Latest revision as of 04:00, 27 September 2012

Click on one of the buttons to get the information on our modules!

Sensor

What does a sensor mean to the Cellular Logbook?

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.

For our proof-of-concept we chose to implement two different promoters as sensors:

Lac-hybrid promoter:

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 E. coli.

In its repressive state, the LacI repressor, an allosteric protein constitutively expressed by E. Coli, 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.

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.

Arabinose promoter:

This promoter is derived from wild-type E. coli and has a modified AraI1 site, which causes this promoter to be less responsive to low concentrations of induction and therefore exhibits a lower maximum response.

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, AraC binds to it and changes its conformation such that it interacts with the AraI1 and AraI2 operator sites, allowing transcription.

For the characterization of our system, we intentionally chose a weak version of the promoter, the pBAD-weak (BBa_K206001), from the parts registry.

@@ Line 3: / Line 3: @@
 {{Team:Amsterdam/Header}}
 {{Team:Amsterdam/Sidebar1}}
+__NOTOC__
 <div id="content-area">
 <div id="sub-menu" class="content-block">
-=== Molecular Design ===
+<center>'''Click on one of the buttons to get the information on our modules!'''</center>
-== Sensing ==
-Every project needs a start, in our case this is the sensor or sensors of ours Cellular Logbook. Our system can be hooked up to any sensor that results in an transcriptional event. This is much in the same fashion as more classical reporters (e.g. GFP) weren't it for the possibility to measure multiple signals at the same time and the theoretical properties that can be derived from a methylation based system (time of signal onset or event intensity/ concentration indication). These theoretical properties where studied in great detail (in silicon). Sadly due to time and manpower constraints we where not able to implement this multi-sensor. More about how we would like to implement this can be found in the Expansion section.
-= Considerations =
-For our proof of concept we chose to individual implement two sensors. Our first sensor utilises the Lac hybrid (R0011) together with a medium/weak ribosomal binding site (R0032) in order to sense IPTG in the medium. This sensor was chose with the following properties in mind:
-<ul>
-<li><b>Well characterised promoter</b><br>With over 6 teams that used and characterized the lac hybrid promoter it is one of the most widely used promoters in the registry.</li>
-</ul>
 <html>
 <script type='text/javascript' src='https://2012.igem.org/Template:Team:Amsterdam/scripts/mapper.js?action=raw'></script>
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 function sensorText(){
      $('#moldesign-content').empty()
-     $('#moldesign-content').append("<h1>Sensor</h1> The Cellular Logbook is build on the natural occurring epigenetic phenomenon of methylation. Each sensor is able to expres a zinc finger and methyltransferase fusion protein (ZnF-Mtase). The ZnF-Mtase binds to its Memory Part (MP), which consists of a restriction site that is targeted by the methyltransferase, and two binding sites that are specific to the zinc finger. Upon binding, the methyltransferase will methylate the restriction site. The methylated restriction site is inaccessible to restriction enzymes. By digesting the Memory Part with the restriction enzyme and performing gel electrophoresis, it can be read out if the ZnF-Mtase has come to expression and as such if the related signal has been present.")
+     $('#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>")
 }
-</script>
+function writerText(){
+    $('#moldesign-content').empty()
+    $('#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>")
+}
+function readerText(){
+    $('#moldesign-content').empty()
+    $('#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>")
+}
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+<center>[https://2012.igem.org/Team:Amsterdam/software/logbook_designer/webtool '''Design your own logbook!''']</center><br\><br\>
 <div id='moldesign-content'>
+<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>
 </div>
 </div>

Team:Amsterdam/project/molecular design

From 2012.igem.org

Latest revision as of 04:00, 27 September 2012

Sensor

What does a sensor mean to the Cellular Logbook?

Lac-hybrid promoter:

Arabinose promoter: