular on the fraction of methylated plasmids in absence of the inducing signal: <br />
* the leaky transcription rate of the mRNA for the writer, denoted by $k_{c\text{MW}}$ and<br />
* the catalysis rate of the writer, the rate with which it methylates umethylated plasmids<br />
<br />
== Introduction == <br />
<br />
Before starting the experiments, a lot of crucial parameters on the molecular system were still unknown. <br />
Both the leaky expression rate of the sensor and the catalysis rate of the writer were hard to model because of this. <br />
Sensible predictions on how the molecular system we were designing was going to function exactly therefore seemed impossible. <br />
We did expect the promoter/sensor we were going to test, pLac, to show some leaky expression. As to how much background activity this was going to cause, we couldn't predict and thus we proceeded to test out the construct in the lab. The [[Team:Amsterdam/Lab|laboratory results]] seemed to suffer from two problems: <br />
* A basal methylation activity that was always recorded even in absence of the signal. This background noise reduces the dynamic range of our system; there is no way to tell whether observed band intensities are due to the signal having passed the ''Cellular Logbook'' [[Team:Amsterdam/modelling/timeinferrance|some time ago]] or simply because of the background noise. We conclude that storing a signal using the ''Cellular Logbook'' requires a transcriptional regulator that has a high repressing quality in order to diminish the background activity.<br />
* A lack of a fully induced state for the pLac operon, but not for the later tested pBAD operon. We suspect this problem to be caused by a low uptake rate of the signalling compound (lac operon inducer IPTG) compared to the amount of repressor LacI present in the system controlling the operons. Especially for the part characterization experiments in which the pLac-MTase construct was cloned into a high copy number plasmid, this could be a problem. This problem is solved by adding the ''LacY''-gene that codes for permease to the existing construct. Inclusion of this gene might also lead to higher background methylation percentages in the negative controls, however.<br />
<br />
==== Stochastic Differential Equations ====<br />
We expected the basal activity of the used sensors to result in small amounts of proteins and thus thought a stochastic modelling approach would be suitable.<br />
When trying to accurately model these small amounts of proteins the thermodynamic equilibrium assumption that characterizes macroscopic ODE-models might not be maintainable.<br />
In these cases a finer-grained modelling method is required: the mesoscopic Stochastic Simulation Algorithm by Gillespie.<br />
By modelling each individual molecular reaction separately, the discreteness of this small amounts system is accounted for. <br />
Despite the small amounts of species present in the system analyzed here, we found no qualitative differences between the ODE and stochastic versions of the model.<br />
<br />
==== Comparison of SSA implementations ====<br />
We've investigated many stochastic simulation packages with implementations of the direct algorithms and some more coarse-grained optimizations thereof (e.g. tau-leaping).<br />
Most notable are [http://www.xlr8r.info/SSA/ xSSA] for Mathematica and [http://www.stompy.sourceforge.net StochPy] for Python.<br />
The former seems to be a great tool, but at the time of writing seems to lack the ability to process third order reactions (reactions with three reactants).<br />
The latter has been developed at the Free University Amsterdam by Timo Maarleveld, currently PhD-student there.<br />
In using this package, we've kept in close contact with Timo and submitted a lot of bug reports, helping the software to grow.<br />
Additionally, we've also extended StochPy by writing a supplementary tool [https://2012.igem.org/Team:Amsterdam/extra/software MDL2LaTeX], that eases the publishing of models developed with StochPy by converting the model input files to LaTeX representations.<br />
<br />
=Leakiness and background noise=<br />
<br />
This model has as its main goal to identify how much the expression rate of the uninduced sensor and the catalysis rate of the writer affect the methylation status of the plasmids.<br />
Each plasmid is assumed to have one bit and one operon that controls the gene for the writer.<br />
Running the stochastic version of this model, we noticed that keeping the amount of equations to a minimum is paramount to success, as the computation time seems to increase exponentially with the amount of reactions in the system.<br />
Focussing on the leakiness rate instead of the repressor-operon interactions that would require a lot of equations, we will therefore make one bold assumption:<br />
all operons present in a single cell are permanently bound to a transcriptional repressor.<br />
This frees us from having to code the signal entering the cell and binding the repressing TF, granting focus to the leaky expression rate and the catalysis rate of the fusion protein. <br />
For these models to have any forecastable value therefore, the amount of repressor molecules present in the system has to be much greater than the amount required to cover all operons.<br />
<br />
In order to not exclude the possible qualitative difference in model behaviour when all molecular reactions are computed individually, the system has also been modelled stochastically. To make the two systems perfectly comparable, the used rates are qualitatively identical to the ones used in the ODE system. <br />
Furthermore, the same parameter values have been used.<br />
<br />
==ODE Model definition==<br />
<table align="right"><br />
<tr><th>Parameter</th><th>Value</th></tr><br />
<tr><td>Ca</td> <td>$200\ \text{or}\ 40$</td><br />
<tr><br />
<td>ksW</td> <td>$30$</td><br />
</tr><br />
<tr><br />
<td>lMW</td> <td>$0.462$</td><br />
</tr><br />
<tr><br />
<td>lW</td> <td>$0.2$</td><br />
</tr><br />
<tr><br />
<td>kPlas</td> <td>$0.00866434 \cdot Ca$</td><br />
</tr><br />
<tr><br />
<td> lPlas </td> <td> $0.00866434$ </td><br />
</tr><br />
<tr><br />
<td colspan="2" align="center">Parameter values used to <br>plot the ODE-system</td><br />
</tr><br />
</table><br />
<br />
[[File:Combinedode.png|thumb|right|300px|Time trajectories of ODE-model in which the construct has been inserted in a high copy number plasmid (top, $Ca = 200$) and a low copy number plasmid (bottom, $Ca = 40$). All initial species values are set to $0$, except for the intial value of unmethylated plasmids which is equal to $Ca$. With these parameters, the dynamic range of the system is completely taken up by the background noise. No qualitative difference is observed between the high and low copy number cases]]<br />
<br />
Writer-mRNA results only from leaky expression from the operons $O$ with leakiness rate $k_{\text{sMW}}$ and is degraded with rate $\lambda_{\text{MW}}$:<br />
$$<br />
\frac{d\text{mW}}{dt} = k_{\text{sMW}} \cdot \text{O} - \lambda_{\text{MW}} \cdot \text{MW}<br />
$$<br />
Writer is created from mRNA and with rate $k_{\text{sW}}$ and degraded with rate $\lambda_{\text{W}}$:<br />
$$<br />
\frac{d\text{W}}{dt} = k_{\text{sW}} \cdot \text{MW} - \lambda_{\text{W}} \cdot \text{W}<br />
$$<br />
Unmethylated plasmids are created from both unmethylated and methylated plasmids; methylation is not transferred to daughter plasmid during plasmid replication in prokaryotes. Plasmid growth continues until the plasmid copy number $Ca$ has been reached:<br />
$$<br />
\frac{d\text{PlasU}}{dt} = k_{\text{Plas}} (1 - \frac{(\text{PlasU} + \text{PlasM})}{Ca}) - k_{cW} \cdot \text{W} \cdot \text{PlasU} - \lambda_{\text{Plas}} \cdot \text{PlasU},<br />
$$<br />
Methylated plasmids result from the fusion protein finding an umethylated plasmid and methylating it:<br />
$$<br />
\frac{d\text{PlasM}}{dt} = k_{c\text{W}} \cdot \text{W} \cdot \text{PlasU} - \lambda_{\text{PlasM}} \cdot \text{PlasM}<br />
$$<br />
The amount of (repressed) operons is identical to the amount of plasmids currently in the system:<br />
$$<br />
\text{O} = \text{PlasU} + \text{PlasM}<br />
$$<br />
<br />
== Stochastic model definition == <br />
<br />
The equations in the stochastic model are qualitatively equal to the ones in the differential equation model.<br />
The equations, parameter and initial species values can be viewed on [[Team:Amsterdam/achievements/stochastic_model_definition|this page]].<br />
Analyzing the behaviour of this model by looking at the time-lapse plots, we see a similar trend as in the ODE-model:<br />
all plasmids are methylated within a short amount of time. The fusion protein amounts are shown to be widely varying, but this does not alter the fraction of methylated plasmids much.<br />
<br />
[[File:Stoch_single.png|thumb|200px|Single trajectory of stochastic model. Just as in the deterministic model, all plasmids are methylated within a short amount of time due to the leaky expression with the used values for $ k_{cat} $ and $ k_{cW} $]]<br />
<br />
== Retrieving sensible parameter values ==<br />
==== Leaky expression rate ====<br />
There are discrepancies between the definitions used in the literature on what leaky expression exactly is:<br />
* The most favoured definition seems to be the that leaky operons show low expression rates even when bound by a negative transcriptional regulator. In this perspective the transcriptional regulator is regarded to be unable to completely silence gene expression. In this case the only way to get a value for leaky expression is to measure it experimentally. In this process, care should be taken to be specific about what version of the operon exactly is being used. Some repressors are known to regulate transcription by binding to DNA regions that are very distant from the controlled operon. 'Plasmid versions' of these operons might not contain these distantly located binding sites and are therefore less likely to be optimally repressed.<br />
* Another definition is that the TF ''can'' fully silence gene expression and that leaky expression is caused by seldomly occurring dissociation events between between the TF and the DNA operator sequence.<br />
During these short time windows of opportunity, the RNA polymerases would then be able to quickly transcribe a small amount of RNA.<br />
Multiplying the maximal rate of transcription by the time fraction during which the polymerase is able to bind gives an indication of the leaky transcription rate defined this way.<br />
<br />
The following relationship must always be true: $[O_{\text{Total}}] = [O_{\text{Free}}] + [R_{2}O]$ with $R_{2}O$ denoting the operon bound by a dimerized repressor (as LacI, the repressor of the Lac operon, functions). $R_{2}O$ is defined as $\frac{[R_{2}][O]}{K_{d}}$. Solving for $O_\text{Free}$:<br />
<br />
$$<br />
[O_{\text{Free}}] = \frac{[O_{\text{Total}}]}{1 + \frac{[R_{2}]}{K_{d}}}<br />
$$<br />
<br />
We immediately see that the $[O_{\text{Free}}]$ depends on $[R_{2}]$ and $O_{\text{Total}}$.<br />
The dissociation constant of the dimerized LacI-repressor for its operator sequence has been determined to be $10^{-12} M $ and the volume of ''E. coli'' is $0.7\ \mu m^{3}$. Assuming $O_{\text{Total}} = 200$ and $R_{2} = 200$, the time fraction during which $O$ is free is $2.10815 \cdot 10^{-6}$.<br />
<br />
More simply, the leaky expression rate can also be retrieved from this [http://oregonstate.edu/instruction/bb492/lectures/Regulation.html website], which states a 1000-fold decrease in expression of the repressor-bound operon compared to the free operon.<br />
<br />
==== Fusion protein catalysis rate ====<br />
The catalysis rate $k_{cat}$ has not been determined for the MTase that we are using, M.ScaI. <br />
However, for another MTase with the same [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.1.1.113 EC2.1.1.113 number] as M.ScaI, BamH1, the $k_{cat}$ has been determined to be $0.0175$ ([[#Cheng|Cheng (1999)]]).<br />
<br />
==== Cell growth rate ====<br />
Our own experiments showed that the bacterial strain $\text{DH}5\alpha$ transformed with two of our preliminary constructs had growth rates (<math>\mu</math>) between $80\ min^{-1}$ and $90\ min^{-1}$ ([https://2012.igem.org/Team:Amsterdam/project/growthrates growth rates]).<br />
In these simulations, cellular division was assumed to be either constant at 80.<br />
<br />
Looking at the plots for both the high and the low copy number plasmids, no qualitative difference is observable. All species in the system simply reach a steady state defined as the fraction between their production and degradation rates.<br />
Most notably, in the steady state that this system reaches all plasmids are methylated.<br />
This is very undesirable of course!<br />
<br />
== Steady state parameter scanning ==<br />
In order to deduce what ranges of operon leaky expression and W catalysis rates would yield acceptable leaky expression, a steady state parameter scan has been performed.<br />
<br />
[[File:Ssparmscanode.jpeg|thumb|300px|right|Parameter scan to study the effects of the operon leaky expression rate $k_{s0MW}$ and the W catalysis rate $k_{c\text{W}}$ on the steady state fraction of $ \frac{\text{Methylated plasmids}}{\text{Methylated} + \text{Unmethylated plasmids}}$ in the ODE-model]]<br />
<br />
Our experimental results can thus be explained by any combination of the two parameters which which has an intermediate value in plot.<br />
<br />
A similar parameter scan has been painstakingly performed with the stochastic model. Simulations with 20 trajectories of 200-minute simulations for each parameter combination in a two-dimensional scan for both the $k_{cat}$ and $k_{cW}$ in the same ranges as the ODE parm-scan. Unfortunately, even for these high amount of replications the variability in the resulting methylation fractions is very high, such that no trends are discernible at all in the results (data not shown, python scripts downloadable at the bottom of this page). This variability can probably be reduced by performing even longer simulations and using more trajectories. A much more efficient implementation of the SSA will have to be used to reach this goal within reasonable time scales however. We hope to finish these simulations before the Jamboree. Because the deterministic simulations show similar qualitative behaviour as the more realistic stochastic simulations in the time-lapse plots, one could argue that we will obtain similar results here as the much less computationally expensive ODE-parameter scan.<br />
<br />
The steady state fraction of methylation shows almost no dependence upon the leaky transcription rate in the ODE model. This is likely caused by the steady state value that the writer mRNA reaches fairly quickly. The parameter scan density plots shows a very weak to no influence of the background noise. The fact that the degradation rate of the mRNA is much larger than the leaky transcription rate probably causes this. On the contrary, the steady state fraction of methylated plasmids is shown to be heavily dependent upon the catalysis rate of the fusion protein. This can steer future experiments to research the effects of lowered catalysis rates.<br />
<br />
= Discussion =<br />
Unlike our initial expectations, the ODE-model analyzed here suggests that the catalysis rate of the W is more important to the high background noise we have observed in the experiments than the leaky expression rate. <br />
This yields a new hypothesis to test in the lab: will lowering the catalysis rate significantly lower the background methylation rate?<br />
A few methods to do this come to mind to try and lower the catalysis rate of the writer protein:<br />
* Lower the temperature during the experiments, which will slow down all reactions in the cell including the catalysis rate of the writer<br />
* Mutate amino acid sequence of binding domain, making the affinity of the for the DNA higher<br />
* Pick a different methyltransferase, one that has a lower catalysis rate<br />
* Attach a fluorescent protein to the methyltransferase. This will both increase its diffusion coefficient, decreasing the catalysis rate, and as an added bonus give more insight into the transcription rate of the sensor.<br />
Concluding, we found that the catalysis rate of the writer protein is more important to the high background activity we've observed in the lab. This lead us to suggest several new experiments of which we can hopefully try one or more before the Jamboree!<br />
<br />
=Source files=<br />
Source files for both the SSA and ODE model, including the stochastic and ODE parm scanners, are [https://www.dropbox.com/sh/ijn9smzthdf27t5/rkpRFci6qU available over here].<br />
<br />
=References=<br />
<span id="Cheng"><br />
<sup><br />
Cheng, X., & Blumenthal, R. (1999). S-Adenosylmethionine-Dependent Methyltransferases: Structures and Functions (p. 400). World Scientific. Retrieved from http://books.google.com/books?id=oUCKHnsZuukC&pgis=1<br />
</sup><br />
</span><br />
<br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/data/time_inference_modelTeam:Amsterdam/data/time inference model2012-09-27T03:53:26Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<br />
<h1>Inferring the time of signal onset</h1><br />
<br />
__NOTOC__<br />
Using the here presented model, we will examine how to infer the signal detection time from the amount of methylated plasmids. The cellular division rate determines how long a signal is stored in the ''Cellular Logbook''. <br />
All units are dimensionless in this model, as its sole purpose is clarification of practical usage of the ''Cellular Logbook''.<br />
<div class='clear'></div><br />
<br />
<h2>Methylated bits over time</h2><br />
<br />
Numerous identical plasmids are often present in single cells and plasmids replicate independently of the bacterial chromosome (Scott 1984). A plasmid copy number (PCN) has been determined for all plasmids in the Parts Registry, which indicates a likely amount of copies of the plasmid to be present in each cell. Unlike eukaryotes, prokaryotes do not copy DNA methylation patterns to the newly synthesized strand during DNA replication. This will lead to a dilution of the amount of ‘written’-plasmids over time, mostly due to cell replication and the ensuing binomial division of the plasmids in the parent cell among the two daughter cells. <br />
<br />
[[File:Celldivision.png|thumb|300px|Due to cell division, the amount of methylated plasmids will be approximately halved during each division cycle. In this picture a lower opacity indicates a lower amount of methylated plasmids]]<br />
<br />
The volatilty of this memory design seemed a downside at first, but quickly opened our eyes to a very exciting feature of this system. By analyzing the fraction:<br />
<center><br />
<math>F(t) = \frac{\text{written plasmids}}{\text{written + unwritten plasmids}}</math><br />
</center><br />
at the time of memory read-out, the time at which the signal was registered can be inferred.<br />
<br />
<h2>Model definition</h2><br />
First, let’s model the input signal/compound which is to be reported on. Imagine the to be stationary and positioned along a fluidic stream so that the signal to be registered can pass the ''Cellular Logbook''. Modelling the signal using the piecewise function <math>S(t)</math> now seems appropriate. Here, <math>s_{\text{on}}</math> is defined as the time at which the signal is first encountered and <math>s_{\text{off}}</math> as the time at which the signal is turned off.<br />
<br />
[[File:Signalformula.png|center|frameless|200px]]<br />
[[File:Signal.jpg|image|thumb|300px|Plot of the input signal <math>S(t)</math> with <math>s_{\text{on}}</math> at 3 and <math>s_{{\text{off}}}</math> at 4]]<br />
We will model a single cell with multiple identical plasmids. Each of the plasmid copies contain the gene for the methyltransferase and the so called ''bit region'', which is the region especially purposed to be methylated in presence of a signal. The following assumptions/conditions are made:<br />
<br />
* A well stirred cellular system with no spatial concentrations differences and all species' concentrations large enough to be approximated by continuous functions. Hence we will use a set of ordinary differential equations (ODE’s)<br />
* To ease the analysis of the model, a single unique bit with multiple copies per cell is considered here<br />
* <math>P_{0}</math> denote plasmids that have the single bit set to 0, no write event has taken place in these cells<br />
* <math>P_{1}</math> denote plasmids in which the bit has been flipped to 1 in response to encountering the signalling compound<br />
* <math>P_{\text{T}}</math> denotes the total amount of cells in the sytem, <math>P_{0} + P_{1}</math><br />
* Assumed is a high response rate (3 min) termed <math>\omega</math>, which is the constant rate with which the system responds with methylation of the bit region to the detection of the signal.<br />
* Logistic growth for the plasmid population inside the cell, with a capacity limit of <math>Ca = 40</math>, the copy number of a low copy number plasmid.<br />
* Alternatively, this maximal plasmid number could also be described as the fraction between the plasmid proliferation rate (<math>\beta</math>) and degradation rate (<math>\alpha</math>). The steady state amount of plasmids in the cell will be determined by <math>P_{\text{SS}} = \frac{\beta}{\alpha}</math>, the solution to the differential equation <math>P'(t) = \beta P - \alpha P</math>.<br />
* Accumulation of cells in which the bit has been written is assumed to result in non-written cells; methylation patterns are not copied to the progeny in prokaryotes.<br />
<br />
From these rules, the following system of ODE’s has been constructed:<br />
$$<br />
\begin{aligned}<br />
\frac{dP_{0}}{dt} &=& k\ P_{0+1}\ (1 - \frac{P_{0+1}}{\text{Ca}}) - \omega\ S(t)\ P_{0} - \alpha\ P_{0} \\<br />
\frac{dP_{1}}{dt} &=& \omega\ S(t)\ P_{0} - \alpha\ P_{1}<br />
\end{aligned}<br />
$$<br />
<br />
<table align="right"><br />
<tr><br />
<th align="left">Parameter</th><br />
<th align="right">Value</th><br />
</tr><br />
<tr class="even"><br />
<td align="left">k</td><br />
<td align="right">0.8</td><br />
</tr><br />
<tr class="even"><br />
<td align="left"><math>\alpha</math></td><br />
<td align="right">.06</td><br />
</tr><br />
<tr class="odd"><br />
<td align="left">Ca</td><br />
<td align="right">200</td><br />
</tr><br />
<tr class="even"><br />
<td align="left"><math>\omega</math></td><br />
<td align="right">4</td><br />
</tr><br />
<tr><br />
<td colspan="2"><b>Table 1</b>: Parameter values for the <br> plasmid methylation model</td><br />
</tr><br />
</table><br />
<br />
Using the parameter values of Table 1 a simulation with a duration of 40 time units is shown in Figure 2. The plasmid population within a ''Cellular Logbook'' is shown to be completely converted to methylated plasmids shortly after <math>s_{\text{on}}</math>. As long as the signal is still present – until <math>s_{\text{off}}</math>, – the bit on all newly copied plasmids will be immediately methylated as the signal is still present. After <math>s_{\text{off}}</math>, <math>F(t)</math> will start to decrease. This is mostly due to cell division, during which the cell’s plasmids will be binomially distributed between the two two daughter cells, halving the plasmid amount every division cycle. In this simulation, this degradation due to cell division has been accounted for in the constant degradation rate <math>\alpha</math>. The duration of time after which a small trail of methylated plasmids is still present is related to two factors: positively to the amount of methylated cells at <math>s_{\text{off}}</math> and negatively to the plasmid degradation rate.<br />
<br />
<table align="right"><br />
<tr><br />
<th align="left"><b>Species</b></th><br />
<th align="right"><b>Value</b></th><br />
</tr><br />
<tr class="even"><br />
<td align="left"><math>P_{0}(0)</math></td><br />
<td align="right">10</td><br />
</tr><br />
<tr class="odd"><br />
<td align="left"><math>P_{1}(0)</math></td><br />
<td align="right">0</td><br />
</tr><br />
<tr><br />
<td colspan="2"><b>Table 2</b>: Initial species values for the <br> plasmid methylation model</td><br />
</tr><br />
</table><br />
<br />
To reinforce that:<br />
$$<br />
P_{0} + P_{1} = P_{\text{T}} \le \text{Ca}<br />
$$<br />
is always true in the model, the total amount of plasmids has also been plotted (purple). This clearly shows the limiting value of the plasmid population count, specified by the capacity limit (<math>Ca</math>). This is reached around <math>t=10</math> with the parameter set used here.<br />
<br />
[[File:Timelapse.jpg|frame|center|Time simulation of the system of ODE’s. Input signal <math>S(t)</math> with <math>s_{\text{on}} = 3</math> and <math>s_{{\text{off}}} = 4</math>. Detection of the signal converts all <math>P_{0}</math> (red) to <math>P_{1}</math> (blue) on a short time scale. After the amount <math>P_{1}</math> will start to diminish due to cell division. Eventually, the steady state will be restored once again and the cell’s capacity for plasmids will be completely taken up by <math>P_{0}</math> plasmids.]]<br />
<br />
Unknown variables affecting <math>F(t)</math> in a real-life setting would be the time of signal onset, signal duration and signal strength. Knowing the values for two of these three values, the value of the third can be solved for. Here we will simply assume maximal signal strength during <math>s_{\text{on}}</math> and <math>s_{\text{off}}</math>.<br />
<br />
The response rate <math>\omega</math> of the ''Cellular Logbook'' could limit <math>F(t)</math>, as a low <math>\omega</math> might yield incomplete methylation of all plasmids before <math>s_{\text{off}}</math>. This rate should be experimentally determinable before actual deployment and application of our system and is more closely looked at in the next section. It is likely to be several magnitudes greater than the cellular division rate, however. Every single gene on a plasmid is thus expected to be methylated within at most 5 minutes of registering of the signal.<br />
<br />
Assuming that the plasmid population will have reached its stationary state level before is plausible and eases the analysis somewhat. If the capacity limit has not been reached yet before, a lower value of results than had the capacity limit been reached. This could fool an experimentalist into thinking that the signal was detected relatively long ago, when in fact the amount of plasmids was still very low at <math>s_{\text{on}}</math>, such that total plasmid population <math>P_{\text{T}}</math> has continued to expand since <math>s_{\text{on}}</math>.<br />
<br />
<h2>In theory</h2><br />
<center><br />
<html><br />
<embed src="https://static.igem.org/mediawiki/2012/f/fd/Timeinferranceanim.swf" width="500px" height="350px" fps="50" caption="Inferring the time of signal onset from the amount of methylated bits"><br />
<br><br />
</html><br />
Animation of <math>P_{1}(t)</math> over time with <math>s_{\text{off}}</math> at <math>t = 0</math>. From the measured value of <math>P_{t}</math>, <math>t</math> of measurement can be solved for as is shown using the dashed lines. The value of <math>P_{1}(t)</math> will decrease over time until it becomes 0. An interactive version of this plot has been included in the attached Mathematica file.<br />
</center><br />
<br />
The monotonically decreasing value of <math>F(t) = \frac{\text{methylated plasmids}}{\text{total plasmids}}</math> can be used to infer <math>s_{\text{off}}</math>, given that the degradation rate (<math>\alpha</math>) and capacity constraint <math>Ca</math> are known and constant. Also assumed is that all bits are methylated during signal presence, this implicates <math>\omega</math> is sufficient to methylate all bits during presence of the signal. Irrespective of the initial amount of plasmids, the population of plasmids within the single cell will have reached a steady state value of <math>\frac{\beta}{\alpha}</math>. As we see in the Figure 2, <math>F(t)</math> will start to decrease as a function of the degradation rate after the signal has left the medium following the following function:<br />
<br />
$$<br />
\frac{dP_{1}}{dt} = - \alpha\ P_{1}<br />
$$<br />
<br />
Integrating this differential equation and multiplying by the steady value <math>\frac{\beta}{\alpha}</math> will yield the amount of methylated plasmids at time <math>t</math>, given that there were <math>\frac{\beta}{\alpha}</math> methylated plasmids at <math>t = 0</math>.<br />
<br />
$$<br />
P_{1}(t) = \frac{\beta}{\alpha} e^{-\alpha t} = \frac{\beta}{\alpha} F(t)<br />
$$<br />
<br />
By solving the previous equation, we can calculate the time <math>t</math> that has passed after <math>s_{\text{off}}</math> from <math>F(t)</math>:<br />
<br />
$$<br />
t = \frac{\ln(F(t))}{-\alpha}<br />
$$<br />
<br />
<h2>In practice</h2><br />
[[File:bands.jpeg|center|thumb|500px|Gel representations for a range of different <math>F(t)</math> values. Complete methylation of all bits results in a single, bright band at the top of the gel. This indicates the undigested, linearized plasmid. Decreasing the amount of methylated bits shifts the intensity of the top band away to the two bottom bands. These indicate the linearized &amp; successfully digested plasmid]]<br />
<br />
In a typical laboratory situation, doing a restriction enzyme assay on the miniprep-extracted plasmid DNA out of followed by gel electrophoresis will be the most convenient way to assess the methylation status of the bits. The relative intensities of the gel bands can then be used to infer <math>F(t)</math>. Unmethylated bits will result in successfully digested DNA fragments and thus two bands of shorter DNA fragments. Methylated bits will not be cut and will therefore result in one longer band, shown more to the top of the gel. Thus the top and two bottom gel bands are mutually exclusive as they indicate the same (linearized) plasmid DNA to either be digested, resulting in the two bottom bands, or undigested, resulting in the top band. A high value for <math>F(t)</math> indicates recent detection of the signal, whereas a low value indicates detection to have occurred longer ago. <br />
<br />
To get a hands-on feel of the effects that the plasmid degradation and replication rate have on <math>F(t)</math>, an interactive version in Mathematica is hosted on [https://www.dropbox.com/s/2b32sys01ywdotl/timeinferrance.nb Dropbox].<br />
This file contains the code for all analyses and graphics (except for the cell division scheme) on this page.<br />
<br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/data/experimentalTeam:Amsterdam/data/experimental2012-09-27T03:52:54Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<h1>Experimental Results</h1><br />
__NOTOC__<br />
<div class='clear'></div><br />
<h2>Functionality of the writer-reader module under different sensor modules</h2><br />
We started measuring the first proof-of-concept of the Cellular Logbook by testing the functionality of a part of our writer-reader module in the context of various sensor modules. To this end, we tested the functionality of the synthesized Methyltransferase (MTase) (our writer) cloned under the control of the LacH promoter in the pSB1AT3 backbone and transformed in Library Efficient® DH5α™ competent cells (Invitrogen).<br />
<br />
<h4>Set up of the writer-reader module</h4><br />
[[File:Amsterdam_exp_fig_1.png|300px|right|thumb|Figure 1]]<br />
As mentioned in Molecular design, the ScaI restriction enzyme is unable to cut methylated restriction sites. Therefore, we expect different possible restriction profiles through ScaI restriction digestion since there is one ScaI site residing in the pSB1AT3 backbone and we created one ScaI site via a scar inside our writer module. We expect to find either an off or intermediate methylation state knowing that the LacH promoter driving the MTase has some basal activity.<br />
<br />
Figure 1 shows the result of a ScaI restriction digestion of the pSB1AT3/LacH/MTase construct without IPTG induction. The pattern displayed corresponds to a combination of bands indicating an intermediate state between the ‘off’ and ‘on’ situation. Interpretation of the read-out: absence of MTase expression in ''E. coli'' leads to a complete digestion of our plasmid. Two bands of 2989 bp and 1621 bp are then observed (A). Incomplete methylation of the plasmid at only one of the two ScaI sites shows a linearized plasmid band 4610 bp. Complete methylation of our writer module prevents ScaI to cut and a typical uncut plasmid profile is observed (C).<br />
<br />
<h4>Behavior of the writer-reader module under IPTG induction</h4><br />
[[File:Amsterdam_exp_fig_2.png|300px|right|thumb|Figure 2]]<br />
The next step was to see if IPTG-based induction of MTase expression would modify the methylation state of our writer-reader module thus changing the resulting restriction profile. In the presence of 1mM IPTG, the promoter should be activated providing MTase production inside the cells. Since our model for this experiment suggests that methylation occurs at a fast rate we expect that after 16h of IPTG induction, the read-out will dramatically shift to the uncut profile.<br />
Figure 2<br />
<br />
The writer-reader module after 16h IPTG induction still shows a partial methylation profile, indicating that our writer-reader module is not fully methylated, we observe a gradual shift towards the uncut plasmid form. That means that IPTG induction leads to an increase of methylation of the plasmid and that our writer-reader module is able to store and read this information: Our writer-reader module is in principle working!!<br />
<br />
<h4>Behavior of the writer-reader module in varying growth conditions</h4><br />
[[File:Amsterdam_exp_fig_4.png|300px|right|thumb|Figure 3]]<br />
We aimed to characterize the activity of our writer during bacterial growth. We performed a growth curve experiment (figure 3) and collected samples at different time points to test the occurrence of methylation over different times of growth. <br />
[[File:Amsterdam_exp_fig_5.png|300px|right|thumb|Figure 4]]<br />
<br />
Figure 4 shows ScaI digestion of plasmids extracted from samples activated or not by 10mM IPTG and collected between 0 and 12h. Both conditions reveal an intermediate restriction profile over. However, the two samples taken after 24 hours show a different result, i.e. almost only uncut plasmid is observed as a result of an significant increase of methylation of our reader. <br />
<br />
We aimed to test the functionality of our system in the stationary state. It was previously described in the literature that the Lac promoter upon IPTG induction is performing better in the stationary phase because the lac-permease, encoded by the LacY gene and responsible for the correct IPTG uptake inside the cell, can be quantitatively integrated into the membrane. <br />
<br />
The same bacterial batch was used in both experiments but for the stationary experiment first grown over a longer period of time (at least more than 24 hours), either with or without the inducer. <br />
[[File:Amsterdam_exp_fig_3.png|300px|right|thumb|Figure 5]]<br />
<br />
Figure 5 shows the restriction profile taken from three different time points: 48, 72 and 96 hours. The first three restriction profiles correspond to growth without IPTG induction and the last three with 10 mM IPTG. During the culture no additional IPTG was added since we expect that IPTG is not degraded over time. We do not observe a change in the restriction profile, meaning that the time of culture does not influence our results. <br />
<br />
<br />
In conclusion, the presence of the LacH promoter showed to provide the activity of the Mtase but did not deliver a restriction profile fitting to the ‘off’ and ‘on’ states. Our observations are in line with our predictions as described in the Molecular Design. An alternative way to lower the basal activity of LacH is to perform our experiments at high LacI levels which is suggested to decrease the basal activity of LacH. In addition, LacH in high-copy plasmids could createsmuch more non-specific activity of the LacH than in a low-copy plasmid.<br />
<br />
<br />
<h4>Behavior of the writer-reader module under reduced basal LacH promoter activity using LacIQ E. Coli strain</h4><br />
[[File:Amsterdam_exp_fig_6.png|300px|right|thumb|Figure 6]]<br />
<br />
Both modeling and experimental results show a strong basal activity of the LacH promoter leading to a substantial methylation of our memory module, even without IPTG present in the medium. We aimed to get rid of the basal activity of the LacH promoter without IPTG induction to create a better ‘off’ state and hence a better writer-reader design.<br />
<br />
Since the silencing of the LacH promoter depends on the concentration of the LacI repressor, we chose to replace our standard DH5α E. Coli strain by the LacIQ strain. Investigation done on the LacH promoter revealed that a higher expression of LacI will show a better suppression and decrease of basal activity of the LacH promoter.<br />
<br />
We transformed our pSB1AT3/LacH/Mtase vector in the LacIQ strain and performed a restriction digestion profile experiment after 24h of growth and under 10 mM IPTG induction.<br />
<br />
<br />
<br />
Figure 6 shows the pSB1AT3/LacH/Mtase cultured overnight in LacIQ E. Coli strain and digested with ScaI restriction enzyme.<br />
<br />
The results of the restriction profile for the LacIQ E. Coli strain are inconclusive (figure 6). No change in the ScaI restriction patterns are observed between plasmids isolated from both strains. Higher expression of LacI does not seem to reveal a restriction profile that confirms the ‘off’ state and it does not seem to creates a shift towards the ‘off’ state. <br />
<br />
<br />
<h4>Behavior of the writer-reader module under tight control of and arabinose-regulated promoter</h4><br />
[[File:Amsterdam_exp_fig_7.png|300px|right|thumb|Figure 7]]<br />
The Cellular Logbook was further characterized using the pBad promoter. This promoter was chosen in order to overcome the leakiness observed with the LacH promoter. Expression of any gene cloned behind the pBad promoter is controlled by the AraC repressor and is considered to be fully suppressed in the absence of arabinose. However, in the presence of arabinose the promoter will generate a gradual response leading to gene expression. <br />
[[File:Amsterdam_exp_fig_8.png|300px|right|thumb|Figure 8]]<br />
<br />
The first characterization experiment involved induction of pSB1AT3/pBad/Mtase in Library Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 1 % arabinose. The construct was digested with ScaI restriction enzyme after 24 hours incubation at 37˚C. Surprisingly, the same intermediate digestion profile as the LacH was observed. A combination of different restriction profiles can be inferred from the results (figure 7). In the absence of arabinose, M.ScaI methyltransferase appears to be expressed and a significant number of plasmids present in the culture are fully methylated, accounting for the intense uncut DNA fragment observed. A similar pattern was observed in the presence of arabinose.<br />
<br />
[[File:Amsterdam_exp_fig_9.png|300px|right|thumb|Figure 9]]<br />
<br />
The second characterisation experiment involved the induction of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 2 % arabinose and incubation at 37˚C for 48 hours. Subsequent digestion with ScaI restriction enzyme showed a shift towards the uncut restriction profile (figure 8). This result shows that under these conditions the writer-reader is optimally functional. Succes!<br />
<br />
<br />
The third characterisation experiment concerns the stimulation of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with addition of 2% arabinose daily. Samples were taken after 48, 72 and 96 hours, and digested with ScaI restriction enzyme (figure 9). These results are consistent with the previous results, showing a gradual shift towards the “on-state” with induction of the pBad promoter by addition of arabinose daily.<br />
<br />
[[File:Amsterdam_exp_fig_10.png|300px|right|thumb|Figure 10]]<br />
[[File:Amsterdam_exp_fig_14.png|450px|right|thumb|Figure 11]]<br />
The “off” state was not achieved in the absence of arabinose. All the experiments have been conducted into the high copy number plasmid pSB1AT3. It is known that leaky expression is relative to the copy number of the plasmid.1 Hence, the inability of the Cellular Logbook to show an “off” state in the absence of arabinose could be attributed to the high copy number plasmid used in these experiments as was discussed with the LacH.<br />
<br />
Similarly to the pSB1AT3/LacH/Mtase, a growth curve experiment was conducted to characterize the acquired construct of pSB1AT3/pBad/Mtase (figure 10).<br />
<br />
<br />
Over the course of time the <br\> methylation-dependent restriction profile observed in the gel showed a shift towards the ‘on’ digestion profile in the presence of arabinose (figure 11). This result shows that our Cellular Logbook is able to sense and write an arabinose signal present in the medium in a shorter period of time.<br\><br\><br\><br\><br\><br />
<br />
<h2>Towards the Cellular Logbook</h2><br />
[[File:Amsterdam_exp_fig_11.png|300px|right|thumb|Figure 12]]<br />
[[File:Amsterdam_exp_fig_12.png|300px|right|thumb|Figure 13]]<br />
After months of cloning attempts, it appears that we succeeded in obtaining the final version of our Cellular Logbook: the pSB1AT3/LacH/PZF3838/Mtase/Mem2X (BBa_K874300). Digestion of extracted plasmid DNA with the restriction enzyme BamHI, present in the pSB1AT3 backbone vector and in the reader module (BBa_K874040), ensured that both the Polydactyl Zinc Finger PZF3838 (BBa_K874001) and the reader module (BBa_K874040) were successfully cloned in as shown in Figure 12 (colony 2, 2 bands expected: 3699 and 1769 bp). Preliminary characterization of BBa_K874300 was attempted only once due to time pressure and consisted of a ScaI digestion in the absence of IPTG (figure 13). Compared to the pSB1AT3/LacH/Mtase, BBa_K874300 shows a significant switch to the non-methylated profile, illustrated by the tremendous intensity of the lower bands (cut plasmid) compared to the first one (partially cut plasmid). These results show that the presence of the PZF3838 enhances the specificity of the writer.<br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br />
<br />
<h1>Reference List</h1><br />
<br />
1. Bowers,L.M., Lapoint,K., Anthony,L., Pluciennik,A., & Filutowicz,M. Bacterial expression system with tightly regulated gene expression and plasmid copy number. Gene 340, 11-18 (2004).<br />
<br />
<br />
<br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/project/features_and_applicationsTeam:Amsterdam/project/features and applications2012-09-27T03:52:38Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<br />
<div id="sub-menu" class="content-block"><br />
<h1>Features and Applications</h1><br />
__NOTOC__<br />
The Cellular Logbook regards quite unexplored and fundamental based research. Our projects holds a platform for new technology. But there are already some applications that are feasible in the near future.<br />
<div class='clear'></div><br />
<h1>Facets</h1><br />
<h4>Combining all sensors: the Cellular Logbook</h4><br />
<br />
One of the more popular themes in iGEM projects is the creation of a biosensor for a specific product, and as such the iGEM part registry contains many sensors. Every year a lot of newly developed sensors are added. These sensors are very much needed in today’s world where many new threats and problems arise as unexpected dangers. However, most of these biosensors are fundamentally different in design, making it hard to have multiple sensors in one system. On top of that, many previous iGEM teams used fluorescence, pH or electrical conductance as a readout mechanism.<br />
<br />
Our Cellular Logbook aims to create a single microorganism with the ability to sense a wide range of different signals and register them efficiently. The Cellular Logbook allows for the incorporation of many standardized sensors of the parts registry database in the same system using the biobrick assembly system. In addition it also provides a unique standardized sensing mechanism that can be used by any sensor that is compatible with the host cell.<br />
<br />
Our system can be linked to any other sensory system introduced into a microorganism, therefore creating a '''multiple-sensor-microorganism'''. Since the registration of a signal occurs via methylation of a specific DNA sequence called Memory Part (MP), a specific signal can be stored effectively for either a short or a longer period of time, to eventually be read-out in an easy digestion providing a simple yes or no answer.<br />
<br />
<h4>Time Indicator</h4><br />
<br />
[[File:Celldivision.png|thumb|right|300px|Figure 1: Due to cell division, the amount of methylated plasmids will be approximately halved during each division cycle. In this picture a lower opacity indicates a lower amount of methylated plasmids]]<br />
Numerous identical plasmids are often present in single cells and plasmids replicate independently of the bacterial chromosome (Scott 1984). A plasmid copy number (PCN) has been determined for all plasmids in the Parts Registry, which indicates a likely amount of copies of the plasmid to be present in each cell. Unlike eukaryotes, prokaryotes do not copy DNA methylation patterns to the newly synthesized strand during DNA replication. This will lead to a dilution of the amount of ‘written’-plasmids over time, mostly due to cell replication and the ensuing binomial division of the plasmids in the parent cell among the two daughter cells.<br />
<br />
The volatilty of this memory design seemed a downside at first, but quickly opened our eyes to a very exciting feature of this system. By analyzing the fraction at the time of memory read-out, the time at which the signal was registered can be inferred.<br />
<br />
<h4>Concentration Indicator</h4><br />
On and off detection of a signal's presence is useful, but often in biology gaining insight in the quantity of a signal is even more relevant. By coupling different promoters with different strengths as responses to the same signal and assigning each promoter its own unique Memory Part, the range in which the strength of the signal is located can be measured.<br />
<br />
<h4>Easy and Quick</h4><br />
A perk and very welcomed bonus of our system is that it is easy. It is easy in use, easy to read and straight forward to understand. For any circumstance specialized systems can be made which can be used in any condition for fast insight.<br />
<br />
The most stripped down version, that could only sense a few signals of our system, could even be used on site. All you need is: our specialized bacteria, a centrifuge, a mini-prep kit, restriction enzymes, a gel and a UV tray. Bring a PCR machine and even the fully customized bacteria can be used!<br />
<br />
<h1>Applications</h1><br />
Taking in mind the above facets, many applications are possible. Below are a few highlighted applications.<br />
<br />
<h4>Compound Detection</h4><br />
<h5>Clean Water Supply Detection</h5><br />
If we are able to use our bacteria in the environment, our Cellular Logbook can be used to sense toxic signals in the environment. One of the places in need for toxic detection are the water supplies, ponds and rivers in the Netherlands. We had a talk with toxicologist Ron van der Oost from Waternet [https://www.waternet.nl/about-waternet/]. During this talk we came to the conclusion that a multi sensor that is able to detect 20 different toxic groups, and is able to detect if the concentration has surpassed a specific amount, would be greatly cost reducing compared to the current setup for detecting if a water supply is clean of toxics, which can cost up to 40.000 euros / place. To achieve this we need a system that contains the bacteria, so no bacteria will roam in the environment. The KWR Water Cycle Research Institute[http://www.kwrwater.nl/] has developed a flow-trough sensor that can also serve as a container for our sensor[http://www.kwrwater.nl/uploadedFiles/Website_KWR/Publicaties_%40_Producten/Posters/Development%20of%20a%20water%20toxicity%20sensor%20based%20on%20genetically%20modified%20bacteria.pdf]. <br />
<br />
<h5>Compound Emission Detection at Industrial Sites</h5><br />
The Cellular Logbook can be used at fabrics to measure compound emission. During a talk with dr. Bart van der Burg, Chief Scientific Officer at BioDetection Systems b.v.[http://www.bds.nl/], who currently use bioassays to detect compounds in samples, we came to the realization that the Cellular Logbook can be used as a cheap detection system for compound emission of industrial sites. If the Cellular Logbook cells are captured in a system that prevents them from being released in the environment, the Cellular Logbook can be placed at multiple locations, after which they are able tell us where, when and in what concentration a certain chemical has been detected.<br />
<br />
<h4>Debugger</h4><br />
Science relies on experimentations. Besides simply measuring or tracking a substance or concentration biologists can encounter unexplainable or vague findings, especially when experimenting with modified or synthetically engineered proteins or genetic networks. Many general questions rise: Is the pathway I’m working with still active? Does my introduced or suspected protein accurately activate my gene?<br />
<br />
The system of our Cellular Logbook can help to answer these questions. Our methylation based system only requires a zinc finger and methyltransferase fusion protein (ZnF-Mtase) and our specific Memory Part. Any promoter can be set before our Mtase and can thus be effectively tested. And since the memory plasmid is so expandable not just one but multiple promoters can be tested in the same experiment if a unique zinc finger is linked to a methyltransferase for every sensor. This allows any scientist to simultaneously test all parts of a pathway or '''multiple pathways''' at the same time, creating a fuller understanding of any complex mechanism.<br />
<br />
<h4>Do It Yourself</h4><br />
The Cellular Logbook is quick and easy to use. A centrifuge, gel electrophoresis setup and pcr machine are all the machines needed to get the Cellular Logbook to work, bringing great promise for the Do It Yourself biologist. This form of biology is one that is gaining in popularity, and we will definately see more of it in the future. We suspect that our Cellular Logbook system can greatly attribute to this form of biology in the future. A farmer that might be worried about the fertility of his land before planting his crops can use the Cellular Logbook to detect for favorable and unfavorable conditions. In the Do It Yourself manner, the Cellular Logbook can also be used as a health check at home, or the detection of spoiled food.<br />
<br />
<h1>Global Challenges</h1><br />
We took the global challenges as given by The Millenium Project[http://www.millennium-project.org/millennium/challeng.html] to show what the Cellular Logbook can contribute to worldwide societal problems. As a result we have identified four challenges where the Cellular Logbook is able to significantly contribute to the solution.<br />
<br />
<h4>How can everyone have sufficient clean water without conflict?</h4><br />
One of the main problems in 3rd world countries concerning clean water supplies, apart from the cleaning of the water, is the detection of contaminated water. The Cellular Logbook can serve as a multi-sensor that comprises all the common causes for contaminated water in the 3rd world. Using this multi-sensor we have a cheap and efficient way of detecting contaminated water that is affordable in 3rd world countries.<br />
<br />
<h4>How can the threat of new and reemerging diseases and immune micro-organisms be reduced?</h4><br />
Our multi-sensor can be used to detect these diseases but also to monitor places at risk. Since it can be adapted to use any sensor it can be used to effectively scan for several threats. And being inside a live organism our logbook provides a longer measurement instead of just capturing a single moment. Thus giving a much better insight into the situation at hand.<br />
<br />
<h4>How can growing energy demands be met safely and efficiently?</h4><br />
Using the Cellular logbook adapted to any specific waste or other suspected threats produced when this energy demand is met, it can efficiently provide a means for safer outcome.<br />
<br />
<h4>How can scientific and technological breakthroughs be accelerated to improve the human condition?</h4><br />
By using our Cellular logbook platform as described for the Debugger. Using the Debugger can provide much faster insight when testing pathways or if you just want to find out whether or not your system is working. These fast means of insight can generate a new bundle of information and save precious time for any researcher.<br />
<br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/project/biobricksTeam:Amsterdam/project/biobricks2012-09-27T03:52:21Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div class="content-block"><br />
<h1>BioBricks</h1><br />
<groupparts>iGEM12 Amsterdam</groupparts><br />
__NOTOC__<br />
<html><div class='clear'/></html><br />
<br />
<h2>Favourites</h2><br />
<br />
<h4>M.ScaI Methyltransferase [http://partsregistry.org/wiki/index.php?title=Part:BBa_K874000 BBa_K874000]</h4><br />
<p><br />
This part codes for the [http://rebase.neb.com/rebase/enz/M.ScaI.html M.ScaI] methyltransferase protein. M.ScaI is a type II methyltransferase (subtype beta) that recognizes site on the<br />
DNA of the following sequence <b>5..AGTACT..3</b>. It methylates this site at the 5th (Cytosine) nucleotide leaving an N4-methylcytosine (m4). This methylation type (m4) is not found in native <i>E. coli</i> nor is the recognition site methylated by any of <i>E. coli</i>'s native methylation systems (Dam, Dcm). Also this specific methylation inhibits restriction by M.ScaI's prototype restriction enzyme ([http://rebase.neb.com/rebase/enz/ScaI.html ScaI]).<br />
</p><br />
<br />
<p><br />
Data-mining of the [http://rebase.neb.com/rebase/rebms.html REBASE (m4) methyltransferase database] revealed that M.ScaI was the best candidate based on the following parameters:<br />
<ul><br />
<li>Methylation (m4) done by this M.ScaI inhibits its prototype (ScaI) restriction enzyme ability to restrict the site</li><br />
<li><i>E. coli's</i> native <b>methylation</b> systems do not methylate the recognition site and thus can not interfere with systems using M.ScaI</li><br />
<li><i>E. coli's</i> native <b>restriction</b> systems do not restrict the recognition site (in either methylated and unmethylated form) and thus can not interfere with systems using M.ScaI</li><br />
<li>Recognition site has high specificity (1 in 4048 random sequences)</li><br />
<li>Its prototype restriction enzyme is commercially available</li><br />
</ul><br />
</p><br />
<br />
<p><br />
Our experiments show that M.ScaI functions as expected in <i>E. coli</i> by showing that it is indeed able to methylate. However it might be worth noting that the protein is natively found in <i>[http://rebase.neb.com/rebase/enz/M.ScaI.html Streptomyces caespitosus]</i> which is a bacteria that has an optimal growth temperature of 26C and thus might not be expressed optimally in <i>E. coli</i>.<br><br />
</p><br />
<br />
<h4>IPTG inducible expression of M.ScaI methyltransferase<br>(IPTG -> M.ScaI) [http://partsregistry.org/wiki/index.php?title=Part:BBa_K874100 BBa_K874100]</h4><br />
<p>This BioBrick contains the first proof of concept and contains the both the <b>Reader</b> and the <b>Sensor</b> described in our molecular design. Therefore this is also the most extensively studied BioBrick in our project, for an in dept view of the experiments we performed using this BioBrick you can look at the [[Team:Amsterdam/data/experimental |Experimental Setup]] section. We managed to insert this BioBrick in both the pSB1AT3 and pSB1C3 backbones but all testing was done in pSB1AT3.</p><br />
<br />
<h4>Arabinose inducible expression of M.ScaI methyltransferase<br> (ARA -> M.ScaI) [http://partsregistry.org/wiki/index.php?title=Part:BBa_K874101 BBa_K874101]</h4><br />
<p>After the assessment of the first BioBrick we deemed it nessesary to change te promotor to the pBAD (Arabinose) promoter. This therefore is still the first proof of concept but contains a different <b>Reader</b> as described in our molecular design. This BioBrick was also extensively studied so for an in dept view of the experiments we performed using this BioBrick you can look at the [[Team:Amsterdam/data/experimental |Experimental Setup]] section. We managed to insert this BioBrick in both the pSB1AT3 and pSB1C3 backbones but all testing was done in pSB1AT3.</p><br />
<br />
<h2>Important</h2><br />
<br />
<h4>Polydactyl Zinc Finger (PZF3838) [http://partsregistry.org/wiki/index.php?title=Part:BBa_K874001 BBa_K874001]</h4><br />
<p>This part codes for the 3838 Polydactyl Zinc Finger (PZF). It consists of 6 individual zinc fingers that together bind to a specific 18pb DNA sequence.<br><br />
The actual binding sequence is <b>5..GGGGCCGGAGCCGCAGTG..3</b> and can be broken to the following codons per zinc finger:<br><br />
<ul><br />
<li><b>Zinc Finger 1:</b> 5..GGG..3</li><br />
<li><b>Zinc Finger 2:</b> 5..GCC..3</li><br />
<li><b>Zinc Finger 3:</b> 5..GGA..3</li><br />
<li><b>Zinc Finger 4:</b> 5..GCC..3</li><br />
<li><b>Zinc Finger 5:</b> 5..GCA..3</li><br />
<li><b>Zinc Finger 6:</b> 5..GTG..3</li><br />
</ul></p><br />
<p><br />
This PZF was chosen because many of the PZF's we found where listed to be partial working or not verified to be working. This seems to be a common problem with PZF's which finnaly led us to enlist the help from Sylvia de Pater in obtaining a working PZF.</p><br />
<p><br />
This PZF was designed in research done by Bert van der Zaal, Paul Hooykaas and Sylvia de Pater in order to recognise the E2C transcription factor binding site in <i>Arabidopsis</i>.[[#Ref1|[1]]] It has thus been tested and verify to fold and function in <i>Arabidopsis</i>.<br><br />
A indication that the PZF functions properly in <i>E. coli</i> (LacIq) is also provided by the iGEM Amsterdam 2012 project. More information can be found on its verification on the [[Part:BBa_K874200 | BBa_K874200]] part page.<br><br />
</p><br />
<p><br />
<b>On a final note!</b> Since the individual Zinc Fingers in this PZF (or any PZF for that matter) are highly similar in sequence we strongly recommender that when doing PCR primers are used that ad-heal outside of the actual PZF..</p><br />
<br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/team/advisorsTeam:Amsterdam/team/advisors2012-09-27T03:51:52Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<h1>Advisors overview</h1><br />
__NOTOC__<br />
Meet our supervisors. We wouldn't have gotten this far without them. <br />
<div class='clear'></div><br />
<br />
<h2>Domenico Bellomo</h2><br />
[[File:Regional_Europe_Domenico.jpg|150px|right]]<br />
'''Name:''' Domenico Bellomo<br\><br />
'''Title:''' PhD<br\><br />
'''University''': Systems Bioinformatics IBIVU, VU University Amsterdam<br\><br />
'''Job:''' Postdoc<br\><br />
'''Advisory role:''' Supervisor concerned with the general overview of the project and the modeling.<br\><br\><br\><br\><br />
<br />
<h2>Frederic Cremazy</h2><br />
[[File:Amsterdam_frederic.jpg|200px|right]]<br />
'''Name:''' Frederic Cremazy<br\><br />
'''Title:''' PhD<br\><br />
'''University:''' University of Amsterdam<br\><br />
'''Job:''' Assistant-Professor and guest at UvA<br\><br />
'''Advisory role:''' My main role is to help students to design, organize and perform experiments during the whole iGEM adventure. I’m also helping them to set up their own scientific environment and guiding them through the lab.<br\><br\><br\><br\><br />
<br />
<h2>Pernette Verschure</h2><br />
[[File:Amsterdam_pernette.jpg|200px|right]]<br />
'''Name:''' Pernette J. Verschure<br\><br />
'''Title:''' PhD<br\><br />
'''University:''' University of Amsterdam<br\><br />
'''Job:''' Assistant Professor and PI of a research team<br\><br />
'''Role:''' Instructor/advisor to assist in setting out the IGEM research line/outreach and keeping the research on the right track. More specifically my role is to guide the students in making the right choices, keeping the science at high standards and advise in sponsoring and PR actions.<br\><br\><br\><br\><br />
<br />
<h2>Wieke Betten</h2><br />
[[File:Amsterdam_wieke.jpeg|200px|right]]<br />
'''Name:''' Wieke Betten<br\><br />
'''Title:''' MSc<br\><br />
'''University:''' VU University<br\><br />
'''Job:''' PhD student 'valorization of synthetic biology' and lecturer<br\><br />
'''Advisory role:''' My role is to share insights from the field of science, technology & society and help to incorporate ethical, legal and social aspects into the project.<br\><br />
<br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/team/membersTeam:Amsterdam/team/members2012-09-27T03:51:37Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<h1>Members overview</h1><br />
__NOTOC__<br />
<div class='clear'></div><br />
<h2>Ernst Bank</h2><br />
[[File:Amsterdam_ernst.jpg|300px|right|border]]<br />
<br />
<b>Name:</b> Ernst Bank<br><br />
<br />
<b>Study:</b> Msc. Biomolecular Siences, VU Amsterdam<br><br />
<br />
<b>iGEM job:</b> Knowledge master, wiki design, graphical designer, software guru<br />
<br />
<b>About:</b> Jack of all trades, put him behind a computer to ‘make it fly’ put him in the lab and it will also fly followed by alot of crashing and burning (better in theory than in practice). Next to studying Biomolecular Siences (1e year) also interested in computers, programming, physics, graphical design and not to be seen as a total nerd also likes swimming, biking and partying :P.<br><br />
<br />
<b>Motivation for iGEM:</b> Opportunity to do an ambitions project in a much shorter time span than a normal internship, also the chance to work on improving synthetic biology and the prospect of increasing teamwork skills by working in an interdisciplinary team.<br><br />
<br />
<b>Future perspectives:</b> Hopes to work in a social setting on ambitious bioinformatics projects where team effort is paramount to success.<br><br />
<br />
<h2>Glenn Groenewegen</h2><br />
[[File:Amsterdam_glenn.jpg|300px|right|border]]<br />
<br />
<b>Name:</b> Glenn Groenewegen<br><br />
<br />
<b>Study:</b> Biomolecular Sciences, VU<br><br />
<br />
<b>iGEM job:</b> Chief of Laboratorial Relations and Experiments<br />
<br />
<b>About:</b> First years master student with a focus on cell biology. Besides studying very much into music and producing his own. Otherwise sports and cooking belong to his interests.<br><br />
<br />
<b>Motivation for iGEM:</b> Wants to grab the chance on creating something real for himself and actually realizing this on the boundary of science.<br><br />
<br />
<b>Future perspectives:</b> Sees himself working with companies on equally innovative projects.<br><br />
<br />
<h2>Maarten Reijnders</h2><br />
[[File:Amsterdam_Maarten.jpg|300px|right|border]]<br />
<br />
<b>Name:</b> Maarten Reijnders<br><br />
<br />
<b>Study:</b> MSc Bioinformatics, VU University Amsterdam<br><br />
<br />
<b>iGEM job:</b> Wiki Chief Editor, software guru, poster/presentation manager<br />
<br />
<b>About yourself:</b> BSc Bioinformatician and currently doing a Masters in the same subject, with the focus on the Artificial Intelligence side. Can be characterized as a nerd. Likes to visit concerts and music festivals and is also interested in (watching) sports.<br><br />
<br />
<b>Motivation for iGEM:</b> iGEM is the perfect preparation for a career in science. Doing a normal internship requires you to help in a PhD students project, or at best have your own sub-project inside an existing project. Doing iGEM as a replacement for your internship gives the added bonus of having to think about every stage of a project, from brainstorming to eventually getting results, and of course human outreach. As a Bioinformatician it is also very important to be able to adapt to biologists in a project environment, for which iGEM is a perfect learning ground.<br><br />
<br />
<b>Future perspectives:</b> After iGEM i will do something completely different, starting my internship in Wageningen where i will study protein homology using machine learning techniques. The plan is to start my PhD after my study, but direction and place is still unknown.<br><br />
<br />
<h2>Maarten Slagter</h2><br />
[[File:Amsterdam_slagter.jpg|300px|right|border]]<br />
<br />
<b>Name: Maarten Slagter</b><br />
<br />
<b>Study:</b> MSc Systems Biology & Bioinformatics<br><br />
<br />
<b>iGEM job:</b> Modeling master<br />
<br />
<b>About yourself:</b> Broadly interested guy, enjoys all the usual stuff: sports (rowing, biking, running), reading, tasty food, some partying, watching series etc. Fascinated by the effects of music on people and effective harmony, studied at a Jazz-conservatory for a year.<br><br />
<br />
<b>Motivation for iGEM:</b> Got excited about the idea of thinking about biological engineering and having the Parts Registry readily available to be able to construct virtually anything. Turns out modifying an E.coli cell is not as straight-forward as programming a computer! But fortunately all modelling theory and software is easily accessible and I’m learning a lot of microbiology and mathematics as we go.<br><br />
<br />
<b>Future perspectives:</b> I’d like to research cell signalling either in collaboration with experimentalists or in a more fundamentally-oriented environment.<br><br />
<br />
<h2>Matias Mendeville</h2><br />
[[File:Amsterdam_matias.jpg|300px|right|border]]<br />
<br />
<b>Name:</b> Matias Mendeville<br><br />
<br />
<b>Study:</b> Msc Bioinformatics/Systems Biology<br><br />
<br />
<b>iGEM job:</b> iGEM Amsterdam 2012 CEO, funding, human outreach and practices, public relations<br />
<b>About yourself:</b> Born in Amsterdam, son of musicians, since a kid a lot into football. At the moment spending most of his time on investigating the complex and beautiful world of biology. <br><br />
<br />
<b>Motivation for iGEM:</b> The right kind of project to experience science from a new perspective, compared to the usual academic way of reading books and attending lectures. Likes the challenge of working in a team. <br><br />
<br />
<b>Future perspectives:</b> Will work on a bioinformatics internship after iGEM. Will follow developments in biotechnology and will consider different job-opportunities or doing a PhD. Curious on where he will find himself in 5 years. Perhaps coaching a football team =)<br><br />
<br />
<h2>Tania Quirin</h2><br />
[[File:Amsterdam_tania.jpg|300px|border|right]]<br />
<br />
<b>Name:</b> Marie Ann Christine Tania Quirin<br><br />
<br />
<b>Study:</b> MSc Biomolecular Science (2nd Year)<br><br />
<br />
<b>iGEM job:</b> Safety manager, lab guru<br><br />
<b>About yourself:</b> Easy-going personality, enjoys all types of discoveries through travelling. Oh and, haven’t you heard about my parties?!?<br><br />
<br />
<b>Motivation for iGEM:</b> I like the concept of iGEM because it delves into other aspects of the scientific society. It differs tremendously from a ‘traditional’ internship in terms of societal impact and also include equally important activities such as the human outreach and funding. Hence, it develops the personality, enhances knowledge and creates a useful list of contacts for a future career. Nonetheless, my main contribution to our iGEM 2012 Amsterdam team........being one of the two “lab heroes” :)<br><br />
<br />
<b>Future perspectives:</b> Would like to pursue a PhD in Virology.<br><br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/software/logbook_designer/webtoolTeam:Amsterdam/software/logbook designer/webtool2012-09-27T03:51:02Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
{{Team:Amsterdam/toolStyleBase}}<br />
{{Team:Amsterdam/toolStyleSunburst}}<br />
<html><br />
<head><br />
<style><br />
p{<br />
text-align: center;<br />
}<br />
</style><br />
<link type="text/css" href="https://2012.igem.org/Team:Amsterdam/maarten/basecss?action=raw" rel="stylesheet" /><br />
<link type="text/css" href="https://2012.igem.org/Team:Amsterdam/maarten/sunburstcss?action=raw" rel="stylesheet" /><br />
<script type="text/javascript" src="https://2012.igem.org/Team:Amsterdam/maarten/toolscripts?action=raw"></script><br />
<script type="text/javascript" src="https://2012.igem.org/Team:Amsterdam/maarten/jit?action=raw"></script><br />
</head><br />
</html><br />
<div id="content-area" align="center" style="height: 4000px; "><br />
<div id="sub-menu" class="content-block"><br />
<br\><br />
<h1>Logbook Designer: Ready, Steady, Build!</h1><br />
[[File:Amsterdam_logbook_designer_logo.png|200px|]]<br />
<br />
{{team:Amsterdam/toolForm}}<br />
<html><br />
<br />
</div><br />
</div><br />
</html><br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/software/logbook_designer/webtoolTeam:Amsterdam/software/logbook designer/webtool2012-09-27T03:50:06Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
{{Team:Amsterdam/toolStyleBase}}<br />
{{Team:Amsterdam/toolStyleSunburst}}<br />
<html><br />
<head><br />
<style><br />
p{<br />
text-align: center;<br />
}<br />
</style><br />
<link type="text/css" href="https://2012.igem.org/Team:Amsterdam/maarten/basecss?action=raw" rel="stylesheet" /><br />
<link type="text/css" href="https://2012.igem.org/Team:Amsterdam/maarten/sunburstcss?action=raw" rel="stylesheet" /><br />
<script type="text/javascript" src="https://2012.igem.org/Team:Amsterdam/maarten/toolscripts?action=raw"></script><br />
<script type="text/javascript" src="https://2012.igem.org/Team:Amsterdam/maarten/jit?action=raw"></script><br />
</head><br />
</html><br />
<div id="content-area" align="center" style="height: 4000px; "><br />
<div id="sub-menu" class="content-block"><br />
<br\><br />
<h1>Logbook Designer: Ready, Steady, Build!</h1><br />
[[File:Amsterdam_logbook_designer_logo.png|200px|]]<br />
[https://2012.igem.org/Team:Amsterdam/software/logbook_designer/setup Logbook Designer Setup]<br\><br />
[https://2012.igem.org/Team:Amsterdam/software/logbook_designer/manual Logbook Designer Manual]<br\><br />
[https://2012.igem.org/Team:Amsterdam/software/logbook_designer/future_perspective Logbook Designer Future Perspective]<br />
{{team:Amsterdam/toolForm}}<br />
<html><br />
<br />
</div><br />
</div><br />
</html><br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/software/logbook_designer/webtoolTeam:Amsterdam/software/logbook designer/webtool2012-09-27T03:49:48Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
{{Team:Amsterdam/toolStyleBase}}<br />
{{Team:Amsterdam/toolStyleSunburst}}<br />
<html><br />
<head><br />
<style><br />
p{<br />
text-align: center;<br />
}<br />
</style><br />
<link type="text/css" href="https://2012.igem.org/Team:Amsterdam/maarten/basecss?action=raw" rel="stylesheet" /><br />
<link type="text/css" href="https://2012.igem.org/Team:Amsterdam/maarten/sunburstcss?action=raw" rel="stylesheet" /><br />
<script type="text/javascript" src="https://2012.igem.org/Team:Amsterdam/maarten/toolscripts?action=raw"></script><br />
<script type="text/javascript" src="https://2012.igem.org/Team:Amsterdam/maarten/jit?action=raw"></script><br />
</head><br />
</html><br />
<div id="content-area" align="center" style="height: 4000px; "><br />
<div id="sub-menu" class="content-block"><br />
<br\><br />
<h1>Logbook Designer: Ready, Steady, Build!</h1><br />
[https://2012.igem.org/Team:Amsterdam/software/logbook_designer/setup Logbook Designer Setup]<br\><br />
[https://2012.igem.org/Team:Amsterdam/software/logbook_designer/manual Logbook Designer Manual]<br\><br />
[https://2012.igem.org/Team:Amsterdam/software/logbook_designer/future_perspective Logbook Designer Future Perspective]<br />
[[File:Amsterdam_logbook_designer_logo.png|200px|]]<br />
{{team:Amsterdam/toolForm}}<br />
<html><br />
<br />
</div><br />
</div><br />
</html><br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/software/logbook_designer/webtoolTeam:Amsterdam/software/logbook designer/webtool2012-09-27T03:49:26Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
{{Team:Amsterdam/toolStyleBase}}<br />
{{Team:Amsterdam/toolStyleSunburst}}<br />
<html><br />
<head><br />
<style><br />
p{<br />
text-align: center;<br />
}<br />
</style><br />
<link type="text/css" href="https://2012.igem.org/Team:Amsterdam/maarten/basecss?action=raw" rel="stylesheet" /><br />
<link type="text/css" href="https://2012.igem.org/Team:Amsterdam/maarten/sunburstcss?action=raw" rel="stylesheet" /><br />
<script type="text/javascript" src="https://2012.igem.org/Team:Amsterdam/maarten/toolscripts?action=raw"></script><br />
<script type="text/javascript" src="https://2012.igem.org/Team:Amsterdam/maarten/jit?action=raw"></script><br />
</head><br />
</html><br />
<div id="content-area" align="center" style="height: 4000px; "><br />
<div id="sub-menu" class="content-block"><br />
[https://2012.igem.org/Team:Amsterdam/software/logbook_designer/setup Logbook Designer Setup]<br\><br />
[https://2012.igem.org/Team:Amsterdam/software/logbook_designer/manual Logbook Designer Manual]<br\><br />
[https://2012.igem.org/Team:Amsterdam/software/logbook_designer/future_perspective Logbook Designer Future Perspective]<br\><br />
<h1>Logbook Designer: Ready, Steady, Build!</h1><br />
[[File:Amsterdam_logbook_designer_logo.png|200px|]]<br />
{{team:Amsterdam/toolForm}}<br />
<html><br />
<br />
</div><br />
</div><br />
</html><br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/achievementsTeam:Amsterdam/achievements2012-09-27T03:47:50Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<html><br />
<table><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/experimental#Behavior_of_the_writer-reader_module_under_IPTG_induction"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/experimental#Behavior_of_the_writer-reader_module_under_IPTG_induction">Demonstrated the functionality of M.ScaI introduced in E. coli</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/experimental#Behavior_of_the_writer-reader_module_under_reduced_basal_LacH_promoter_activity_using_LacIQ_E._Coli_strain"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/experimental#Behavior_of_the_writer-reader_module_under_reduced_basal_LacH_promoter_activity_using_LacIQ_E._Coli_strain">Succesfully sensing, writing and reading of an arabinose signal</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/time_inference_model"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/time_inference_model">We developed the theory behind inferring the time of signal registration by way of a model</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/background_activity"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/background_activity">We devised grounded suggestions on how to improve the molecular design of our system using a model</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/software/logbook_designer/webtool"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/software/logbook_designer/webtool">We developed software that is able to generate a Cellular Logbook plasmid that enhances our project in such a way that it can be used on a bigger scale</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/practices/overview"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/practices/overview">We designed and outlined a new responsible development-approach for iGEM projects and applied it to our Cellular Logbook</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/practices/results#Human_Outreach"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/practices/results#Human_Outreach">As part of Human Outreach we collaborated with Dutch iGEM teams to organize part of the Discovery Festival in 3 major Dutch cities</a><br />
</td><br />
</tr><br />
<br />
</table><br />
</html><br />
</div><br />
</div><br />
<br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/achievementsTeam:Amsterdam/achievements2012-09-27T03:47:33Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<html><br />
<table><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/experimental#Behavior_of_the_writer-reader_module_under_IPTG_induction"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/experimental#Behavior_of_the_writer-reader_module_under_IPTG_induction">Demonstrated the functionality of M.ScaI introduced in E. coli</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/experimental#Behavior_of_the_writer-reader_module_under_reduced_basal_LacH_promoter_activity_using_LacIQ_E._Coli_strain"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/experimental#Behavior_of_the_writer-reader_module_under_reduced_basal_LacH_promoter_activity_using_LacIQ_E._Coli_strain">Succesfully sensing, writing and reading of an arabinose signal</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/time_inference_model"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/time_inference_model">We developed the theory behind inferring the time of signal registration by way of a model</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/background_activity"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/background_activity">We devised grounded suggestions on how to improve the molecular design of our system using a model</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/software/logbook_designer/webtool"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/software/logbook_designer/webtool">We developed software that is able to generate a Cellular Logbook plasmid that enhances our project in such a way that it can be used on a bigger scale</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/practices/overview"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/practices/overview">We designed and outlined a new responsible development-approach for iGEM projects and applied it to our Cellular Logbook</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/practices/results#Human_Outreach"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/practices/results#Human_Outreach">As part of we collaborated with Dutch iGEM teams to organize part of the Discovery Festival in 3 major Dutch cities</a><br />
</td><br />
</tr><br />
<br />
</table><br />
</html><br />
</div><br />
</div><br />
<br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/achievementsTeam:Amsterdam/achievements2012-09-27T03:46:06Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<html><br />
<table><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/experimental#Behavior_of_the_writer-reader_module_under_IPTG_induction"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/experimental#Behavior_of_the_writer-reader_module_under_IPTG_induction">Demonstrated the functionality of M.ScaI introduced in E. coli</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/experimental#Behavior_of_the_writer-reader_module_under_reduced_basal_LacH_promoter_activity_using_LacIQ_E._Coli_strain"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/experimental#Behavior_of_the_writer-reader_module_under_reduced_basal_LacH_promoter_activity_using_LacIQ_E._Coli_strain">Succesfully sensing, writing and reading of an arabinose signal</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/time_inference_model"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/time_inference_model">We developed the theory behind inferring the time of signal registration by way of a model</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/background_activity"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/data/background_activity">We devised grounded suggestions on how to improve the molecular design of our system using a model</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/software/logbook_designer/webtool"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/software/logbook_designer/webtool">We developed software that is able to generate a Cellular Logbook plasmid that enhances our project in such a way that it can be used on a bigger scale</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/practices/overview"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/practices/overview">We designed and outlined a new responsible development-approach for iGEM projects and applied it to our Cellular Logbook</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/practices/results#Human_Outreach"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/practices/results#Human_Outreach">As part of [https://2012.igem.org/Team:Amsterdam/practices/results#Human_Outreach Human Outreach] we collaborated with Dutch iGEM teams to organize part of the Discovery Festival in 3 major Dutch cities</a><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/practices/results#Human_Outreach"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/practices/results#Human_Outreach">We collaborated with Dutch iGEM teams to organize part of the Discovery Festival in 3 major Dutch cities.</a></td><br />
</tr><br />
</table><br />
</html><br />
</div><br />
</div><br />
<br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/achievementsTeam:Amsterdam/achievements2012-09-27T03:41:02Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<html><br />
<table><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
Demonstrated the functionality of M.ScaI introduced in E. coli<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
Succesfully sensing, writing and reading of an arabinose signal<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We developed the theory behind inferring the time of signal registration by way of a model<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We devised grounded suggestions on how to improve the molecular design of our system using a model<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We developed software that is able to generate a Cellular Logbook plasmid that enhances our project in such a way that it can be used on a bigger scale<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We designed and outlined a new responsible development-approach for iGEM projects and applied it to our Cellular Logbook<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
As part of [https://2012.igem.org/Team:Amsterdam/practices/results#Human_Outreach Human Outreach] we collaborated with Dutch iGEM teams to organize part of the Discovery Festival in 3 major Dutch cities<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/practices/results#Human_Outreach"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a href="https://2012.igem.org/Team:Amsterdam/practices/results#Human_Outreach">We collaborated with Dutch iGEM teams to organize part of the Discovery Festival in 3 major Dutch cities.</a></td><br />
</tr><br />
</table><br />
</html><br />
</div><br />
</div><br />
<br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/achievementsTeam:Amsterdam/achievements2012-09-27T03:40:36Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<html><br />
<table><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
Demonstrated the functionality of M.ScaI introduced in E. coli<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
Succesfully sensing, writing and reading of an arabinose signal<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We developed the theory behind inferring the time of signal registration by way of a model<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We devised grounded suggestions on how to improve the molecular design of our system using a model<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We developed software that is able to generate a Cellular Logbook plasmid that enhances our project in such a way that it can be used on a bigger scale<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We designed and outlined a new responsible development-approach for iGEM projects and applied it to our Cellular Logbook<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
As part of [https://2012.igem.org/Team:Amsterdam/practices/results#Human_Outreach Human Outreach] we collaborated with Dutch iGEM teams to organize part of the Discovery Festival in 3 major Dutch cities<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<a src="https://2012.igem.org/Team:Amsterdam/practices/results#Human_Outreach"><img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \></a><br />
</td><br />
<td><br />
<a src="https://2012.igem.org/Team:Amsterdam/practices/results#Human_Outreach">We collaborated with Dutch iGEM teams to organize part of the Discovery Festival in 3 major Dutch cities.</a></td><br />
</tr><br />
</table><br />
</html><br />
</div><br />
</div><br />
<br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/achievementsTeam:Amsterdam/achievements2012-09-27T03:38:40Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<html><br />
<table><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
Demonstrated the functionality of M.ScaI introduced in E. coli<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
Succesfully sensing, writing and reading of an arabinose signal<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We developed the theory behind inferring the time of signal registration by way of a model<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We devised grounded suggestions on how to improve the molecular design of our system using a model<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We developed software that is able to generate a Cellular Logbook plasmid that enhances our project in such a way that it can be used on a bigger scale<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We designed and outlined a new responsible development-approach for iGEM projects and applied it to our Cellular Logbook<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
As part of [https://2012.igem.org/Team:Amsterdam/practices/results#Human_Outreach Human Outreach] we collaborated with Dutch iGEM teams to organize part of the Discovery Festival in 3 major Dutch cities<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
Matias: We collaborated with Dutch iGEM teams to organize part of the Discovery Festival in 3 major Dutch cities.</td><br />
</tr><br />
</table><br />
</html><br />
</div><br />
</div><br />
<br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Template:Team:Amsterdam/Sidebar1Template:Team:Amsterdam/Sidebar12012-09-27T03:38:17Z<p>MaartenR: </p>
<hr />
<div><html><br />
<div id="sidebar-area"><br />
<div class="content-block"><br />
<div id="navmenu"><br />
<ul><br />
<li><a href='/Team:Amsterdam'>Home</a></li><br />
<li><a href='#'>Team</a><br />
<ul><br />
<li><a href='/Team:Amsterdam/team/members'>Members</a></li><br />
<li><a href='/Team:Amsterdam/team/advisors'>Advisors</a></li><br />
</ul><br />
</li><br />
<li><a href='/Team:Amsterdam/achievements'>Achievements</a></li><br />
<li><a href='#'>Project</a><br />
<ul><br />
<li><a href='/Team:Amsterdam/project/molecular_design'>Molecular Design</a></li><br />
<li><a href='/Team:Amsterdam/project/biobricks'>BioBricks</a></li><br />
<li><a href='/Team:Amsterdam/project/features_and_applications'>Features and Applications</a></li><br />
</ul><br />
</li><br />
<li><a href='#'>Data</a><br />
<ul><br />
<li><a href='/Team:Amsterdam/data/experimental'>Experimental Results</a></li><br />
<li><a href='/Team:Amsterdam/data/time_inference_model'>Time Inference Model</a></li><br />
<li><a href='/Team:Amsterdam/data/background_activity'>Background activity</a></li><br />
</ul><br />
</li><br />
<li><a href='/Team:Amsterdam/project/diary/'>Logbook Designer</a><br />
<ul><br />
<li><a href='/Team:Amsterdam/software/logbook_designer/webtool'>Webtool</a></li><br />
<li><a href='/Team:Amsterdam/software/logbook_designer/setup'>Setup</a></li><br />
<li><a href='/Team:Amsterdam/software/logbook_designer/manual'>Manual</a></li><br />
<li><a href='/Team:Amsterdam/software/logbook_designer/future_perspective'>Future Perspective</a></li><br />
</ul><br />
</li><br />
<li><a href='#'>Human Practices</a><br />
<ul><br />
<li><a href='/Team:Amsterdam/practices/overview'>Overview</a></li><br />
<li><a href='/Team:Amsterdam/practices/methods'>Methods</a></li><br />
<li><a href='/Team:Amsterdam/practices/results'>Results</a></li><br />
<li><a href='/Team:Amsterdam/practices/conclusion'>Conclusion</a></li><br />
</ul><br />
</li><br />
<li><a href='#'>Safety</a><br />
<ul><br />
<li><a href='/Team:Amsterdam/safety/questions'>Questions</a></li><br />
</ul><br />
</li><br />
<li><a href='#'>Extra</a><br />
<ul><br />
<li><a href='/Team:Amsterdam/extra/protocols'>Protocols</a></li><br />
<li><a href='/Team:Amsterdam/extra/diary'>Lab Diary</a></li><br />
<li><a href='/Team:Amsterdam/extra/faq'>FAQ</a></li><br />
<li><a href='/Team:Amsterdam/extra/software'>Software</a></li><br />
</ul><br />
</li><br />
</div><br />
</div><br />
<br />
<div class="content-block"><br />
<div id="social-feeds"><br />
<div id="feed-selection"><br />
<a href='https://twitter.com/igemamsterd2012'><img src='https://static.igem.org/mediawiki/2012/b/bc/Amsterdam_twitter.png' width='50px /></a><a href='http://www.facebook.com/IgemAmsterdam2012'><img src='https://static.igem.org/mediawiki/2012/8/89/Amsterdam_facebook.png' width='50px' /></a><br />
</div><br />
<div id="feed-fb"></div><br />
</div><br />
</div><br />
<br />
</div><br />
</html></div>MaartenRhttp://2012.igem.org/Team:Amsterdam/data/experimentalTeam:Amsterdam/data/experimental2012-09-27T03:37:56Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<h1>Experimental Results</h1><br />
__TOC__<br />
<div class='clear'></div><br />
<h2>Functionality of the writer-reader module under different sensor modules</h2><br />
We started measuring the first proof-of-concept of the Cellular Logbook by testing the functionality of a part of our writer-reader module in the context of various sensor modules. To this end, we tested the functionality of the synthesized Methyltransferase (MTase) (our writer) cloned under the control of the LacH promoter in the pSB1AT3 backbone and transformed in Library Efficient® DH5α™ competent cells (Invitrogen).<br />
<br />
<h4>Set up of the writer-reader module</h4><br />
[[File:Amsterdam_exp_fig_1.png|300px|right|thumb|Figure 1]]<br />
As mentioned in Molecular design, the ScaI restriction enzyme is unable to cut methylated restriction sites. Therefore, we expect different possible restriction profiles through ScaI restriction digestion since there is one ScaI site residing in the pSB1AT3 backbone and we created one ScaI site via a scar inside our writer module. We expect to find either an off or intermediate methylation state knowing that the LacH promoter driving the MTase has some basal activity.<br />
<br />
Figure 1 shows the result of a ScaI restriction digestion of the pSB1AT3/LacH/MTase construct without IPTG induction. The pattern displayed corresponds to a combination of bands indicating an intermediate state between the ‘off’ and ‘on’ situation. Interpretation of the read-out: absence of MTase expression in ''E. coli'' leads to a complete digestion of our plasmid. Two bands of 2989 bp and 1621 bp are then observed (A). Incomplete methylation of the plasmid at only one of the two ScaI sites shows a linearized plasmid band 4610 bp. Complete methylation of our writer module prevents ScaI to cut and a typical uncut plasmid profile is observed (C).<br />
<br />
<h4>Behavior of the writer-reader module under IPTG induction</h4><br />
[[File:Amsterdam_exp_fig_2.png|300px|right|thumb|Figure 2]]<br />
The next step was to see if IPTG-based induction of MTase expression would modify the methylation state of our writer-reader module thus changing the resulting restriction profile. In the presence of 1mM IPTG, the promoter should be activated providing MTase production inside the cells. Since our model for this experiment suggests that methylation occurs at a fast rate we expect that after 16h of IPTG induction, the read-out will dramatically shift to the uncut profile.<br />
Figure 2<br />
<br />
The writer-reader module after 16h IPTG induction still shows a partial methylation profile, indicating that our writer-reader module is not fully methylated, we observe a gradual shift towards the uncut plasmid form. That means that IPTG induction leads to an increase of methylation of the plasmid and that our writer-reader module is able to store and read this information: Our writer-reader module is in principle working!!<br />
<br />
<h4>Behavior of the writer-reader module in varying growth conditions</h4><br />
[[File:Amsterdam_exp_fig_4.png|300px|right|thumb|Figure 3]]<br />
We aimed to characterize the activity of our writer during bacterial growth. We performed a growth curve experiment (figure 3) and collected samples at different time points to test the occurrence of methylation over different times of growth. <br />
[[File:Amsterdam_exp_fig_5.png|300px|right|thumb|Figure 4]]<br />
<br />
Figure 4 shows ScaI digestion of plasmids extracted from samples activated or not by 10mM IPTG and collected between 0 and 12h. Both conditions reveal an intermediate restriction profile over. However, the two samples taken after 24 hours show a different result, i.e. almost only uncut plasmid is observed as a result of an significant increase of methylation of our reader. <br />
<br />
We aimed to test the functionality of our system in the stationary state. It was previously described in the literature that the Lac promoter upon IPTG induction is performing better in the stationary phase because the lac-permease, encoded by the LacY gene and responsible for the correct IPTG uptake inside the cell, can be quantitatively integrated into the membrane. <br />
<br />
The same bacterial batch was used in both experiments but for the stationary experiment first grown over a longer period of time (at least more than 24 hours), either with or without the inducer. <br />
[[File:Amsterdam_exp_fig_3.png|300px|right|thumb|Figure 5]]<br />
<br />
Figure 5 shows the restriction profile taken from three different time points: 48, 72 and 96 hours. The first three restriction profiles correspond to growth without IPTG induction and the last three with 10 mM IPTG. During the culture no additional IPTG was added since we expect that IPTG is not degraded over time. We do not observe a change in the restriction profile, meaning that the time of culture does not influence our results. <br />
<br />
<br />
In conclusion, the presence of the LacH promoter showed to provide the activity of the Mtase but did not deliver a restriction profile fitting to the ‘off’ and ‘on’ states. Our observations are in line with our predictions as described in the Molecular Design. An alternative way to lower the basal activity of LacH is to perform our experiments at high LacI levels which is suggested to decrease the basal activity of LacH. In addition, LacH in high-copy plasmids could createsmuch more non-specific activity of the LacH than in a low-copy plasmid.<br />
<br />
<br />
<h4>Behavior of the writer-reader module under reduced basal LacH promoter activity using LacIQ E. Coli strain</h4><br />
[[File:Amsterdam_exp_fig_6.png|300px|right|thumb|Figure 6]]<br />
<br />
Both modeling and experimental results show a strong basal activity of the LacH promoter leading to a substantial methylation of our memory module, even without IPTG present in the medium. We aimed to get rid of the basal activity of the LacH promoter without IPTG induction to create a better ‘off’ state and hence a better writer-reader design.<br />
<br />
Since the silencing of the LacH promoter depends on the concentration of the LacI repressor, we chose to replace our standard DH5α E. Coli strain by the LacIQ strain. Investigation done on the LacH promoter revealed that a higher expression of LacI will show a better suppression and decrease of basal activity of the LacH promoter.<br />
<br />
We transformed our pSB1AT3/LacH/Mtase vector in the LacIQ strain and performed a restriction digestion profile experiment after 24h of growth and under 10 mM IPTG induction.<br />
<br />
<br />
<br />
Figure 6 shows the pSB1AT3/LacH/Mtase cultured overnight in LacIQ E. Coli strain and digested with ScaI restriction enzyme.<br />
<br />
The results of the restriction profile for the LacIQ E. Coli strain are inconclusive (figure 6). No change in the ScaI restriction patterns are observed between plasmids isolated from both strains. Higher expression of LacI does not seem to reveal a restriction profile that confirms the ‘off’ state and it does not seem to creates a shift towards the ‘off’ state. <br />
<br />
<br />
<h4>Behavior of the writer-reader module under tight control of and arabinose-regulated promoter</h4><br />
[[File:Amsterdam_exp_fig_7.png|300px|right|thumb|Figure 7]]<br />
The Cellular Logbook was further characterized using the pBad promoter. This promoter was chosen in order to overcome the leakiness observed with the LacH promoter. Expression of any gene cloned behind the pBad promoter is controlled by the AraC repressor and is considered to be fully suppressed in the absence of arabinose. However, in the presence of arabinose the promoter will generate a gradual response leading to gene expression. <br />
[[File:Amsterdam_exp_fig_8.png|300px|right|thumb|Figure 8]]<br />
<br />
The first characterization experiment involved induction of pSB1AT3/pBad/Mtase in Library Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 1 % arabinose. The construct was digested with ScaI restriction enzyme after 24 hours incubation at 37˚C. Surprisingly, the same intermediate digestion profile as the LacH was observed. A combination of different restriction profiles can be inferred from the results (figure 7). In the absence of arabinose, M.ScaI methyltransferase appears to be expressed and a significant number of plasmids present in the culture are fully methylated, accounting for the intense uncut DNA fragment observed. A similar pattern was observed in the presence of arabinose.<br />
<br />
[[File:Amsterdam_exp_fig_9.png|300px|right|thumb|Figure 9]]<br />
<br />
The second characterisation experiment involved the induction of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 2 % arabinose and incubation at 37˚C for 48 hours. Subsequent digestion with ScaI restriction enzyme showed a shift towards the uncut restriction profile (figure 8). This result shows that under these conditions the writer-reader is optimally functional. Succes!<br />
<br />
<br />
The third characterisation experiment concerns the stimulation of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with addition of 2% arabinose daily. Samples were taken after 48, 72 and 96 hours, and digested with ScaI restriction enzyme (figure 9). These results are consistent with the previous results, showing a gradual shift towards the “on-state” with induction of the pBad promoter by addition of arabinose daily.<br />
<br />
[[File:Amsterdam_exp_fig_10.png|300px|right|thumb|Figure 10]]<br />
[[File:Amsterdam_exp_fig_14.png|450px|right|thumb|Figure 11]]<br />
The “off” state was not achieved in the absence of arabinose. All the experiments have been conducted into the high copy number plasmid pSB1AT3. It is known that leaky expression is relative to the copy number of the plasmid.1 Hence, the inability of the Cellular Logbook to show an “off” state in the absence of arabinose could be attributed to the high copy number plasmid used in these experiments as was discussed with the LacH.<br />
<br />
Similarly to the pSB1AT3/LacH/Mtase, a growth curve experiment was conducted to characterize the acquired construct of pSB1AT3/pBad/Mtase (figure 10).<br />
<br />
<br />
Over the course of time the <br\> methylation-dependent restriction profile observed in the gel showed a shift towards the ‘on’ digestion profile in the presence of arabinose (figure 11). This result shows that our Cellular Logbook is able to sense and write an arabinose signal present in the medium in a shorter period of time.<br\><br\><br\><br\><br\><br />
<br />
<h2>Towards the Cellular Logbook</h2><br />
[[File:Amsterdam_exp_fig_11.png|300px|right|thumb|Figure 12]]<br />
[[File:Amsterdam_exp_fig_12.png|300px|right|thumb|Figure 13]]<br />
After months of cloning attempts, it appears that we succeeded in obtaining the final version of our Cellular Logbook: the pSB1AT3/LacH/PZF3838/Mtase/Mem2X (BBa_K874300). Digestion of extracted plasmid DNA with the restriction enzyme BamHI, present in the pSB1AT3 backbone vector and in the reader module (BBa_K874040), ensured that both the Polydactyl Zinc Finger PZF3838 (BBa_K874001) and the reader module (BBa_K874040) were successfully cloned in as shown in Figure 12 (colony 2, 2 bands expected: 3699 and 1769 bp). Preliminary characterization of BBa_K874300 was attempted only once due to time pressure and consisted of a ScaI digestion in the absence of IPTG (figure 13). Compared to the pSB1AT3/LacH/Mtase, BBa_K874300 shows a significant switch to the non-methylated profile, illustrated by the tremendous intensity of the lower bands (cut plasmid) compared to the first one (partially cut plasmid). These results show that the presence of the PZF3838 enhances the specificity of the writer.<br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br />
<br />
<h1>Reference List</h1><br />
<br />
1. Bowers,L.M., Lapoint,K., Anthony,L., Pluciennik,A., & Filutowicz,M. Bacterial expression system with tightly regulated gene expression and plasmid copy number. Gene 340, 11-18 (2004).<br />
<br />
<br />
<br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/data/experimentalTeam:Amsterdam/data/experimental2012-09-27T03:37:26Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<h1>Experimental results</h1><br />
__TOC__<br />
<div class='clear'></div><br />
<h2>Functionality of the writer-reader module under different sensor modules</h2><br />
We started measuring the first proof-of-concept of the Cellular Logbook by testing the functionality of a part of our writer-reader module in the context of various sensor modules. To this end, we tested the functionality of the synthesized Methyltransferase (MTase) (our writer) cloned under the control of the LacH promoter in the pSB1AT3 backbone and transformed in Library Efficient® DH5α™ competent cells (Invitrogen).<br />
<br />
<h4>Set up of the writer-reader module</h4><br />
[[File:Amsterdam_exp_fig_1.png|300px|right|thumb|Figure 1]]<br />
As mentioned in Molecular design, the ScaI restriction enzyme is unable to cut methylated restriction sites. Therefore, we expect different possible restriction profiles through ScaI restriction digestion since there is one ScaI site residing in the pSB1AT3 backbone and we created one ScaI site via a scar inside our writer module. We expect to find either an off or intermediate methylation state knowing that the LacH promoter driving the MTase has some basal activity.<br />
<br />
Figure 1 shows the result of a ScaI restriction digestion of the pSB1AT3/LacH/MTase construct without IPTG induction. The pattern displayed corresponds to a combination of bands indicating an intermediate state between the ‘off’ and ‘on’ situation. Interpretation of the read-out: absence of MTase expression in ''E. coli'' leads to a complete digestion of our plasmid. Two bands of 2989 bp and 1621 bp are then observed (A). Incomplete methylation of the plasmid at only one of the two ScaI sites shows a linearized plasmid band 4610 bp. Complete methylation of our writer module prevents ScaI to cut and a typical uncut plasmid profile is observed (C).<br />
<br />
<h4>Behavior of the writer-reader module under IPTG induction</h4><br />
[[File:Amsterdam_exp_fig_2.png|300px|right|thumb|Figure 2]]<br />
The next step was to see if IPTG-based induction of MTase expression would modify the methylation state of our writer-reader module thus changing the resulting restriction profile. In the presence of 1mM IPTG, the promoter should be activated providing MTase production inside the cells. Since our model for this experiment suggests that methylation occurs at a fast rate we expect that after 16h of IPTG induction, the read-out will dramatically shift to the uncut profile.<br />
Figure 2<br />
<br />
The writer-reader module after 16h IPTG induction still shows a partial methylation profile, indicating that our writer-reader module is not fully methylated, we observe a gradual shift towards the uncut plasmid form. That means that IPTG induction leads to an increase of methylation of the plasmid and that our writer-reader module is able to store and read this information: Our writer-reader module is in principle working!!<br />
<br />
<h4>Behavior of the writer-reader module in varying growth conditions</h4><br />
[[File:Amsterdam_exp_fig_4.png|300px|right|thumb|Figure 3]]<br />
We aimed to characterize the activity of our writer during bacterial growth. We performed a growth curve experiment (figure 3) and collected samples at different time points to test the occurrence of methylation over different times of growth. <br />
[[File:Amsterdam_exp_fig_5.png|300px|right|thumb|Figure 4]]<br />
<br />
Figure 4 shows ScaI digestion of plasmids extracted from samples activated or not by 10mM IPTG and collected between 0 and 12h. Both conditions reveal an intermediate restriction profile over. However, the two samples taken after 24 hours show a different result, i.e. almost only uncut plasmid is observed as a result of an significant increase of methylation of our reader. <br />
<br />
We aimed to test the functionality of our system in the stationary state. It was previously described in the literature that the Lac promoter upon IPTG induction is performing better in the stationary phase because the lac-permease, encoded by the LacY gene and responsible for the correct IPTG uptake inside the cell, can be quantitatively integrated into the membrane. <br />
<br />
The same bacterial batch was used in both experiments but for the stationary experiment first grown over a longer period of time (at least more than 24 hours), either with or without the inducer. <br />
[[File:Amsterdam_exp_fig_3.png|300px|right|thumb|Figure 5]]<br />
<br />
Figure 5 shows the restriction profile taken from three different time points: 48, 72 and 96 hours. The first three restriction profiles correspond to growth without IPTG induction and the last three with 10 mM IPTG. During the culture no additional IPTG was added since we expect that IPTG is not degraded over time. We do not observe a change in the restriction profile, meaning that the time of culture does not influence our results. <br />
<br />
<br />
In conclusion, the presence of the LacH promoter showed to provide the activity of the Mtase but did not deliver a restriction profile fitting to the ‘off’ and ‘on’ states. Our observations are in line with our predictions as described in the Molecular Design. An alternative way to lower the basal activity of LacH is to perform our experiments at high LacI levels which is suggested to decrease the basal activity of LacH. In addition, LacH in high-copy plasmids could createsmuch more non-specific activity of the LacH than in a low-copy plasmid.<br />
<br />
<br />
<h4>Behavior of the writer-reader module under reduced basal LacH promoter activity using LacIQ E. Coli strain</h4><br />
[[File:Amsterdam_exp_fig_6.png|300px|right|thumb|Figure 6]]<br />
<br />
Both modeling and experimental results show a strong basal activity of the LacH promoter leading to a substantial methylation of our memory module, even without IPTG present in the medium. We aimed to get rid of the basal activity of the LacH promoter without IPTG induction to create a better ‘off’ state and hence a better writer-reader design.<br />
<br />
Since the silencing of the LacH promoter depends on the concentration of the LacI repressor, we chose to replace our standard DH5α E. Coli strain by the LacIQ strain. Investigation done on the LacH promoter revealed that a higher expression of LacI will show a better suppression and decrease of basal activity of the LacH promoter.<br />
<br />
We transformed our pSB1AT3/LacH/Mtase vector in the LacIQ strain and performed a restriction digestion profile experiment after 24h of growth and under 10 mM IPTG induction.<br />
<br />
<br />
<br />
Figure 6 shows the pSB1AT3/LacH/Mtase cultured overnight in LacIQ E. Coli strain and digested with ScaI restriction enzyme.<br />
<br />
The results of the restriction profile for the LacIQ E. Coli strain are inconclusive (figure 6). No change in the ScaI restriction patterns are observed between plasmids isolated from both strains. Higher expression of LacI does not seem to reveal a restriction profile that confirms the ‘off’ state and it does not seem to creates a shift towards the ‘off’ state. <br />
<br />
<br />
<h4>Behavior of the writer-reader module under tight control of and arabinose-regulated promoter</h4><br />
[[File:Amsterdam_exp_fig_7.png|300px|right|thumb|Figure 7]]<br />
The Cellular Logbook was further characterized using the pBad promoter. This promoter was chosen in order to overcome the leakiness observed with the LacH promoter. Expression of any gene cloned behind the pBad promoter is controlled by the AraC repressor and is considered to be fully suppressed in the absence of arabinose. However, in the presence of arabinose the promoter will generate a gradual response leading to gene expression. <br />
[[File:Amsterdam_exp_fig_8.png|300px|right|thumb|Figure 8]]<br />
<br />
The first characterization experiment involved induction of pSB1AT3/pBad/Mtase in Library Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 1 % arabinose. The construct was digested with ScaI restriction enzyme after 24 hours incubation at 37˚C. Surprisingly, the same intermediate digestion profile as the LacH was observed. A combination of different restriction profiles can be inferred from the results (figure 7). In the absence of arabinose, M.ScaI methyltransferase appears to be expressed and a significant number of plasmids present in the culture are fully methylated, accounting for the intense uncut DNA fragment observed. A similar pattern was observed in the presence of arabinose.<br />
<br />
[[File:Amsterdam_exp_fig_9.png|300px|right|thumb|Figure 9]]<br />
<br />
The second characterisation experiment involved the induction of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 2 % arabinose and incubation at 37˚C for 48 hours. Subsequent digestion with ScaI restriction enzyme showed a shift towards the uncut restriction profile (figure 8). This result shows that under these conditions the writer-reader is optimally functional. Succes!<br />
<br />
<br />
The third characterisation experiment concerns the stimulation of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with addition of 2% arabinose daily. Samples were taken after 48, 72 and 96 hours, and digested with ScaI restriction enzyme (figure 9). These results are consistent with the previous results, showing a gradual shift towards the “on-state” with induction of the pBad promoter by addition of arabinose daily.<br />
<br />
[[File:Amsterdam_exp_fig_10.png|300px|right|thumb|Figure 10]]<br />
[[File:Amsterdam_exp_fig_14.png|450px|right|thumb|Figure 11]]<br />
The “off” state was not achieved in the absence of arabinose. All the experiments have been conducted into the high copy number plasmid pSB1AT3. It is known that leaky expression is relative to the copy number of the plasmid.1 Hence, the inability of the Cellular Logbook to show an “off” state in the absence of arabinose could be attributed to the high copy number plasmid used in these experiments as was discussed with the LacH.<br />
<br />
Similarly to the pSB1AT3/LacH/Mtase, a growth curve experiment was conducted to characterize the acquired construct of pSB1AT3/pBad/Mtase (figure 10).<br />
<br />
<br />
Over the course of time the <br\> methylation-dependent restriction profile observed in the gel showed a shift towards the ‘on’ digestion profile in the presence of arabinose (figure 11). This result shows that our Cellular Logbook is able to sense and write an arabinose signal present in the medium in a shorter period of time.<br\><br\><br\><br\><br\><br />
<br />
<h2>Towards the Cellular Logbook</h2><br />
[[File:Amsterdam_exp_fig_11.png|300px|right|thumb|Figure 12]]<br />
[[File:Amsterdam_exp_fig_12.png|300px|right|thumb|Figure 13]]<br />
After months of cloning attempts, it appears that we succeeded in obtaining the final version of our Cellular Logbook: the pSB1AT3/LacH/PZF3838/Mtase/Mem2X (BBa_K874300). Digestion of extracted plasmid DNA with the restriction enzyme BamHI, present in the pSB1AT3 backbone vector and in the reader module (BBa_K874040), ensured that both the Polydactyl Zinc Finger PZF3838 (BBa_K874001) and the reader module (BBa_K874040) were successfully cloned in as shown in Figure 12 (colony 2, 2 bands expected: 3699 and 1769 bp). Preliminary characterization of BBa_K874300 was attempted only once due to time pressure and consisted of a ScaI digestion in the absence of IPTG (figure 13). Compared to the pSB1AT3/LacH/Mtase, BBa_K874300 shows a significant switch to the non-methylated profile, illustrated by the tremendous intensity of the lower bands (cut plasmid) compared to the first one (partially cut plasmid). These results show that the presence of the PZF3838 enhances the specificity of the writer.<br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br />
<br />
<h1>Reference List</h1><br />
<br />
1. Bowers,L.M., Lapoint,K., Anthony,L., Pluciennik,A., & Filutowicz,M. Bacterial expression system with tightly regulated gene expression and plasmid copy number. Gene 340, 11-18 (2004).<br />
<br />
<br />
<br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/data/experimentalTeam:Amsterdam/data/experimental2012-09-27T03:37:12Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<h1>Experimental results</h1><br />
__TOC__<br />
<div class='clear'></div><br />
<h2>Functionality of the writer-reader module under different sensor modules</h2><br />
We started measuring the first proof-of-concept of the Cellular Logbook by testing the functionality of a part of our writer-reader module in the context of various sensor modules. To this end, we tested the functionality of the synthesized Methyltransferase (MTase) (our writer) cloned under the control of the LacH promoter in the pSB1AT3 backbone and transformed in Library Efficient® DH5α™ competent cells (Invitrogen).<br />
<br />
<h4>Set up of the writer-reader module</h4><br />
[[File:Amsterdam_exp_fig_1.png|300px|right|thumb|Figure 1]]<br />
As mentioned in Molecular design, the ScaI restriction enzyme is unable to cut methylated restriction sites. Therefore, we expect different possible restriction profiles through ScaI restriction digestion since there is one ScaI site residing in the pSB1AT3 backbone and we created one ScaI site via a scar inside our writer module. We expect to find either an off or intermediate methylation state knowing that the LacH promoter driving the MTase has some basal activity.<br />
<br />
Figure 1 shows the result of a ScaI restriction digestion of the pSB1AT3/LacH/MTase construct without IPTG induction. The pattern displayed corresponds to a combination of bands indicating an intermediate state between the ‘off’ and ‘on’ situation. Interpretation of the read-out: absence of MTase expression in E. coli leads to a complete digestion of our plasmid. Two bands of 2989 bp and 1621 bp are then observed (A). Incomplete methylation of the plasmid at only one of the two ScaI sites shows a linearized plasmid band 4610 bp. Complete methylation of our writer module prevents ScaI to cut and a typical uncut plasmid profile is observed (C).<br />
<br />
<h4>Behavior of the writer-reader module under IPTG induction</h4><br />
[[File:Amsterdam_exp_fig_2.png|300px|right|thumb|Figure 2]]<br />
The next step was to see if IPTG-based induction of MTase expression would modify the methylation state of our writer-reader module thus changing the resulting restriction profile. In the presence of 1mM IPTG, the promoter should be activated providing MTase production inside the cells. Since our model for this experiment suggests that methylation occurs at a fast rate we expect that after 16h of IPTG induction, the read-out will dramatically shift to the uncut profile.<br />
Figure 2<br />
<br />
The writer-reader module after 16h IPTG induction still shows a partial methylation profile, indicating that our writer-reader module is not fully methylated, we observe a gradual shift towards the uncut plasmid form. That means that IPTG induction leads to an increase of methylation of the plasmid and that our writer-reader module is able to store and read this information: Our writer-reader module is in principle working!!<br />
<br />
<h4>Behavior of the writer-reader module in varying growth conditions</h4><br />
[[File:Amsterdam_exp_fig_4.png|300px|right|thumb|Figure 3]]<br />
We aimed to characterize the activity of our writer during bacterial growth. We performed a growth curve experiment (figure 3) and collected samples at different time points to test the occurrence of methylation over different times of growth. <br />
[[File:Amsterdam_exp_fig_5.png|300px|right|thumb|Figure 4]]<br />
<br />
Figure 4 shows ScaI digestion of plasmids extracted from samples activated or not by 10mM IPTG and collected between 0 and 12h. Both conditions reveal an intermediate restriction profile over. However, the two samples taken after 24 hours show a different result, i.e. almost only uncut plasmid is observed as a result of an significant increase of methylation of our reader. <br />
<br />
We aimed to test the functionality of our system in the stationary state. It was previously described in the literature that the Lac promoter upon IPTG induction is performing better in the stationary phase because the lac-permease, encoded by the LacY gene and responsible for the correct IPTG uptake inside the cell, can be quantitatively integrated into the membrane. <br />
<br />
The same bacterial batch was used in both experiments but for the stationary experiment first grown over a longer period of time (at least more than 24 hours), either with or without the inducer. <br />
[[File:Amsterdam_exp_fig_3.png|300px|right|thumb|Figure 5]]<br />
<br />
Figure 5 shows the restriction profile taken from three different time points: 48, 72 and 96 hours. The first three restriction profiles correspond to growth without IPTG induction and the last three with 10 mM IPTG. During the culture no additional IPTG was added since we expect that IPTG is not degraded over time. We do not observe a change in the restriction profile, meaning that the time of culture does not influence our results. <br />
<br />
<br />
In conclusion, the presence of the LacH promoter showed to provide the activity of the Mtase but did not deliver a restriction profile fitting to the ‘off’ and ‘on’ states. Our observations are in line with our predictions as described in the Molecular Design. An alternative way to lower the basal activity of LacH is to perform our experiments at high LacI levels which is suggested to decrease the basal activity of LacH. In addition, LacH in high-copy plasmids could createsmuch more non-specific activity of the LacH than in a low-copy plasmid.<br />
<br />
<br />
<h4>Behavior of the writer-reader module under reduced basal LacH promoter activity using LacIQ E. Coli strain</h4><br />
[[File:Amsterdam_exp_fig_6.png|300px|right|thumb|Figure 6]]<br />
<br />
Both modeling and experimental results show a strong basal activity of the LacH promoter leading to a substantial methylation of our memory module, even without IPTG present in the medium. We aimed to get rid of the basal activity of the LacH promoter without IPTG induction to create a better ‘off’ state and hence a better writer-reader design.<br />
<br />
Since the silencing of the LacH promoter depends on the concentration of the LacI repressor, we chose to replace our standard DH5α E. Coli strain by the LacIQ strain. Investigation done on the LacH promoter revealed that a higher expression of LacI will show a better suppression and decrease of basal activity of the LacH promoter.<br />
<br />
We transformed our pSB1AT3/LacH/Mtase vector in the LacIQ strain and performed a restriction digestion profile experiment after 24h of growth and under 10 mM IPTG induction.<br />
<br />
<br />
<br />
Figure 6 shows the pSB1AT3/LacH/Mtase cultured overnight in LacIQ E. Coli strain and digested with ScaI restriction enzyme.<br />
<br />
The results of the restriction profile for the LacIQ E. Coli strain are inconclusive (figure 6). No change in the ScaI restriction patterns are observed between plasmids isolated from both strains. Higher expression of LacI does not seem to reveal a restriction profile that confirms the ‘off’ state and it does not seem to creates a shift towards the ‘off’ state. <br />
<br />
<br />
<h4>Behavior of the writer-reader module under tight control of and arabinose-regulated promoter</h4><br />
[[File:Amsterdam_exp_fig_7.png|300px|right|thumb|Figure 7]]<br />
The Cellular Logbook was further characterized using the pBad promoter. This promoter was chosen in order to overcome the leakiness observed with the LacH promoter. Expression of any gene cloned behind the pBad promoter is controlled by the AraC repressor and is considered to be fully suppressed in the absence of arabinose. However, in the presence of arabinose the promoter will generate a gradual response leading to gene expression. <br />
[[File:Amsterdam_exp_fig_8.png|300px|right|thumb|Figure 8]]<br />
<br />
The first characterization experiment involved induction of pSB1AT3/pBad/Mtase in Library Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 1 % arabinose. The construct was digested with ScaI restriction enzyme after 24 hours incubation at 37˚C. Surprisingly, the same intermediate digestion profile as the LacH was observed. A combination of different restriction profiles can be inferred from the results (figure 7). In the absence of arabinose, M.ScaI methyltransferase appears to be expressed and a significant number of plasmids present in the culture are fully methylated, accounting for the intense uncut DNA fragment observed. A similar pattern was observed in the presence of arabinose.<br />
<br />
[[File:Amsterdam_exp_fig_9.png|300px|right|thumb|Figure 9]]<br />
<br />
The second characterisation experiment involved the induction of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 2 % arabinose and incubation at 37˚C for 48 hours. Subsequent digestion with ScaI restriction enzyme showed a shift towards the uncut restriction profile (figure 8). This result shows that under these conditions the writer-reader is optimally functional. Succes!<br />
<br />
<br />
The third characterisation experiment concerns the stimulation of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with addition of 2% arabinose daily. Samples were taken after 48, 72 and 96 hours, and digested with ScaI restriction enzyme (figure 9). These results are consistent with the previous results, showing a gradual shift towards the “on-state” with induction of the pBad promoter by addition of arabinose daily.<br />
<br />
[[File:Amsterdam_exp_fig_10.png|300px|right|thumb|Figure 10]]<br />
[[File:Amsterdam_exp_fig_14.png|450px|right|thumb|Figure 11]]<br />
The “off” state was not achieved in the absence of arabinose. All the experiments have been conducted into the high copy number plasmid pSB1AT3. It is known that leaky expression is relative to the copy number of the plasmid.1 Hence, the inability of the Cellular Logbook to show an “off” state in the absence of arabinose could be attributed to the high copy number plasmid used in these experiments as was discussed with the LacH.<br />
<br />
Similarly to the pSB1AT3/LacH/Mtase, a growth curve experiment was conducted to characterize the acquired construct of pSB1AT3/pBad/Mtase (figure 10).<br />
<br />
<br />
Over the course of time the <br\> methylation-dependent restriction profile observed in the gel showed a shift towards the ‘on’ digestion profile in the presence of arabinose (figure 11). This result shows that our Cellular Logbook is able to sense and write an arabinose signal present in the medium in a shorter period of time.<br\><br\><br\><br\><br\><br />
<br />
<h2>Towards the Cellular Logbook</h2><br />
[[File:Amsterdam_exp_fig_11.png|300px|right|thumb|Figure 12]]<br />
[[File:Amsterdam_exp_fig_12.png|300px|right|thumb|Figure 13]]<br />
After months of cloning attempts, it appears that we succeeded in obtaining the final version of our Cellular Logbook: the pSB1AT3/LacH/PZF3838/Mtase/Mem2X (BBa_K874300). Digestion of extracted plasmid DNA with the restriction enzyme BamHI, present in the pSB1AT3 backbone vector and in the reader module (BBa_K874040), ensured that both the Polydactyl Zinc Finger PZF3838 (BBa_K874001) and the reader module (BBa_K874040) were successfully cloned in as shown in Figure 12 (colony 2, 2 bands expected: 3699 and 1769 bp). Preliminary characterization of BBa_K874300 was attempted only once due to time pressure and consisted of a ScaI digestion in the absence of IPTG (figure 13). Compared to the pSB1AT3/LacH/Mtase, BBa_K874300 shows a significant switch to the non-methylated profile, illustrated by the tremendous intensity of the lower bands (cut plasmid) compared to the first one (partially cut plasmid). These results show that the presence of the PZF3838 enhances the specificity of the writer.<br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br />
<br />
<h1>Reference List</h1><br />
<br />
1. Bowers,L.M., Lapoint,K., Anthony,L., Pluciennik,A., & Filutowicz,M. Bacterial expression system with tightly regulated gene expression and plasmid copy number. Gene 340, 11-18 (2004).<br />
<br />
<br />
<br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/data/experimentalTeam:Amsterdam/data/experimental2012-09-27T03:36:48Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<h1>Experimental results</h1><br />
__TOC__<br />
<div class='clear'></div><br />
<h2>Functionality of the writer-reader module under different sensor modules</h2><br />
We started measuring the first proof-of-concept of the Cellular Logbook by testing the functionality of a part of our writer-reader module in the context of various sensor modules. To this end, we tested the functionality of the synthesized Methyltransferase (MTase) (our writer) cloned under the control of the LacH promoter in the pSB1AT3 backbone and transformed in Library Efficient® DH5α™ competent cells (Invitrogen).<br />
<br />
<h4>Set up of the writer-reader module</h4><br />
[[File:Amsterdam_exp_fig_1.png|300px|right|thumb|Figure 1]]<br />
As mentioned in Molecular design, the ScaI restriction enzyme is unable to cut methylated restriction sites. Therefore, we expect different possible restriction profiles through ScaI restriction digestion since there is one ScaI site residing in the pSB1AT3 backbone and we created one ScaI site via a scar inside our writer module. We expect to find either an off or intermediate methylation state knowing that the LacH promoter driving the MTase has some basal activity.<br />
<br />
Figure 1 shows the result of a ScaI restriction digestion of the pSB1AT3/LacH/MTase construct without IPTG induction. The pattern displayed corresponds to a combination of bands indicating an intermediate state between the ‘off’ and ‘on’ situation. Interpretation of the read-out: absence of MTase expression in E. Coli leads to a complete digestion of our plasmid. Two bands of 2989 bp and 1621 bp are then observed (A). Incomplete methylation of the plasmid at only one of the two ScaI sites shows a linearized plasmid band 4610 bp. Complete methylation of our writer module prevents ScaI to cut and a typical uncut plasmid profile is observed (C).<br />
<br />
<h4>Behavior of the writer-reader module under IPTG induction</h4><br />
[[File:Amsterdam_exp_fig_2.png|300px|right|thumb|Figure 2]]<br />
The next step was to see if IPTG-based induction of MTase expression would modify the methylation state of our writer-reader module thus changing the resulting restriction profile. In the presence of 1mM IPTG, the promoter should be activated providing MTase production inside the cells. Since our model for this experiment suggests that methylation occurs at a fast rate we expect that after 16h of IPTG induction, the read-out will dramatically shift to the uncut profile.<br />
Figure 2<br />
<br />
The writer-reader module after 16h IPTG induction still shows a partial methylation profile, indicating that our writer-reader module is not fully methylated, we observe a gradual shift towards the uncut plasmid form. That means that IPTG induction leads to an increase of methylation of the plasmid and that our writer-reader module is able to store and read this information: Our writer-reader module is in principle working!!<br />
<br />
<h4>Behavior of the writer-reader module in varying growth conditions</h4><br />
[[File:Amsterdam_exp_fig_4.png|300px|right|thumb|Figure 3]]<br />
We aimed to characterize the activity of our writer during bacterial growth. We performed a growth curve experiment (figure 3) and collected samples at different time points to test the occurrence of methylation over different times of growth. <br />
[[File:Amsterdam_exp_fig_5.png|300px|right|thumb|Figure 4]]<br />
<br />
Figure 4 shows ScaI digestion of plasmids extracted from samples activated or not by 10mM IPTG and collected between 0 and 12h. Both conditions reveal an intermediate restriction profile over. However, the two samples taken after 24 hours show a different result, i.e. almost only uncut plasmid is observed as a result of an significant increase of methylation of our reader. <br />
<br />
We aimed to test the functionality of our system in the stationary state. It was previously described in the literature that the Lac promoter upon IPTG induction is performing better in the stationary phase because the lac-permease, encoded by the LacY gene and responsible for the correct IPTG uptake inside the cell, can be quantitatively integrated into the membrane. <br />
<br />
The same bacterial batch was used in both experiments but for the stationary experiment first grown over a longer period of time (at least more than 24 hours), either with or without the inducer. <br />
[[File:Amsterdam_exp_fig_3.png|300px|right|thumb|Figure 5]]<br />
<br />
Figure 5 shows the restriction profile taken from three different time points: 48, 72 and 96 hours. The first three restriction profiles correspond to growth without IPTG induction and the last three with 10 mM IPTG. During the culture no additional IPTG was added since we expect that IPTG is not degraded over time. We do not observe a change in the restriction profile, meaning that the time of culture does not influence our results. <br />
<br />
<br />
In conclusion, the presence of the LacH promoter showed to provide the activity of the Mtase but did not deliver a restriction profile fitting to the ‘off’ and ‘on’ states. Our observations are in line with our predictions as described in the Molecular Design. An alternative way to lower the basal activity of LacH is to perform our experiments at high LacI levels which is suggested to decrease the basal activity of LacH. In addition, LacH in high-copy plasmids could createsmuch more non-specific activity of the LacH than in a low-copy plasmid.<br />
<br />
<br />
<h4>Behavior of the writer-reader module under reduced basal LacH promoter activity using LacIQ E. Coli strain</h4><br />
[[File:Amsterdam_exp_fig_6.png|300px|right|thumb|Figure 6]]<br />
<br />
Both modeling and experimental results show a strong basal activity of the LacH promoter leading to a substantial methylation of our memory module, even without IPTG present in the medium. We aimed to get rid of the basal activity of the LacH promoter without IPTG induction to create a better ‘off’ state and hence a better writer-reader design.<br />
<br />
Since the silencing of the LacH promoter depends on the concentration of the LacI repressor, we chose to replace our standard DH5α E. Coli strain by the LacIQ strain. Investigation done on the LacH promoter revealed that a higher expression of LacI will show a better suppression and decrease of basal activity of the LacH promoter.<br />
<br />
We transformed our pSB1AT3/LacH/Mtase vector in the LacIQ strain and performed a restriction digestion profile experiment after 24h of growth and under 10 mM IPTG induction.<br />
<br />
<br />
<br />
Figure 6 shows the pSB1AT3/LacH/Mtase cultured overnight in LacIQ E. Coli strain and digested with ScaI restriction enzyme.<br />
<br />
The results of the restriction profile for the LacIQ E. Coli strain are inconclusive (figure 6). No change in the ScaI restriction patterns are observed between plasmids isolated from both strains. Higher expression of LacI does not seem to reveal a restriction profile that confirms the ‘off’ state and it does not seem to creates a shift towards the ‘off’ state. <br />
<br />
<br />
<h4>Behavior of the writer-reader module under tight control of and arabinose-regulated promoter</h4><br />
[[File:Amsterdam_exp_fig_7.png|300px|right|thumb|Figure 7]]<br />
The Cellular Logbook was further characterized using the pBad promoter. This promoter was chosen in order to overcome the leakiness observed with the LacH promoter. Expression of any gene cloned behind the pBad promoter is controlled by the AraC repressor and is considered to be fully suppressed in the absence of arabinose. However, in the presence of arabinose the promoter will generate a gradual response leading to gene expression. <br />
[[File:Amsterdam_exp_fig_8.png|300px|right|thumb|Figure 8]]<br />
<br />
The first characterization experiment involved induction of pSB1AT3/pBad/Mtase in Library Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 1 % arabinose. The construct was digested with ScaI restriction enzyme after 24 hours incubation at 37˚C. Surprisingly, the same intermediate digestion profile as the LacH was observed. A combination of different restriction profiles can be inferred from the results (figure 7). In the absence of arabinose, M.ScaI methyltransferase appears to be expressed and a significant number of plasmids present in the culture are fully methylated, accounting for the intense uncut DNA fragment observed. A similar pattern was observed in the presence of arabinose.<br />
<br />
[[File:Amsterdam_exp_fig_9.png|300px|right|thumb|Figure 9]]<br />
<br />
The second characterisation experiment involved the induction of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 2 % arabinose and incubation at 37˚C for 48 hours. Subsequent digestion with ScaI restriction enzyme showed a shift towards the uncut restriction profile (figure 8). This result shows that under these conditions the writer-reader is optimally functional. Succes!<br />
<br />
<br />
The third characterisation experiment concerns the stimulation of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with addition of 2% arabinose daily. Samples were taken after 48, 72 and 96 hours, and digested with ScaI restriction enzyme (figure 9). These results are consistent with the previous results, showing a gradual shift towards the “on-state” with induction of the pBad promoter by addition of arabinose daily.<br />
<br />
[[File:Amsterdam_exp_fig_10.png|300px|right|thumb|Figure 10]]<br />
[[File:Amsterdam_exp_fig_14.png|450px|right|thumb|Figure 11]]<br />
The “off” state was not achieved in the absence of arabinose. All the experiments have been conducted into the high copy number plasmid pSB1AT3. It is known that leaky expression is relative to the copy number of the plasmid.1 Hence, the inability of the Cellular Logbook to show an “off” state in the absence of arabinose could be attributed to the high copy number plasmid used in these experiments as was discussed with the LacH.<br />
<br />
Similarly to the pSB1AT3/LacH/Mtase, a growth curve experiment was conducted to characterize the acquired construct of pSB1AT3/pBad/Mtase (figure 10).<br />
<br />
<br />
Over the course of time the <br\> methylation-dependent restriction profile observed in the gel showed a shift towards the ‘on’ digestion profile in the presence of arabinose (figure 11). This result shows that our Cellular Logbook is able to sense and write an arabinose signal present in the medium in a shorter period of time.<br\><br\><br\><br\><br\><br />
<br />
<h2>Towards the Cellular Logbook</h2><br />
[[File:Amsterdam_exp_fig_11.png|300px|right|thumb|Figure 12]]<br />
[[File:Amsterdam_exp_fig_12.png|300px|right|thumb|Figure 13]]<br />
After months of cloning attempts, it appears that we succeeded in obtaining the final version of our Cellular Logbook: the pSB1AT3/LacH/PZF3838/Mtase/Mem2X (BBa_K874300). Digestion of extracted plasmid DNA with the restriction enzyme BamHI, present in the pSB1AT3 backbone vector and in the reader module (BBa_K874040), ensured that both the Polydactyl Zinc Finger PZF3838 (BBa_K874001) and the reader module (BBa_K874040) were successfully cloned in as shown in Figure 12 (colony 2, 2 bands expected: 3699 and 1769 bp). Preliminary characterization of BBa_K874300 was attempted only once due to time pressure and consisted of a ScaI digestion in the absence of IPTG (figure 13). Compared to the pSB1AT3/LacH/Mtase, BBa_K874300 shows a significant switch to the non-methylated profile, illustrated by the tremendous intensity of the lower bands (cut plasmid) compared to the first one (partially cut plasmid). These results show that the presence of the PZF3838 enhances the specificity of the writer.<br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br />
<br />
<h1>Reference List</h1><br />
<br />
1. Bowers,L.M., Lapoint,K., Anthony,L., Pluciennik,A., & Filutowicz,M. Bacterial expression system with tightly regulated gene expression and plasmid copy number. Gene 340, 11-18 (2004).<br />
<br />
<br />
<br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/data/experimentalTeam:Amsterdam/data/experimental2012-09-27T03:35:19Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<h1>Experimental results</h1><br />
__TOC__<br />
<div class='clear'></div><br />
<h2>Functionality of the writer-reader module under different sensor modules</h2><br />
We started measuring the first proof-of-concept of the Cellular Logbook by testing the functionality of a part of our writer-reader module in the context of various sensor modules. To this end, we tested the functionality of the synthesized Methyltransferase (MTase) (our writer) cloned under the control of the LacH promoter in the pSB1AT3 backbone and transformed in Library Efficient® DH5α™ competent cells (Invitrogen).<br />
<br />
<h4>Set up of the writer-reader module</h4><br />
[[File:Amsterdam_exp_fig_1.png|300px|right|thumb|Figure 1]]<br />
As mentioned in Molecular design, the ScaI restriction enzyme is unable to cut methylated restriction sites. Therefore, we expect different possible restriction profiles through ScaI restriction digestion since there is one ScaI site residing in the pSB1AT3 backbone and we created one ScaI site via a scar inside our writer module. We expect to find either an off or intermediate methylation state knowing that the LacH promoter driving the MTase has some basal activity.<br />
<br />
Figure 1 shows the result of a ScaI restriction digestion of the pSB1AT3/LacH/MTase construct without IPTG induction. The pattern displayed corresponds to a combination of bands indicating an intermediate state between the ‘off’ and ‘on’ situation. Interpretation of the read-out: absence of MTase expression in E. Coli leads to a complete digestion of our plasmid. Two bands of 2989 bp and 1621 bp are then observed (A). Incomplete methylation of the plasmid at only one of the two ScaI sites shows a linearized plasmid band 4610 bp. Complete methylation of our writer module prevents ScaI to cut and a typical uncut plasmid profile is observed (C).<br />
<br />
<h4>Behavior of the writer-reader module under IPTG induction</h4><br />
[[File:Amsterdam_exp_fig_2.png|300px|right|thumb|Figure 2]]<br />
The next step was to see if IPTG-based induction of MTase expression would modify the methylation state of our writer-reader module thus changing the resulting restriction profile. In the presence of 1mM IPTG, the promoter should be activated providing MTase production inside the cells. Since our model for this experiment suggests that methylation occurs at a fast rate we expect that after 16h of IPTG induction, the read-out will dramatically shift to the uncut profile.<br />
Figure 2<br />
<br />
The writer-reader module after 16h IPTG induction still shows a partial methylation profile, indicating that our writer-reader module is not fully methylated, we observe a gradual shift towards the uncut plasmid form. That means that IPTG induction leads to an increase of methylation of the plasmid and that our writer-reader module is able to store and read this information: Our writer-reader module is in principle working!!<br />
<br />
<h4>Behavior of the writer-reader module in varying growth conditions</h4><br />
[[File:Amsterdam_exp_fig_4.png|300px|right|thumb|Figure 3]]<br />
We aimed to characterize the activity of our writer during bacterial growth. We performed a growth curve experiment (figure 3) and collected samples at different time points to test the occurrence of methylation over different times of growth. <br />
[[File:Amsterdam_exp_fig_5.png|300px|right|thumb|Figure 4]]<br />
<br />
Figure 4 shows ScaI digestion of plasmids extracted from samples activated or not by 10mM IPTG and collected between 0 and 12h. Both conditions reveal an intermediate restriction profile over. However, the two samples taken after 24 hours show a different result, i.e. almost only uncut plasmid is observed as a result of an significant increase of methylation of our reader. <br />
<br />
We aimed to test the functionality of our system in the stationary state. It was previously described in the literature that the Lac promoter upon IPTG induction is performing better in the stationary phase because the lac-permease, encoded by the LacY gene and responsible for the correct IPTG uptake inside the cell, can be quantitatively integrated into the membrane. <br />
<br />
The same bacterial batch was used in both experiments but for the stationary experiment first grown over a longer period of time (at least more than 24 hours), either with or without the inducer. <br />
[[File:Amsterdam_exp_fig_3.png|300px|right|thumb|Figure 5]]<br />
<br />
Figure 5 shows the restriction profile taken from three different time points: 48, 72 and 96 hours. The first three restriction profiles correspond to growth without IPTG induction and the last three with 10 mM IPTG. During the culture no additional IPTG was added since we expect that IPTG is not degraded over time. We do not observe a change in the restriction profile, meaning that the time of culture does not influence our results. <br />
<br />
<br />
In conclusion, the presence of the LacH promoter showed to provide the activity of the Mtase but did not deliver a restriction profile fitting to the ‘off’ and ‘on’ states. Our observations are in line with our predictions as described in the Molecular Design. An alternative way to lower the basal activity of LacH is to perform our experiments at high LacI levels which is suggested to decrease the basal activity of LacH. In addition, LacH in high-copy plasmids could createsmuch more non-specific activity of the LacH than in a low-copy plasmid.<br />
<br />
<br />
<h4>Behavior of the writer-reader module under reduced basal LacH promoter activity using LacIQ E. Coli strain</h4><br />
[[File:Amsterdam_exp_fig_6.png|300px|right|thumb|Figure 6]]<br />
<br />
Both modeling and experimental results show a strong basal activity of the LacH promoter leading to a substantial methylation of our memory module, even without IPTG present in the medium. We aimed to get rid of the basal activity of the LacH promoter without IPTG induction to create a better ‘off’ state and hence a better writer-reader design.<br />
<br />
Since the silencing of the LacH promoter depends on the concentration of the LacI repressor, we chose to replace our standard DH5α E. Coli strain by the LacIQ strain. Investigation done on the LacH promoter revealed that a higher expression of LacI will show a better suppression and decrease of basal activity of the LacH promoter.<br />
<br />
We transformed our pSB1AT3/LacH/Mtase vector in the LacIQ strain and performed a restriction digestion profile experiment after 24h of growth and under 10 mM IPTG induction.<br />
<br />
<br />
<br />
Figure 6 shows the pSB1AT3/LacH/Mtase cultured overnight in LacIQ E. Coli strain and digested with ScaI restriction enzyme.<br />
<br />
The results of the restriction profile for the LacIQ E. Coli strain are inconclusive (figure 6). No change in the ScaI restriction patterns are observed between plasmids isolated from both strains. Higher expression of LacI does not seem to reveal a restriction profile that confirms the ‘off’ state and it does not seem to creates a shift towards the ‘off’ state. <br />
<br />
<br />
<h4>Behavior of the writer-reader module under tight control of and arabinose-regulated promoter</h4><br />
[[File:Amsterdam_exp_fig_7.png|300px|right|thumb|Figure 7]]<br />
The Cellular Logbook was further characterized using the pBad promoter. This promoter was chosen in order to overcome the leakiness observed with the LacH promoter. Expression of any gene cloned behind the pBad promoter is controlled by the AraC repressor and is considered to be fully suppressed in the absence of arabinose. However, in the presence of arabinose the promoter will generate a gradual response leading to gene expression. <br />
[[File:Amsterdam_exp_fig_8.png|300px|right|thumb|Figure 8]]<br />
<br />
The first characterization experiment involved induction of pSB1AT3/pBad/Mtase in Library Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 1 % arabinose. The construct was digested with ScaI restriction enzyme after 24 hours incubation at 37˚C. Surprisingly, the same intermediate digestion profile as the LacH was observed. A combination of different restriction profiles can be inferred from the results (figure 7). In the absence of arabinose, M.ScaI methyltransferase appears to be expressed and a significant number of plasmids present in the culture are fully methylated, accounting for the intense uncut DNA fragment observed. A similar pattern was observed in the presence of arabinose.<br />
<br />
[[File:Amsterdam_exp_fig_9.png|300px|right|thumb|Figure 9]]<br />
<br />
The second characterisation experiment involved the induction of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 2 % arabinose and incubation at 37˚C for 48 hours. Subsequent digestion with ScaI restriction enzyme showed a shift towards the uncut restriction profile (figure 8). This result shows that under these conditions the writer-reader is optimally functional. Succes!<br />
<br />
<br />
The third characterisation experiment concerns the stimulation of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with addition of 2% arabinose daily. Samples were taken after 48, 72 and 96 hours, and digested with ScaI restriction enzyme (figure 9). These results are consistent with the previous results, showing a gradual shift towards the “on-state” with induction of the pBad promoter by addition of arabinose daily.<br />
<br />
[[File:Amsterdam_exp_fig_10.png|300px|right|thumb|Figure 10]]<br />
[[File:Amsterdam_exp_fig_14.png|450px|right|thumb|Figure 11]]<br />
The “off” state was not achieved in the absence of arabinose. All the experiments have been conducted into the high copy number plasmid pSB1AT3. It is known that leaky expression is relative to the copy number of the plasmid.1 Hence, the inability of the Cellular Logbook to show an “off” state in the absence of arabinose could be attributed to the high copy number plasmid used in these experiments as was discussed with the LacH.<br />
<br />
Similarly to the pSB1AT3/LacH/Mtase, a growth curve experiment was conducted to characterize the acquired construct of pSB1AT3/pBad/Mtase.<br />
<br />
<br />
Over the course of time the <br\> methylation-dependent restriction profile observed in the gel showed a shift towards the ‘on’ digestion profile in the presence of arabinose. This result shows that our Cellular Logbook is able to sense and write an arabinose signal present in the medium in a shorter period of time.<br\><br\><br\><br\><br\><br />
<br />
<h2>Towards the Cellular Logbook</h2><br />
[[File:Amsterdam_exp_fig_11.png|300px|right|thumb|Figure 12]]<br />
[[File:Amsterdam_exp_fig_12.png|300px|right|thumb|Figure 13]]<br />
After months of cloning attempts, it appears that we succeeded in obtaining the final version of our Cellular Logbook: the pSB1AT3/LacH/PZF3838/Mtase/Mem2X (BBa_K874300). Digestion of extracted plasmid DNA with the restriction enzyme BamHI, present in the pSB1AT3 backbone vector and in the reader module (BBa_K874040), ensured that both the Polydactyl Zinc Finger PZF3838 (BBa_K874001) and the reader module (BBa_K874040) were successfully cloned in as shown in Figure 12 (colony 2, 2 bands expected: 3699 and 1769 bp). Preliminary characterization of BBa_K874300 was attempted only once due to time pressure and consisted of a ScaI digestion in the absence of IPTG (figure 13). Compared to the pSB1AT3/LacH/Mtase, BBa_K874300 shows a significant switch to the non-methylated profile, illustrated by the tremendous intensity of the lower bands (cut plasmid) compared to the first one (partially cut plasmid). These results show that the presence of the PZF3838 enhances the specificity of the writer.<br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br />
<br />
<h1>Reference List</h1><br />
<br />
1. Bowers,L.M., Lapoint,K., Anthony,L., Pluciennik,A., & Filutowicz,M. Bacterial expression system with tightly regulated gene expression and plasmid copy number. Gene 340, 11-18 (2004).<br />
<br />
<br />
<br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/data/experimentalTeam:Amsterdam/data/experimental2012-09-27T03:33:40Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<h1>Experimental results</h1><br />
__TOC__<br />
<div class='clear'></div><br />
<h2>Functionality of the writer-reader module under different sensor modules</h2><br />
We started measuring the first proof-of-concept of the Cellular Logbook by testing the functionality of a part of our writer-reader module in the context of various sensor modules. To this end, we tested the functionality of the synthesized Methyltransferase (MTase) (our writer) cloned under the control of the LacH promoter in the pSB1AT3 backbone and transformed in Library Efficient® DH5α™ competent cells (Invitrogen).<br />
<br />
<h4>Set up of the writer-reader module</h4><br />
[[File:Amsterdam_exp_fig_1.png|300px|right|thumb|Figure 1]]<br />
As mentioned in Molecular design, the ScaI restriction enzyme is unable to cut methylated restriction sites. Therefore, we expect different possible restriction profiles through ScaI restriction digestion since there is one ScaI site residing in the pSB1AT3 backbone and we created one ScaI site via a scar inside our writer module. We expect to find either an off or intermediate methylation state knowing that the LacH promoter driving the MTase has some basal activity.<br />
<br />
Figure 1 shows the result of a ScaI restriction digestion of the pSB1AT3/LacH/MTase construct without IPTG induction. The pattern displayed corresponds to a combination of bands indicating an intermediate state between the ‘off’ and ‘on’ situation. Interpretation of the read-out: absence of MTase expression in E. Coli leads to a complete digestion of our plasmid. Two bands of 2989 bp and 1621 bp are then observed (A). Incomplete methylation of the plasmid at only one of the two ScaI sites shows a linearized plasmid band 4610 bp. Complete methylation of our writer module prevents ScaI to cut and a typical uncut plasmid profile is observed (C).<br />
<br />
<h4>Behavior of the writer-reader module under IPTG induction</h4><br />
[[File:Amsterdam_exp_fig_2.png|300px|right|thumb|Figure 2]]<br />
The next step was to see if IPTG-based induction of MTase expression would modify the methylation state of our writer-reader module thus changing the resulting restriction profile. In the presence of 1mM IPTG, the promoter should be activated providing MTase production inside the cells. Since our model for this experiment suggests that methylation occurs at a fast rate we expect that after 16h of IPTG induction, the read-out will dramatically shift to the uncut profile.<br />
Figure 2<br />
<br />
The writer-reader module after 16h IPTG induction still shows a partial methylation profile, indicating that our writer-reader module is not fully methylated, we observe a gradual shift towards the uncut plasmid form. That means that IPTG induction leads to an increase of methylation of the plasmid and that our writer-reader module is able to store and read this information: Our writer-reader module is in principle working!!<br />
<br />
<h4>Behavior of the writer-reader module in varying growth conditions</h4><br />
[[File:Amsterdam_exp_fig_4.png|300px|right|thumb|Figure 3]]<br />
We aimed to characterize the activity of our writer during bacterial growth. We performed a growth curve experiment (figure 3) and collected samples at different time points to test the occurrence of methylation over different times of growth. <br />
[[File:Amsterdam_exp_fig_5.png|300px|right|thumb|Figure 4]]<br />
<br />
Figure 4 shows ScaI digestion of plasmids extracted from samples activated or not by 10mM IPTG and collected between 0 and 12h. Both conditions reveal an intermediate restriction profile over. However, the two samples taken after 24 hours show a different result, i.e. almost only uncut plasmid is observed as a result of an significant increase of methylation of our reader. <br />
<br />
We aimed to test the functionality of our system in the stationary state. It was previously described in the literature that the Lac promoter upon IPTG induction is performing better in the stationary phase because the lac-permease, encoded by the LacY gene and responsible for the correct IPTG uptake inside the cell, can be quantitatively integrated into the membrane. <br />
<br />
The same bacterial batch was used in both experiments but for the stationary experiment first grown over a longer period of time (at least more than 24 hours), either with or without the inducer. <br />
[[File:Amsterdam_exp_fig_3.png|300px|right|thumb|Figure 5]]<br />
<br />
Figure 5 shows the restriction profile taken from three different time points: 48, 72 and 96 hours. The first three restriction profiles correspond to growth without IPTG induction and the last three with 10 mM IPTG. During the culture no additional IPTG was added since we expect that IPTG is not degraded over time. We do not observe a change in the restriction profile, meaning that the time of culture does not influence our results. <br />
<br />
<br />
In conclusion, the presence of the LacH promoter showed to provide the activity of the Mtase but did not deliver a restriction profile fitting to the ‘off’ and ‘on’ states. Our observations are in line with our predictions as described in the Molecular Design. An alternative way to lower the basal activity of LacH is to perform our experiments at high LacI levels which is suggested to decrease the basal activity of LacH. In addition, LacH in high-copy plasmids could createsmuch more non-specific activity of the LacH than in a low-copy plasmid.<br />
<br />
<br />
<h4>Behavior of the writer-reader module under reduced basal LacH promoter activity using LacIQ E. Coli strain</h4><br />
[[File:Amsterdam_exp_fig_6.png|300px|right|thumb|Figure 6]]<br />
<br />
Both modeling and experimental results show a strong basal activity of the LacH promoter leading to a substantial methylation of our memory module, even without IPTG present in the medium. We aimed to get rid of the basal activity of the LacH promoter without IPTG induction to create a better ‘off’ state and hence a better writer-reader design.<br />
<br />
Since the silencing of the LacH promoter depends on the concentration of the LacI repressor, we chose to replace our standard DH5α E. Coli strain by the LacIQ strain. Investigation done on the LacH promoter revealed that a higher expression of LacI will show a better suppression and decrease of basal activity of the LacH promoter.<br />
<br />
We transformed our pSB1AT3/LacH/Mtase vector in the LacIQ strain and performed a restriction digestion profile experiment after 24h of growth and under 10 mM IPTG induction.<br />
<br />
<br />
<br />
Figure 6 shows the pSB1AT3/LacH/Mtase cultured overnight in LacIQ E. Coli strain and digested with ScaI restriction enzyme.<br />
<br />
The results of the restriction profile for the LacIQ E. Coli strain are inconclusive (figure 6). No change in the ScaI restriction patterns are observed between plasmids isolated from both strains. Higher expression of LacI does not seem to reveal a restriction profile that confirms the ‘off’ state and it does not seem to creates a shift towards the ‘off’ state. <br />
<br />
<br />
<h4>Behavior of the writer-reader module under tight control of and arabinose-regulated promoter</h4><br />
[[File:Amsterdam_exp_fig_7.png|300px|right|thumb|Figure 7]]<br />
The Cellular Logbook was further characterized using the pBad promoter. This promoter was chosen in order to overcome the leakiness observed with the LacH promoter. Expression of any gene cloned behind the pBad promoter is controlled by the AraC repressor and is considered to be fully suppressed in the absence of arabinose. However, in the presence of arabinose the promoter will generate a gradual response leading to gene expression. <br />
[[File:Amsterdam_exp_fig_8.png|300px|right|thumb|Figure 8]]<br />
<br />
The first characterization experiment involved induction of pSB1AT3/pBad/Mtase in Library Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 1 % arabinose. The construct was digested with ScaI restriction enzyme after 24 hours incubation at 37˚C. Surprisingly, the same intermediate digestion profile as the LacH was observed. A combination of different restriction profiles can be inferred from the results (figure 7). In the absence of arabinose, M.ScaI methyltransferase appears to be expressed and a significant number of plasmids present in the culture are fully methylated, accounting for the intense uncut DNA fragment observed. A similar pattern was observed in the presence of arabinose.<br />
<br />
[[File:Amsterdam_exp_fig_9.png|300px|right|thumb|Figure 9]]<br />
<br />
The second characterisation experiment involved the induction of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 2 % arabinose and incubation at 37˚C for 48 hours. Subsequent digestion with ScaI restriction enzyme showed a shift towards the uncut restriction profile (figure 8). This result shows that under these conditions the writer-reader is optimally functional. Succes!<br />
<br />
<br />
The third characterisation experiment concerns the stimulation of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with addition of 2% arabinose daily. Samples were taken after 48, 72 and 96 hours, and digested with ScaI restriction enzyme (figure 9). These results are consistent with the previous results, showing a gradual shift towards the “on-state” with induction of the pBad promoter by addition of arabinose daily.<br />
<br />
[[File:Amsterdam_exp_fig_10.png|300px|right|thumb|Figure 10]]<br />
[[File:Amsterdam_exp_fig_14.png|450px|right|thumb|Figure 11]]<br />
The “off” state was not achieved in the absence of arabinose. All the experiments have been conducted into the high copy number plasmid pSB1AT3. It is known that leaky expression is relative to the copy number of the plasmid.1 Hence, the inability of the Cellular Logbook to show an “off” state in the absence of arabinose could be attributed to the high copy number plasmid used in these experiments as was discussed with the LacH.<br />
<br />
Similarly to the pSB1AT3/LacH/Mtase, a growth curve experiment was conducted to characterize the acquired construct of pSB1AT3/pBad/Mtase.<br />
<br />
<br />
Over the course of time the methylation-dependent restriction profile observed in the gel showed a shift towards the ‘on’ digestion profile in the presence of arabinose. This result shows that our Cellular Logbook is able to sense and write an arabinose signal present in the medium in a shorter period of time.<br\><br\><br\><br />
<br />
<h2>Towards the Cellular Logbook</h2><br />
[[File:Amsterdam_exp_fig_11.png|300px|right|thumb|Figure 12]]<br />
[[File:Amsterdam_exp_fig_12.png|300px|right|thumb|Figure 13]]<br />
After months of cloning attempts, it appears that we succeeded in obtaining the final version of our Cellular Logbook: the pSB1AT3/LacH/PZF3838/Mtase/Mem2X (BBa_K874300). Digestion of extracted plasmid DNA with the restriction enzyme BamHI, present in the pSB1AT3 backbone vector and in the reader module (BBa_K874040), ensured that both the Polydactyl Zinc Finger PZF3838 (BBa_K874001) and the reader module (BBa_K874040) were successfully cloned in as shown in Figure 11 (colony 2, 2 bands expected: 3699 and 1769 bp). Preliminary characterization of BBa_K874300 was attempted only once due to time pressure and consisted of a ScaI digestion in the absence of IPTG (figure 12). Compared to the pSB1AT3/LacH/Mtase, BBa_K874300 shows a significant switch to the non-methylated profile, illustrated by the tremendous intensity of the lower bands (cut plasmid) compared to the first one (partially cut plasmid). These results show that the presence of the PZF3838 enhances the specificity of the writer.<br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br />
<br />
<h1>Reference List</h1><br />
<br />
1. Bowers,L.M., Lapoint,K., Anthony,L., Pluciennik,A., & Filutowicz,M. Bacterial expression system with tightly regulated gene expression and plasmid copy number. Gene 340, 11-18 (2004).<br />
<br />
<br />
<br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/data/experimentalTeam:Amsterdam/data/experimental2012-09-27T03:32:29Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<h1>Experimental results</h1><br />
__TOC__<br />
<div class='clear'></div><br />
<h2>Functionality of the writer-reader module under different sensor modules</h2><br />
We started measuring the first proof-of-concept of the Cellular Logbook by testing the functionality of a part of our writer-reader module in the context of various sensor modules. To this end, we tested the functionality of the synthesized Methyltransferase (MTase) (our writer) cloned under the control of the LacH promoter in the pSB1AT3 backbone and transformed in in Library Efficient® DH5α™ competent cells (Invitrogen).<br />
<br />
<h4>Set up of the writer-reader module</h4><br />
[[File:Amsterdam_exp_fig_1.png|300px|right|thumb|Figure 1]]<br />
As mentioned in Molecular design, the ScaI restriction enzyme is unable to cut methylated restriction sites. Therefore, we expect different possible restriction profiles through ScaI restriction digestion since there is one ScaI site residing in the pSB1AT3 backbone and we created one ScaI site via a scar inside our writer module. We expect to find either an off or intermediate methylation state knowing that the LacH promoter driving the MTase has some basal activity.<br />
<br />
Figure 1 shows the result of a ScaI restriction digestion of the pSB1AT3/LacH/MTase construct without IPTG induction. The pattern displayed corresponds to a combination of bands indicating an intermediate state between the ‘off’ and ‘on’ situation. Interpretation of the read-out: absence of MTase expression in E. Coli leads to a complete digestion of our plasmid. Two bands of 2989 bp and 1621 bp are then observed (A). Incomplete methylation of the plasmid at only one of the two ScaI sites shows a linearized plasmid band 4610 bp. Complete methylation of our writer module prevents ScaI to cut and a typical uncut plasmid profile is observed (C).<br />
<br />
<h4>Behavior of the writer-reader module under IPTG induction</h4><br />
[[File:Amsterdam_exp_fig_2.png|300px|right|thumb|Figure 2]]<br />
The next step was to see if IPTG-based induction of MTase expression would modify the methylation state of our writer-reader module thus changing the resulting restriction profile. In the presence of 1mM IPTG, the promoter should be activated providing MTase production inside the cells. Since our model for this experiment suggests that methylation occurs at a fast rate we expect that after 16h of IPTG induction, the read-out will dramatically shift to the uncut profile.<br />
Figure 2<br />
<br />
The writer-reader module after 16h IPTG induction still shows a partial methylation profile, indicating that our writer-reader module is not fully methylated, we observe a gradual shift towards the uncut plasmid form. That means that IPTG induction leads to an increase of methylation of the plasmid and that our writer-reader module is able to store and read this information: Our writer-reader module is in principle working!!<br />
<br />
<h4>Behavior of the writer-reader module in varying growth conditions</h4><br />
[[File:Amsterdam_exp_fig_4.png|300px|right|thumb|Figure 3]]<br />
We aimed to characterize the activity of our writer during bacterial growth. We performed a growth curve experiment (figure 3) and collected samples at different time points to test the occurrence of methylation over different times of growth. <br />
[[File:Amsterdam_exp_fig_5.png|300px|right|thumb|Figure 4]]<br />
<br />
Figure 4 shows ScaI digestion of plasmids extracted from samples activated or not by 10mM IPTG and collected between 0 and 12h. Both conditions reveal an intermediate restriction profile over. However, the two samples taken after 24 hours show a different result, i.e. almost only uncut plasmid is observed as a result of an significant increase of methylation of our reader. <br />
<br />
We aimed to test the functionality of our system in the stationary state. It was previously described in the literature that the Lac promoter upon IPTG induction is performing better in the stationary phase because the lac-permease, encoded by the LacY gene and responsible for the correct IPTG uptake inside the cell, can be quantitatively integrated into the membrane. <br />
<br />
The same bacterial batch was used in both experiments but for the stationary experiment first grown over a longer period of time (at least more than 24 hours), either with or without the inducer. <br />
[[File:Amsterdam_exp_fig_3.png|300px|right|thumb|Figure 5]]<br />
<br />
Figure 5 shows the restriction profile taken from three different time points: 48, 72 and 96 hours. The first three restriction profiles correspond to growth without IPTG induction and the last three with 10 mM IPTG. During the culture no additional IPTG was added since we expect that IPTG is not degraded over time. We do not observe a change in the restriction profile, meaning that the time of culture does not influence our results. <br />
<br />
<br />
In conclusion, the presence of the LacH promoter showed to provide the activity of the Mtase but did not deliver a restriction profile fitting to the ‘off’ and ‘on’ states. Our observations are in line with our predictions as described in the Molecular Design. An alternative way to lower the basal activity of LacH is to perform our experiments at high LacI levels which is suggested to decrease the basal activity of LacH. In addition, LacH in high-copy plasmids could createsmuch more non-specific activity of the LacH than in a low-copy plasmid.<br />
<br />
<br />
<h4>Behavior of the writer-reader module under reduced basal LacH promoter activity using LacIQ E. Coli strain</h4><br />
[[File:Amsterdam_exp_fig_6.png|300px|right|thumb|Figure 6]]<br />
<br />
Both modeling and experimental results show a strong basal activity of the LacH promoter leading to a substantial methylation of our memory module, even without IPTG present in the medium. We aimed to get rid of the basal activity of the LacH promoter without IPTG induction to create a better ‘off’ state and hence a better writer-reader design.<br />
<br />
Since the silencing of the LacH promoter depends on the concentration of the LacI repressor, we chose to replace our standard DH5α E. Coli strain by the LacIQ strain. Investigation done on the LacH promoter revealed that a higher expression of LacI will show a better suppression and decrease of basal activity of the LacH promoter.<br />
<br />
We transformed our pSB1AT3/LacH/Mtase vector in the LacIQ strain and performed a restriction digestion profile experiment after 24h of growth and under 10 mM IPTG induction.<br />
<br />
<br />
<br />
Figure 6 shows the pSB1AT3/LacH/Mtase cultured overnight in LacIQ E. Coli strain and digested with ScaI restriction enzyme.<br />
<br />
The results of the restriction profile for the LacIQ E. Coli strain are inconclusive (figure 6). No change in the ScaI restriction patterns are observed between plasmids isolated from both strains. Higher expression of LacI does not seem to reveal a restriction profile that confirms the ‘off’ state and it does not seem to creates a shift towards the ‘off’ state. <br />
<br />
<br />
<h4>Behavior of the writer-reader module under tight control of and arabinose-regulated promoter</h4><br />
[[File:Amsterdam_exp_fig_7.png|300px|right|thumb|Figure 7]]<br />
The Cellular Logbook was further characterized using the pBad promoter. This promoter was chosen in order to overcome the leakiness observed with the LacH promoter. Expression of any gene cloned behind the pBad promoter is controlled by the AraC repressor and is considered to be fully suppressed in the absence of arabinose. However, in the presence of arabinose the promoter will generate a gradual response leading to gene expression. <br />
[[File:Amsterdam_exp_fig_8.png|300px|right|thumb|Figure 8]]<br />
<br />
The first characterization experiment involved induction of pSB1AT3/pBad/Mtase in Library Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 1 % arabinose. The construct was digested with ScaI restriction enzyme after 24 hours incubation at 37˚C. Surprisingly, the same intermediate digestion profile as the LacH was observed. A combination of different restriction profiles can be inferred from the results (figure 7). In the absence of arabinose, M.ScaI methyltransferase appears to be expressed and a significant number of plasmids present in the culture are fully methylated, accounting for the intense uncut DNA fragment observed. A similar pattern was observed in the presence of arabinose.<br />
<br />
[[File:Amsterdam_exp_fig_9.png|300px|right|thumb|Figure 9]]<br />
<br />
The second characterisation experiment involved the induction of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 2 % arabinose and incubation at 37˚C for 48 hours. Subsequent digestion with ScaI restriction enzyme showed a shift towards the uncut restriction profile (figure 8). This result shows that under these conditions the writer-reader is optimally functional. Succes!<br />
<br />
<br />
The third characterisation experiment concerns the stimulation of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with addition of 2% arabinose daily. Samples were taken after 48, 72 and 96 hours, and digested with ScaI restriction enzyme (figure 9). These results are consistent with the previous results, showing a gradual shift towards the “on-state” with induction of the pBad promoter by addition of arabinose daily.<br />
<br />
[[File:Amsterdam_exp_fig_10.png|300px|right|thumb|Figure 10]]<br />
[[File:Amsterdam_exp_fig_14.png|300px|right|thumb|Figure 11]]<br />
The “off” state was not achieved in the absence of arabinose. All the experiments have been conducted into the high copy number plasmid pSB1AT3. It is known that leaky expression is relative to the copy number of the plasmid.1 Hence, the inability of the Cellular Logbook to show an “off” state in the absence of arabinose could be attributed to the high copy number plasmid used in these experiments as was discussed with the LacH.<br />
<br />
Similarly to the pSB1AT3/LacH/Mtase, a growth curve experiment was conducted to characterize the acquired construct of pSB1AT3/pBad/Mtase.<br />
<br />
<br />
Over the course of time the methylation-dependent restriction profile observed in the gel showed a shift towards the ‘on’ digestion profile in the presence of arabinose. This result shows that our Cellular Logbook is able to sense and write an arabinose signal present in the medium in a shorter period of time.<br\><br\><br\><br />
<br />
<h2>Towards the Cellular Logbook</h2><br />
[[File:Amsterdam_exp_fig_11.png|300px|right|thumb|Figure 12]]<br />
[[File:Amsterdam_exp_fig_12.png|300px|right|thumb|Figure 13]]<br />
After months of cloning attempts, it appears that we succeeded in obtaining the final version of our Cellular Logbook: the pSB1AT3/LacH/PZF3838/Mtase/Mem2X (BBa_K874300). Digestion of extracted plasmid DNA with the restriction enzyme BamHI, present in the pSB1AT3 backbone vector and in the reader module (BBa_K874040), ensured that both the Polydactyl Zinc Finger PZF3838 (BBa_K874001) and the reader module (BBa_K874040) were successfully cloned in as shown in Figure 11 (colony 2, 2 bands expected: 3699 and 1769 bp). Preliminary characterization of BBa_K874300 was attempted only once due to time pressure and consisted of a ScaI digestion in the absence of IPTG (figure 12). Compared to the pSB1AT3/LacH/Mtase, BBa_K874300 shows a significant switch to the non-methylated profile, illustrated by the tremendous intensity of the lower bands (cut plasmid) compared to the first one (partially cut plasmid). These results show that the presence of the PZF3838 enhances the specificity of the writer.<br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br />
<br />
<h1>Reference List</h1><br />
<br />
1. Bowers,L.M., Lapoint,K., Anthony,L., Pluciennik,A., & Filutowicz,M. Bacterial expression system with tightly regulated gene expression and plasmid copy number. Gene 340, 11-18 (2004).<br />
<br />
<br />
<br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/File:Amsterdam_exp_fig_14.pngFile:Amsterdam exp fig 14.png2012-09-27T03:31:27Z<p>MaartenR: </p>
<hr />
<div></div>MaartenRhttp://2012.igem.org/Team:Amsterdam/data/experimentalTeam:Amsterdam/data/experimental2012-09-27T03:29:40Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<h1>Experimental results</h1><br />
__TOC__<br />
<div class='clear'></div><br />
<h2>Functionality of the writer-reader module under different sensor modules</h2><br />
We started measuring the first proof-of-concept of the Cellular Logbook by testing the functionality of a part of our writer-reader module in the context of various sensor modules. To this end, we tested the functionality of the synthesized Methyltransferase (MTase) (our writer) cloned under the control of the LacH promoter in the pSB1AT3 backbone and transformed in in Library Efficient® DH5α™ competent cells (Invitrogen).<br />
<br />
<h4>Set up of the writer-reader module</h4><br />
[[File:Amsterdam_exp_fig_1.png|300px|right|thumb|Figure 1]]<br />
As mentioned in Molecular design, the ScaI restriction enzyme is unable to cut methylated restriction sites. Therefore, we expect different possible restriction profiles through ScaI restriction digestion since there is one ScaI site residing in the pSB1AT3 backbone and we created one ScaI site via a scar inside our writer module. We expect to find either an off or intermediate methylation state knowing that the LacH promoter driving the MTase has some basal activity.<br />
<br />
Figure 1 shows the result of a ScaI restriction digestion of the pSB1AT3/LacH/MTase construct without IPTG induction. The pattern displayed corresponds to a combination of bands indicating an intermediate state between the ‘off’ and ‘on’ situation. Interpretation of the read-out: absence of MTase expression in E. Coli leads to a complete digestion of our plasmid. Two bands of 2989 bp and 1621 bp are then observed (A). Incomplete methylation of the plasmid at only one of the two ScaI sites shows a linearized plasmid band 4610 bp. Complete methylation of our writer module prevents ScaI to cut and a typical uncut plasmid profile is observed (C).<br />
<br />
<h4>Behavior of the writer-reader module under IPTG induction</h4><br />
[[File:Amsterdam_exp_fig_2.png|300px|right|thumb|Figure 2]]<br />
The next step was to see if IPTG-based induction of MTase expression would modify the methylation state of our writer-reader module thus changing the resulting restriction profile. In the presence of 1mM IPTG, the promoter should be activated providing MTase production inside the cells. Since our model for this experiment suggests that methylation occurs at a fast rate we expect that after 16h of IPTG induction, the read-out will dramatically shift to the uncut profile.<br />
Figure 2<br />
<br />
The writer-reader module after 16h IPTG induction still shows a partial methylation profile, indicating that our writer-reader module is not fully methylated, we observe a gradual shift towards the uncut plasmid form. That means that IPTG induction leads to an increase of methylation of the plasmid and that our writer-reader module is able to store and read this information: Our writer-reader module is in principle working!!<br />
<br />
<h4>Behavior of the writer-reader module in varying growth conditions</h4><br />
[[File:Amsterdam_exp_fig_4.png|300px|right|thumb|Figure 3]]<br />
We aimed to characterize the activity of our writer during bacterial growth. We performed a growth curve experiment (figure 3) and collected samples at different time points to test the occurrence of methylation over different times of growth. <br />
[[File:Amsterdam_exp_fig_5.png|300px|right|thumb|Figure 4]]<br />
<br />
Figure 4 shows ScaI digestion of plasmids extracted from samples activated or not by 10mM IPTG and collected between 0 and 12h. Both conditions reveal an intermediate restriction profile over. However, the two samples taken after 24 hours show a different result, i.e. almost only uncut plasmid is observed as a result of an significant increase of methylation of our reader. <br />
<br />
We aimed to test the functionality of our system in the stationary state. It was previously described in the literature that the Lac promoter upon IPTG induction is performing better in the stationary phase because the lac-permease, encoded by the LacY gene and responsible for the correct IPTG uptake inside the cell, can be quantitatively integrated into the membrane. <br />
<br />
The same bacterial batch was used in both experiments but for the stationary experiment first grown over a longer period of time (at least more than 24 hours), either with or without the inducer. <br />
[[File:Amsterdam_exp_fig_3.png|300px|right|thumb|Figure 5]]<br />
<br />
Figure 5 shows the restriction profile taken from three different time points: 48, 72 and 96 hours. The first three restriction profiles correspond to growth without IPTG induction and the last three with 10 mM IPTG. During the culture no additional IPTG was added since we expect that IPTG is not degraded over time. We do not observe a change in the restriction profile, meaning that the time of culture does not influence our results. <br />
<br />
<br />
In conclusion, the presence of the LacH promoter showed to provide the activity of the Mtase but did not deliver a restriction profile fitting to the ‘off’ and ‘on’ states. Our observations are in line with our predictions as described in the Molecular Design. An alternative way to lower the basal activity of LacH is to perform our experiments at high LacI levels which is suggested to decrease the basal activity of LacH. In addition, LacH in high-copy plasmids could createsmuch more non-specific activity of the LacH than in a low-copy plasmid.<br />
<br />
<br />
<h4>Behavior of the writer-reader module under reduced basal LacH promoter activity using LacIQ E. Coli strain</h4><br />
[[File:Amsterdam_exp_fig_6.png|300px|right|thumb|Figure 6]]<br />
<br />
Both modeling and experimental results show a strong basal activity of the LacH promoter leading to a substantial methylation of our memory module, even without IPTG present in the medium. We aimed to get rid of the basal activity of the LacH promoter without IPTG induction to create a better ‘off’ state and hence a better writer-reader design.<br />
<br />
Since the silencing of the LacH promoter depends on the concentration of the LacI repressor, we chose to replace our standard DH5α E. Coli strain by the LacIQ strain. Investigation done on the LacH promoter revealed that a higher expression of LacI will show a better suppression and decrease of basal activity of the LacH promoter.<br />
<br />
We transformed our pSB1AT3/LacH/Mtase vector in the LacIQ strain and performed a restriction digestion profile experiment after 24h of growth and under 10 mM IPTG induction.<br />
<br />
<br />
<br />
Figure 6 shows the pSB1AT3/LacH/Mtase cultured overnight in LacIQ E. Coli strain and digested with ScaI restriction enzyme.<br />
<br />
The results of the restriction profile for the LacIQ E. Coli strain are inconclusive (figure 6). No change in the ScaI restriction patterns are observed between plasmids isolated from both strains. Higher expression of LacI does not seem to reveal a restriction profile that confirms the ‘off’ state and it does not seem to creates a shift towards the ‘off’ state. <br />
<br />
<br />
<h4>Behavior of the writer-reader module under tight control of and arabinose-regulated promoter</h4><br />
[[File:Amsterdam_exp_fig_7.png|300px|right|thumb|Figure 7]]<br />
The Cellular Logbook was further characterized using the pBad promoter. This promoter was chosen in order to overcome the leakiness observed with the LacH promoter. Expression of any gene cloned behind the pBad promoter is controlled by the AraC repressor and is considered to be fully suppressed in the absence of arabinose. However, in the presence of arabinose the promoter will generate a gradual response leading to gene expression. <br />
[[File:Amsterdam_exp_fig_8.png|300px|right|thumb|Figure 8]]<br />
<br />
The first characterization experiment involved induction of pSB1AT3/pBad/Mtase in Library Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 1 % arabinose. The construct was digested with ScaI restriction enzyme after 24 hours incubation at 37˚C. Surprisingly, the same intermediate digestion profile as the LacH was observed. A combination of different restriction profiles can be inferred from the results (figure 7). In the absence of arabinose, M.ScaI methyltransferase appears to be expressed and a significant number of plasmids present in the culture are fully methylated, accounting for the intense uncut DNA fragment observed. A similar pattern was observed in the presence of arabinose.<br />
<br />
[[File:Amsterdam_exp_fig_9.png|300px|right|thumb|Figure 9]]<br />
<br />
The second characterisation experiment involved the induction of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 2 % arabinose and incubation at 37˚C for 48 hours. Subsequent digestion with ScaI restriction enzyme showed a shift towards the uncut restriction profile (figure 8). This result shows that under these conditions the writer-reader is optimally functional. Succes!<br />
<br />
<br />
The third characterisation experiment concerns the stimulation of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with addition of 2% arabinose daily. Samples were taken after 48, 72 and 96 hours, and digested with ScaI restriction enzyme (figure 9). These results are consistent with the previous results, showing a gradual shift towards the “on-state” with induction of the pBad promoter by addition of arabinose daily.<br />
<br />
[[File:Amsterdam_exp_fig_10.png|300px|right|thumb|Figure 10]]<br />
<br />
The “off” state was not achieved in the absence of arabinose. All the experiments have been conducted into the high copy number plasmid pSB1AT3. It is known that leaky expression is relative to the copy number of the plasmid.1 Hence, the inability of the Cellular Logbook to show an “off” state in the absence of arabinose could be attributed to the high copy number plasmid used in these experiments as was discussed with the LacH.<br />
<br />
Similarly to the pSB1AT3/LacH/Mtase, a growth curve experiment was conducted to characterize the acquired construct of pSB1AT3/pBad/Mtase.<br />
<br />
<br />
Over the course of time the methylation-dependent restriction profile observed in the gel showed a shift towards the ‘on’ digestion profile in the presence of arabinose. This result shows that our Cellular Logbook is able to sense and write an arabinose signal present in the medium in a shorter period of time.<br\><br\><br\><br />
<br />
<h2>Towards the Cellular Logbook</h2><br />
[[File:Amsterdam_exp_fig_11.png|300px|right|thumb|Figure 11]]<br />
[[File:Amsterdam_exp_fig_12.png|300px|right|thumb|Figure 12]]<br />
After months of cloning attempts, it appears that we succeeded in obtaining the final version of our Cellular Logbook: the pSB1AT3/LacH/PZF3838/Mtase/Mem2X (BBa_K874300). Digestion of extracted plasmid DNA with the restriction enzyme BamHI, present in the pSB1AT3 backbone vector and in the reader module (BBa_K874040), ensured that both the Polydactyl Zinc Finger PZF3838 (BBa_K874001) and the reader module (BBa_K874040) were successfully cloned in as shown in Figure 11 (colony 2, 2 bands expected: 3699 and 1769 bp). Preliminary characterization of BBa_K874300 was attempted only once due to time pressure and consisted of a ScaI digestion in the absence of IPTG (figure 12). Compared to the pSB1AT3/LacH/Mtase, BBa_K874300 shows a significant switch to the non-methylated profile, illustrated by the tremendous intensity of the lower bands (cut plasmid) compared to the first one (partially cut plasmid). These results show that the presence of the PZF3838 enhances the specificity of the writer.<br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br />
<br />
<h1>Reference List</h1><br />
<br />
1. Bowers,L.M., Lapoint,K., Anthony,L., Pluciennik,A., & Filutowicz,M. Bacterial expression system with tightly regulated gene expression and plasmid copy number. Gene 340, 11-18 (2004).<br />
<br />
<br />
<br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/File:Amsterdam_exp_fig_10.pngFile:Amsterdam exp fig 10.png2012-09-27T03:29:02Z<p>MaartenR: uploaded a new version of &quot;File:Amsterdam exp fig 10.png&quot;</p>
<hr />
<div></div>MaartenRhttp://2012.igem.org/Team:Amsterdam/data/experimentalTeam:Amsterdam/data/experimental2012-09-27T03:25:33Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<h1>Experimental results</h1><br />
__TOC__<br />
<div class='clear'></div><br />
<h2>Functionality of the writer-reader module under different sensor modules</h2><br />
We started measuring the first proof-of-concept of the Cellular Logbook by testing the functionality of a part of our writer-reader module in the context of various sensor modules. To this end, we tested the functionality of the synthesized Methyltransferase (MTase) (our writer) cloned under the control of the LacH promoter in the pSB1AT3 backbone and transformed in in Library Efficient® DH5α™ competent cells (Invitrogen).<br />
<br />
<h4>Set up of the writer-reader module</h4><br />
[[File:Amsterdam_exp_fig_1.png|300px|right|thumb|Figure 1]]<br />
As mentioned in Molecular design, the ScaI restriction enzyme is unable to cut methylated restriction sites. Therefore, we expect different possible restriction profiles through ScaI restriction digestion since there is one ScaI site residing in the pSB1AT3 backbone and we created one ScaI site via a scar inside our writer module. We expect to find either an off or intermediate methylation state knowing that the LacH promoter driving the MTase has some basal activity.<br />
<br />
Figure 1 shows the result of a ScaI restriction digestion of the pSB1AT3/LacH/MTase construct without IPTG induction. The pattern displayed corresponds to a combination of bands indicating an intermediate state between the ‘off’ and ‘on’ situation. Interpretation of the read-out: absence of MTase expression in E. Coli leads to a complete digestion of our plasmid. Two bands of 2989 bp and 1621 bp are then observed (A). Incomplete methylation of the plasmid at only one of the two ScaI sites shows a linearized plasmid band 4610 bp. Complete methylation of our writer module prevents ScaI to cut and a typical uncut plasmid profile is observed (C).<br />
<br />
<h4>Behavior of the writer-reader module under IPTG induction</h4><br />
[[File:Amsterdam_exp_fig_2.png|300px|right|thumb|Figure 2]]<br />
The next step was to see if IPTG-based induction of MTase expression would modify the methylation state of our writer-reader module thus changing the resulting restriction profile. In the presence of 1mM IPTG, the promoter should be activated providing MTase production inside the cells. Since our model for this experiment suggests that methylation occurs at a fast rate we expect that after 16h of IPTG induction, the read-out will dramatically shift to the uncut profile.<br />
Figure 2<br />
<br />
The writer-reader module after 16h IPTG induction still shows a partial methylation profile, indicating that our writer-reader module is not fully methylated, we observe a gradual shift towards the uncut plasmid form. That means that IPTG induction leads to an increase of methylation of the plasmid and that our writer-reader module is able to store and read this information: Our writer-reader module is in principle working!!<br />
<br />
<h4>Behavior of the writer-reader module in varying growth conditions</h4><br />
[[File:Amsterdam_exp_fig_4.png|300px|right|thumb|Figure 3]]<br />
We aimed to characterize the activity of our writer during bacterial growth. We performed a growth curve experiment (figure 3) and collected samples at different time points to test the occurrence of methylation over different times of growth. <br />
[[File:Amsterdam_exp_fig_5.png|300px|right|thumb|Figure 4]]<br />
<br />
Figure 4 shows ScaI digestion of plasmids extracted from samples activated or not by 10mM IPTG and collected between 0 and 12h. Both conditions reveal an intermediate restriction profile over. However, the two samples taken after 24 hours show a different result, i.e. almost only uncut plasmid is observed as a result of an significant increase of methylation of our reader. <br />
<br />
We aimed to test the functionality of our system in the stationary state. It was previously described in the literature that the Lac promoter upon IPTG induction is performing better in the stationary phase because the lac-permease, encoded by the LacY gene and responsible for the correct IPTG uptake inside the cell, can be quantitatively integrated into the membrane. <br />
<br />
The same bacterial batch was used in both experiments but for the stationary experiment first grown over a longer period of time (at least more than 24 hours), either with or without the inducer. <br />
[[File:Amsterdam_exp_fig_3.png|300px|right|thumb|Figure 5]]<br />
<br />
Figure 5 shows the restriction profile taken from three different time points: 48, 72 and 96 hours. The first three restriction profiles correspond to growth without IPTG induction and the last three with 10 mM IPTG. During the culture no additional IPTG was added since we expect that IPTG is not degraded over time. We do not observe a change in the restriction profile, meaning that the time of culture does not influence our results. <br />
<br />
<br />
In conclusion, the presence of the LacH promoter showed to provide the activity of the Mtase but did not deliver a restriction profile fitting to the ‘off’ and ‘on’ states. Our observations are in line with our predictions as described in the Molecular Design. An alternative way to lower the basal activity of LacH is to perform our experiments at high LacI levels which is suggested to decrease the basal activity of LacH. In addition, LacH in high-copy plasmids could createsmuch more non-specific activity of the LacH than in a low-copy plasmid.<br />
<br />
<br />
<h4>Behavior of the writer-reader module under reduced basal LacH promoter activity using LacIQ E. Coli strain</h4><br />
[[File:Amsterdam_exp_fig_6.png|300px|right|thumb|Figure 6]]<br />
<br />
Both modeling and experimental results show a strong basal activity of the LacH promoter leading to a substantial methylation of our memory module, even without IPTG present in the medium. We aimed to get rid of the basal activity of the LacH promoter without IPTG induction to create a better ‘off’ state and hence a better writer-reader design.<br />
<br />
Since the silencing of the LacH promoter depends on the concentration of the LacI repressor, we chose to replace our standard DH5α E. Coli strain by the LacIQ strain. Investigation done on the LacH promoter revealed that a higher expression of LacI will show a better suppression and decrease of basal activity of the LacH promoter.<br />
<br />
We transformed our pSB1AT3/LacH/Mtase vector in the LacIQ strain and performed a restriction digestion profile experiment after 24h of growth and under 10 mM IPTG induction.<br />
<br />
<br />
<br />
Figure 6 shows the pSB1AT3/LacH/Mtase cultured overnight in LacIQ E. Coli strain and digested with ScaI restriction enzyme.<br />
<br />
The results of the restriction profile for the LacIQ E. Coli strain are inconclusive (figure 6). No change in the ScaI restriction patterns are observed between plasmids isolated from both strains. Higher expression of LacI does not seem to reveal a restriction profile that confirms the ‘off’ state and it does not seem to creates a shift towards the ‘off’ state. <br />
<br />
<br />
<h4>Behavior of the writer-reader module under tight control of and arabinose-regulated promoter</h4><br />
[[File:Amsterdam_exp_fig_7.png|300px|right|thumb|Figure 7]]<br />
The Cellular Logbook was further characterized using the pBad promoter. This promoter was chosen in order to overcome the leakiness observed with the LacH promoter. Expression of any gene cloned behind the pBad promoter is controlled by the AraC repressor and is considered to be fully suppressed in the absence of arabinose. However, in the presence of arabinose the promoter will generate a gradual response leading to gene expression. <br />
[[File:Amsterdam_exp_fig_8.png|300px|right|thumb|Figure 8]]<br />
<br />
The first characterization experiment involved induction of pSB1AT3/pBad/Mtase in Library Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 1 % arabinose. The construct was digested with ScaI restriction enzyme after 24 hours incubation at 37˚C. Surprisingly, the same intermediate digestion profile as the LacH was observed. A combination of different restriction profiles can be inferred from the results (figure 7). In the absence of arabinose, M.ScaI methyltransferase appears to be expressed and a significant number of plasmids present in the culture are fully methylated, accounting for the intense uncut DNA fragment observed. A similar pattern was observed in the presence of arabinose.<br />
<br />
[[File:Amsterdam_exp_fig_9.png|300px|right|thumb|Figure 9]]<br />
<br />
The second characterisation experiment involved the induction of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 2 % arabinose and incubation at 37˚C for 48 hours. Subsequent digestion with ScaI restriction enzyme showed a shift towards the uncut restriction profile (figure 8). This result shows that under these conditions the writer-reader is optimally functional. Succes!<br />
<br />
<br />
The third characterisation experiment concerns the stimulation of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with addition of 2% arabinose daily. Samples were taken after 48, 72 and 96 hours, and digested with ScaI restriction enzyme (figure 9). These results are consistent with the previous results, showing a gradual shift towards the “on-state” with induction of the pBad promoter by addition of arabinose daily.<br />
<br />
[[File:Amsterdam_exp_fig_7.png|300px|right|thumb|Figure 10]]<br />
<br />
The “off” state was not achieved in the absence of arabinose. All the experiments have been conducted into the high copy number plasmid pSB1AT3. It is known that leaky expression is relative to the copy number of the plasmid.1 Hence, the inability of the Cellular Logbook to show an “off” state in the absence of arabinose could be attributed to the high copy number plasmid used in these experiments as was discussed with the LacH.<br />
<br />
Similarly to the pSB1AT3/LacH/Mtase, a growth curve experiment was conducted to characterize the acquired construct of pSB1AT3/pBad/Mtase.<br />
<br />
<br />
Over the course of time the methylation-dependent restriction profile observed in the gel showed a shift towards the ‘on’ digestion profile in the presence of arabinose. This result shows that our Cellular Logbook is able to sense and write an arabinose signal present in the medium in a shorter period of time.<br\><br\><br\><br />
<br />
<h2>Towards the Cellular Logbook</h2><br />
[[File:Amsterdam_exp_fig_11.png|300px|right|thumb|Figure 11]]<br />
[[File:Amsterdam_exp_fig_12.png|300px|right|thumb|Figure 12]]<br />
After months of cloning attempts, it appears that we succeeded in obtaining the final version of our Cellular Logbook: the pSB1AT3/LacH/PZF3838/Mtase/Mem2X (BBa_K874300). Digestion of extracted plasmid DNA with the restriction enzyme BamHI, present in the pSB1AT3 backbone vector and in the reader module (BBa_K874040), ensured that both the Polydactyl Zinc Finger PZF3838 (BBa_K874001) and the reader module (BBa_K874040) were successfully cloned in as shown in Figure 11 (colony 2, 2 bands expected: 3699 and 1769 bp). Preliminary characterization of BBa_K874300 was attempted only once due to time pressure and consisted of a ScaI digestion in the absence of IPTG (figure 12). Compared to the pSB1AT3/LacH/Mtase, BBa_K874300 shows a significant switch to the non-methylated profile, illustrated by the tremendous intensity of the lower bands (cut plasmid) compared to the first one (partially cut plasmid). These results show that the presence of the PZF3838 enhances the specificity of the writer.<br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br />
<br />
<h1>Reference List</h1><br />
<br />
1. Bowers,L.M., Lapoint,K., Anthony,L., Pluciennik,A., & Filutowicz,M. Bacterial expression system with tightly regulated gene expression and plasmid copy number. Gene 340, 11-18 (2004).<br />
<br />
<br />
<br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/data/experimentalTeam:Amsterdam/data/experimental2012-09-27T03:24:53Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<h1>Experimental results</h1><br />
__TOC__<br />
<div class='clear'></div><br />
<h2>Functionality of the writer-reader module under different sensor modules</h2><br />
We started measuring the first proof-of-concept of the Cellular Logbook by testing the functionality of a part of our writer-reader module in the context of various sensor modules. To this end, we tested the functionality of the synthesized Methyltransferase (MTase) (our writer) cloned under the control of the LacH promoter in the pSB1AT3 backbone and transformed in in Library Efficient® DH5α™ competent cells (Invitrogen).<br />
<br />
<h4>Set up of the writer-reader module</h4><br />
[[File:Amsterdam_exp_fig_1.png|300px|right|thumb|Figure 1]]<br />
As mentioned in Molecular design, the ScaI restriction enzyme is unable to cut methylated restriction sites. Therefore, we expect different possible restriction profiles through ScaI restriction digestion since there is one ScaI site residing in the pSB1AT3 backbone and we created one ScaI site via a scar inside our writer module. We expect to find either an off or intermediate methylation state knowing that the LacH promoter driving the MTase has some basal activity.<br />
<br />
Figure 1 shows the result of a ScaI restriction digestion of the pSB1AT3/LacH/MTase construct without IPTG induction. The pattern displayed corresponds to a combination of bands indicating an intermediate state between the ‘off’ and ‘on’ situation. Interpretation of the read-out: absence of MTase expression in E. Coli leads to a complete digestion of our plasmid. Two bands of 2989 bp and 1621 bp are then observed (A). Incomplete methylation of the plasmid at only one of the two ScaI sites shows a linearized plasmid band 4610 bp. Complete methylation of our writer module prevents ScaI to cut and a typical uncut plasmid profile is observed (C).<br />
<br />
<h4>Behavior of the writer-reader module under IPTG induction</h4><br />
[[File:Amsterdam_exp_fig_2.png|300px|right|thumb|Figure 2]]<br />
The next step was to see if IPTG-based induction of MTase expression would modify the methylation state of our writer-reader module thus changing the resulting restriction profile. In the presence of 1mM IPTG, the promoter should be activated providing MTase production inside the cells. Since our model for this experiment suggests that methylation occurs at a fast rate we expect that after 16h of IPTG induction, the read-out will dramatically shift to the uncut profile.<br />
Figure 2<br />
<br />
The writer-reader module after 16h IPTG induction still shows a partial methylation profile, indicating that our writer-reader module is not fully methylated, we observe a gradual shift towards the uncut plasmid form. That means that IPTG induction leads to an increase of methylation of the plasmid and that our writer-reader module is able to store and read this information: Our writer-reader module is in principle working!!<br />
<br />
<h4>Behavior of the writer-reader module in varying growth conditions</h4><br />
[[File:Amsterdam_exp_fig_4.png|300px|right|thumb|Figure 3]]<br />
We aimed to characterize the activity of our writer during bacterial growth. We performed a growth curve experiment (figure 3) and collected samples at different time points to test the occurrence of methylation over different times of growth. <br />
[[File:Amsterdam_exp_fig_3.png|300px|right|thumb|Figure 4]]<br />
<br />
Figure 4 shows ScaI digestion of plasmids extracted from samples activated or not by 10mM IPTG and collected between 0 and 12h. Both conditions reveal an intermediate restriction profile over. However, the two samples taken after 24 hours show a different result, i.e. almost only uncut plasmid is observed as a result of an significant increase of methylation of our reader. <br />
<br />
We aimed to test the functionality of our system in the stationary state. It was previously described in the literature that the Lac promoter upon IPTG induction is performing better in the stationary phase because the lac-permease, encoded by the LacY gene and responsible for the correct IPTG uptake inside the cell, can be quantitatively integrated into the membrane. <br />
<br />
The same bacterial batch was used in both experiments but for the stationary experiment first grown over a longer period of time (at least more than 24 hours), either with or without the inducer. <br />
<br />
[[File:Amsterdam_exp_fig_5.png|300px|right|thumb|Figure 5]]<br />
<br />
Figure 5 shows the restriction profile taken from three different time points: 48, 72 and 96 hours. The first three restriction profiles correspond to growth without IPTG induction and the last three with 10 mM IPTG. During the culture no additional IPTG was added since we expect that IPTG is not degraded over time. We do not observe a change in the restriction profile, meaning that the time of culture does not influence our results. <br />
<br />
<br />
In conclusion, the presence of the LacH promoter showed to provide the activity of the Mtase but did not deliver a restriction profile fitting to the ‘off’ and ‘on’ states. Our observations are in line with our predictions as described in the Molecular Design. An alternative way to lower the basal activity of LacH is to perform our experiments at high LacI levels which is suggested to decrease the basal activity of LacH. In addition, LacH in high-copy plasmids could createsmuch more non-specific activity of the LacH than in a low-copy plasmid.<br />
<br />
<br />
<h4>Behavior of the writer-reader module under reduced basal LacH promoter activity using LacIQ E. Coli strain</h4><br />
[[File:Amsterdam_exp_fig_6.png|300px|right|thumb|Figure 6]]<br />
<br />
Both modeling and experimental results show a strong basal activity of the LacH promoter leading to a substantial methylation of our memory module, even without IPTG present in the medium. We aimed to get rid of the basal activity of the LacH promoter without IPTG induction to create a better ‘off’ state and hence a better writer-reader design.<br />
<br />
Since the silencing of the LacH promoter depends on the concentration of the LacI repressor, we chose to replace our standard DH5α E. Coli strain by the LacIQ strain. Investigation done on the LacH promoter revealed that a higher expression of LacI will show a better suppression and decrease of basal activity of the LacH promoter.<br />
<br />
We transformed our pSB1AT3/LacH/Mtase vector in the LacIQ strain and performed a restriction digestion profile experiment after 24h of growth and under 10 mM IPTG induction.<br />
<br />
<br />
<br />
Figure 6 shows the pSB1AT3/LacH/Mtase cultured overnight in LacIQ E. Coli strain and digested with ScaI restriction enzyme.<br />
<br />
The results of the restriction profile for the LacIQ E. Coli strain are inconclusive (figure 6). No change in the ScaI restriction patterns are observed between plasmids isolated from both strains. Higher expression of LacI does not seem to reveal a restriction profile that confirms the ‘off’ state and it does not seem to creates a shift towards the ‘off’ state. <br />
<br />
<br />
<h4>Behavior of the writer-reader module under tight control of and arabinose-regulated promoter</h4><br />
[[File:Amsterdam_exp_fig_7.png|300px|right|thumb|Figure 7]]<br />
The Cellular Logbook was further characterized using the pBad promoter. This promoter was chosen in order to overcome the leakiness observed with the LacH promoter. Expression of any gene cloned behind the pBad promoter is controlled by the AraC repressor and is considered to be fully suppressed in the absence of arabinose. However, in the presence of arabinose the promoter will generate a gradual response leading to gene expression. <br />
[[File:Amsterdam_exp_fig_8.png|300px|right|thumb|Figure 8]]<br />
<br />
The first characterization experiment involved induction of pSB1AT3/pBad/Mtase in Library Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 1 % arabinose. The construct was digested with ScaI restriction enzyme after 24 hours incubation at 37˚C. Surprisingly, the same intermediate digestion profile as the LacH was observed. A combination of different restriction profiles can be inferred from the results (figure 7). In the absence of arabinose, M.ScaI methyltransferase appears to be expressed and a significant number of plasmids present in the culture are fully methylated, accounting for the intense uncut DNA fragment observed. A similar pattern was observed in the presence of arabinose.<br />
<br />
[[File:Amsterdam_exp_fig_9.png|300px|right|thumb|Figure 9]]<br />
<br />
The second characterisation experiment involved the induction of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with 2 % arabinose and incubation at 37˚C for 48 hours. Subsequent digestion with ScaI restriction enzyme showed a shift towards the uncut restriction profile (figure 8). This result shows that under these conditions the writer-reader is optimally functional. Succes!<br />
<br />
<br />
The third characterisation experiment concerns the stimulation of pSB1AT3/pBad/Mtase in Efficient® DH5α™ competent cells (Invitrogen) in stationary phase with addition of 2% arabinose daily. Samples were taken after 48, 72 and 96 hours, and digested with ScaI restriction enzyme (figure 9). These results are consistent with the previous results, showing a gradual shift towards the “on-state” with induction of the pBad promoter by addition of arabinose daily.<br />
<br />
[[File:Amsterdam_exp_fig_7.png|300px|right|thumb|Figure 10]]<br />
<br />
The “off” state was not achieved in the absence of arabinose. All the experiments have been conducted into the high copy number plasmid pSB1AT3. It is known that leaky expression is relative to the copy number of the plasmid.1 Hence, the inability of the Cellular Logbook to show an “off” state in the absence of arabinose could be attributed to the high copy number plasmid used in these experiments as was discussed with the LacH.<br />
<br />
Similarly to the pSB1AT3/LacH/Mtase, a growth curve experiment was conducted to characterize the acquired construct of pSB1AT3/pBad/Mtase.<br />
<br />
<br />
Over the course of time the methylation-dependent restriction profile observed in the gel showed a shift towards the ‘on’ digestion profile in the presence of arabinose. This result shows that our Cellular Logbook is able to sense and write an arabinose signal present in the medium in a shorter period of time.<br\><br\><br\><br />
<br />
<h2>Towards the Cellular Logbook</h2><br />
[[File:Amsterdam_exp_fig_11.png|300px|right|thumb|Figure 11]]<br />
[[File:Amsterdam_exp_fig_12.png|300px|right|thumb|Figure 12]]<br />
After months of cloning attempts, it appears that we succeeded in obtaining the final version of our Cellular Logbook: the pSB1AT3/LacH/PZF3838/Mtase/Mem2X (BBa_K874300). Digestion of extracted plasmid DNA with the restriction enzyme BamHI, present in the pSB1AT3 backbone vector and in the reader module (BBa_K874040), ensured that both the Polydactyl Zinc Finger PZF3838 (BBa_K874001) and the reader module (BBa_K874040) were successfully cloned in as shown in Figure 11 (colony 2, 2 bands expected: 3699 and 1769 bp). Preliminary characterization of BBa_K874300 was attempted only once due to time pressure and consisted of a ScaI digestion in the absence of IPTG (figure 12). Compared to the pSB1AT3/LacH/Mtase, BBa_K874300 shows a significant switch to the non-methylated profile, illustrated by the tremendous intensity of the lower bands (cut plasmid) compared to the first one (partially cut plasmid). These results show that the presence of the PZF3838 enhances the specificity of the writer.<br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br\><br />
<br />
<h1>Reference List</h1><br />
<br />
1. Bowers,L.M., Lapoint,K., Anthony,L., Pluciennik,A., & Filutowicz,M. Bacterial expression system with tightly regulated gene expression and plasmid copy number. Gene 340, 11-18 (2004).<br />
<br />
<br />
<br />
</div><br />
</div><br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/achievementsTeam:Amsterdam/achievements2012-09-27T03:21:05Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<html><br />
<table><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
Demonstrated the functionality of M.ScaI introduced in E. coli<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
Succesfully sensing, writing and reading of an arabinose signal<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We developed the theory behind inferring the time of signal registration by way of a model<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We devised grounded suggestions on how to improve the molecular design of our system using a model<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We developed a software that is able to generate a Cellular Logbook plasmid that enhances our project in such a way that it can be used on a bigger scale<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We designed and outlined a new responsible development-approach for iGEM projects and applied it to our Cellular Logbook<br />
</td><br />
</tr><br />
<br />
</table><br />
</html><br />
</div><br />
</div><br />
<br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/achievementsTeam:Amsterdam/achievements2012-09-27T03:20:10Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<html><br />
<table><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
Demonstrated the functionality of M.ScaI introduced in E. coli<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
Succesfully sensing, writing and reading of an arabinose signal<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We developed the theory behind inferring the time of signal registration by way of a model<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We devised grounded suggestions on how to improve the molecular design of our system using a model<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We designed and outlined a new responsible development-approach for iGEM projects and applied it to our Cellular Logbook<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We developed a software that is able to generate a Cellular Logbook plasmid that enhances our project in such a way that it can be used on a bigger scale<br />
</td><br />
</tr><br />
</table><br />
</html><br />
</div><br />
</div><br />
<br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/achievementsTeam:Amsterdam/achievements2012-09-27T03:18:56Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<html><br />
<table><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
Demonstrated the functionality of M.ScaI introduced in E. coli<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
Succesfully sensing, writing and reading of an arabinose signal<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We developed the theory behind inferring the time of signal registration by way of a model<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We devised grounded suggestions on how to improve the molecular design of our system using a model<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We designed and outlined a new responsible development-approach for iGEM projects and applied it to our Cellular Logbook<br />
</td><br />
</tr><br />
</table><br />
</html><br />
</div><br />
</div><br />
<br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/achievementsTeam:Amsterdam/achievements2012-09-27T03:18:11Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<html><br />
<table><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
Demonstrated the functionality of M.ScaI introduced in E. coli<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
Succesfully sensing, writing and reading of an arabinose signal<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We developed the theory behind inferring the time of signal registration by way of a model<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
We devised grounded suggestions on how to improve the molecular design of our system using a model<br />
</td><br />
</tr><br />
</table><br />
</html><br />
</div><br />
</div><br />
<br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/achievementsTeam:Amsterdam/achievements2012-09-27T03:17:14Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<html><br />
<table><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
Demonstrated the functionality of M.ScaI introduced in E. coli<br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
Succesfully sensing, writing and reading of an arabinose signal<br />
</td><br />
</tr><br />
</table><br />
</html><br />
</div><br />
</div><br />
<br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/achievementsTeam:Amsterdam/achievements2012-09-27T03:16:21Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<html><br />
<table><br />
<tr><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Amsterdam_checkmark.png" width="50px" \><br />
</td><br />
<td><br />
Demonstrated the functionality of M.ScaI introduced in E. coli<br />
</td><br />
</tr><br />
</table><br />
</html><br />
</div><br />
</div><br />
<br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenRhttp://2012.igem.org/Team:Amsterdam/achievementsTeam:Amsterdam/achievements2012-09-27T03:15:46Z<p>MaartenR: </p>
<hr />
<div>{{Team:Amsterdam/stylesheet}}<br />
{{Team:Amsterdam/scripts}}<br />
{{Team:Amsterdam/Header}}<br />
{{Team:Amsterdam/Sidebar1}}<br />
<br />
<div id="content-area"><br />
<div id="sub-menu" class="content-block"><br />
<html><br />
<table><br />
<tr><br />
<td><br />
[[File:Amsterdam_checkmark.png|50px]]<br />
</td><br />
<td><br />
Demonstrated the functionality of M.ScaI introduced in E. coli<br />
</td><br />
</tr><br />
</table><br />
</html><br />
</div><br />
</div><br />
<br />
<br />
{{Team:Amsterdam/Foot}}</div>MaartenR