Team:Amsterdam

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<h2>Cellular Logbook - A methylation-based reporter system</h2>
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<h1>Parts</h1>
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'''Multi-sensing genetic devices''' offer great future perspectives for biotechnology, environmental monitoring and medical diagnostics. In light of this we have created an innovative '''epigenetic DNA-methylation''' based '''detection system''' in <i>E. coli</i>, named '''Cellular Logbook''', that has the potential of simultaneously reporting on significantly more signals than current fluorescence-based systems (eg. GFP). The Cellular Logbook can be used to '''detect (Sensor module) and store (Writer module)''' the presence of '''any compound''' linked to a transcriptional regulator. This system allows for '''offline monitoring''' by functioning as a '''memory module (Writer module)'''. Assessment of the memory status is performed by digesting with restriction endonucleases followed by gel electrophoresis '''(Reader Module)'''. Furthermore, the Cellular Logbook is able to infer the '''time of signal-onset''' or '''signal-intensity''' using the natural dilution of the registered signal’s due to cell division. In shourt our novel epigenetic memory module system could potentially be utilized as a '''mulit-sensor/time indicating platform''' for many groundbreaking technologies and applications (eg. to measure environmental changes such as toxic compounds).
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<h2>The Lac-Hybrid</h2>
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<h2>Sponsors</h2>
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We chose this promoter because it had a lot of reference on the parts registry page and it seems like a straightforward and easy to use promoter. It has been used many times without problems and seems to be a commonly used promoter for many researchers. Suppressed by LacI and allosteric induced by IPTG it creates an on/off situation, being a strong promoter, for us to test our platform.
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<a href="http://www.aimms.vu.nl/en/index.asp"><img src="https://static.igem.org/mediawiki/2012/1/12/Amsterdam_Logo_AIMMS.png" height="35" align="left" style="padding:10px; padding-top:20px;"></a>
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Side note; added IPTG is not degraded by bacteria and has a long degradation time.
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<a href="http://www.genscript.com/"><img src="https://static.igem.org/mediawiki/2012/6/6e/Amsterdam_Logo_Genscript.jpeg" height="35" align="left" style="padding:10px; padding-top:20px;"></a>
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<a href="http://nbv.kncv.nl/"><img src="https://static.igem.org/mediawiki/2012/8/8e/Amsterdam_Logo_nbv.gif" height="35" align="left" style="padding:10px; padding-top:20px;"></a>
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<a href="http://www.sysbio.nl/"><img src="https://static.igem.org/mediawiki/2012/0/01/Amsterdam_Logo_NISB.png" height="35" align="left" style="padding:10px; padding-top:20px;"></a>
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<a href="http://www.uva-alumni.nl/page.aspx?pid=460"><img src="https://static.igem.org/mediawiki/2012/3/3f/Amsterdam_Logo_universiteitfonds.png" height="35" align="left" style="padding:10px; padding-top:20px;"></a>
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<a href="http://eu.idtdna.com/site"><img src="https://static.igem.org/mediawiki/2012/c/c4/Xxidt.png" height="35" align="left" style="padding:10px; padding-top:20px;"></a>
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<h2>The Arabinose promoter</h2>
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<a href="http://www.vu.nl/nl/index.asp"><img src="https://static.igem.org/mediawiki/2012/8/81/Amsterdam2012VuLogoHeader.png" height="35" align="left" style="padding:10px; padding-top:20px;"></a>
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Just like the Lac-Hybrid promoter we wanted to use another reliable promoter that has been used extensively and creates another on and off situation by an inducible substrate. Induced only by arabinose it should give a clear signal with and without the presence of arabinose.  
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<a href="http://www.uva.nl/"><img src="https://static.igem.org/mediawiki/2012/e/e4/Amsterdam2012UvaLogoHeader.png" height="35" align="left" style="padding:10px; padding-top:20px;"></a>
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<h2>The methyltransferase</h2>
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Our biggest and most essential part. The methyltransferase protein, not endogenous to E. coli, we chose to introduce in E. coli. Completely synthesized since it is a very rare protein, we actually took from N. Meningitis, and not easy to come by. It qualifies as a N-4-methyltransferase and has no direct link to E. coli, other than its ability to methylate ScaI sites. This gives us no indication that the E. coli should suffer by the presence of this protein. Using … as a substrate.
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We have to oblige to the iGEM rules of forbidden and avoidable sites and for that reason we had to make some adjustments to the protein. We don’t suspect this will have any influence on the protein folding or activity.
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<h2>The mycosin-linker</h2>
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In order to create a fusion protein of our own we need to link the two together. When checking up on our literature we found that in eukaryotes Bas van Steensel used a myc-linker for his fusion protein. After searching for some more information we found that a myc-linker is actually commonly used and seems reliable. As a bonus feature the myc part can also be used as a myc-tag for immune-blotting, providing information about protein presence and degradation. We ended up using a 39 bp linker to generate enough flexibility for the fusion protein to sustain and to be able to reach and find its specific sites.
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<h2>The double terminators</h2>
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We chose to put two terminators together that both occur in the part registry but not together. The both terminators did not have any bad commentary and look like reliable terminators. We chose to have these synthesized to save time and eliminate a cloning step.
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<h2>The Zinc Finger</h2>
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Choosing a zinc finger presented itself as a much harder task. We discovered that nature and science both give a wide selection of zinc-fingers that all have their own specialization, plus the part registry does not supply a zinc finger. To gain more information and knowledge about zinc fingers we contacted some specialists and eventually got in contact with prof. Dr. P. Hooykaas, who was interested in our project and could supply us with a zinc finger. His institute specializes in constructing and using zinc fingers fused to Nucleases. It was Dr. S. de Pater that could eventually send us two of the zinc finger nucleases she was working on to try for ourselves. Both consisting of 6 fused zinc fingers, creating a high level of specificity.
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<h2>The Backbones</h2>
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Using standard backbones provided by iGEM and backbones that allready contained some elements of the parts we wanted to put together we ended up using a wide array of different backbones. Using these backbones not just because they are required but also because they differ in copy-number, creating different situations inside the cells for us to compare.
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<h2>Ribosomal binding site</h2>
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We took this one from the part registry. It is a medium RBS which should be enough for our purpose. In later situation it might be an option to change to either a weak or strong RBS.
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<h2>The memory part</h2>
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Every memory system needs a memory module. We designed our own to meet our specific demands. Incorporation a ScaI site next to either one or two Zinc-finger sites. In the case of two sites the other side is anti-sense to the first since the ScaI site is blunt and the methylation can occur on either sides. Of course all lengths of DNA material between the sites is adjusted to the linker length. For the parts of ‘junk’ DNA we used a tool to generate these sequences to ensure that no genes, restriction sites or other problematic pieces of sequences could be generated.
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<h2>Summary of used parts</h2>
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<h4>Used parts</h4>
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-LacH (BBa_R0011)
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-pBAD (BBa_K206001)
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-terminators (B0014 containing B0012 and B0011)
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<h4>Synthesized parts</h4>
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-Methyltransferase with myc-linker and double terminator
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-Memory parts 1x Znf and 2x Znf
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<h4>Externally received parts</h4>
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-Zinc Finger
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<h4>Backbones</h4>
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-pSB1AT3
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<h4>Final parts in all mentioned backbones</h4>
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-LacH + Mtase + MemP 1x
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-pBAD + Mtase + MemP 1x
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-LacH + Znf + Mtase + MemP 1x
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-LacH + Znf + Mtase + MemP 2x
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-pBAD + Znf + Mtase + MemP 1x
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-pBAD + Znf + Mtase + MemP 2x
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<h4>Intermediate parts</h4>
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-LacH + Mtase
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-pBAD + Mtase
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{{Team:Amsterdam/Foot}}
{{Team:Amsterdam/Foot}}

Latest revision as of 03:56, 27 September 2012

Cellular Logbook - A methylation-based reporter system

Multi-sensing genetic devices offer great future perspectives for biotechnology, environmental monitoring and medical diagnostics. In light of this we have created an innovative epigenetic DNA-methylation based detection system in E. coli, named Cellular Logbook, that has the potential of simultaneously reporting on significantly more signals than current fluorescence-based systems (eg. GFP). The Cellular Logbook can be used to detect (Sensor module) and store (Writer module) the presence of any compound linked to a transcriptional regulator. This system allows for offline monitoring by functioning as a memory module (Writer module). Assessment of the memory status is performed by digesting with restriction endonucleases followed by gel electrophoresis (Reader Module). Furthermore, the Cellular Logbook is able to infer the time of signal-onset or signal-intensity using the natural dilution of the registered signal’s due to cell division. In shourt our novel epigenetic memory module system could potentially be utilized as a mulit-sensor/time indicating platform for many groundbreaking technologies and applications (eg. to measure environmental changes such as toxic compounds).

Sponsors

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