http://2013.igem.org/wiki/index.php?title=Special:Contributions/Hezscha&feed=atom&limit=50&target=Hezscha&year=&month=2013.igem.org - User contributions [en]2024-03-29T11:23:20ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:DTU-DenmarkTeam:DTU-Denmark2013-10-03T08:45:46Z<p>Hezscha: /* Requiem for a Stream:From Ammonia Pollution to Energy Production via Denitrification */</p>
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== Requiem for a Stream:<br>From Ammonia Pollution to Energy Production via Denitrification ==<br />
<br />
Intense agriculture is the base of feeding a growing world population. However it comes at a cost for the environment. Part of this is the growing problem of ammonia pollution. As the demand for food articles is steadily increasing farmers turn to extensive use of fertilizers and nitrogen-enriched animal feeds to increase their yield. Much of this nitrogen comes in the form of ammonia and accumulates in the argicultural runoff, which pollutes surface bodies of water like lakes and streams but also the groundwater. <br />
Our project is a requiem for those streams. Our goal is to remove surplus ammonia from wastewater, and via two ''E. coli'' mutants turn it into nitrous oxide.<br />
<br />
TODO: There should either be a link to our video or it should be embedded here. Alternatively, we can use the scanned pictures.<br />
<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
<br />
Ammonia is a common pollutant in agricultural runoff, and excess ammonia in the environment leads to increased algae growth and eutrophication, which starves other water-borne organisms for oxygen. Our project reverses nitrogen fixation, and is a means to clean up polluted streams and wastewater. The final product is nitrous oxide, which can be sold as an industrial product or reclaimed and used as a source of small scale sustainable energy.<br />
<br />
==Highlights==<br />
<br />
For more information on the mutants, please see [[Team:DTU-Denmark/Project| project details]], and for information on the experiments we did to characterize the mutants, see [[Team:DTU-Denmark/Experiments|an overview of the experiments]] we conducted. Also be sure to check out our [[Team:DTU-Denmark/Notebook| online lab notebook]], and the [[Team:DTU-Denmark/Human_Practices | human practices events]] that we hosted. <br />
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<!-- {{:Team:DTU-Denmark/Templates/Footer}} --></div>Hezschahttp://2013.igem.org/Team:DTU-DenmarkTeam:DTU-Denmark2013-10-03T08:45:29Z<p>Hezscha: /* Requiem for a Stream:From Ammonia Pollution to Energy Production via Denitrification */</p>
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<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
== Requiem for a Stream:<br>From Ammonia Pollution to Energy Production via Denitrification ==<br />
<br />
Intense agriculture is the base of feeding a growing world population. However it comes at a cost for the environment. Part of this is the growing problem of ammonia pollution. As the demand for food articles is steadily increasing farmers turn to extensive use of fertilizers and nitrogen-enriched animal feeds to increase their yield. Much of this nitrogen comes in the form of ammonia and accumulates in the argicultural runoff, which pollutes surface bodies of water like lakes and streams but also the groundwater. <br />
Our project is a requiem for those streams. Our goal is to remove surplus ammonia from wastewater, and via two ''E. coli'' mutants turn it into nitrous oxide.<br />
<br />
There should eith be a link to our video or it should be embedded here. Alternatively, we can use the scanned pictures.<br />
<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
<br />
Ammonia is a common pollutant in agricultural runoff, and excess ammonia in the environment leads to increased algae growth and eutrophication, which starves other water-borne organisms for oxygen. Our project reverses nitrogen fixation, and is a means to clean up polluted streams and wastewater. The final product is nitrous oxide, which can be sold as an industrial product or reclaimed and used as a source of small scale sustainable energy.<br />
<br />
==Highlights==<br />
<br />
For more information on the mutants, please see [[Team:DTU-Denmark/Project| project details]], and for information on the experiments we did to characterize the mutants, see [[Team:DTU-Denmark/Experiments|an overview of the experiments]] we conducted. Also be sure to check out our [[Team:DTU-Denmark/Notebook| online lab notebook]], and the [[Team:DTU-Denmark/Human_Practices | human practices events]] that we hosted. <br />
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<!-- {{:Team:DTU-Denmark/Templates/Footer}} --></div>Hezschahttp://2013.igem.org/Team:DTU-DenmarkTeam:DTU-Denmark2013-10-03T08:41:09Z<p>Hezscha: /* Requiem for a Stream:From Ammonia Pollution to Energy Production via Denitrification */</p>
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<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
== Requiem for a Stream:<br>From Ammonia Pollution to Energy Production via Denitrification ==<br />
<br />
Intense agriculture is the base of feeding a growing world population. However it comes at a cost for the environment. Part of this is the growing problem of ammonia pollution. As the demand for food articles is steadily increasing farmers turn to extensive use of fertilizers and nitrogen-enriched animal feeds to increase their yield. Much of this nitrogen comes in the form of ammonia and accumulates in the argicultural runoff, which pollutes surface bodies of water like lakes and streams but also the groundwater. <br />
Our project is a requiem for those streams. Our goal is to remove surplus ammonia from wastewater, and via two ''E. coli'' mutants turn it into nitrous oxide.<br />
<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
<br />
Ammonia is a common pollutant in agricultural runoff, and excess ammonia in the environment leads to increased algae growth and eutrophication, which starves other water-borne organisms for oxygen. Our project reverses nitrogen fixation, and is a means to clean up polluted streams and wastewater. The final product is nitrous oxide, which can be sold as an industrial product or reclaimed and used as a source of small scale sustainable energy.<br />
<br />
==Highlights==<br />
<br />
For more information on the mutants, please see [[Team:DTU-Denmark/Project| project details]], and for information on the experiments we did to characterize the mutants, see [[Team:DTU-Denmark/Experiments|an overview of the experiments]] we conducted. Also be sure to check out our [[Team:DTU-Denmark/Notebook| online lab notebook]], and the [[Team:DTU-Denmark/Human_Practices | human practices events]] that we hosted. <br />
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<!-- {{:Team:DTU-Denmark/Templates/Footer}} --></div>Hezschahttp://2013.igem.org/Team:DTU-DenmarkTeam:DTU-Denmark2013-10-03T08:39:53Z<p>Hezscha: /* Requiem for a Stream:From Ammonia Pollution to Energy Production via Denitrification */</p>
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<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
== Requiem for a Stream:<br>From Ammonia Pollution to Energy Production via Denitrification ==<br />
<br />
Intense agriculture is the base of feeding a growing world population. However it comes at a cost for the environment. Part of this is the growing problem of ammonia pollution. As the demand for food articles is steadily increasing farmers turn to extensive use of fertilizers and nitrogen-enriched animal feeds to increase their yield. Much of this nitrogen comes in the form of ammonia and accumulates in the argicultural runoff, which pollutes surface bodies of water like lakes and streams but also the groundwater. <br />
Our project is a requiem for those streams. Our goal is to remove surplus ammonia from wastewater, and via two ''E. coli'' mutants turn it into nitrous oxide.<br />
<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
<br />
Ammonia is a common pollutant in agricultural runoff, and excess ammonia in the environment leads to increased algae growth and eutrophication, which starves other water-borne organisms for oxygen. Our project reverses nitrogen fixation, and is a means to clean up polluted streams and wastewater. The final product is nitrous oxide, which can be sold as an industrial product or reclaimed and used as a source of small scale sustainable energy.<br />
<br />
For more information on the mutants, please see [[Team:DTU-Denmark/Project| project details]], and for information on the experiments we did to characterize the mutants, see [[Team:DTU-Denmark/Experiments|an overview of the experiments]] we conducted. Also be sure to check out our [[Team:DTU-Denmark/Notebook| online lab notebook]], and the [[Team:DTU-Denmark/Human_Practices | human practices events]] that we hosted. <br />
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<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
== Requiem for a Stream:<br>From Ammonia Pollution to Energy Production via Denitrification ==<br />
<br />
Intense agriculture is the base of feeding a growing world population. However it comes at a cost for the environment. As the demand for food articles is steadily increasing farmers turn to extensive use of fertilizers and nitrogen-enriched animal feeds to increase their yield. Much of this nitrogen comes in the form of ammonia and accumulates in the argicultural runoff, which pollutes surface bodies of water like lakes and streams but also the groundwater. <br />
Our project is a requiem for those streams. Our goal is to remove surplus ammonia from wastewater, and via two ''E. coli'' mutants turn it into nitrous oxide.<br />
<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
<br />
Ammonia is a common pollutant in agricultural runoff, and excess ammonia in the environment leads to increased algae growth and eutrophication, which starves other water-borne organisms for oxygen. Our project reverses nitrogen fixation, and is a means to clean up polluted streams and wastewater. The final product is nitrous oxide, which can be sold as an industrial product or reclaimed and used as a source of small scale sustainable energy.<br />
<br />
For more information on the mutants, please see [[Team:DTU-Denmark/Project| project details]], and for information on the experiments we did to characterize the mutants, see [[Team:DTU-Denmark/Experiments|an overview of the experiments]] we conducted. Also be sure to check out our [[Team:DTU-Denmark/Notebook| online lab notebook]], and the [[Team:DTU-Denmark/Human_Practices | human practices events]] that we hosted. <br />
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== Requiem for a Stream:<br>From Ammonia Pollution to Energy Production via Denitrification ==<br />
<br />
Intense agriculture is the base of feeding a growing world population. However it comes at a cost for the environment. As the demand for food articles is steadily increasing farmers turn to extensive use of fertilizers and nitrogen-enriched animal feeds to increase their yield. Much of this nitrogen comes in the form of ammonia and accumulates in the argicultural runoff, which pollutes streams and the groundwater. <br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants turns it into nitrous oxide.<br />
<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
<br />
Ammonia is a common pollutant in agricultural runoff, and excess ammonia in the environment leads to increased algae growth and eutrophication, which starves other water-borne organisms for oxygen. Our project reverses nitrogen fixation, and is a means to clean up polluted streams and wastewater. The final product is nitrous oxide, which can be sold as an industrial product or reclaimed and used as a source of small scale sustainable energy.<br />
<br />
For more information on the mutants, please see [[Team:DTU-Denmark/Project| project details]], and for information on the experiments we did to characterize the mutants, see [[Team:DTU-Denmark/Experiments|an overview of the experiments]] we conducted. Also be sure to check out our [[Team:DTU-Denmark/Notebook| online lab notebook]], and the [[Team:DTU-Denmark/Human_Practices | human practices events]] that we hosted. <br />
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<!-- {{:Team:DTU-Denmark/Templates/Footer}} --></div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-02T15:57:17Z<p>Hezscha: </p>
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__NOTOC__<br />
==Project Description==<br />
<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
Global demand for fixed nitrogen has increased to the point that half the human population now relies on chemical fertilizer to grow their food. While fertilizer is a requirement for modern life, runoff from overfertilized farmland can cause eutrophication. In the presence of abundant ammonia, algae overgrow and consume the much of the available oxygen in the water. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
Using two ''E. coli'' mutants built with genes from ''Nitrosomonas europaea'' and ''Pseudomonas aeruginosa'', we provide a system to reverse nitrogen fixation. Our mutants consume ammonia and produce nitrous oxide, releases a sustainable source of energy when decomposed into nitrogen and oxygen. We also provide a prototype of a bioreactor that could be scaled up and deployed in the field to simultaneously clean the water and produce energy.<br />
<br />
==Details==<br />
<br />
=== Mutant 1: Aerobic ===<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
The first mutant incorporates [[Team:DTU-Denmark/Protein_Models#AMO|AMO]], [[Team:DTU-Denmark/Protein_Models#Hao|HAO]] and two cytochromes [[Team:DTU-Denmark/Protein_Models#Cc554|c554]] and [[Team:DTU-Denmark/Protein_Models#Ccm552|cm552]] from ''Nitrosomonas europaea'' (shown in blue). AMO is a 3 subunit protein which converts ammonia (NH<sub>4</sub>) to an intermediate called hydroxylamine (NH<sub>2</sub>OH). HAO then converts the hydroxylamine to nitrite (NO<sub>2</sub><sup>-</sup>). During this conversion process, cytochrome c554 accepts an electron from HAO, and then passes this electron on to ccm552. The terminal electron acceptor in this chain is quinone. AMO and ccm552 are embedded in the cytoplasmic membrane, and all other proteins in this process are found in the periplasm. <br />
<br />
=== Mutant 2: Anaerobic ===<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
The second mutant incorporates the Nir region from ''Pseudomonas aeruginosa'' (shown in yellow). [[Team:DTU-Denmark/Protein_Models#NirS|NirS]] converts nitrite to nitric oxide (NO), while removing an electron from [[Team:DTU-Denmark/Protein_Models#NirM|NirM]]. The remainder of the Nir region is necessary for the synthesis of NirS, and so we have included these genes as well. [[Team:DTU-Denmark/Protein_Models#NOR|NOR]], which is present natively in ''E. coli'', converts nitric oxide to nitrous oxide (N<sub>2</sub>O). In contrast to ''N. europaea'', ''E. coli'' is not a nitrifying bacterium and so does not convert nitrous oxide into nitrogen to complete the denitrification process.<br />
<br />
== Plasmids ==<br />
<br />
=== Mutant 1 ===<br />
<br />
The final plasmid for Mutant 1 should be the following which includes all genes on one plasmid:<br />
<br />
[[File:dtu_Plasmid_mutant_1-01.png|300px]]<br />
<br />
In order to simplify and to be able to debug each part of this pathway separately, we first implemented the genes individually. Unfortunately, we did not have time to combine all the genes into the final plasmid.<br />
<br />
[[File:dtu-mutant1-implementation.png|600px]]<br />
<br />
=== Mutant 2 ===<br />
<br />
The final plasmid for Mutant 2 is the following:<br />
<br />
[[File:Dtu Plasmid mutant 2-01.png|300px]]<br />
<br />
Since we had difficulty extracting Nir from ''Pseudomonas aeruginosa'' in one piece, we split it into two pieces to be extracted seperately and then re-combined them with USER cloning.<br />
<br />
== User Cloning ==<br />
<br />
To assemble our plasmids we employ uracil-specific excision reagent (USER) cloning as described by Nour-Eldin et al. This technique enables us to speed up the cloning process, to clone seamlessly and to assemble many fragments in one reaction. The steps are:<br />
<br />
# PCR: The backbone and all fragments are amplified as linear products containing a uracil instead of a thymine at a specific position. This requires specifically designed primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
# Optional - If circular DNA was used as PCR template a DpnI digestion can be performed to eliminate this DNA.<br />
# USER-reaction: Digesting the linearized PCR fragment with the USER enzyme removes the uracil-insertions, which will produce the required overhangs.<br />
# Transformation: The mix is immediately transformed into competent E. coli cells where the plasmid assembles itself by base pairing between the complementary overhangs.<br />
<br />
See our "Bricks of Knowledge" [https://www.youtube.com/watch?v=7EiVttJpXH4 video on USER cloning] and the [https://2011.igem.org/Team:DTU-Denmark-2 wiki of the 2011 DTU-2 iGEM team] for more information.<br />
<br />
==References== <br />
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<ul><br />
<li>Kanehisa, M., Goto, S., Sato, Y., Furumichi, M., and Tanabe, M.; KEGG for integration and interpretation of large-scale molecular datasets. Nucleic Acids Res. 40, D109-D114 (2012).</li><br />
<li>Kanehisa, M. and Goto, S.; KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27-30 (2000).</li><br />
<li>Zumft, Walter G. (1997) Cell Biology and Molecular Basis of Denitrification. Microbiology and Molecular Biology Reviews, December 1997 p. 533–616</li><br />
<li>Ingledew, WJ and Poole, RK (1984) The Respiratory Chains of Escherichia coli. Microbiological Reviews, Sept. 1984, p. 222-271</li><br />
<li>Nour-Eldin, HH et al (2010). USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories.<br />
Methods Mol Biol. 2010;643:185-200. doi: 10.1007/978-1-60761-723-5_13.</li><br />
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== pBAD synthetic promoter library ==<br />
<br />
As a tool for expressing lethal proteins in ''E. coli'' we made a synthetic promoter library (SPL, [http://dspace.mit.edu/handle/1721.1/60080 RFC 63]) with the pBAD arabinose inducible promoter. The concept was taken from the [https://2010.igem.org/Team:DTU-Denmark/SPL DTU iGEM team from 2010].<br />
<br />
== Methods ==<br />
<br />
=== Experimental procedure ===<br />
<br />
# Random promoter sequences were ordered matching the sequence CTGACGNNNNNNNNNNNNNNNNNNTAWWATNNNNA.<br />
# USER cloning to add RFP downstream of promoter.<br />
# Colonies were plated.<br />
# Plates were induced by spraying them with an aqueous arabinose solution.<br />
# Colonies that were not red prior to induction with arabinose but that did turn red after induction with arabinose were selected and re-innoculated as liquid cultures.<br />
# Biolector: Wells were inoculated from overnight cultures of each of the selected colonies. All wells were run in duplicate.<br />
# All duplicate colonies were run twice -- once with arabinose added at t=0, and again without arabinose. <br />
# The constitutive promoter TODO:which was used as a reference.<br />
<br />
=== Data analysis ===<br />
<br />
# Data was collected from the Biolector, and analyzed using a series of R scripts written by Chris Workman (unpublished). <br />
#* The maturation and degradation times for mCherry were both assumed to be 40 min. TODOref<br />
#* The growth rate, mu, was estimated to be 1.28 (from an average of all wells on all plates) since we expect each strain to grow at the same rate.<br />
#* A time window representing exponential growth was selected (between 1 and 4.5 hours).<br />
# The RFP measurement for a constitutively expressed strain was used as a standard measure of growth. This is plotted on the x-axis in the detailed plots per colony below. <br />
# Figures were plotted using R.<br />
<br />
== Results ==<br />
<br />
=== Summary ===<br />
<br />
Promoter activity when induced (with arabinose added) plotted vs basal activity (without arabinose; ie leakiness of the promoter). The colonies that we selected all show less activity than the the constitutive promoter, and when induced, show higher activity than the constitutive promoter. <br />
[[File:Induced_vs_basal.png]]<br />
<br />
=== Details ===<br />
<br />
Promoter strengths for two trials of each colony with and without arabinose. <br />
{| class="wikitable"<br />
!Colony Number<br />
!With arabinose 1<br />
!With arabinose 2<br />
!Without arabinose 1<br />
!Without arabinose 2<br />
|-<br />
|Col2||8.9025||7.8699||0.727||0.9552<br />
|-<br />
|Col3||9.2724||12.1142||0.5248||0.6982<br />
|-<br />
|Col4||9.4571||11.4522||0.5231||0.2508<br />
|-<br />
|Col12||6.3641||10.5389||0.5897||0.6869<br />
|-<br />
|Col10||7.9697||9.7949||0.3392||0.733<br />
|-<br />
|Col9||7.8563||20.1094||0.6995||0.7432<br />
|-<br />
|Col8||12.2318||15.4548||0.4203||0.4538<br />
|-<br />
|Col13||11.0377||7.3343||0.482||0.4641<br />
|-<br />
|Col15||15.6817||8.2707||0.8169||0.1343<br />
|-<br />
|Col18||14.7916||15.5736||0.6674||0.6745<br />
|-<br />
|Col19||14.2126||16.4898||0.4545||0.3566<br />
|-<br />
|Col29||7.1853||16.3467||0.5445||0.5013<br />
|-<br />
|Col26||9.7724||9.6269||0.7118||0.7865<br />
|-<br />
|Col22||8.4168||5.5958||0.6049||0.5645<br />
|-<br />
|Col33||9.1982||8.9987||0.6508||1.374<br />
|-<br />
|Col34||10.6987||7.883||0.5067||0.5031<br />
|-<br />
|Col35||13.8427||7.5469||0.4363||0.6281<br />
|-<br />
|ConRef||6.506||7.9323||8.7811||7.9323<br />
|}<br />
<br />
[[File:dtu-Fss-plot-col2.png|300px]]<br />
[[File:dtu-Fss-plot-col3.png|300px]]<br />
[[File:dtu-Fss-plot-col4.png|300px]]<br />
[[File:dtu-Fss-plot-col12.png|300px]]<br />
[[File:dtu-Fss-plot-col10.png|300px]]<br />
[[File:dtu-Fss-plot-col9.png|300px]]<br />
[[File:dtu-Fss-plot-col8.png|300px]]<br />
[[File:dtu-Fss-plot-col13.png|300px]]<br />
[[File:dtu-Fss-plot-col15.png|300px]]<br />
[[File:dtu-Fss-plot-col18.png|300px]]<br />
[[File:dtu-Fss-plot-col19.png|300px]]<br />
[[File:dtu-Fss-plot-col29.png|300px]]<br />
[[File:dut-Fss-plot-col26.png|300px]]<br />
[[File:dtu-Fss-plot-col22.png|300px]]<br />
[[File:dtu-Fss-plot-col33.png|300px]]<br />
[[File:dtu-Fss-plot-col34.png|300px]]<br />
[[File:dtu-Fss-plot-col25.png|300px]]<br />
[[File:dtu-Fss-plot-conref.png|300px]]<br />
<br />
== Example of use ==<br />
<br />
The tight inducible pBAD promoter was used in our [https://2013.igem.org/Team:DTU-Denmark/HelloWorld "Hello World project"] to regulate the expression of GFP SF, which was tagged with a signal peptide to direct it into the periplasm. Production and folding of GFP SF is faster than the transport system of ''E. coli'', which leads to undesired accumulation of GFP SF in the cytoplasm. Only when using a promoter with low leakiness it is possible to translocate a significant fraction of GFP SF after its production has been switched off. Thereby we get a clear signal from the periplasm with low interference from the cytoplasm.<br />
<br />
[[File:GFP in perimplasm RFP in cytoplasm.png|650px|thumbnail|upright=4|left|alt=Alt text|Overview microscopic pciture showing "Hello World" transformants.]]<br />
[[File:GFP in perimplasm RFP in cytoplasm close up.png|300px|thumbnail|upright=2|left|alt=Alt text|High resolution picture of "Hello World" transformants, showing a clear seperation of green and red fluorescence. GFP is primarily located in the periplasm while RFP is located in the cytoplasm. Fluorescence intensity measurements are taken along the cross-section indicated by the white line. Picture taken with a confocal microscope and ??? filter.]]<br />
[[File:Graf.PNG|290px|thumbnail|upright=2|left|alt=Alt text|Graph of flourescence measurement (or is this the model???) on green and red channel. It can be seen that the intensity of the red flourescence is restricted to the cytoplasm while green flourescence has it's peaks on the egdes. A weak green signal is measured for the cytoplasmic region because the periplasm envelopes the cytoplasm.]]<br />
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See also [https://2013.igem.org/Team:DTU-Denmark/HelloWorld "Hello World project"].<br />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/pBAD_SPLTeam:DTU-Denmark/pBAD SPL2013-10-02T12:23:56Z<p>Hezscha: /* Example of use */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|pBAD SPL}}<br />
<br />
== pBAD synthetic promoter library ==<br />
<br />
As a tool for expressing lethal proteins in ''E. coli'' we made a synthetic promoter library (SPL, [http://dspace.mit.edu/handle/1721.1/60080 RFC 63]) with the pBAD arabinose inducible promoter. The concept was taken from the [https://2010.igem.org/Team:DTU-Denmark/SPL DTU iGEM team from 2010].<br />
<br />
== Methods ==<br />
<br />
=== Experimental procedure ===<br />
<br />
# Random promoter sequences were ordered matching the sequence CTGACGNNNNNNNNNNNNNNNNNNTAWWATNNNNA.<br />
# USER cloning to add RFP downstream of promoter.<br />
# Colonies were plated.<br />
# Plates were induced by spraying them with an aqueous arabinose solution.<br />
# Colonies that were not red prior to induction with arabinose but that did turn red after induction with arabinose were selected and re-innoculated as liquid cultures.<br />
# Biolector: Wells were inoculated from overnight cultures of each of the selected colonies. All wells were run in duplicate.<br />
# All duplicate colonies were run twice -- once with arabinose added at t=0, and again without arabinose. <br />
# The constitutive promoter TODO:which was used as a reference.<br />
<br />
=== Data analysis ===<br />
<br />
# Data was collected from the Biolector, and analyzed using a series of R scripts written by Chris Workman (unpublished). <br />
#* The maturation and degradation times for mCherry were both assumed to be 40 min. TODOref<br />
#* The growth rate, mu, was estimated to be 1.28 (from an average of all wells on all plates) since we expect each strain to grow at the same rate.<br />
#* A time window representing exponential growth was selected (between 1 and 4.5 hours).<br />
# The RFP measurement for a constitutively expressed strain was used as a standard measure of growth. This is plotted on the x-axis in the detailed plots per colony below. <br />
# Figures were plotted using R.<br />
<br />
== Results ==<br />
<br />
=== Summary ===<br />
<br />
Promoter activity when induced (with arabinose added) plotted vs basal activity (without arabinose; ie leakiness of the promoter). The colonies that we selected all show less activity than the the constitutive promoter, and when induced, show higher activity than the constitutive promoter. <br />
[[File:Induced_vs_basal.png]]<br />
<br />
=== Details ===<br />
<br />
Promoter strengths for two trials of each colony with and without arabinose. <br />
{| class="wikitable"<br />
!Colony Number<br />
!With arabinose 1<br />
!With arabinose 2<br />
!Without arabinose 1<br />
!Without arabinose 2<br />
|-<br />
|Col2||8.9025||7.8699||0.727||0.9552<br />
|-<br />
|Col3||9.2724||12.1142||0.5248||0.6982<br />
|-<br />
|Col4||9.4571||11.4522||0.5231||0.2508<br />
|-<br />
|Col12||6.3641||10.5389||0.5897||0.6869<br />
|-<br />
|Col10||7.9697||9.7949||0.3392||0.733<br />
|-<br />
|Col9||7.8563||20.1094||0.6995||0.7432<br />
|-<br />
|Col8||12.2318||15.4548||0.4203||0.4538<br />
|-<br />
|Col13||11.0377||7.3343||0.482||0.4641<br />
|-<br />
|Col15||15.6817||8.2707||0.8169||0.1343<br />
|-<br />
|Col18||14.7916||15.5736||0.6674||0.6745<br />
|-<br />
|Col19||14.2126||16.4898||0.4545||0.3566<br />
|-<br />
|Col29||7.1853||16.3467||0.5445||0.5013<br />
|-<br />
|Col26||9.7724||9.6269||0.7118||0.7865<br />
|-<br />
|Col22||8.4168||5.5958||0.6049||0.5645<br />
|-<br />
|Col33||9.1982||8.9987||0.6508||1.374<br />
|-<br />
|Col34||10.6987||7.883||0.5067||0.5031<br />
|-<br />
|Col35||13.8427||7.5469||0.4363||0.6281<br />
|-<br />
|ConRef||6.506||7.9323||8.7811||7.9323<br />
|}<br />
<br />
[[File:dtu-Fss-plot-col2.png|300px]]<br />
[[File:dtu-Fss-plot-col3.png|300px]]<br />
[[File:dtu-Fss-plot-col4.png|300px]]<br />
[[File:dtu-Fss-plot-col12.png|300px]]<br />
[[File:dtu-Fss-plot-col10.png|300px]]<br />
[[File:dtu-Fss-plot-col9.png|300px]]<br />
[[File:dtu-Fss-plot-col8.png|300px]]<br />
[[File:dtu-Fss-plot-col13.png|300px]]<br />
[[File:dtu-Fss-plot-col15.png|300px]]<br />
[[File:dtu-Fss-plot-col18.png|300px]]<br />
[[File:dtu-Fss-plot-col19.png|300px]]<br />
[[File:dtu-Fss-plot-col29.png|300px]]<br />
[[File:dut-Fss-plot-col26.png|300px]]<br />
[[File:dtu-Fss-plot-col22.png|300px]]<br />
[[File:dtu-Fss-plot-col33.png|300px]]<br />
[[File:dtu-Fss-plot-col34.png|300px]]<br />
[[File:dtu-Fss-plot-col25.png|300px]]<br />
[[File:dtu-Fss-plot-conref.png|300px]]<br />
<br />
== Example of use ==<br />
<br />
The tight inducible pBAD promoter was used in our [https://2013.igem.org/Team:DTU-Denmark/HelloWorld "Hello World project"] to regulate the expression of GFP SF, which was tagged with a signal peptide to direct it into the periplasm. Production and folding of GFP SF is faster than the transport system of ''E. coli'', which leads to undesired accumulation of GFP SF in the cytoplasm. Only when using a promoter with low leakiness it is possible to translocate a significant fraction of GFP SF after its production has been switched off. Thereby we get a clear signal from the periplasm with low interference from the cytoplasm.<br />
<br />
[[File:GFP in perimplasm RFP in cytoplasm.png|650px|thumbnail|upright=4|left|alt=Alt text|Overview microscopic pciture showing "Hello World" transformants.]]<br />
[[File:GFP in perimplasm RFP in cytoplasm close up.png|300px|thumbnail|upright=2|left|alt=Alt text|High resolution picture of "Hello World" transformants, showing a clear seperation of green and red fluorescence. GFP is primarily located in the periplasm while RFP is located in the cytoplasm. Fluorescence intensity measurements are taken along the cross-section indicated by the white line. Picture taken with a confocal microscope and ??? filter.]]<br />
[[File:Graf.PNG|290px|thumbnail|upright=2|left|alt=Alt text|Graph of flourescence measurement (or is this the model???) on green and red channel. It can be seen that the intensity of the red flourescence is restricted to the cytoplasm while green flourescence has it's peaks on the egdes. A weak green signal is measured for the cytoplasmic region because the periplasm envelopes the cytoplasm.]]<br />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/pBAD_SPLTeam:DTU-Denmark/pBAD SPL2013-10-02T12:23:35Z<p>Hezscha: /* Example of use */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|pBAD SPL}}<br />
<br />
== pBAD synthetic promoter library ==<br />
<br />
As a tool for expressing lethal proteins in ''E. coli'' we made a synthetic promoter library (SPL, [http://dspace.mit.edu/handle/1721.1/60080 RFC 63]) with the pBAD arabinose inducible promoter. The concept was taken from the [https://2010.igem.org/Team:DTU-Denmark/SPL DTU iGEM team from 2010].<br />
<br />
== Methods ==<br />
<br />
=== Experimental procedure ===<br />
<br />
# Random promoter sequences were ordered matching the sequence CTGACGNNNNNNNNNNNNNNNNNNTAWWATNNNNA.<br />
# USER cloning to add RFP downstream of promoter.<br />
# Colonies were plated.<br />
# Plates were induced by spraying them with an aqueous arabinose solution.<br />
# Colonies that were not red prior to induction with arabinose but that did turn red after induction with arabinose were selected and re-innoculated as liquid cultures.<br />
# Biolector: Wells were inoculated from overnight cultures of each of the selected colonies. All wells were run in duplicate.<br />
# All duplicate colonies were run twice -- once with arabinose added at t=0, and again without arabinose. <br />
# The constitutive promoter TODO:which was used as a reference.<br />
<br />
=== Data analysis ===<br />
<br />
# Data was collected from the Biolector, and analyzed using a series of R scripts written by Chris Workman (unpublished). <br />
#* The maturation and degradation times for mCherry were both assumed to be 40 min. TODOref<br />
#* The growth rate, mu, was estimated to be 1.28 (from an average of all wells on all plates) since we expect each strain to grow at the same rate.<br />
#* A time window representing exponential growth was selected (between 1 and 4.5 hours).<br />
# The RFP measurement for a constitutively expressed strain was used as a standard measure of growth. This is plotted on the x-axis in the detailed plots per colony below. <br />
# Figures were plotted using R.<br />
<br />
== Results ==<br />
<br />
=== Summary ===<br />
<br />
Promoter activity when induced (with arabinose added) plotted vs basal activity (without arabinose; ie leakiness of the promoter). The colonies that we selected all show less activity than the the constitutive promoter, and when induced, show higher activity than the constitutive promoter. <br />
[[File:Induced_vs_basal.png]]<br />
<br />
=== Details ===<br />
<br />
Promoter strengths for two trials of each colony with and without arabinose. <br />
{| class="wikitable"<br />
!Colony Number<br />
!With arabinose 1<br />
!With arabinose 2<br />
!Without arabinose 1<br />
!Without arabinose 2<br />
|-<br />
|Col2||8.9025||7.8699||0.727||0.9552<br />
|-<br />
|Col3||9.2724||12.1142||0.5248||0.6982<br />
|-<br />
|Col4||9.4571||11.4522||0.5231||0.2508<br />
|-<br />
|Col12||6.3641||10.5389||0.5897||0.6869<br />
|-<br />
|Col10||7.9697||9.7949||0.3392||0.733<br />
|-<br />
|Col9||7.8563||20.1094||0.6995||0.7432<br />
|-<br />
|Col8||12.2318||15.4548||0.4203||0.4538<br />
|-<br />
|Col13||11.0377||7.3343||0.482||0.4641<br />
|-<br />
|Col15||15.6817||8.2707||0.8169||0.1343<br />
|-<br />
|Col18||14.7916||15.5736||0.6674||0.6745<br />
|-<br />
|Col19||14.2126||16.4898||0.4545||0.3566<br />
|-<br />
|Col29||7.1853||16.3467||0.5445||0.5013<br />
|-<br />
|Col26||9.7724||9.6269||0.7118||0.7865<br />
|-<br />
|Col22||8.4168||5.5958||0.6049||0.5645<br />
|-<br />
|Col33||9.1982||8.9987||0.6508||1.374<br />
|-<br />
|Col34||10.6987||7.883||0.5067||0.5031<br />
|-<br />
|Col35||13.8427||7.5469||0.4363||0.6281<br />
|-<br />
|ConRef||6.506||7.9323||8.7811||7.9323<br />
|}<br />
<br />
[[File:dtu-Fss-plot-col2.png|300px]]<br />
[[File:dtu-Fss-plot-col3.png|300px]]<br />
[[File:dtu-Fss-plot-col4.png|300px]]<br />
[[File:dtu-Fss-plot-col12.png|300px]]<br />
[[File:dtu-Fss-plot-col10.png|300px]]<br />
[[File:dtu-Fss-plot-col9.png|300px]]<br />
[[File:dtu-Fss-plot-col8.png|300px]]<br />
[[File:dtu-Fss-plot-col13.png|300px]]<br />
[[File:dtu-Fss-plot-col15.png|300px]]<br />
[[File:dtu-Fss-plot-col18.png|300px]]<br />
[[File:dtu-Fss-plot-col19.png|300px]]<br />
[[File:dtu-Fss-plot-col29.png|300px]]<br />
[[File:dut-Fss-plot-col26.png|300px]]<br />
[[File:dtu-Fss-plot-col22.png|300px]]<br />
[[File:dtu-Fss-plot-col33.png|300px]]<br />
[[File:dtu-Fss-plot-col34.png|300px]]<br />
[[File:dtu-Fss-plot-col25.png|300px]]<br />
[[File:dtu-Fss-plot-conref.png|300px]]<br />
<br />
== Example of use ==<br />
<br />
The tight inducible pBAD promoter was used in our [https://2013.igem.org/Team:DTU-Denmark/HelloWorld "Hello World project"] to regulate the expression of GFP SF, which was tagged with a signal peptide to direct it into the periplasm. Production and folding of GFP SF is faster than the transport system of ''E. coli'', which leads to undesired accumulation of GFP SF in the cytoplasm. Only when using a promoter with low leakiness it is possible to translocate a significant fraction of GFP SF after its production has been switched off. Thereby we get a clear signal from the periplasm with low interference from the cytoplasm.<br />
<br />
[[File:GFP in perimplasm RFP in cytoplasm.png|650px|thumbnail|upright=4|left|alt=Alt text|Overview microscopic pciture showing "Hello World" transformants.]]<br />
[[File:GFP in perimplasm RFP in cytoplasm close up.png|300px|thumbnail|upright=2|left|alt=Alt text|High resolution picture of "Hello World" transformants, showing a clear seperation of green and red fluorescence. GFP is primarily located in the periplasm while RFP is located in the cytoplasm. Fluorescence intensity measurements are taken along the cross-section indicated by the white line. Picture taken with a confocal microscope and ??? filter.]]<br />
[[File:Graf.PNG|290px|thumbnail|upright=2|left|alt=Alt text|Graph of flourescence measurement (or is this the model???) on green and red channel. It can be seen that the intensity of the red flourescence is restricted to the cytoplasm while green flourescence has it's peaks on the egdes. A weak green signal is measured for the cytoplasmic region because the periplasm envelopes the cytoplasm.]]<br />
<br />
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<br />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/pBAD_SPLTeam:DTU-Denmark/pBAD SPL2013-10-02T12:22:19Z<p>Hezscha: /* Example of use */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|pBAD SPL}}<br />
<br />
== pBAD synthetic promoter library ==<br />
<br />
As a tool for expressing lethal proteins in ''E. coli'' we made a synthetic promoter library (SPL, [http://dspace.mit.edu/handle/1721.1/60080 RFC 63]) with the pBAD arabinose inducible promoter. The concept was taken from the [https://2010.igem.org/Team:DTU-Denmark/SPL DTU iGEM team from 2010].<br />
<br />
== Methods ==<br />
<br />
=== Experimental procedure ===<br />
<br />
# Random promoter sequences were ordered matching the sequence CTGACGNNNNNNNNNNNNNNNNNNTAWWATNNNNA.<br />
# USER cloning to add RFP downstream of promoter.<br />
# Colonies were plated.<br />
# Plates were induced by spraying them with an aqueous arabinose solution.<br />
# Colonies that were not red prior to induction with arabinose but that did turn red after induction with arabinose were selected and re-innoculated as liquid cultures.<br />
# Biolector: Wells were inoculated from overnight cultures of each of the selected colonies. All wells were run in duplicate.<br />
# All duplicate colonies were run twice -- once with arabinose added at t=0, and again without arabinose. <br />
# The constitutive promoter TODO:which was used as a reference.<br />
<br />
=== Data analysis ===<br />
<br />
# Data was collected from the Biolector, and analyzed using a series of R scripts written by Chris Workman (unpublished). <br />
#* The maturation and degradation times for mCherry were both assumed to be 40 min. TODOref<br />
#* The growth rate, mu, was estimated to be 1.28 (from an average of all wells on all plates) since we expect each strain to grow at the same rate.<br />
#* A time window representing exponential growth was selected (between 1 and 4.5 hours).<br />
# The RFP measurement for a constitutively expressed strain was used as a standard measure of growth. This is plotted on the x-axis in the detailed plots per colony below. <br />
# Figures were plotted using R.<br />
<br />
== Results ==<br />
<br />
=== Summary ===<br />
<br />
Promoter activity when induced (with arabinose added) plotted vs basal activity (without arabinose; ie leakiness of the promoter). The colonies that we selected all show less activity than the the constitutive promoter, and when induced, show higher activity than the constitutive promoter. <br />
[[File:Induced_vs_basal.png]]<br />
<br />
=== Details ===<br />
<br />
Promoter strengths for two trials of each colony with and without arabinose. <br />
{| class="wikitable"<br />
!Colony Number<br />
!With arabinose 1<br />
!With arabinose 2<br />
!Without arabinose 1<br />
!Without arabinose 2<br />
|-<br />
|Col2||8.9025||7.8699||0.727||0.9552<br />
|-<br />
|Col3||9.2724||12.1142||0.5248||0.6982<br />
|-<br />
|Col4||9.4571||11.4522||0.5231||0.2508<br />
|-<br />
|Col12||6.3641||10.5389||0.5897||0.6869<br />
|-<br />
|Col10||7.9697||9.7949||0.3392||0.733<br />
|-<br />
|Col9||7.8563||20.1094||0.6995||0.7432<br />
|-<br />
|Col8||12.2318||15.4548||0.4203||0.4538<br />
|-<br />
|Col13||11.0377||7.3343||0.482||0.4641<br />
|-<br />
|Col15||15.6817||8.2707||0.8169||0.1343<br />
|-<br />
|Col18||14.7916||15.5736||0.6674||0.6745<br />
|-<br />
|Col19||14.2126||16.4898||0.4545||0.3566<br />
|-<br />
|Col29||7.1853||16.3467||0.5445||0.5013<br />
|-<br />
|Col26||9.7724||9.6269||0.7118||0.7865<br />
|-<br />
|Col22||8.4168||5.5958||0.6049||0.5645<br />
|-<br />
|Col33||9.1982||8.9987||0.6508||1.374<br />
|-<br />
|Col34||10.6987||7.883||0.5067||0.5031<br />
|-<br />
|Col35||13.8427||7.5469||0.4363||0.6281<br />
|-<br />
|ConRef||6.506||7.9323||8.7811||7.9323<br />
|}<br />
<br />
[[File:dtu-Fss-plot-col2.png|300px]]<br />
[[File:dtu-Fss-plot-col3.png|300px]]<br />
[[File:dtu-Fss-plot-col4.png|300px]]<br />
[[File:dtu-Fss-plot-col12.png|300px]]<br />
[[File:dtu-Fss-plot-col10.png|300px]]<br />
[[File:dtu-Fss-plot-col9.png|300px]]<br />
[[File:dtu-Fss-plot-col8.png|300px]]<br />
[[File:dtu-Fss-plot-col13.png|300px]]<br />
[[File:dtu-Fss-plot-col15.png|300px]]<br />
[[File:dtu-Fss-plot-col18.png|300px]]<br />
[[File:dtu-Fss-plot-col19.png|300px]]<br />
[[File:dtu-Fss-plot-col29.png|300px]]<br />
[[File:dut-Fss-plot-col26.png|300px]]<br />
[[File:dtu-Fss-plot-col22.png|300px]]<br />
[[File:dtu-Fss-plot-col33.png|300px]]<br />
[[File:dtu-Fss-plot-col34.png|300px]]<br />
[[File:dtu-Fss-plot-col25.png|300px]]<br />
[[File:dtu-Fss-plot-conref.png|300px]]<br />
<br />
== Example of use ==<br />
<br />
The tight inducible pBAD promoter was used in our [https://2013.igem.org/Team:DTU-Denmark/HelloWorld "Hello World project"] to regulate the expression of GFP SF, which was tagged with a signal peptide to direct it into the periplasm. Production and folding of GFP SF is faster than the transport system of ''E. coli'', which leads to undesired accumulation of GFP SF in the cytoplasm. Only when using a promoter with low leakiness it is possible to translocate a significant fraction of GFP SF after its production has been switched off. Thereby we get a clear signal from the periplasm with low interference from the cytoplasm.<br />
<br />
[[File:GFP in perimplasm RFP in cytoplasm.png|650px|thumbnail|upright=4|left|alt=Alt text|Overview microscopic pciture showing "Hello World" transformants.]]<br />
[[File:GFP in perimplasm RFP in cytoplasm close up.png|300px|thumbnail|upright=2|left|alt=Alt text|High resolution picture of "Hello World" transformants, showing a clear seperation of green and red fluorescence. GFP is primarily located in the periplasm while RFP is located in the cytoplasm. Fluorescence intensity measurements are taken along the cross-section indicated by the white line. Picture taken with a confocal microscope and ??? filter.]]<br />
[[File:Graf.PNG|290px|thumbnail|upright=2|left|alt=Alt text|Graph of flourescence measurement (or is this the model???) on green and red channel. It can be seen that the intensity of the red flourescence is restricted to the cytoplasm while green flourescence has it's peaks on the egdes. A weak green signal is measured for the cytoplasmic region because the periplasm envelopes the cytoplasm.]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/pBAD_SPLTeam:DTU-Denmark/pBAD SPL2013-10-02T12:02:23Z<p>Hezscha: /* Example of use */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|pBAD SPL}}<br />
<br />
== pBAD synthetic promoter library ==<br />
<br />
As a tool for expressing lethal proteins in ''E. coli'' we made a synthetic promoter library (SPL, [http://dspace.mit.edu/handle/1721.1/60080 RFC 63]) with the pBAD arabinose inducible promoter. The concept was taken from the [https://2010.igem.org/Team:DTU-Denmark/SPL DTU iGEM team from 2010].<br />
<br />
== Methods ==<br />
<br />
=== Experimental procedure ===<br />
<br />
# Random promoter sequences were ordered matching the sequence CTGACGNNNNNNNNNNNNNNNNNNTAWWATNNNNA.<br />
# USER cloning to add RFP downstream of promoter.<br />
# Colonies were plated.<br />
# Plates were induced by spraying them with an aqueous arabinose solution.<br />
# Colonies that were not red prior to induction with arabinose but that did turn red after induction with arabinose were selected and re-innoculated as liquid cultures.<br />
# Biolector: Wells were inoculated from overnight cultures of each of the selected colonies. All wells were run in duplicate.<br />
# All duplicate colonies were run twice -- once with arabinose added at t=0, and again without arabinose. <br />
# The constitutive promoter TODO:which was used as a reference.<br />
<br />
=== Data analysis ===<br />
<br />
# Data was collected from the Biolector, and analyzed using a series of R scripts written by Chris Workman (unpublished). <br />
#* The maturation and degradation times for mCherry were both assumed to be 40 min. TODOref<br />
#* The growth rate, mu, was estimated to be 1.28 (from an average of all wells on all plates) since we expect each strain to grow at the same rate.<br />
#* A time window representing exponential growth was selected (between 1 and 4.5 hours).<br />
# The RFP measurement for a constitutively expressed strain was used as a standard measure of growth. This is plotted on the x-axis in the detailed plots per colony below. <br />
# Figures were plotted using R.<br />
<br />
== Results ==<br />
<br />
=== Summary ===<br />
<br />
Promoter activity when induced (with arabinose added) plotted vs basal activity (without arabinose; ie leakiness of the promoter). The colonies that we selected all show less activity than the the constitutive promoter, and when induced, show higher activity than the constitutive promoter. <br />
[[File:Induced_vs_basal.png]]<br />
<br />
=== Details ===<br />
<br />
Promoter strengths for two trials of each colony with and without arabinose. <br />
{| class="wikitable"<br />
!Colony Number<br />
!With arabinose 1<br />
!With arabinose 2<br />
!Without arabinose 1<br />
!Without arabinose 2<br />
|-<br />
|Col2||8.9025||7.8699||0.727||0.9552<br />
|-<br />
|Col3||9.2724||12.1142||0.5248||0.6982<br />
|-<br />
|Col4||9.4571||11.4522||0.5231||0.2508<br />
|-<br />
|Col12||6.3641||10.5389||0.5897||0.6869<br />
|-<br />
|Col10||7.9697||9.7949||0.3392||0.733<br />
|-<br />
|Col9||7.8563||20.1094||0.6995||0.7432<br />
|-<br />
|Col8||12.2318||15.4548||0.4203||0.4538<br />
|-<br />
|Col13||11.0377||7.3343||0.482||0.4641<br />
|-<br />
|Col15||15.6817||8.2707||0.8169||0.1343<br />
|-<br />
|Col18||14.7916||15.5736||0.6674||0.6745<br />
|-<br />
|Col19||14.2126||16.4898||0.4545||0.3566<br />
|-<br />
|Col29||7.1853||16.3467||0.5445||0.5013<br />
|-<br />
|Col26||9.7724||9.6269||0.7118||0.7865<br />
|-<br />
|Col22||8.4168||5.5958||0.6049||0.5645<br />
|-<br />
|Col33||9.1982||8.9987||0.6508||1.374<br />
|-<br />
|Col34||10.6987||7.883||0.5067||0.5031<br />
|-<br />
|Col35||13.8427||7.5469||0.4363||0.6281<br />
|-<br />
|ConRef||6.506||7.9323||8.7811||7.9323<br />
|}<br />
<br />
[[File:dtu-Fss-plot-col2.png|300px]]<br />
[[File:dtu-Fss-plot-col3.png|300px]]<br />
[[File:dtu-Fss-plot-col4.png|300px]]<br />
[[File:dtu-Fss-plot-col12.png|300px]]<br />
[[File:dtu-Fss-plot-col10.png|300px]]<br />
[[File:dtu-Fss-plot-col9.png|300px]]<br />
[[File:dtu-Fss-plot-col8.png|300px]]<br />
[[File:dtu-Fss-plot-col13.png|300px]]<br />
[[File:dtu-Fss-plot-col15.png|300px]]<br />
[[File:dtu-Fss-plot-col18.png|300px]]<br />
[[File:dtu-Fss-plot-col19.png|300px]]<br />
[[File:dtu-Fss-plot-col29.png|300px]]<br />
[[File:dut-Fss-plot-col26.png|300px]]<br />
[[File:dtu-Fss-plot-col22.png|300px]]<br />
[[File:dtu-Fss-plot-col33.png|300px]]<br />
[[File:dtu-Fss-plot-col34.png|300px]]<br />
[[File:dtu-Fss-plot-col25.png|300px]]<br />
[[File:dtu-Fss-plot-conref.png|300px]]<br />
<br />
== Example of use ==<br />
<br />
The tight inducible pBAD promoter was used in our [https://2013.igem.org/Team:DTU-Denmark/HelloWorld "Hello World project"] to regulate the expression of GFP SF, which was tagged with a signal peptide to direct it into the periplasm. Production and folding of GFP SF is faster than the transport system of ''E. coli'', which leads to undesired accumulation of GFP SF in the cytoplasm. Only when using a promoter with low leakiness it is possible to translocate a significant fraction of GFP SF after its production has been switched off. Thereby we get a clear signal from the periplasm with low interference from the cytoplasm.<br />
<br />
[[File:GFP in perimplasm RFP in cytoplasm.png|650px|thumbnail|upright=4|left|alt=Alt text|Overview microscopic pciture showing "Hello World" transformants.]]<br />
[[File:GFP in perimplasm RFP in cytoplasm close up.png|300px|thumbnail|upright=2|left|alt=Alt text|High resolution picture of "Hello World" transformants, showing a clear seperation of green and red fluorescence. GFP is primarily located in the periplasm while RFP is located in the cytoplasm. Fluorescence intensity measurements are taken along the cross-section indicated by the white line. Picture taken with a confocal microscope and ??? filter.]]<br />
[[File:Graf.PNG|290px|thumbnail|upright=2|left|alt=Alt text|Graph of flourescence measurement (or is this the model???) on green and red channel. It can be seen that the intensity of the red flourescence is restricted to the cytoplasm while green flourescence has it's peaks on the egdes. A weak green signal is measured for the cytoplasmic region because the periplasm envelopes the cytoplasm.]]<br />
<br />
<br />
<br />
See also [https://2013.igem.org/Team:DTU-Denmark/HelloWorld "Hello World project"].<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/pBAD_SPLTeam:DTU-Denmark/pBAD SPL2013-10-02T11:55:05Z<p>Hezscha: /* Example of use */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|pBAD SPL}}<br />
<br />
== pBAD synthetic promoter library ==<br />
<br />
As a tool for expressing lethal proteins in ''E. coli'' we made a synthetic promoter library (SPL, [http://dspace.mit.edu/handle/1721.1/60080 RFC 63]) with the pBAD arabinose inducible promoter. The concept was taken from the [https://2010.igem.org/Team:DTU-Denmark/SPL DTU iGEM team from 2010].<br />
<br />
== Methods ==<br />
<br />
=== Experimental procedure ===<br />
<br />
# Random promoter sequences were ordered matching the sequence CTGACGNNNNNNNNNNNNNNNNNNTAWWATNNNNA.<br />
# USER cloning to add RFP downstream of promoter.<br />
# Colonies were plated.<br />
# Plates were induced by spraying them with an aqueous arabinose solution.<br />
# Colonies that were not red prior to induction with arabinose but that did turn red after induction with arabinose were selected and re-innoculated as liquid cultures.<br />
# Biolector: Wells were inoculated from overnight cultures of each of the selected colonies. All wells were run in duplicate.<br />
# All duplicate colonies were run twice -- once with arabinose added at t=0, and again without arabinose. <br />
# The constitutive promoter TODO:which was used as a reference.<br />
<br />
=== Data analysis ===<br />
<br />
# Data was collected from the Biolector, and analyzed using a series of R scripts written by Chris Workman (unpublished). <br />
#* The maturation and degradation times for mCherry were both assumed to be 40 min. TODOref<br />
#* The growth rate, mu, was estimated to be 1.28 (from an average of all wells on all plates) since we expect each strain to grow at the same rate.<br />
#* A time window representing exponential growth was selected (between 1 and 4.5 hours).<br />
# The RFP measurement for a constitutively expressed strain was used as a standard measure of growth. This is plotted on the x-axis in the detailed plots per colony below. <br />
# Figures were plotted using R.<br />
<br />
== Results ==<br />
<br />
=== Summary ===<br />
<br />
Promoter activity when induced (with arabinose added) plotted vs basal activity (without arabinose; ie leakiness of the promoter). The colonies that we selected all show less activity than the the constitutive promoter, and when induced, show higher activity than the constitutive promoter. <br />
[[File:Induced_vs_basal.png]]<br />
<br />
=== Details ===<br />
<br />
Promoter strengths for two trials of each colony with and without arabinose. <br />
{| class="wikitable"<br />
!Colony Number<br />
!With arabinose 1<br />
!With arabinose 2<br />
!Without arabinose 1<br />
!Without arabinose 2<br />
|-<br />
|Col2||8.9025||7.8699||0.727||0.9552<br />
|-<br />
|Col3||9.2724||12.1142||0.5248||0.6982<br />
|-<br />
|Col4||9.4571||11.4522||0.5231||0.2508<br />
|-<br />
|Col12||6.3641||10.5389||0.5897||0.6869<br />
|-<br />
|Col10||7.9697||9.7949||0.3392||0.733<br />
|-<br />
|Col9||7.8563||20.1094||0.6995||0.7432<br />
|-<br />
|Col8||12.2318||15.4548||0.4203||0.4538<br />
|-<br />
|Col13||11.0377||7.3343||0.482||0.4641<br />
|-<br />
|Col15||15.6817||8.2707||0.8169||0.1343<br />
|-<br />
|Col18||14.7916||15.5736||0.6674||0.6745<br />
|-<br />
|Col19||14.2126||16.4898||0.4545||0.3566<br />
|-<br />
|Col29||7.1853||16.3467||0.5445||0.5013<br />
|-<br />
|Col26||9.7724||9.6269||0.7118||0.7865<br />
|-<br />
|Col22||8.4168||5.5958||0.6049||0.5645<br />
|-<br />
|Col33||9.1982||8.9987||0.6508||1.374<br />
|-<br />
|Col34||10.6987||7.883||0.5067||0.5031<br />
|-<br />
|Col35||13.8427||7.5469||0.4363||0.6281<br />
|-<br />
|ConRef||6.506||7.9323||8.7811||7.9323<br />
|}<br />
<br />
[[File:dtu-Fss-plot-col2.png|300px]]<br />
[[File:dtu-Fss-plot-col3.png|300px]]<br />
[[File:dtu-Fss-plot-col4.png|300px]]<br />
[[File:dtu-Fss-plot-col12.png|300px]]<br />
[[File:dtu-Fss-plot-col10.png|300px]]<br />
[[File:dtu-Fss-plot-col9.png|300px]]<br />
[[File:dtu-Fss-plot-col8.png|300px]]<br />
[[File:dtu-Fss-plot-col13.png|300px]]<br />
[[File:dtu-Fss-plot-col15.png|300px]]<br />
[[File:dtu-Fss-plot-col18.png|300px]]<br />
[[File:dtu-Fss-plot-col19.png|300px]]<br />
[[File:dtu-Fss-plot-col29.png|300px]]<br />
[[File:dut-Fss-plot-col26.png|300px]]<br />
[[File:dtu-Fss-plot-col22.png|300px]]<br />
[[File:dtu-Fss-plot-col33.png|300px]]<br />
[[File:dtu-Fss-plot-col34.png|300px]]<br />
[[File:dtu-Fss-plot-col25.png|300px]]<br />
[[File:dtu-Fss-plot-conref.png|300px]]<br />
<br />
== Example of use ==<br />
<br />
The tight inducible pBAD promoter was used in our [https://2013.igem.org/Team:DTU-Denmark/HelloWorld "Hello World project"] to regulate the expression of GFP SF, which was tagged with a signal peptide to direct it into the periplasm. Production and folding of GFP SF is faster than the transport system of ''E. coli'', which leads to undesired accumulation of GFP SF in the cytoplasm. Only when using a promoter with low leakiness it is possible to translocate a significant fraction of GFP SF after its production has been switched off. Thereby we get a clear signal from the periplasm with low interference from the cytoplasm.<br />
<br />
[[File:GFP in perimplasm RFP in cytoplasm.png|650px|thumbnail|upright=4|left|alt=Alt text|Overview microscopic pciture showing "Hello World" transformants.]]<br />
[[File:GFP in perimplasm RFP in cytoplasm close up.png|300px|thumbnail|upright=2|left|alt=Alt text|High resolution picture of "Hello World" transformants, showing a clear seperation of green and red fluoresence. GFP is primarily located in the periplasm while RFP is located in the cytoplasm. Picture taken with a confocal microscope and ??? filter.]]<br />
[[File:Graf.PNG|290px|thumbnail|upright=2|left|alt=Alt text|Caption text]]<br />
<br />
<br />
<br />
See also [https://2013.igem.org/Team:DTU-Denmark/HelloWorld "Hello World project"].<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/pBAD_SPLTeam:DTU-Denmark/pBAD SPL2013-10-02T11:52:41Z<p>Hezscha: /* Example of use */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|pBAD SPL}}<br />
<br />
== pBAD synthetic promoter library ==<br />
<br />
As a tool for expressing lethal proteins in ''E. coli'' we made a synthetic promoter library (SPL, [http://dspace.mit.edu/handle/1721.1/60080 RFC 63]) with the pBAD arabinose inducible promoter. The concept was taken from the [https://2010.igem.org/Team:DTU-Denmark/SPL DTU iGEM team from 2010].<br />
<br />
== Methods ==<br />
<br />
=== Experimental procedure ===<br />
<br />
# Random promoter sequences were ordered matching the sequence CTGACGNNNNNNNNNNNNNNNNNNTAWWATNNNNA.<br />
# USER cloning to add RFP downstream of promoter.<br />
# Colonies were plated.<br />
# Plates were induced by spraying them with an aqueous arabinose solution.<br />
# Colonies that were not red prior to induction with arabinose but that did turn red after induction with arabinose were selected and re-innoculated as liquid cultures.<br />
# Biolector: Wells were inoculated from overnight cultures of each of the selected colonies. All wells were run in duplicate.<br />
# All duplicate colonies were run twice -- once with arabinose added at t=0, and again without arabinose. <br />
# The constitutive promoter TODO:which was used as a reference.<br />
<br />
=== Data analysis ===<br />
<br />
# Data was collected from the Biolector, and analyzed using a series of R scripts written by Chris Workman (unpublished). <br />
#* The maturation and degradation times for mCherry were both assumed to be 40 min. TODOref<br />
#* The growth rate, mu, was estimated to be 1.28 (from an average of all wells on all plates) since we expect each strain to grow at the same rate.<br />
#* A time window representing exponential growth was selected (between 1 and 4.5 hours).<br />
# The RFP measurement for a constitutively expressed strain was used as a standard measure of growth. This is plotted on the x-axis in the detailed plots per colony below. <br />
# Figures were plotted using R.<br />
<br />
== Results ==<br />
<br />
=== Summary ===<br />
<br />
Promoter activity when induced (with arabinose added) plotted vs basal activity (without arabinose; ie leakiness of the promoter). The colonies that we selected all show less activity than the the constitutive promoter, and when induced, show higher activity than the constitutive promoter. <br />
[[File:Induced_vs_basal.png]]<br />
<br />
=== Details ===<br />
<br />
Promoter strengths for two trials of each colony with and without arabinose. <br />
{| class="wikitable"<br />
!Colony Number<br />
!With arabinose 1<br />
!With arabinose 2<br />
!Without arabinose 1<br />
!Without arabinose 2<br />
|-<br />
|Col2||8.9025||7.8699||0.727||0.9552<br />
|-<br />
|Col3||9.2724||12.1142||0.5248||0.6982<br />
|-<br />
|Col4||9.4571||11.4522||0.5231||0.2508<br />
|-<br />
|Col12||6.3641||10.5389||0.5897||0.6869<br />
|-<br />
|Col10||7.9697||9.7949||0.3392||0.733<br />
|-<br />
|Col9||7.8563||20.1094||0.6995||0.7432<br />
|-<br />
|Col8||12.2318||15.4548||0.4203||0.4538<br />
|-<br />
|Col13||11.0377||7.3343||0.482||0.4641<br />
|-<br />
|Col15||15.6817||8.2707||0.8169||0.1343<br />
|-<br />
|Col18||14.7916||15.5736||0.6674||0.6745<br />
|-<br />
|Col19||14.2126||16.4898||0.4545||0.3566<br />
|-<br />
|Col29||7.1853||16.3467||0.5445||0.5013<br />
|-<br />
|Col26||9.7724||9.6269||0.7118||0.7865<br />
|-<br />
|Col22||8.4168||5.5958||0.6049||0.5645<br />
|-<br />
|Col33||9.1982||8.9987||0.6508||1.374<br />
|-<br />
|Col34||10.6987||7.883||0.5067||0.5031<br />
|-<br />
|Col35||13.8427||7.5469||0.4363||0.6281<br />
|-<br />
|ConRef||6.506||7.9323||8.7811||7.9323<br />
|}<br />
<br />
[[File:dtu-Fss-plot-col2.png|300px]]<br />
[[File:dtu-Fss-plot-col3.png|300px]]<br />
[[File:dtu-Fss-plot-col4.png|300px]]<br />
[[File:dtu-Fss-plot-col12.png|300px]]<br />
[[File:dtu-Fss-plot-col10.png|300px]]<br />
[[File:dtu-Fss-plot-col9.png|300px]]<br />
[[File:dtu-Fss-plot-col8.png|300px]]<br />
[[File:dtu-Fss-plot-col13.png|300px]]<br />
[[File:dtu-Fss-plot-col15.png|300px]]<br />
[[File:dtu-Fss-plot-col18.png|300px]]<br />
[[File:dtu-Fss-plot-col19.png|300px]]<br />
[[File:dtu-Fss-plot-col29.png|300px]]<br />
[[File:dut-Fss-plot-col26.png|300px]]<br />
[[File:dtu-Fss-plot-col22.png|300px]]<br />
[[File:dtu-Fss-plot-col33.png|300px]]<br />
[[File:dtu-Fss-plot-col34.png|300px]]<br />
[[File:dtu-Fss-plot-col25.png|300px]]<br />
[[File:dtu-Fss-plot-conref.png|300px]]<br />
<br />
== Example of use ==<br />
<br />
The tight inducible pBAD promoter was used in our [https://2013.igem.org/Team:DTU-Denmark/HelloWorld "Hello World project"] to regulate the expression of GFP SF, which was tagged with a signal peptide to direct it into the periplasm. Production and folding of GFP SF is faster than the transport system of ''E. coli'', which leads to undesired accumulation of GFP SF in the cytoplasm. Only when using a promoter with low leakiness it is possible to translocate a significant fraction of GFP SF after its production has been switched off. Thereby we get a clear signal from the periplasm with low interference from the cytoplasm.<br />
<br />
[[File:GFP in perimplasm RFP in cytoplasm.png|650px|thumbnail|upright=4|left|alt=Alt text|Overview showing "Hello World" transformants.]]<br />
[[File:GFP in perimplasm RFP in cytoplasm close up.png|300px|thumbnail|upright=2|left|alt=Alt text|High resolution picture of "Hello World" transformants, showing a clear seperation of green and red fluoresence. GFP is primarily located in the periplasm while RFP is located in the cytoplasm. Picture taken with a confocal microscope and ??? filter.]]<br />
[[File:Graf.PNG|290px|thumbnail|upright=2|left|alt=Alt text|Caption text]]<br />
<br />
<br />
<br />
See also [https://2013.igem.org/Team:DTU-Denmark/HelloWorld "Hello World project"].<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/pBAD_SPLTeam:DTU-Denmark/pBAD SPL2013-10-02T11:42:34Z<p>Hezscha: /* Example of use */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|pBAD SPL}}<br />
<br />
== pBAD synthetic promoter library ==<br />
<br />
As a tool for expressing lethal proteins in ''E. coli'' we made a synthetic promoter library (SPL, [http://dspace.mit.edu/handle/1721.1/60080 RFC 63]) with the pBAD arabinose inducible promoter. The concept was taken from the [https://2010.igem.org/Team:DTU-Denmark/SPL DTU iGEM team from 2010].<br />
<br />
== Methods ==<br />
<br />
=== Experimental procedure ===<br />
<br />
# Random promoter sequences were ordered matching the sequence CTGACGNNNNNNNNNNNNNNNNNNTAWWATNNNNA.<br />
# USER cloning to add RFP downstream of promoter.<br />
# Colonies were plated.<br />
# Plates were induced by spraying them with an aqueous arabinose solution.<br />
# Colonies that were not red prior to induction with arabinose but that did turn red after induction with arabinose were selected and re-innoculated as liquid cultures.<br />
# Biolector: Wells were inoculated from overnight cultures of each of the selected colonies. All wells were run in duplicate.<br />
# All duplicate colonies were run twice -- once with arabinose added at t=0, and again without arabinose. <br />
# The constitutive promoter TODO:which was used as a reference.<br />
<br />
=== Data analysis ===<br />
<br />
# Data was collected from the Biolector, and analyzed using a series of R scripts written by Chris Workman (unpublished). <br />
#* The maturation and degradation times for mCherry were both assumed to be 40 min. TODOref<br />
#* The growth rate, mu, was estimated to be 1.28 (from an average of all wells on all plates) since we expect each strain to grow at the same rate.<br />
#* A time window representing exponential growth was selected (between 1 and 4.5 hours).<br />
# The RFP measurement for a constitutively expressed strain was used as a standard measure of growth. This is plotted on the x-axis in the detailed plots per colony below. <br />
# Figures were plotted using R.<br />
<br />
== Results ==<br />
<br />
=== Summary ===<br />
<br />
Promoter activity when induced (with arabinose added) plotted vs basal activity (without arabinose; ie leakiness of the promoter). The colonies that we selected all show less activity than the the constitutive promoter, and when induced, show higher activity than the constitutive promoter. <br />
[[File:Induced_vs_basal.png]]<br />
<br />
=== Details ===<br />
<br />
Promoter strengths for two trials of each colony with and without arabinose. <br />
{| class="wikitable"<br />
!Colony Number<br />
!With arabinose 1<br />
!With arabinose 2<br />
!Without arabinose 1<br />
!Without arabinose 2<br />
|-<br />
|Col2||8.9025||7.8699||0.727||0.9552<br />
|-<br />
|Col3||9.2724||12.1142||0.5248||0.6982<br />
|-<br />
|Col4||9.4571||11.4522||0.5231||0.2508<br />
|-<br />
|Col12||6.3641||10.5389||0.5897||0.6869<br />
|-<br />
|Col10||7.9697||9.7949||0.3392||0.733<br />
|-<br />
|Col9||7.8563||20.1094||0.6995||0.7432<br />
|-<br />
|Col8||12.2318||15.4548||0.4203||0.4538<br />
|-<br />
|Col13||11.0377||7.3343||0.482||0.4641<br />
|-<br />
|Col15||15.6817||8.2707||0.8169||0.1343<br />
|-<br />
|Col18||14.7916||15.5736||0.6674||0.6745<br />
|-<br />
|Col19||14.2126||16.4898||0.4545||0.3566<br />
|-<br />
|Col29||7.1853||16.3467||0.5445||0.5013<br />
|-<br />
|Col26||9.7724||9.6269||0.7118||0.7865<br />
|-<br />
|Col22||8.4168||5.5958||0.6049||0.5645<br />
|-<br />
|Col33||9.1982||8.9987||0.6508||1.374<br />
|-<br />
|Col34||10.6987||7.883||0.5067||0.5031<br />
|-<br />
|Col35||13.8427||7.5469||0.4363||0.6281<br />
|-<br />
|ConRef||6.506||7.9323||8.7811||7.9323<br />
|}<br />
<br />
[[File:dtu-Fss-plot-col2.png|300px]]<br />
[[File:dtu-Fss-plot-col3.png|300px]]<br />
[[File:dtu-Fss-plot-col4.png|300px]]<br />
[[File:dtu-Fss-plot-col12.png|300px]]<br />
[[File:dtu-Fss-plot-col10.png|300px]]<br />
[[File:dtu-Fss-plot-col9.png|300px]]<br />
[[File:dtu-Fss-plot-col8.png|300px]]<br />
[[File:dtu-Fss-plot-col13.png|300px]]<br />
[[File:dtu-Fss-plot-col15.png|300px]]<br />
[[File:dtu-Fss-plot-col18.png|300px]]<br />
[[File:dtu-Fss-plot-col19.png|300px]]<br />
[[File:dtu-Fss-plot-col29.png|300px]]<br />
[[File:dut-Fss-plot-col26.png|300px]]<br />
[[File:dtu-Fss-plot-col22.png|300px]]<br />
[[File:dtu-Fss-plot-col33.png|300px]]<br />
[[File:dtu-Fss-plot-col34.png|300px]]<br />
[[File:dtu-Fss-plot-col25.png|300px]]<br />
[[File:dtu-Fss-plot-conref.png|300px]]<br />
<br />
== Example of use ==<br />
<br />
The tight inducible pBAD promoter was used in our [https://2013.igem.org/Team:DTU-Denmark/HelloWorld "Hello World project"] to regulate the expression of GFP SF, which was tagged with a signal peptide to direct it into the periplasm. Production and folding of GFP SF is faster than the transport system of ''E. coli'', which leads to undesired accumulation of GFP SF in the cytoplasm. Only when using a promoter with low leakiness it is possible to translocate a significant fraction of GFP SF after its production has been switched off. Thereby we get a clear signal from the periplasm with low interference from the cytoplasm.<br />
<br />
[[File:GFP in perimplasm RFP in cytoplasm.png|650px|thumbnail|upright=4|left|alt=Alt text|Caption text]]<br />
[[File:GFP in perimplasm RFP in cytoplasm close up.png|300px|thumbnail|upright=2|left|alt=Alt text|Caption text]]<br />
[[File:Graf.PNG|290px|thumbnail|upright=2|left|alt=Alt text|Caption text]]<br />
<br />
<br />
<br />
See also [https://2013.igem.org/Team:DTU-Denmark/HelloWorld "Hello World project"].<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/pBAD_SPLTeam:DTU-Denmark/pBAD SPL2013-10-02T11:41:42Z<p>Hezscha: /* Example of use */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|pBAD SPL}}<br />
<br />
== pBAD synthetic promoter library ==<br />
<br />
As a tool for expressing lethal proteins in ''E. coli'' we made a synthetic promoter library (SPL, [http://dspace.mit.edu/handle/1721.1/60080 RFC 63]) with the pBAD arabinose inducible promoter. The concept was taken from the [https://2010.igem.org/Team:DTU-Denmark/SPL DTU iGEM team from 2010].<br />
<br />
== Methods ==<br />
<br />
=== Experimental procedure ===<br />
<br />
# Random promoter sequences were ordered matching the sequence CTGACGNNNNNNNNNNNNNNNNNNTAWWATNNNNA.<br />
# USER cloning to add RFP downstream of promoter.<br />
# Colonies were plated.<br />
# Plates were induced by spraying them with an aqueous arabinose solution.<br />
# Colonies that were not red prior to induction with arabinose but that did turn red after induction with arabinose were selected and re-innoculated as liquid cultures.<br />
# Biolector: Wells were inoculated from overnight cultures of each of the selected colonies. All wells were run in duplicate.<br />
# All duplicate colonies were run twice -- once with arabinose added at t=0, and again without arabinose. <br />
# The constitutive promoter TODO:which was used as a reference.<br />
<br />
=== Data analysis ===<br />
<br />
# Data was collected from the Biolector, and analyzed using a series of R scripts written by Chris Workman (unpublished). <br />
#* The maturation and degradation times for mCherry were both assumed to be 40 min. TODOref<br />
#* The growth rate, mu, was estimated to be 1.28 (from an average of all wells on all plates) since we expect each strain to grow at the same rate.<br />
#* A time window representing exponential growth was selected (between 1 and 4.5 hours).<br />
# The RFP measurement for a constitutively expressed strain was used as a standard measure of growth. This is plotted on the x-axis in the detailed plots per colony below. <br />
# Figures were plotted using R.<br />
<br />
== Results ==<br />
<br />
=== Summary ===<br />
<br />
Promoter activity when induced (with arabinose added) plotted vs basal activity (without arabinose; ie leakiness of the promoter). The colonies that we selected all show less activity than the the constitutive promoter, and when induced, show higher activity than the constitutive promoter. <br />
[[File:Induced_vs_basal.png]]<br />
<br />
=== Details ===<br />
<br />
Promoter strengths for two trials of each colony with and without arabinose. <br />
{| class="wikitable"<br />
!Colony Number<br />
!With arabinose 1<br />
!With arabinose 2<br />
!Without arabinose 1<br />
!Without arabinose 2<br />
|-<br />
|Col2||8.9025||7.8699||0.727||0.9552<br />
|-<br />
|Col3||9.2724||12.1142||0.5248||0.6982<br />
|-<br />
|Col4||9.4571||11.4522||0.5231||0.2508<br />
|-<br />
|Col12||6.3641||10.5389||0.5897||0.6869<br />
|-<br />
|Col10||7.9697||9.7949||0.3392||0.733<br />
|-<br />
|Col9||7.8563||20.1094||0.6995||0.7432<br />
|-<br />
|Col8||12.2318||15.4548||0.4203||0.4538<br />
|-<br />
|Col13||11.0377||7.3343||0.482||0.4641<br />
|-<br />
|Col15||15.6817||8.2707||0.8169||0.1343<br />
|-<br />
|Col18||14.7916||15.5736||0.6674||0.6745<br />
|-<br />
|Col19||14.2126||16.4898||0.4545||0.3566<br />
|-<br />
|Col29||7.1853||16.3467||0.5445||0.5013<br />
|-<br />
|Col26||9.7724||9.6269||0.7118||0.7865<br />
|-<br />
|Col22||8.4168||5.5958||0.6049||0.5645<br />
|-<br />
|Col33||9.1982||8.9987||0.6508||1.374<br />
|-<br />
|Col34||10.6987||7.883||0.5067||0.5031<br />
|-<br />
|Col35||13.8427||7.5469||0.4363||0.6281<br />
|-<br />
|ConRef||6.506||7.9323||8.7811||7.9323<br />
|}<br />
<br />
[[File:dtu-Fss-plot-col2.png|300px]]<br />
[[File:dtu-Fss-plot-col3.png|300px]]<br />
[[File:dtu-Fss-plot-col4.png|300px]]<br />
[[File:dtu-Fss-plot-col12.png|300px]]<br />
[[File:dtu-Fss-plot-col10.png|300px]]<br />
[[File:dtu-Fss-plot-col9.png|300px]]<br />
[[File:dtu-Fss-plot-col8.png|300px]]<br />
[[File:dtu-Fss-plot-col13.png|300px]]<br />
[[File:dtu-Fss-plot-col15.png|300px]]<br />
[[File:dtu-Fss-plot-col18.png|300px]]<br />
[[File:dtu-Fss-plot-col19.png|300px]]<br />
[[File:dtu-Fss-plot-col29.png|300px]]<br />
[[File:dut-Fss-plot-col26.png|300px]]<br />
[[File:dtu-Fss-plot-col22.png|300px]]<br />
[[File:dtu-Fss-plot-col33.png|300px]]<br />
[[File:dtu-Fss-plot-col34.png|300px]]<br />
[[File:dtu-Fss-plot-col25.png|300px]]<br />
[[File:dtu-Fss-plot-conref.png|300px]]<br />
<br />
== Example of use ==<br />
<br />
The tight inducible pBAD promoter was used in our [https://2013.igem.org/Team:DTU-Denmark/HelloWorld "Hello World project"] to regulate the expression of GFP SF, which was tagged with a signal peptide to direct it into the periplasm. Production and folding of GFP SF is faster than the transport system of ''E. coli'', which leads to undesired accumulation of GFP SF in the cytoplasm. Only when using a promoter with low leakiness it is possible to translocate a significant fraction of GFP SF and get a clear signal from the periplasm with low interference from the cytoplasm.<br />
<br />
[[File:GFP in perimplasm RFP in cytoplasm.png|650px|thumbnail|upright=4|left|alt=Alt text|Caption text]]<br />
[[File:GFP in perimplasm RFP in cytoplasm close up.png|300px|thumbnail|upright=2|left|alt=Alt text|Caption text]]<br />
[[File:Graf.PNG|290px|thumbnail|upright=2|left|alt=Alt text|Caption text]]<br />
<br />
<br />
<br />
See also [https://2013.igem.org/Team:DTU-Denmark/HelloWorld "Hello World project"].<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/pBAD_SPLTeam:DTU-Denmark/pBAD SPL2013-10-02T11:40:58Z<p>Hezscha: /* Example of use */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|pBAD SPL}}<br />
<br />
== pBAD synthetic promoter library ==<br />
<br />
As a tool for expressing lethal proteins in ''E. coli'' we made a synthetic promoter library (SPL, [http://dspace.mit.edu/handle/1721.1/60080 RFC 63]) with the pBAD arabinose inducible promoter. The concept was taken from the [https://2010.igem.org/Team:DTU-Denmark/SPL DTU iGEM team from 2010].<br />
<br />
== Methods ==<br />
<br />
=== Experimental procedure ===<br />
<br />
# Random promoter sequences were ordered matching the sequence CTGACGNNNNNNNNNNNNNNNNNNTAWWATNNNNA.<br />
# USER cloning to add RFP downstream of promoter.<br />
# Colonies were plated.<br />
# Plates were induced by spraying them with an aqueous arabinose solution.<br />
# Colonies that were not red prior to induction with arabinose but that did turn red after induction with arabinose were selected and re-innoculated as liquid cultures.<br />
# Biolector: Wells were inoculated from overnight cultures of each of the selected colonies. All wells were run in duplicate.<br />
# All duplicate colonies were run twice -- once with arabinose added at t=0, and again without arabinose. <br />
# The constitutive promoter TODO:which was used as a reference.<br />
<br />
=== Data analysis ===<br />
<br />
# Data was collected from the Biolector, and analyzed using a series of R scripts written by Chris Workman (unpublished). <br />
#* The maturation and degradation times for mCherry were both assumed to be 40 min. TODOref<br />
#* The growth rate, mu, was estimated to be 1.28 (from an average of all wells on all plates) since we expect each strain to grow at the same rate.<br />
#* A time window representing exponential growth was selected (between 1 and 4.5 hours).<br />
# The RFP measurement for a constitutively expressed strain was used as a standard measure of growth. This is plotted on the x-axis in the detailed plots per colony below. <br />
# Figures were plotted using R.<br />
<br />
== Results ==<br />
<br />
=== Summary ===<br />
<br />
Promoter activity when induced (with arabinose added) plotted vs basal activity (without arabinose; ie leakiness of the promoter). The colonies that we selected all show less activity than the the constitutive promoter, and when induced, show higher activity than the constitutive promoter. <br />
[[File:Induced_vs_basal.png]]<br />
<br />
=== Details ===<br />
<br />
Promoter strengths for two trials of each colony with and without arabinose. <br />
{| class="wikitable"<br />
!Colony Number<br />
!With arabinose 1<br />
!With arabinose 2<br />
!Without arabinose 1<br />
!Without arabinose 2<br />
|-<br />
|Col2||8.9025||7.8699||0.727||0.9552<br />
|-<br />
|Col3||9.2724||12.1142||0.5248||0.6982<br />
|-<br />
|Col4||9.4571||11.4522||0.5231||0.2508<br />
|-<br />
|Col12||6.3641||10.5389||0.5897||0.6869<br />
|-<br />
|Col10||7.9697||9.7949||0.3392||0.733<br />
|-<br />
|Col9||7.8563||20.1094||0.6995||0.7432<br />
|-<br />
|Col8||12.2318||15.4548||0.4203||0.4538<br />
|-<br />
|Col13||11.0377||7.3343||0.482||0.4641<br />
|-<br />
|Col15||15.6817||8.2707||0.8169||0.1343<br />
|-<br />
|Col18||14.7916||15.5736||0.6674||0.6745<br />
|-<br />
|Col19||14.2126||16.4898||0.4545||0.3566<br />
|-<br />
|Col29||7.1853||16.3467||0.5445||0.5013<br />
|-<br />
|Col26||9.7724||9.6269||0.7118||0.7865<br />
|-<br />
|Col22||8.4168||5.5958||0.6049||0.5645<br />
|-<br />
|Col33||9.1982||8.9987||0.6508||1.374<br />
|-<br />
|Col34||10.6987||7.883||0.5067||0.5031<br />
|-<br />
|Col35||13.8427||7.5469||0.4363||0.6281<br />
|-<br />
|ConRef||6.506||7.9323||8.7811||7.9323<br />
|}<br />
<br />
[[File:dtu-Fss-plot-col2.png|300px]]<br />
[[File:dtu-Fss-plot-col3.png|300px]]<br />
[[File:dtu-Fss-plot-col4.png|300px]]<br />
[[File:dtu-Fss-plot-col12.png|300px]]<br />
[[File:dtu-Fss-plot-col10.png|300px]]<br />
[[File:dtu-Fss-plot-col9.png|300px]]<br />
[[File:dtu-Fss-plot-col8.png|300px]]<br />
[[File:dtu-Fss-plot-col13.png|300px]]<br />
[[File:dtu-Fss-plot-col15.png|300px]]<br />
[[File:dtu-Fss-plot-col18.png|300px]]<br />
[[File:dtu-Fss-plot-col19.png|300px]]<br />
[[File:dtu-Fss-plot-col29.png|300px]]<br />
[[File:dut-Fss-plot-col26.png|300px]]<br />
[[File:dtu-Fss-plot-col22.png|300px]]<br />
[[File:dtu-Fss-plot-col33.png|300px]]<br />
[[File:dtu-Fss-plot-col34.png|300px]]<br />
[[File:dtu-Fss-plot-col25.png|300px]]<br />
[[File:dtu-Fss-plot-conref.png|300px]]<br />
<br />
== Example of use ==<br />
<br />
The tight inducible pBAD promoter was used in our [https://2013.igem.org/Team:DTU-Denmark/HelloWorld "Hello World project"] to regulate the expression of GFP SF, which was tagged with a signal peptide to direct it into the periplasm. Production and folding of GFP SF is faster than the transport system of E. coli, which leads to undesired accumulation of GFP SF in the cytoplasm. Only when using a promoter with low leakiness it is possible to translocate a significant fraction of GFP SF and get a clear signal from the periplasm with low interference from the cytoplasm.<br />
<br />
[[File:GFP in perimplasm RFP in cytoplasm.png|650px|thumbnail|upright=4|left|alt=Alt text|Caption text]]<br />
[[File:GFP in perimplasm RFP in cytoplasm close up.png|300px|thumbnail|upright=2|left|alt=Alt text|Caption text]]<br />
[[File:Graf.PNG|290px|thumbnail|upright=2|left|alt=Alt text|Caption text]]<br />
<br />
<br />
<br />
See also [https://2013.igem.org/Team:DTU-Denmark/HelloWorld "Hello World project"].<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/NotebookTeam:DTU-Denmark/Notebook2013-10-02T11:34:49Z<p>Hezscha: </p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|Notebook}}<br />
__NOTOC__<br />
<br />
Our team decided to keep an online notebook for our lab work. Click in the calendar to see the entry for a specific date.<br />
<br />
<div class="overviewPage"><br />
==Lab Book Entries==<br />
<br />
{| style="width: 60%; height: 200px;" border="1" <br />
|-<br />
| <center> {{#calendar: title=Team:DTU-Denmark/Notebook |year=2013 | month=06}} </center><br />
| <center> {{#calendar: title=Team:DTU-Denmark/Notebook |year=2013 | month=07}} </center><br />
| <center> {{#calendar: title=Team:DTU-Denmark/Notebook |year=2013 | month=08}} </center><br />
| <center> {{#calendar: title=Team:DTU-Denmark/Notebook |year=2013 | month=09}} </center><br />
|}<br />
<br />
<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-02T11:32:58Z<p>Hezscha: /* Plasmids */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
Global demand for fixed nitrogen has increased to the point that half the human population now relies on chemical fertilizer to grow their food. While fertilizer is a requirement for modern life, runoff from overfertilized farmland can cause eutrophication. In the presence of abundant ammonia, algae overgrow and consume the much of the available oxygen in the water. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
Using two ''E. coli'' mutants built with genes from ''Nitrosomonas europaea'' and ''Pseudomonas aeruginosa'', we provide a system to reverse nitrogen fixation. Our mutants consume ammonia and produce nitrous oxide, releases a sustainable source of energy when decomposed into nitrogen and oxygen. We also provide a prototype of a bioreactor that could be scaled up and deployed in the field to simultaneously clean the water and produce energy.<br />
<br />
==Details==<br />
<br />
=== Mutant 1: Aerobic ===<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
The first mutant incorporates [[Team:DTU-Denmark/Protein_Models#AMO|AMO]], [[Team:DTU-Denmark/Protein_Models#Hao|HAO]] and two cytochromes [[Team:DTU-Denmark/Protein_Models#Cc554|c554]] and [[Team:DTU-Denmark/Protein_Models#Ccm552|cm552]] from ''Nitrosomonas europaea'' (shown in blue). AMO is a 3 subunit protein which converts ammonia (NH<sub>4</sub> to an intermediate called hydroxylamine (NH<sub>2</sub>OH). HAO then converts the hydroxylamine to nitrite (NO<sub>2</sub><sup>-</sup>. During this conversion process, cytochrome c554 accepts an electron from HAO, and then passes this electron on to ccm552. The terminal electron acceptor in this chain is quinone. AMO and ccm552 are embedded in the cytoplasmic membrane, and all other proteins in this process are found in the periplasm. <br />
<br />
=== Mutant 2: Anaerobic ===<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
The second mutant incorporates the Nir region from ''Pseudomonas aeruginosa'' (shown in yellow). [[Team:DTU-Denmark/Protein_Models#NirS|NirS]] converts nitrite to nitric oxide (NO), while removing an electron from [[Team:DTU-Denmark/Protein_Models#NirM|NirM]]. The remainder of the Nir region is necessary for the synthesis of NirS, and so we have included these genes as well. [[Team:DTU-Denmark/Protein_Models#NOR|NOR]], which is present natively in ''E. coli'', converts nitric oxide to nitrous oxide (N<sub>2</sub>O). In contrast to ''N. europaea'', ''E. coli'' is not a nitrifying bacterium and so does not convert nitrous oxide into nitrogen to complete the denitrification process.<br />
<br />
== Plasmids ==<br />
<br />
=== Mutant 1 ===<br />
<br />
The final plasmid for Mutant 1 should be the following which includes all genes on one plasmid:<br />
<br />
[[File:dtu_Plasmid_mutant_1-01.png|200px]]<br />
<br />
In order to simplify and to be able to debug each part of this pathway separately, we first implemented the genes individually. Unfortunately, we did not have time to combine all the genes into the final plasmid.<br />
<br />
[[File:Dtu Plasmids amo.png|120px]] [[File:Dtu Plasmids hao-01.png|150px]][[File:Dtu_Plasmids_cyt-01.png|150px]]<br />
<br />
=== Mutant 2 ===<br />
<br />
The final plasmid for Mutant 2 should be the following:<br />
<br />
[[File:Dtu Plasmid mutant 2-01.png|200px]]<br />
<br />
Since we had difficulty extracting Nir from ''Pseudomonas aeruginosa'' in one piece, we split it into two pieces to be extracted seperately and then re-combined them with USER cloning.<br />
<br />
== User Cloning ==<br />
<br />
To assemble our plasmids we employ uracil-specific excision reagent (USER) cloning as described by Nour-Eldin et al. This technique enables us to speed up the cloning process, to clone seamlessly and to assemble many fragments in one reaction. The steps are:<br />
<br />
# PCR: The backbone and all fragments are amplified as linear products containing a uracil instead of a thymine at a specific position. This requires specifically designed primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
# Optional - If circular DNA was used as PCR template a DpnI digestion can be performed to eliminate this DNA.<br />
# USER-reaction: Digesting the linearized PCR fragment with the USER enzyme removes the uracil-insertions, which will produce the required overhangs.<br />
# Transformation: The mix is immediately transformed into competent E. coli cells where the plasmid assembles itself by base pairing between the complementary overhangs.<br />
<br />
See our "Bricks of Knowledge" [https://www.youtube.com/watch?v=7EiVttJpXH4 video on USER cloning] and the [https://2011.igem.org/Team:DTU-Denmark-2 wiki of the 2011 DTU-2 iGEM team] for more information.<br />
<br />
==References== <br />
<html><br />
<br />
<ul><br />
<li>Nour-Eldin, HH et al (2010). USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories.<br />
Methods Mol Biol. 2010;643:185-200. doi: 10.1007/978-1-60761-723-5_13.<br />
</ul><br />
</td><br />
<td width="163px" height="100%" valign="top"><br />
</td><br />
</tr><br />
</font><br />
</table><br />
<br />
<!-- Main content area --><br />
<br />
</body><br />
<br />
</html><br />
<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-02T11:12:03Z<p>Hezscha: /* References */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
Global demand for fixed nitrogen has increased to the point that half the human population now relies on chemical fertilizer to grow their food. While fertilizer is a requirement for modern life, runoff from overfertilized farmland can cause eutrophication. In the presence of abundant ammonia, algae overgrow and consume the much of the available oxygen in the water. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
Using two ''E. coli'' mutants built with genes from ''Nitrosomonas europaea'' and ''Pseudomonas aeruginosa'', we provide a system to reverse nitrogen fixation. Our mutants consume ammonia and produce nitrous oxide, releases a sustainable source of energy when decomposed into nitrogen and oxygen. We also provide a prototype of a bioreactor that could be scaled up and deployed in the field to simultaneously clean the water and produce energy.<br />
<br />
==Details==<br />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
The first mutant incorporates [[Team:DTU-Denmark/Protein_Models#AMO|AMO]], [[Team:DTU-Denmark/Protein_Models#Hao|HAO]] and two cytochromes [[Team:DTU-Denmark/Protein_Models#Cc554|c554]] and [[Team:DTU-Denmark/Protein_Models#Ccm552|cm552]] from ''Nitrosomonas europaea'' (shown in blue). AMO is a 3 subunit protein which converts ammonia (NH<sub>4</sub> to an intermediate called hydroxylamine (NH<sub>2</sub>OH). HAO then converts the hydroxylamine to nitrite (NO<sub>2</sub><sup>-</sup>. During this conversion process, cytochrome c554 accepts an electron from HAO, and then passes this electron on to ccm552. The terminal electron acceptor in this chain is quinone. AMO and ccm552 are embedded in the cytoplasmic membrane, and all other proteins in this process are found in the periplasm. <br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
The second mutant incorporates the Nir region from ''Pseudomonas aeruginosa'' (shown in yellow). [[Team:DTU-Denmark/Protein_Models#NirS|NirS]] converts nitrite to nitric oxide (NO), while removing an electron from [[Team:DTU-Denmark/Protein_Models#NirM|NirM]]. The remainder of the Nir region is necessary for the synthesis of NirM, and so we have included these genes as well. [[Team:DTU-Denmark/Protein_Models#NOR|NOR]], which is present natively in ''E. coli'', converts nitric oxide to nitrous oxide (N<sub>2</sub>O). In contrast to ''N. europaea'', ''E. coli'' is not a nitrifying bacterium and so does not convert nitrous oxide into nitrogen to complete the denitrification process.<br />
<br />
== Plasmids ==<br />
<br />
=== Mutant 1 ===<br />
<br />
The final plasmid for Mutant 1 should be the following:<br />
<br />
[[File:dtu_Plasmid_mutant_1-01.png|200px]]<br />
<br />
In order to simplify and to be able to debug each part of this pathway separately, we implemented the genes individually.<br />
<br />
Unfortunately, we did not have time to combine all the genes into the final plasmid.<br />
<br />
[[File:Dtu Plasmids amo.png|100px]] [[File:Dtu Plasmids hao-01.png|150px]][[File:Dtu_Plasmids_cyt-01.png|150px]]<br />
<br />
=== Mutant 2 ===<br />
<br />
The final plasmid for Mutant 2 should be the following:<br />
<br />
[[File:Dtu Plasmid mutant 2-01.png|200px]]<br />
<br />
Since we had difficulty extracting Nir from ''Pseudomonas aeruginosa'' in one piece, we split it into two pieces to be extracted seperately and then ligated with USER cloning.<br />
<br />
== User Cloning ==<br />
<br />
To assemble our plasmids we employ uracil-specific excision reagent (USER) cloning as described by Nour-Eldin et al. This technique enables us to speed up the cloning process, to clone seamlessly and to assemble many fragments in one reaction. The steps are:<br />
<br />
# PCR: The backbone and all fragments are amplified as linear products containing a uracil instead of a thymine at a specific position. This requires specifically designed primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
# Optional - If circular DNA was used as PCR template a DpnI digestion can be performed to eliminate this DNA.<br />
# USER-reaction: Digesting the linearized PCR fragment with the USER enzyme removes the uracil-insertions, which will produce the required overhangs.<br />
# Transformation: The mix is immediately transformed into competent E. coli cells where the plasmid assembles itself by base pairing between the complementary overhangs.<br />
<br />
See our "Bricks of Knowledge" [https://www.youtube.com/watch?v=7EiVttJpXH4 video on USER cloning] and the [https://2011.igem.org/Team:DTU-Denmark-2 wiki of the 2011 DTU-2 iGEM team] for more information.<br />
<br />
==References== <br />
<html><br />
<br />
<ul><br />
<li>Nour-Eldin, HH et al (2010). USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories.<br />
Methods Mol Biol. 2010;643:185-200. doi: 10.1007/978-1-60761-723-5_13.<br />
</ul><br />
</td><br />
<td width="163px" height="100%" valign="top"><br />
</td><br />
</tr><br />
</font><br />
</table><br />
<br />
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<br />
</body><br />
<br />
</html><br />
<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/HelloWorldTeam:DTU-Denmark/HelloWorld2013-10-02T11:11:27Z<p>Hezscha: /* Visualization */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|Hello World Pilot Project}}<br />
__TOC__<br />
<br />
== Introduction ==<br />
<br />
‘Hello World!’ are the first words a programmer prints when learning a new programming language. In analogy to this our team decided to do a ‘Hello World’ project in order to familiarize ourselves with lab techniques that we used later on to construct plasmids. Specifically we were performing PCR with uracil-containing primers, purifying PCR products and ligating them by means of USER cloning (Nour-Eldin, H. H.).<br />
<br />
Since we are working with many periplasmic proteins, we wanted to try to target proteins to the periplasm. To do this, we used periplasmic signal peptides from the TAT and Sec pathways, and with a translational fusion of the signal peptide to GFP, we expressed GFP in the periplasm. Simultaneously, we expressed RFP in the cytoplasm as a background color, inspired by Skoog, Karl, et al.<br />
<br />
<br />
== Methods ==<br />
===Construction===<br />
The overal goal was to test the twin arginine pathway (TAT) and whether this signal peptide could transport GFP SF into the periplasm. The reason why we choose to use GFP superfolder (SF) was that this variant has been shown to fold faster than the E. coli transport system is at translocation, (Fisher, Adam C., and Matthew P. DeLisa.). This assures that the GFP will form its fluorophore before translocation to the more reductive periplasmic space. Thus it will not be inactivated by the inhibition of fluorophore formation as seen in other oxidative compartments like ER (Aronson, Deborah E., Lindsey M. Costantini, and Erik L. Snapp.). <br />
<br />
Construction of the plasmids was done with USER-cloning and assembly of 3 fragments. The starting point was a plasmid construct with RFP and GFP SF respectively. The RFP and GFP SF were amplified out with their associated RBS (the same in both cases). <br />
[[File:Both.png|680px]]<br />
<br />
<br />
The TAT signal peptide was bought as a gBlock from IDT. All fragments were assembled into an expression vector specially designed to have a tight on/off mechanism ([http://parts.igem.org/Part:BBa_K1067007 BBa_K1067007]). Primers were design by the program [http://www.cbs.dtu.dk/services/PHUSER-2.0/web/ PHUSER] (Olsen, Lars Rønn, et al.) so that we got a seamless assembly.<br />
<br />
[[File:PZA21-MCS TAT araBAD.png|680px|alt=Alt|"Hello World"" construct]]<br />
<br />
===Visualization===<br />
To visualize that GFP SF actually is exported we constructed a procedure for growing the cells and tracing the GFP SF to the periplasm (Skoog, Karl, et al.). It turns out that if you just grow cells in LB and induce the GFP SF/RFP expression there will be too much GFP SF in the cytoplasm to get a good resolution between cytoplasm and periplasm. That is why it’s important to devise a procedure for stopping the production for GFP SP and thereafter tracing it from the cytoplasm into the periplasm. This underlines the importance of having a non-leaky inducible expression system; when expression is stopped by removing the inducer it would spoil the procedure if the system kept leaking out GFP SF into the cytoplasm.<br />
<br />
After optimizing the procedure we made a final version which can be found in the [https://2013.igem.org/Team:DTU-Denmark/Methods/Visualizing_GFP_in_the_periplasm methods page]. This also includes how to use background subtraction to get a better resolution.<br />
<br />
===Modeling===<br />
<br />
gfdssssss<br />
<br />
== Results ==<br />
===Fluorescent microscope images=== <br />
[[File:GFP in perimplasm RFP in cytoplasm.png|400px|Transformed ''E. coli'' taken with a confocal microscope]]<br />
<br />
<br />
[[File:GFP in perimplasm RFP in cytoplasm close up.png|400px|Close up of several cells, showing GFP expression in the periplasm]]<br />
<br />
<br />
[[File:Graf.PNG|400px|Fluorescence spectrum cross section of one transformed ''E. coli'' cell]]<br />
<br />
<br />
<br />
===Model of "Hello Wolrd" construct===<br />
<br />
== Conclusions ==<br />
<br />
Biobrick [[Team:DTU-Denmark/Parts|BBa_K1067009]] successfully directs proteins to the periplasm in ''E. coli''.<br />
<br />
<br />
<br />
<br />
==References== <br />
<html><br />
<br />
<ul><br />
<li>Skoog, Karl, et al. "Sequential Closure of the Cytoplasm and Then the Periplasm during Cell Division in Escherichia coli." Journal of bacteriology 194.3 (2012): 584-586.<br />
<li>Nour-Eldin, H. H., Geu-Flores, F., & Halkier, B. A. (2010). USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories. In Plant Secondary Metabolism Engineering (pp. 185-200). Humana Press.<br />
<li>Olsen, Lars Rønn, et al. "PHUSER (Primer Help for USER): a novel tool for USER fusion primer design." Nucleic acids research 39.suppl 2 (2011): W61-W67.<br />
<li>Fisher, Adam C., and Matthew P. DeLisa. "Laboratory evolution of fast-folding green fluorescent protein using secretory pathway quality control." PLoS One 3.6 (2008): e2351.<br />
<li>Aronson, Deborah E., Lindsey M. Costantini, and Erik L. Snapp. "Superfolder GFP is fluorescent in oxidizing environments when targeted via the Sec translocon." Traffic 12.5 (2011): 543-548.<br />
</ul><br />
</td><br />
<td width="163px" height="100%" valign="top"><br />
</td><br />
</tr><br />
</font><br />
</table><br />
<br />
<!-- Main content area --><br />
<br />
</body><br />
<br />
</html><br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/HelloWorldTeam:DTU-Denmark/HelloWorld2013-10-02T11:07:25Z<p>Hezscha: /* Construction */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|Hello World Pilot Project}}<br />
__TOC__<br />
<br />
== Introduction ==<br />
<br />
‘Hello World!’ are the first words a programmer prints when learning a new programming language. In analogy to this our team decided to do a ‘Hello World’ project in order to familiarize ourselves with lab techniques that we used later on to construct plasmids. Specifically we were performing PCR with uracil-containing primers, purifying PCR products and ligating them by means of USER cloning (Nour-Eldin, H. H.).<br />
<br />
Since we are working with many periplasmic proteins, we wanted to try to target proteins to the periplasm. To do this, we used periplasmic signal peptides from the TAT and Sec pathways, and with a translational fusion of the signal peptide to GFP, we expressed GFP in the periplasm. Simultaneously, we expressed RFP in the cytoplasm as a background color, inspired by Skoog, Karl, et al.<br />
<br />
<br />
== Methods ==<br />
===Construction===<br />
The overal goal was to test the twin arginine pathway (TAT) and whether this signal peptide could transport GFP SF into the periplasm. The reason why we choose to use GFP superfolder (SF) was that this variant has been shown to fold faster than the E. coli transport system is at translocation, (Fisher, Adam C., and Matthew P. DeLisa.). This assures that the GFP will form its fluorophore before translocation to the more reductive periplasmic space. Thus it will not be inactivated by the inhibition of fluorophore formation as seen in other oxidative compartments like ER (Aronson, Deborah E., Lindsey M. Costantini, and Erik L. Snapp.). <br />
<br />
Construction of the plasmids was done with USER-cloning and assembly of 3 fragments. The starting point was a plasmid construct with RFP and GFP SF respectively. The RFP and GFP SF were amplified out with their associated RBS (the same in both cases). <br />
[[File:Both.png|680px]]<br />
<br />
<br />
The TAT signal peptide was bought as a gBlock from IDT. All fragments were assembled into an expression vector specially designed to have a tight on/off mechanism ([http://parts.igem.org/Part:BBa_K1067007 BBa_K1067007]). Primers were design by the program [http://www.cbs.dtu.dk/services/PHUSER-2.0/web/ PHUSER] (Olsen, Lars Rønn, et al.) so that we got a seamless assembly.<br />
<br />
[[File:PZA21-MCS TAT araBAD.png|680px|alt=Alt|"Hello World"" construct]]<br />
<br />
===Visualization===<br />
To visualize that GFP SF actually is exported we constructed a procedure for growing the cells and tracing the GFP SF to the periplasm (Skoog, Karl, et al.). It turns out that if you just grow cells in LB and induce the GFP SF/RFP expression there will be too much GFP SF in the cytoplasm still to get a good resolution between cytoplasm and periplasm. That is why it’s important with a procedure for stopping the production for GFP SP and thereafter tracing it from the cytoplasm into the periplasm. This underlines the importance of having a non-leaky inducible expression system; if expression is stopped by removing the inducer it would spoil the procedure if the system kept leaking out GFP SF to the cytoplasm.<br />
<br />
After optimizing the procedure we made a final version which can be found in the [https://2013.igem.org/Team:DTU-Denmark/Methods/Visualizing_GFP_in_the_periplasm methods page]. This also includes how to use background subtraction to get a better resolution.<br />
<br />
<br />
===Modeling===<br />
<br />
gfdssssss<br />
<br />
== Results ==<br />
===Fluorescent microscope images=== <br />
[[File:GFP in perimplasm RFP in cytoplasm.png|400px|Transformed ''E. coli'' taken with a confocal microscope]]<br />
<br />
<br />
[[File:GFP in perimplasm RFP in cytoplasm close up.png|400px|Close up of several cells, showing GFP expression in the periplasm]]<br />
<br />
<br />
[[File:Graf.PNG|400px|Fluorescence spectrum cross section of one transformed ''E. coli'' cell]]<br />
<br />
<br />
<br />
===Model of "Hello Wolrd" construct===<br />
<br />
== Conclusions ==<br />
<br />
Biobrick [[Team:DTU-Denmark/Parts|BBa_K1067009]] successfully directs proteins to the periplasm in ''E. coli''.<br />
<br />
<br />
<br />
<br />
==References== <br />
<html><br />
<br />
<ul><br />
<li>Skoog, Karl, et al. "Sequential Closure of the Cytoplasm and Then the Periplasm during Cell Division in Escherichia coli." Journal of bacteriology 194.3 (2012): 584-586.<br />
<li>Nour-Eldin, H. H., Geu-Flores, F., & Halkier, B. A. (2010). USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories. In Plant Secondary Metabolism Engineering (pp. 185-200). Humana Press.<br />
<li>Olsen, Lars Rønn, et al. "PHUSER (Primer Help for USER): a novel tool for USER fusion primer design." Nucleic acids research 39.suppl 2 (2011): W61-W67.<br />
<li>Fisher, Adam C., and Matthew P. DeLisa. "Laboratory evolution of fast-folding green fluorescent protein using secretory pathway quality control." PLoS One 3.6 (2008): e2351.<br />
<li>Aronson, Deborah E., Lindsey M. Costantini, and Erik L. Snapp. "Superfolder GFP is fluorescent in oxidizing environments when targeted via the Sec translocon." Traffic 12.5 (2011): 543-548.<br />
</ul><br />
</td><br />
<td width="163px" height="100%" valign="top"><br />
</td><br />
</tr><br />
</font><br />
</table><br />
<br />
<!-- Main content area --><br />
<br />
</body><br />
<br />
</html><br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/HelloWorldTeam:DTU-Denmark/HelloWorld2013-10-02T11:04:03Z<p>Hezscha: </p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|Hello World Pilot Project}}<br />
__TOC__<br />
<br />
== Introduction ==<br />
<br />
‘Hello World!’ are the first words a programmer prints when learning a new programming language. In analogy to this our team decided to do a ‘Hello World’ project in order to familiarize ourselves with lab techniques that we used later on to construct plasmids. Specifically we were performing PCR with uracil-containing primers, purifying PCR products and ligating them by means of USER cloning (Nour-Eldin, H. H.).<br />
<br />
Since we are working with many periplasmic proteins, we wanted to try to target proteins to the periplasm. To do this, we used periplasmic signal peptides from the TAT and Sec pathways, and with a translational fusion of the signal peptide to GFP, we expressed GFP in the periplasm. Simultaneously, we expressed RFP in the cytoplasm as a background color, inspired by Skoog, Karl, et al.<br />
<br />
<br />
== Methods ==<br />
===Construction===<br />
The overal goal was to test the twin arginine pathway (TAT) and whether this signal peptide could transport GFP SF into the periplasm. The reason why we choose to use GFP superfolder (SF) was that this variant has been shown to fold faster than the E. coli transport system is at translocation, (Fisher, Adam C., and Matthew P. DeLisa.). This assures that the GFP will form its fluorophore before translocation to the more reductive periplasmic space. Thus it will not be inactivated by the inhibition of fluorophore formation as seen in other oxidative compartments like ER (Aronson, Deborah E., Lindsey M. Costantini, and Erik L. Snapp.). <br />
<br />
Construction of the plasmids was done with USER-cloning and assembled from 3 fragments. The starting point was a plasmid construct with RFP and GFP SF respectively. The RFP and GFP SF were amplified out with their associated RBS (the same in both cases). <br />
[[File:Both.png|680px]]<br />
<br />
<br />
The TAT signal peptide was bought as a gBlock from IDT. All fragments were assembled into an expression vector specially designed to have a tight on/off mechanism ([http://parts.igem.org/Part:BBa_K1067007 BBa_K1067007]). Primers were design by the program [http://www.cbs.dtu.dk/services/PHUSER-2.0/web/ PHUSER] (Olsen, Lars Rønn, et al.) so that we got a seamless assembly.<br />
<br />
[[File:PZA21-MCS TAT araBAD.png|680px|alt=Alt|"Hello World"" construct]]<br />
<br />
<br />
===Visualization===<br />
To visualize that GFP SF actually is exported we constructed a procedure for growing the cells and tracing the GFP SF to the periplasm (Skoog, Karl, et al.). It turns out that if you just grow cells in LB and induce the GFP SF/RFP expression there will be too much GFP SF in the cytoplasm still to get a good resolution between cytoplasm and periplasm. That is why it’s important with a procedure for stopping the production for GFP SP and thereafter tracing it from the cytoplasm into the periplasm. This underlines the importance of having a non-leaky inducible expression system; if expression is stopped by removing the inducer it would spoil the procedure if the system kept leaking out GFP SF to the cytoplasm.<br />
<br />
After optimizing the procedure we made a final version which can be found in the [https://2013.igem.org/Team:DTU-Denmark/Methods/Visualizing_GFP_in_the_periplasm methods page]. This also includes how to use background subtraction to get a better resolution.<br />
<br />
<br />
===Modeling===<br />
<br />
gfdssssss<br />
<br />
== Results ==<br />
===Fluorescent microscope images=== <br />
[[File:GFP in perimplasm RFP in cytoplasm.png|400px|Transformed ''E. coli'' taken with a confocal microscope]]<br />
<br />
<br />
[[File:GFP in perimplasm RFP in cytoplasm close up.png|400px|Close up of several cells, showing GFP expression in the periplasm]]<br />
<br />
<br />
[[File:Graf.PNG|400px|Fluorescence spectrum cross section of one transformed ''E. coli'' cell]]<br />
<br />
<br />
<br />
===Model of "Hello Wolrd" construct===<br />
<br />
== Conclusions ==<br />
<br />
Biobrick [[Team:DTU-Denmark/Parts|BBa_K1067009]] successfully directs proteins to the periplasm in ''E. coli''.<br />
<br />
<br />
<br />
<br />
==References== <br />
<html><br />
<br />
<ul><br />
<li>Skoog, Karl, et al. "Sequential Closure of the Cytoplasm and Then the Periplasm during Cell Division in Escherichia coli." Journal of bacteriology 194.3 (2012): 584-586.<br />
<li>Nour-Eldin, H. H., Geu-Flores, F., & Halkier, B. A. (2010). USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories. In Plant Secondary Metabolism Engineering (pp. 185-200). Humana Press.<br />
<li>Olsen, Lars Rønn, et al. "PHUSER (Primer Help for USER): a novel tool for USER fusion primer design." Nucleic acids research 39.suppl 2 (2011): W61-W67.<br />
<li>Fisher, Adam C., and Matthew P. DeLisa. "Laboratory evolution of fast-folding green fluorescent protein using secretory pathway quality control." PLoS One 3.6 (2008): e2351.<br />
<li>Aronson, Deborah E., Lindsey M. Costantini, and Erik L. Snapp. "Superfolder GFP is fluorescent in oxidizing environments when targeted via the Sec translocon." Traffic 12.5 (2011): 543-548.<br />
</ul><br />
</td><br />
<td width="163px" height="100%" valign="top"><br />
</td><br />
</tr><br />
</font><br />
</table><br />
<br />
<!-- Main content area --><br />
<br />
</body><br />
<br />
</html><br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-01T15:34:06Z<p>Hezscha: /* Mutant 2 */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
Global demand for fixed nitrogen has increased to the point that half the human population now relies on chemical fertilizer to grow their food. While fertilizer is a requirement for modern life, runoff from overfertilized farmland can cause eutrophication. In the presence of abundant ammonia, algae overgrow and consume the much of the available oxygen in the water. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
Using two ''E. coli'' mutants built with genes from ''Nitrosomonas europaea'' and ''Pseudomonas aeruginosa'', we provide a system to reverse nitrogen fixation. Our mutants consume ammonia and produce nitrous oxide, releases a sustainable source of energy when decomposed into nitrogen and oxygen. We also provide a prototype of a bioreactor that could be scaled up and deployed in the field to simultaneously clean the water and produce energy.<br />
<br />
==Details==<br />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
The first mutant incorporates AMO, HAO and two cytochromes c554 and cm552 from ''Nitrosomonas europaea'' (shown in blue). AMO is a 3 subunit protein which converts ammonia (NH<sub>4</sub> to an intermediate called hydroxylamine (NH<sub>2</sub>OH). HAO then converts the hydroxylamine to nitrite (NO<sub>2</sub><sup>-</sup>. During this conversion process, cytochrome c554 accepts an electron from HAO, and then passes this electron on to ccm552. The terminal electron acceptor in this chain is quinone. AMO and ccm552 are embedded in the cytoplasmic membrane, and all other proteins in this process are found in the periplasm. <br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
The second mutant incorporates the Nir region from ''Pseudomonas aeruginosa'' (shown in yellow). NirS converts nitrite to nitric oxide (NO), while removing an electron from NirM. The remainder of the Nir region is necessary for the synthesis of NirM, and so we have included these genes as well. NOR, which is present natively in ''E. coli'', converts nitric oxide to nitrous oxide (N<sub>2</sub>O). In contrast to ''N. europaea'', ''E. coli'' is not a nitrifying bacterium and so does not convert nitrous oxide into nitrogen to complete the denitrification process.<br />
<br />
== Plasmids ==<br />
<br />
=== Mutant 1 ===<br />
<br />
The final plasmid for Mutant 1 should be the following:<br />
<br />
[[File:dtu_Plasmid_mutant_1-01.png|200px]]<br />
<br />
In order to simplify and to be able to debug each part of this pathway separately, we implemented the genes individually.<br />
<br />
Unfortunately, we did not have time to combine all the genes into the final plasmid.<br />
<br />
[[File:Dtu Plasmids amo.png|100px]] [[File:Dtu Plasmids hao-01.png|150px]][[File:Dtu_Plasmids_cyt-01.png|150px]]<br />
<br />
=== Mutant 2 ===<br />
<br />
The final plasmid for Mutant 2 should be the following:<br />
<br />
[[File:Dtu Plasmid mutant 2-01.png|200px]]<br />
<br />
Since we had difficulty extracting Nir from ''Pseudomonas aeruginosa'' in one piece, we split it into two pieces to be extracted seperately and then ligated with USER cloning.<br />
<br />
== User Cloning ==<br />
<br />
To assemble our plasmids we employ uracil-specific excision reagent (USER) cloning as described by Nour-Eldin et al. This technique enables us to speed up the cloning process, to clone seamlessly and to assemble many fragments in one reaction. The steps are:<br />
<br />
# PCR: The backbone and all fragments are amplified as linear products containing a uracil instead of a thymine at a specific position. This requires specifically designed primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
# Optional - If circular DNA was used as PCR template a DpnI digestion can be performed to eliminate this DNA.<br />
# USER-reaction: Digesting the linearized PCR fragment with the USER enzyme removes the uracil-insertions, which will produce the required overhangs.<br />
# Transformation: The mix is immediately transformed into competent E. coli cells where the plasmid assembles itself by base pairing between the complementary overhangs.<br />
<br />
See our "Bricks of Knowledge" [https://www.youtube.com/watch?v=7EiVttJpXH4 video on USER cloning] and the [https://2011.igem.org/Team:DTU-Denmark-2 wiki of the 2011 DTU-2 iGEM team] for more information.<br />
<br />
==References== <br />
<html><br />
<br />
<ul><br />
<li>Nour-Eldin, HH et al (2010). USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories.<br />
Methods Mol Biol. 2010;643:185-200. doi: 10.1007/978-1-60761-723-5_13.<br />
<li>Ref2<br />
</ul><br />
</td><br />
<td width="163px" height="100%" valign="top"><br />
</td><br />
</tr><br />
</font><br />
</table><br />
<br />
<!-- Main content area --><br />
<br />
</body><br />
<br />
</html><br />
<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-01T15:24:00Z<p>Hezscha: /* Mutant 1 */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
Global demand for fixed nitrogen has increased to the point that half the human population now relies on chemical fertilizer to grow their food. While fertilizer is a requirement for modern life, runoff from overfertilized farmland can cause eutrophication. In the presence of abundant ammonia, algae overgrow and consume the much of the available oxygen in the water. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
Using two ''E. coli'' mutants built with genes from ''Nitrosomonas europaea'' and ''Pseudomonas aeruginosa'', we provide a system to reverse nitrogen fixation. Our mutants consume ammonia and produce nitrous oxide, releases a sustainable source of energy when decomposed into nitrogen and oxygen. We also provide a prototype of a bioreactor that could be scaled up and deployed in the field to simultaneously clean the water and produce energy.<br />
<br />
==Details==<br />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
The first mutant incorporates AMO, HAO and two cytochromes c554 and cm552 from ''Nitrosomonas europaea'' (shown in blue). AMO is a 3 subunit protein which converts ammonia (NH<sub>4</sub> to an intermediate called hydroxylamine (NH<sub>2</sub>OH). HAO then converts the hydroxylamine to nitrite (NO<sub>2</sub><sup>-</sup>. During this conversion process, cytochrome c554 accepts an electron from HAO, and then passes this electron on to ccm552. The terminal electron acceptor in this chain is quinone. AMO and ccm552 are embedded in the cytoplasmic membrane, and all other proteins in this process are found in the periplasm. <br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
The second mutant incorporates the Nir region from ''Pseudomonas aeruginosa'' (shown in yellow). NirS converts nitrite to nitric oxide (NO), while removing an electron from NirM. The remainder of the Nir region is necessary for the synthesis of NirM, and so we have included these genes as well. NOR, which is present natively in ''E. coli'', converts nitric oxide to nitrous oxide (N<sub>2</sub>O). In contrast to ''N. europaea'', ''E. coli'' is not a nitrifying bacterium and so does not convert nitrous oxide into nitrogen to complete the denitrification process.<br />
<br />
== Plasmids ==<br />
<br />
=== Mutant 1 ===<br />
<br />
The final plasmid for Mutant 1 should be the following:<br />
<br />
[[File:dtu_Plasmid_mutant_1-01.png|200px]]<br />
<br />
In order to simplify and to be able to debug each part of this pathway separately, we implemented the genes individually.<br />
<br />
Unfortunately, we did not have time to combine all the genes into the final plasmid.<br />
<br />
[[File:Dtu Plasmids amo.png|100px]] [[File:Dtu Plasmids hao-01.png|150px]][[File:Dtu_Plasmids_cyt-01.png|150px]]<br />
<br />
=== Mutant 2 ===<br />
<br />
The final plasmid for Mutant 2 should be the following:<br />
<br />
[[File:Dtu Plasmid mutant 2-01.png|200px]]<br />
<br />
Since we had difficulty extracting Nir from ''Pseudomonas aeruginosa'' in one piece, we have implemented TODO<br />
<br />
== User Cloning ==<br />
<br />
To assemble our plasmids we employ uracil-specific excision reagent (USER) cloning as described by Nour-Eldin et al. This technique enables us to speed up the cloning process, to clone seamlessly and to assemble many fragments in one reaction. The steps are:<br />
<br />
# PCR: The backbone and all fragments are amplified as linear products containing a uracil instead of a thymine at a specific position. This requires specifically designed primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
# Optional - If circular DNA was used as PCR template a DpnI digestion can be performed to eliminate this DNA.<br />
# USER-reaction: Digesting the linearized PCR fragment with the USER enzyme removes the uracil-insertions, which will produce the required overhangs.<br />
# Transformation: The mix is immediately transformed into competent E. coli cells where the plasmid assembles itself by base pairing between the complementary overhangs.<br />
<br />
See our "Bricks of Knowledge" [https://www.youtube.com/watch?v=7EiVttJpXH4 video on USER cloning] and the [https://2011.igem.org/Team:DTU-Denmark-2 wiki of the 2011 DTU-2 iGEM team] for more information.<br />
<br />
==References== <br />
<html><br />
<br />
<ul><br />
<li>Nour-Eldin, HH et al (2010). USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories.<br />
Methods Mol Biol. 2010;643:185-200. doi: 10.1007/978-1-60761-723-5_13.<br />
<li>Ref2<br />
</ul><br />
</td><br />
<td width="163px" height="100%" valign="top"><br />
</td><br />
</tr><br />
</font><br />
</table><br />
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<br />
</body><br />
<br />
</html><br />
<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-01T15:04:52Z<p>Hezscha: /* User Cloning */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
Global demand for fixed nitrogen has increased to the point that half the human population now relies on chemical fertilizer to grow their food. While fertilizer is a requirement for modern life, runoff from overfertilized farmland can cause eutrophication. In the presence of abundant ammonia, algae overgrow and consume the much of the available oxygen in the water. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
Using two ''E. coli'' mutants built with genes from ''Nitrosomonas europaea'' and ''Pseudomonas aeruginosa'', we provide a system to reverse nitrogen fixation. Our mutants consume ammonia and produce nitrous oxide, releases a sustainable source of energy when decomposed into nitrogen and oxygen. We also provide a prototype of a bioreactor that could be scaled up and deployed in the field to simultaneously clean the water and produce energy.<br />
<br />
==Details==<br />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
The first mutant incorporates AMO, HAO and two cytochromes c554 and cm552 from ''Nitrosomonas europaea'' (shown in blue). AMO is a 3 subunit protein which converts ammonia (NH<sub>4</sub> to an intermediate called hydroxylamine (NH<sub>2</sub>OH). HAO then converts the hydroxylamine to nitrite (NO<sub>2</sub><sup>-</sup>. During this conversion process, cytochrome c554 accepts an electron from HAO, and then passes this electron on to ccm552. The terminal electron acceptor in this chain is quinone. AMO and ccm552 are embedded in the cytoplasmic membrane, and all other proteins in this process are found in the periplasm. <br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
The second mutant incorporates the Nir region from ''Pseudomonas aeruginosa'' (shown in yellow). NirS converts nitrite to nitric oxide (NO), while removing an electron from NirM. The remainder of the Nir region is necessary for the synthesis of NirM, and so we have included these genes as well. NOR, which is present natively in ''E. coli'', converts nitric oxide to nitrous oxide (N<sub>2</sub>O). In contrast to ''N. europaea'', ''E. coli'' is not a nitrifying bacterium and so does not convert nitrous oxide into nitrogen to complete the denitrification process.<br />
<br />
== Plasmids ==<br />
<br />
=== Mutant 1 ===<br />
<br />
The final plasmid for Mutant 1 should be the following:<br />
<br />
[[File:dtu_Plasmid_mutant_1-01.png|200px]]<br />
<br />
In order to simplify and to be able to debug each part of this pathway separately, we implemented the following.<br />
<br />
Unfortunately, we did not have time to combine all the genes into the final plasmid.<br />
<br />
[[File:Dtu Plasmids amo.png|100px]] [[File:Dtu Plasmids hao-01.png|150px]][[File:Dtu_Plasmids_cyt-01.png|150px]]<br />
<br />
=== Mutant 2 ===<br />
<br />
The final plasmid for Mutant 2 should be the following:<br />
<br />
[[File:Dtu Plasmid mutant 2-01.png|200px]]<br />
<br />
Since we had difficulty extracting Nir from ''Pseudomonas aeruginosa'' in one piece, we have implemented TODO<br />
<br />
== User Cloning ==<br />
<br />
To assemble our plasmids we employ uracil-specific excision reagent (USER) cloning as described by Nour-Eldin et al. This technique enables us to speed up the cloning process, to clone seamlessly and to assemble many fragments in one reaction. The steps are:<br />
<br />
# PCR: The backbone and all fragments are amplified as linear products containing a uracil instead of a thymine at a specific position. This requires specifically designed primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
# Optional - If circular DNA was used as PCR template a DpnI digestion can be performed to eliminate this DNA.<br />
# USER-reaction: Digesting the linearized PCR fragment with the USER enzyme removes the uracil-insertions, which will produce the required overhangs.<br />
# Transformation: The mix is immediately transformed into competent E. coli cells where the plasmid assembles itself by base pairing between the complementary overhangs.<br />
<br />
See our "Bricks of Knowledge" [https://www.youtube.com/watch?v=7EiVttJpXH4 video on USER cloning] and the [https://2011.igem.org/Team:DTU-Denmark-2 wiki of the 2011 DTU-2 iGEM team] for more information.<br />
<br />
==References== <br />
<html><br />
<br />
<ul><br />
<li>Nour-Eldin, HH et al (2010). USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories.<br />
Methods Mol Biol. 2010;643:185-200. doi: 10.1007/978-1-60761-723-5_13.<br />
<li>Ref2<br />
</ul><br />
</td><br />
<td width="163px" height="100%" valign="top"><br />
</td><br />
</tr><br />
</font><br />
</table><br />
<br />
<!-- Main content area --><br />
<br />
</body><br />
<br />
</html><br />
<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-01T15:03:59Z<p>Hezscha: /* References */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
Global demand for fixed nitrogen has increased to the point that half the human population now relies on chemical fertilizer to grow their food. While fertilizer is a requirement for modern life, runoff from overfertilized farmland can cause eutrophication. In the presence of abundant ammonia, algae overgrow and consume the much of the available oxygen in the water. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
Using two ''E. coli'' mutants built with genes from ''Nitrosomonas europaea'' and ''Pseudomonas aeruginosa'', we provide a system to reverse nitrogen fixation. Our mutants consume ammonia and produce nitrous oxide, releases a sustainable source of energy when decomposed into nitrogen and oxygen. We also provide a prototype of a bioreactor that could be scaled up and deployed in the field to simultaneously clean the water and produce energy.<br />
<br />
==Details==<br />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
The first mutant incorporates AMO, HAO and two cytochromes c554 and cm552 from ''Nitrosomonas europaea'' (shown in blue). AMO is a 3 subunit protein which converts ammonia (NH<sub>4</sub> to an intermediate called hydroxylamine (NH<sub>2</sub>OH). HAO then converts the hydroxylamine to nitrite (NO<sub>2</sub><sup>-</sup>. During this conversion process, cytochrome c554 accepts an electron from HAO, and then passes this electron on to ccm552. The terminal electron acceptor in this chain is quinone. AMO and ccm552 are embedded in the cytoplasmic membrane, and all other proteins in this process are found in the periplasm. <br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
The second mutant incorporates the Nir region from ''Pseudomonas aeruginosa'' (shown in yellow). NirS converts nitrite to nitric oxide (NO), while removing an electron from NirM. The remainder of the Nir region is necessary for the synthesis of NirM, and so we have included these genes as well. NOR, which is present natively in ''E. coli'', converts nitric oxide to nitrous oxide (N<sub>2</sub>O). In contrast to ''N. europaea'', ''E. coli'' is not a nitrifying bacterium and so does not convert nitrous oxide into nitrogen to complete the denitrification process.<br />
<br />
== Plasmids ==<br />
<br />
=== Mutant 1 ===<br />
<br />
The final plasmid for Mutant 1 should be the following:<br />
<br />
[[File:dtu_Plasmid_mutant_1-01.png|200px]]<br />
<br />
In order to simplify and to be able to debug each part of this pathway separately, we implemented the following.<br />
<br />
Unfortunately, we did not have time to combine all the genes into the final plasmid.<br />
<br />
[[File:Dtu Plasmids amo.png|100px]] [[File:Dtu Plasmids hao-01.png|150px]][[File:Dtu_Plasmids_cyt-01.png|150px]]<br />
<br />
=== Mutant 2 ===<br />
<br />
The final plasmid for Mutant 2 should be the following:<br />
<br />
[[File:Dtu Plasmid mutant 2-01.png|200px]]<br />
<br />
Since we had difficulty extracting Nir from ''Pseudomonas aeruginosa'' in one piece, we have implemented TODO<br />
<br />
== User Cloning ==<br />
<br />
To assemble our plasmids we employ uracil-specific excision reagent (USER) cloning[there should be a ref here]. This technique enables us to speed up the cloning process, to clone seamlessly and to assemble many fragments in one reaction. The steps are:<br />
<br />
# PCR: The backbone and all fragments are amplified as linear products containing a uracil instead of a thymine at a specific position. This requires specifically designed primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
# Optional - If circular DNA was used as PCR template a DpnI digestion can be performed to eliminate this DNA.<br />
# USER-reaction: Digesting the linearized PCR fragment with the USER enzyme removes the uracil-insertions, which will produce the required overhangs.<br />
# Transformation: The mix is immediately transformed into competent E. coli cells where the plasmid assembles itself by base pairing between the complementary overhangs.<br />
<br />
See our "Bricks of Knowledge" [https://www.youtube.com/watch?v=7EiVttJpXH4 video on USER cloning] and the [https://2011.igem.org/Team:DTU-Denmark-2 wiki of the 2011 DTU-2 iGEM team] for more information.<br />
<br />
<br />
==References== <br />
<html><br />
<br />
<ul><br />
<li>Nour-Eldin, HH et al (2010). USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories.<br />
Methods Mol Biol. 2010;643:185-200. doi: 10.1007/978-1-60761-723-5_13.<br />
<li>Ref2<br />
</ul><br />
</td><br />
<td width="163px" height="100%" valign="top"><br />
</td><br />
</tr><br />
</font><br />
</table><br />
<br />
<!-- Main content area --><br />
<br />
</body><br />
<br />
</html><br />
<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-01T15:00:54Z<p>Hezscha: /* User Cloning */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
Global demand for fixed nitrogen has increased to the point that half the human population now relies on chemical fertilizer to grow their food. While fertilizer is a requirement for modern life, runoff from overfertilized farmland can cause eutrophication. In the presence of abundant ammonia, algae overgrow and consume the much of the available oxygen in the water. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
Using two ''E. coli'' mutants built with genes from ''Nitrosomonas europaea'' and ''Pseudomonas aeruginosa'', we provide a system to reverse nitrogen fixation. Our mutants consume ammonia and produce nitrous oxide, releases a sustainable source of energy when decomposed into nitrogen and oxygen. We also provide a prototype of a bioreactor that could be scaled up and deployed in the field to simultaneously clean the water and produce energy.<br />
<br />
==Details==<br />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
The first mutant incorporates AMO, HAO and two cytochromes c554 and cm552 from ''Nitrosomonas europaea'' (shown in blue). AMO is a 3 subunit protein which converts ammonia (NH<sub>4</sub> to an intermediate called hydroxylamine (NH<sub>2</sub>OH). HAO then converts the hydroxylamine to nitrite (NO<sub>2</sub><sup>-</sup>. During this conversion process, cytochrome c554 accepts an electron from HAO, and then passes this electron on to ccm552. The terminal electron acceptor in this chain is quinone. AMO and ccm552 are embedded in the cytoplasmic membrane, and all other proteins in this process are found in the periplasm. <br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
The second mutant incorporates the Nir region from ''Pseudomonas aeruginosa'' (shown in yellow). NirS converts nitrite to nitric oxide (NO), while removing an electron from NirM. The remainder of the Nir region is necessary for the synthesis of NirM, and so we have included these genes as well. NOR, which is present natively in ''E. coli'', converts nitric oxide to nitrous oxide (N<sub>2</sub>O). In contrast to ''N. europaea'', ''E. coli'' is not a nitrifying bacterium and so does not convert nitrous oxide into nitrogen to complete the denitrification process.<br />
<br />
== Plasmids ==<br />
<br />
=== Mutant 1 ===<br />
<br />
The final plasmid for Mutant 1 should be the following:<br />
<br />
[[File:dtu_Plasmid_mutant_1-01.png|200px]]<br />
<br />
In order to simplify and to be able to debug each part of this pathway separately, we implemented the following.<br />
<br />
Unfortunately, we did not have time to combine all the genes into the final plasmid.<br />
<br />
[[File:Dtu Plasmids amo.png|100px]] [[File:Dtu Plasmids hao-01.png|150px]][[File:Dtu_Plasmids_cyt-01.png|150px]]<br />
<br />
=== Mutant 2 ===<br />
<br />
The final plasmid for Mutant 2 should be the following:<br />
<br />
[[File:Dtu Plasmid mutant 2-01.png|200px]]<br />
<br />
Since we had difficulty extracting Nir from ''Pseudomonas aeruginosa'' in one piece, we have implemented TODO<br />
<br />
== User Cloning ==<br />
<br />
To assemble our plasmids we employ uracil-specific excision reagent (USER) cloning[there should be a ref here]. This technique enables us to speed up the cloning process, to clone seamlessly and to assemble many fragments in one reaction. The steps are:<br />
<br />
# PCR: The backbone and all fragments are amplified as linear products containing a uracil instead of a thymine at a specific position. This requires specifically designed primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
# Optional - If circular DNA was used as PCR template a DpnI digestion can be performed to eliminate this DNA.<br />
# USER-reaction: Digesting the linearized PCR fragment with the USER enzyme removes the uracil-insertions, which will produce the required overhangs.<br />
# Transformation: The mix is immediately transformed into competent E. coli cells where the plasmid assembles itself by base pairing between the complementary overhangs.<br />
<br />
See our "Bricks of Knowledge" [https://www.youtube.com/watch?v=7EiVttJpXH4 video on USER cloning] and the [https://2011.igem.org/Team:DTU-Denmark-2 wiki of the 2011 DTU-2 iGEM team] for more information.<br />
<br />
<br />
==References== <br />
<html><br />
<br />
<ul><br />
<li>Ref1<br />
<li>Ref2<br />
</ul><br />
</td><br />
<td width="163px" height="100%" valign="top"><br />
</td><br />
</tr><br />
</font><br />
</table><br />
<br />
<!-- Main content area --><br />
<br />
</body><br />
<br />
</html><br />
<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-01T14:58:56Z<p>Hezscha: /* User Cloning */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
Global demand for fixed nitrogen has increased to the point that half the human population now relies on chemical fertilizer to grow their food. While fertilizer is a requirement for modern life, runoff from overfertilized farmland can cause eutrophication. In the presence of abundant ammonia, algae overgrow and consume the much of the available oxygen in the water. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
Using two ''E. coli'' mutants built with genes from ''Nitrosomonas europaea'' and ''Pseudomonas aeruginosa'', we provide a system to reverse nitrogen fixation. Our mutants consume ammonia and produce nitrous oxide, releases a sustainable source of energy when decomposed into nitrogen and oxygen. We also provide a prototype of a bioreactor that could be scaled up and deployed in the field to simultaneously clean the water and produce energy.<br />
<br />
==Details==<br />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
The first mutant incorporates AMO, HAO and two cytochromes c554 and cm552 from ''Nitrosomonas europaea'' (shown in blue). AMO is a 3 subunit protein which converts ammonia (NH<sub>4</sub> to an intermediate called hydroxylamine (NH<sub>2</sub>OH). HAO then converts the hydroxylamine to nitrite (NO<sub>2</sub><sup>-</sup>. During this conversion process, cytochrome c554 accepts an electron from HAO, and then passes this electron on to ccm552. The terminal electron acceptor in this chain is quinone. AMO and ccm552 are embedded in the cytoplasmic membrane, and all other proteins in this process are found in the periplasm. <br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
The second mutant incorporates the Nir region from ''Pseudomonas aeruginosa'' (shown in yellow). NirS converts nitrite to nitric oxide (NO), while removing an electron from NirM. The remainder of the Nir region is necessary for the synthesis of NirM, and so we have included these genes as well. NOR, which is present natively in ''E. coli'', converts nitric oxide to nitrous oxide (N<sub>2</sub>O). In contrast to ''N. europaea'', ''E. coli'' is not a nitrifying bacterium and so does not convert nitrous oxide into nitrogen to complete the denitrification process.<br />
<br />
== Plasmids ==<br />
<br />
=== Mutant 1 ===<br />
<br />
The final plasmid for Mutant 1 should be the following:<br />
<br />
[[File:dtu_Plasmid_mutant_1-01.png|200px]]<br />
<br />
In order to simplify and to be able to debug each part of this pathway separately, we implemented the following.<br />
<br />
Unfortunately, we did not have time to combine all the genes into the final plasmid.<br />
<br />
[[File:Dtu Plasmids amo.png|100px]] [[File:Dtu Plasmids hao-01.png|150px]][[File:Dtu_Plasmids_cyt-01.png|150px]]<br />
<br />
=== Mutant 2 ===<br />
<br />
The final plasmid for Mutant 2 should be the following:<br />
<br />
[[File:Dtu Plasmid mutant 2-01.png|200px]]<br />
<br />
Since we had difficulty extracting Nir from ''Pseudomonas aeruginosa'' in one piece, we have implemented TODO<br />
<br />
== User Cloning ==<br />
<br />
To assemble our plasmids we employ uracil-specific excision reagent (USER) cloning[there should be a ref here]. This technique enables us to speed up the cloning process, to clone seamlessly and to assemble many fragments in one reaction. The steps are:<br />
<br />
# PCR: The backbone and all fragments are amplified as linear products containing a uracil instead of a thymine at a specific position. This requires specifically designed primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
# Optional - If circular DNA was used as PCR template a DpnI digestion can be performed to eliminate this DNA.<br />
# USER-reaction: Digesting the linearized PCR fragment with the USER enzyme removes the uracil-insertions, which will produce the required overhangs.<br />
# Transformation: The mix is immediately transformed into competent E. coli cells where the plasmid assembles itself by base pairing between the complementary overhangs.<br />
<br />
See our "Bricks of Knowledge" [https://www.youtube.com/watch?v=7EiVttJpXH4 video on USER cloning] and the [https://2011.igem.org/Team:DTU-Denmark-2 wiki of the 2011 DTU-2 iGEM team] for more information.<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-01T14:53:37Z<p>Hezscha: /* User Cloning */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
Global demand for fixed nitrogen has increased to the point that half the human population now relies on chemical fertilizer to grow their food. While fertilizer is a requirement for modern life, runoff from overfertilized farmland can cause eutrophication. In the presence of abundant ammonia, algae overgrow and consume the much of the available oxygen in the water. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
Using two ''E. coli'' mutants built with genes from ''Nitrosomonas europaea'' and ''Pseudomonas aeruginosa'', we provide a system to reverse nitrogen fixation. Our mutants consume ammonia and produce nitrous oxide, releases a sustainable source of energy when decomposed into nitrogen and oxygen. We also provide a prototype of a bioreactor that could be scaled up and deployed in the field to simultaneously clean the water and produce energy.<br />
<br />
==Details==<br />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
The first mutant incorporates AMO, HAO and two cytochromes c554 and cm552 from ''Nitrosomonas europaea'' (shown in blue). AMO is a 3 subunit protein which converts ammonia (NH<sub>4</sub> to an intermediate called hydroxylamine (NH<sub>2</sub>OH). HAO then converts the hydroxylamine to nitrite (NO<sub>2</sub><sup>-</sup>. During this conversion process, cytochrome c554 accepts an electron from HAO, and then passes this electron on to ccm552. The terminal electron acceptor in this chain is quinone. AMO and ccm552 are embedded in the cytoplasmic membrane, and all other proteins in this process are found in the periplasm. <br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
The second mutant incorporates the Nir region from ''Pseudomonas aeruginosa'' (shown in yellow). NirS converts nitrite to nitric oxide (NO), while removing an electron from NirM. The remainder of the Nir region is necessary for the synthesis of NirM, and so we have included these genes as well. NOR, which is present natively in ''E. coli'', converts nitric oxide to nitrous oxide (N<sub>2</sub>O). In contrast to ''N. europaea'', ''E. coli'' is not a nitrifying bacterium and so does not convert nitrous oxide into nitrogen to complete the denitrification process.<br />
<br />
== Plasmids ==<br />
<br />
=== Mutant 1 ===<br />
<br />
The final plasmid for Mutant 1 should be the following:<br />
<br />
[[File:dtu_Plasmid_mutant_1-01.png|200px]]<br />
<br />
In order to simplify and to be able to debug each part of this pathway separately, we implemented the following.<br />
<br />
Unfortunately, we did not have time to combine all the genes into the final plasmid.<br />
<br />
[[File:Dtu Plasmids amo.png|100px]] [[File:Dtu Plasmids hao-01.png|150px]][[File:Dtu_Plasmids_cyt-01.png|150px]]<br />
<br />
=== Mutant 2 ===<br />
<br />
The final plasmid for Mutant 2 should be the following:<br />
<br />
[[File:Dtu Plasmid mutant 2-01.png|200px]]<br />
<br />
Since we had difficulty extracting Nir from ''Pseudomonas aeruginosa'' in one piece, we have implemented TODO<br />
<br />
== User Cloning ==<br />
<br />
To assemble our plasmids we employ uracil-specific excision reagent (USER) cloning[there should be a ref here]. This technique enables us to speed up the cloning process, to clone seamless and to assemble many fragments in one reaction. The steps are:<br />
<br />
# PCR: The backbone and all fragments are amplified as linear products containing a uracil instead of a thymine at a specific position. Requires special uracil-containing primers and a polymerase that is able to perform uracil-insertion.<br />
# Optional - If circular DNA was used as PCR template a DpnI digestion can be performed to eliminate this DNA.<br />
# USER-reaction: Digesting the linearized PCR fragment with the USER enzyme removes the uracil-insertions, which will produce the required overhangs.<br />
# Transformation: The mix is immediately transformed into competent E. coli cells.<br />
<br />
See our "Bricks of Knowledge" [https://www.youtube.com/watch?v=7EiVttJpXH4 video on USER cloning] and the [https://2011.igem.org/Team:DTU-Denmark-2 wiki of the 2011 DTU-2 iGEM team] for more information.<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-01T14:43:35Z<p>Hezscha: /* User Cloning */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
Global demand for fixed nitrogen has increased to the point that half the human population now relies on chemical fertilizer to grow their food. While fertilizer is a requirement for modern life, runoff from overfertilized farmland can cause eutrophication. In the presence of abundant ammonia, algae overgrow and consume the much of the available oxygen in the water. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
Using two ''E. coli'' mutants built with genes from ''Nitrosomonas europaea'' and ''Pseudomonas aeruginosa'', we provide a system to reverse nitrogen fixation. Our mutants consume ammonia and produce nitrous oxide, releases a sustainable source of energy when decomposed into nitrogen and oxygen. We also provide a prototype of a bioreactor that could be scaled up and deployed in the field to simultaneously clean the water and produce energy.<br />
<br />
==Details==<br />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
The first mutant incorporates AMO, HAO and two cytochromes c554 and cm552 from ''Nitrosomonas europaea'' (shown in blue). AMO is a 3 subunit protein which converts ammonia (NH<sub>4</sub> to an intermediate called hydroxylamine (NH<sub>2</sub>OH). HAO then converts the hydroxylamine to nitrite (NO<sub>2</sub><sup>-</sup>. During this conversion process, cytochrome c554 accepts an electron from HAO, and then passes this electron on to ccm552. The terminal electron acceptor in this chain is quinone. AMO and ccm552 are embedded in the cytoplasmic membrane, and all other proteins in this process are found in the periplasm. <br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
The second mutant incorporates the Nir region from ''Pseudomonas aeruginosa'' (shown in yellow). NirS converts nitrite to nitric oxide (NO), while removing an electron from NirM. The remainder of the Nir region is necessary for the synthesis of NirM, and so we have included these genes as well. NOR, which is present natively in ''E. coli'', converts nitric oxide to nitrous oxide (N<sub>2</sub>O). In contrast to ''N. europaea'', ''E. coli'' is not a nitrifying bacterium and so does not convert nitrous oxide into nitrogen to complete the denitrification process.<br />
<br />
== Plasmids ==<br />
<br />
=== Mutant 1 ===<br />
<br />
The final plasmid for Mutant 1 should be the following:<br />
<br />
[[File:dtu_Plasmid_mutant_1-01.png|200px]]<br />
<br />
In order to simplify and to be able to debug each part of this pathway separately, we implemented the following.<br />
<br />
Unfortunately, we did not have time to combine all the genes into the final plasmid.<br />
<br />
[[File:Dtu Plasmids amo.png|100px]] [[File:Dtu Plasmids hao-01.png|150px]][[File:Dtu_Plasmids_cyt-01.png|150px]]<br />
<br />
=== Mutant 2 ===<br />
<br />
The final plasmid for Mutant 2 should be the following:<br />
<br />
[[File:Dtu Plasmid mutant 2-01.png|200px]]<br />
<br />
Since we had difficulty extracting Nir from ''Pseudomonas aeruginosa'' in one piece, we have implemented TODO<br />
<br />
== User Cloning ==<br />
<br />
To assemble our plasmids we employ USER cloning[there should be a ref here]. This technique enables us to speed up the cloning process, to clone seamless and to assemble many fragments in one reaction. The steps are:<br />
<br />
# PCR: The backbone and all fragments are amplified as linear products containing a uracil instead of a thymine at a specific position. Requires special uracil-containing primers and a polymerase that is able to perform uracil-insertion.<br />
# Optional - If circular DNA was used as PCR template a DpnI digestion can be performed to eliminate this DNA.<br />
# USER-reaction: Digesting the linearized PCR fragment with the USER enzyme removes the uracil-insertions, which will produce the required overhangs.<br />
# Transformation: The mix is immediately transformed into competent E. coli cells.<br />
<br />
See our "Bricks of Knowledge" [https://www.youtube.com/watch?v=7EiVttJpXH4 video on USER cloning] and the [https://2011.igem.org/Team:DTU-Denmark-2 wiki of the 2011 DTU-2 iGEM team] for more information.<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-01T14:22:20Z<p>Hezscha: /* User Cloning */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
Global demand for fixed nitrogen has increased to the point that half the human population now relies on chemical fertilizer to grow their food. While fertilizer is a requirement for modern life, runoff from overfertilized farmland can cause eutrophication. In the presence of abundant ammonia, algae overgrow and consume the much of the available oxygen in the water. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
Using two ''E. coli'' mutants built with genes from ''Nitrosomonas europaea'' and ''Pseudomonas aeruginosa'', we provide a system to reverse nitrogen fixation. Our mutants consume ammonia and produce nitrous oxide, releases a sustainable source of energy when decomposed into nitrogen and oxygen. We also provide a prototype of a bioreactor that could be scaled up and deployed in the field to simultaneously clean the water and produce energy.<br />
<br />
==Details==<br />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
The first mutant incorporates AMO, HAO and two cytochromes c554 and cm552 from ''Nitrosomonas europaea'' (shown in blue). AMO is a 3 subunit protein which converts ammonia (NH<sub>4</sub> to an intermediate called hydroxylamine (NH<sub>2</sub>OH). HAO then converts the hydroxylamine to nitrite (NO<sub>2</sub><sup>-</sup>. During this conversion process, cytochrome c554 accepts an electron from HAO, and then passes this electron on to ccm552. The terminal electron acceptor in this chain is quinone. AMO and ccm552 are embedded in the cytoplasmic membrane, and all other proteins in this process are found in the periplasm. <br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
The second mutant incorporates the Nir region from ''Pseudomonas aeruginosa'' (shown in yellow). NirS converts nitrite to nitric oxide (NO), while removing an electron from NirM. The remainder of the Nir region is necessary for the synthesis of NirM, and so we have included these genes as well. NOR, which is present natively in ''E. coli'', converts nitric oxide to nitrous oxide (N<sub>2</sub>O). In contrast to ''N. europaea'', ''E. coli'' is not a nitrifying bacterium and so does not convert nitrous oxide into nitrogen to complete the denitrification process.<br />
<br />
== Plasmids ==<br />
<br />
=== Mutant 1 ===<br />
<br />
The final plasmid for Mutant 1 should be the following:<br />
<br />
[[File:dtu_Plasmid_mutant_1-01.png|200px]]<br />
<br />
In order to simplify and to be able to debug each part of this pathway separately, we implemented the following.<br />
<br />
Unfortunately, we did not have time to combine all the genes into the final plasmid.<br />
<br />
[[File:Dtu Plasmids amo.png|100px]] [[File:Dtu Plasmids hao-01.png|150px]][[File:Dtu_Plasmids_cyt-01.png|150px]]<br />
<br />
=== Mutant 2 ===<br />
<br />
The final plasmid for Mutant 2 should be the following:<br />
<br />
[[File:Dtu Plasmid mutant 2-01.png|200px]]<br />
<br />
Since we had difficulty extracting Nir from ''Pseudomonas aeruginosa'' in one piece, we have implemented TODO<br />
<br />
== User Cloning ==<br />
<br />
To assemble our plasmids we employ USER cloning[there should be a ref here]. This technique enables us to speed up the cloning process, to clone seamless and to assemble many fragments in one reaction. The steps are:<br />
<br />
# PCR: The backbone and all fragments are amplified as linear products containing a uracil instead of a thymine at a specific position. Requires special uracil-containing primers and a polymerase that is able to perform uracil-insertion.<br />
# Optional - If circular DNA was used as PCR template a DpnI digestion can be performed to eliminate this DNA.<br />
# USER-reaction: Digesting the linearized PCR fragment with the USER enzyme removes the uracil-insertions, which will produce the required overhangs.<br />
# Ligation: Ligation is performed in chemically compentent E. coli, making use of this bacterium's repair system to assemble the plasmid.<br />
<br />
See our "Bricks of Knowledge" [https://www.youtube.com/watch?v=7EiVttJpXH4 video on USER cloning] and the [https://2011.igem.org/Team:DTU-Denmark-2 wiki of the 2011 DTU-2 iGEM team] for more information.<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-01T14:21:49Z<p>Hezscha: /* User Cloning */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
Global demand for fixed nitrogen has increased to the point that half the human population now relies on chemical fertilizer to grow their food. While fertilizer is a requirement for modern life, runoff from overfertilized farmland can cause eutrophication. In the presence of abundant ammonia, algae overgrow and consume the much of the available oxygen in the water. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
Using two ''E. coli'' mutants built with genes from ''Nitrosomonas europaea'' and ''Pseudomonas aeruginosa'', we provide a system to reverse nitrogen fixation. Our mutants consume ammonia and produce nitrous oxide, releases a sustainable source of energy when decomposed into nitrogen and oxygen. We also provide a prototype of a bioreactor that could be scaled up and deployed in the field to simultaneously clean the water and produce energy.<br />
<br />
==Details==<br />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
The first mutant incorporates AMO, HAO and two cytochromes c554 and cm552 from ''Nitrosomonas europaea'' (shown in blue). AMO is a 3 subunit protein which converts ammonia (NH<sub>4</sub> to an intermediate called hydroxylamine (NH<sub>2</sub>OH). HAO then converts the hydroxylamine to nitrite (NO<sub>2</sub><sup>-</sup>. During this conversion process, cytochrome c554 accepts an electron from HAO, and then passes this electron on to ccm552. The terminal electron acceptor in this chain is quinone. AMO and ccm552 are embedded in the cytoplasmic membrane, and all other proteins in this process are found in the periplasm. <br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
The second mutant incorporates the Nir region from ''Pseudomonas aeruginosa'' (shown in yellow). NirS converts nitrite to nitric oxide (NO), while removing an electron from NirM. The remainder of the Nir region is necessary for the synthesis of NirM, and so we have included these genes as well. NOR, which is present natively in ''E. coli'', converts nitric oxide to nitrous oxide (N<sub>2</sub>O). In contrast to ''N. europaea'', ''E. coli'' is not a nitrifying bacterium and so does not convert nitrous oxide into nitrogen to complete the denitrification process.<br />
<br />
== Plasmids ==<br />
<br />
=== Mutant 1 ===<br />
<br />
The final plasmid for Mutant 1 should be the following:<br />
<br />
[[File:dtu_Plasmid_mutant_1-01.png|200px]]<br />
<br />
In order to simplify and to be able to debug each part of this pathway separately, we implemented the following.<br />
<br />
Unfortunately, we did not have time to combine all the genes into the final plasmid.<br />
<br />
[[File:Dtu Plasmids amo.png|100px]] [[File:Dtu Plasmids hao-01.png|150px]][[File:Dtu_Plasmids_cyt-01.png|150px]]<br />
<br />
=== Mutant 2 ===<br />
<br />
The final plasmid for Mutant 2 should be the following:<br />
<br />
[[File:Dtu Plasmid mutant 2-01.png|200px]]<br />
<br />
Since we had difficulty extracting Nir from ''Pseudomonas aeruginosa'' in one piece, we have implemented TODO<br />
<br />
== User Cloning ==<br />
<br />
To assemble our plasmids we employ USER cloning[there should be a ref here]. This technique enables us to speed up the cloning process, to clone seamless and to assemble many fragments in one reaction. The steps are:<br />
<br />
# PCR: The backbone and all fragments are amplified as linear products containing a uracil instead of a thymine at a specific position. Requires special uracil-containing primers and a polymerase that is able to perform uracil-insertion.<br />
# Optional - If circular DNA was used as PCR template a DpnI digestion can be performed to eliminate this DNA.<br />
# USER-reaction: Digesting the linearized PCR fragment with the USER enzyme removes the uracil-insertions, which will produce the required overhangs.<br />
# Ligation: Ligation is performed in chemically compentent E. coli, making use of this bacterium's repair system to assemble the plasmid.<br />
<br />
See our "Bricks of Knowledge" [https://www.youtube.com/watch?v=7EiVttJpXH4 video on USER cloning] and the [https://2011.igem.org/Team:DTU-Denmark-2| wiki of the 2011 DTU-2 iGEM team] for more information.<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-01T14:21:29Z<p>Hezscha: /* User Cloning */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
Global demand for fixed nitrogen has increased to the point that half the human population now relies on chemical fertilizer to grow their food. While fertilizer is a requirement for modern life, runoff from overfertilized farmland can cause eutrophication. In the presence of abundant ammonia, algae overgrow and consume the much of the available oxygen in the water. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
Using two ''E. coli'' mutants built with genes from ''Nitrosomonas europaea'' and ''Pseudomonas aeruginosa'', we provide a system to reverse nitrogen fixation. Our mutants consume ammonia and produce nitrous oxide, releases a sustainable source of energy when decomposed into nitrogen and oxygen. We also provide a prototype of a bioreactor that could be scaled up and deployed in the field to simultaneously clean the water and produce energy.<br />
<br />
==Details==<br />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
The first mutant incorporates AMO, HAO and two cytochromes c554 and cm552 from ''Nitrosomonas europaea'' (shown in blue). AMO is a 3 subunit protein which converts ammonia (NH<sub>4</sub> to an intermediate called hydroxylamine (NH<sub>2</sub>OH). HAO then converts the hydroxylamine to nitrite (NO<sub>2</sub><sup>-</sup>. During this conversion process, cytochrome c554 accepts an electron from HAO, and then passes this electron on to ccm552. The terminal electron acceptor in this chain is quinone. AMO and ccm552 are embedded in the cytoplasmic membrane, and all other proteins in this process are found in the periplasm. <br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
The second mutant incorporates the Nir region from ''Pseudomonas aeruginosa'' (shown in yellow). NirS converts nitrite to nitric oxide (NO), while removing an electron from NirM. The remainder of the Nir region is necessary for the synthesis of NirM, and so we have included these genes as well. NOR, which is present natively in ''E. coli'', converts nitric oxide to nitrous oxide (N<sub>2</sub>O). In contrast to ''N. europaea'', ''E. coli'' is not a nitrifying bacterium and so does not convert nitrous oxide into nitrogen to complete the denitrification process.<br />
<br />
== Plasmids ==<br />
<br />
=== Mutant 1 ===<br />
<br />
The final plasmid for Mutant 1 should be the following:<br />
<br />
[[File:dtu_Plasmid_mutant_1-01.png|200px]]<br />
<br />
In order to simplify and to be able to debug each part of this pathway separately, we implemented the following.<br />
<br />
Unfortunately, we did not have time to combine all the genes into the final plasmid.<br />
<br />
[[File:Dtu Plasmids amo.png|100px]] [[File:Dtu Plasmids hao-01.png|150px]][[File:Dtu_Plasmids_cyt-01.png|150px]]<br />
<br />
=== Mutant 2 ===<br />
<br />
The final plasmid for Mutant 2 should be the following:<br />
<br />
[[File:Dtu Plasmid mutant 2-01.png|200px]]<br />
<br />
Since we had difficulty extracting Nir from ''Pseudomonas aeruginosa'' in one piece, we have implemented TODO<br />
<br />
== User Cloning ==<br />
<br />
To assemble our plasmids we employ USER cloning[there should be a ref here]. This technique enables us to speed up the cloning process, to clone seamless and to assemble many fragments in one reaction. The steps are:<br />
<br />
# PCR: The backbone and all fragments are amplified as linear products containing a uracil instead of a thymine at a specific position. Requires special uracil-containing primers and a polymerase that is able to perform uracil-insertion.<br />
# Optional - If circular DNA was used as PCR template a DpnI digestion can be performed to eliminate this DNA.<br />
# USER-reaction: Digesting the linearized PCR fragment with the USER enzyme removes the uracil-insertions, which will produce the required overhangs.<br />
# Ligation: Ligation is performed in chemically compentent E. coli, making use of this bacterium's repair system to assemble the plasmid.<br />
<br />
See our "Bricks of Knowledge" [https://www.youtube.com/watch?v=7EiVttJpXH4 video on USER cloning] and the [https://2011.igem.org/Team:DTU-Denmark-2|wiki of the 2011 DTU-2 iGEM team] for more information.<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/NotebookTeam:DTU-Denmark/Notebook2013-10-01T14:19:11Z<p>Hezscha: </p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|Notebook}}<br />
__NOTOC__<br />
<br />
Our team decided to keep an online notebook for our lab work. Click in the calendar to see the entry for a specific day.<br />
<br />
<div class="overviewPage"><br />
==Lab Book Entries==<br />
<br />
{| style="width: 60%; height: 200px;" border="1" <br />
|-<br />
| <center> {{#calendar: title=Team:DTU-Denmark/Notebook |year=2013 | month=06}} </center><br />
| <center> {{#calendar: title=Team:DTU-Denmark/Notebook |year=2013 | month=07}} </center><br />
| <center> {{#calendar: title=Team:DTU-Denmark/Notebook |year=2013 | month=08}} </center><br />
| <center> {{#calendar: title=Team:DTU-Denmark/Notebook |year=2013 | month=09}} </center><br />
|}<br />
<br />
<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-01T14:18:36Z<p>Hezscha: /* User Cloning */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
Global demand for fixed nitrogen has increased to the point that half the human population now relies on chemical fertilizer to grow their food. While fertilizer is a requirement for modern life, runoff from overfertilized farmland can cause eutrophication. In the presence of abundant ammonia, algae overgrow and consume the much of the available oxygen in the water. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
Using two ''E. coli'' mutants built with genes from ''Nitrosomonas europaea'' and ''Pseudomonas aeruginosa'', we provide a system to reverse nitrogen fixation. Our mutants consume ammonia and produce nitrous oxide, releases a sustainable source of energy when decomposed into nitrogen and oxygen. We also provide a prototype of a bioreactor that could be scaled up and deployed in the field to simultaneously clean the water and produce energy.<br />
<br />
==Details==<br />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
The first mutant incorporates AMO, HAO and two cytochromes c554 and cm552 from ''Nitrosomonas europaea'' (shown in blue). AMO is a 3 subunit protein which converts ammonia (NH<sub>4</sub> to an intermediate called hydroxylamine (NH<sub>2</sub>OH). HAO then converts the hydroxylamine to nitrite (NO<sub>2</sub><sup>-</sup>. During this conversion process, cytochrome c554 accepts an electron from HAO, and then passes this electron on to ccm552. The terminal electron acceptor in this chain is quinone. AMO and ccm552 are embedded in the cytoplasmic membrane, and all other proteins in this process are found in the periplasm. <br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
The second mutant incorporates the Nir region from ''Pseudomonas aeruginosa'' (shown in yellow). NirS converts nitrite to nitric oxide (NO), while removing an electron from NirM. The remainder of the Nir region is necessary for the synthesis of NirM, and so we have included these genes as well. NOR, which is present natively in ''E. coli'', converts nitric oxide to nitrous oxide (N<sub>2</sub>O). In contrast to ''N. europaea'', ''E. coli'' is not a nitrifying bacterium and so does not convert nitrous oxide into nitrogen to complete the denitrification process.<br />
<br />
== Plasmids ==<br />
<br />
=== Mutant 1 ===<br />
<br />
The final plasmid for Mutant 1 should be the following:<br />
<br />
[[File:dtu_Plasmid_mutant_1-01.png|200px]]<br />
<br />
In order to simplify and to be able to debug each part of this pathway separately, we implemented the following.<br />
<br />
Unfortunately, we did not have time to combine all the genes into the final plasmid.<br />
<br />
[[File:Dtu Plasmids amo.png|100px]] [[File:Dtu Plasmids hao-01.png|150px]][[File:Dtu_Plasmids_cyt-01.png|150px]]<br />
<br />
=== Mutant 2 ===<br />
<br />
The final plasmid for Mutant 2 should be the following:<br />
<br />
[[File:Dtu Plasmid mutant 2-01.png|200px]]<br />
<br />
Since we had difficulty extracting Nir from ''Pseudomonas aeruginosa'' in one piece, we have implemented TODO<br />
<br />
== User Cloning ==<br />
<br />
To assemble our plasmids we employ USER cloning[there should be a ref here]. This technique enables us to speed up the cloning process, to clone seamless and to assemble many fragments in one reaction. The steps are:<br />
<br />
# PCR: The backbone and all fragments are amplified as linear products containing a uracil instead of a thymine at a specific position. Requires special uracil-containing primers and a polymerase that is able to perform uracil-insertion.<br />
# Optional - If circular DNA was used as PCR template a DpnI digestion can be performed to eliminate this DNA.<br />
# USER-reaction: Digesting the linearized PCR fragment with the USER enzyme removes the uracil-insertions, which will produce the required overhangs.<br />
# Ligation: Ligation is performed in chemically compentent E. coli, making use of this bacterium's repair system to assemble the plasmid.<br />
<br />
See our "Bricks of Knowledge" [https://www.youtube.com/watch?v=7EiVttJpXH4 video on USER cloning] and the wiki of the 2011 DTU-2 iGEM team for more information.<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-01T14:18:00Z<p>Hezscha: /* Plasmids */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
Global demand for fixed nitrogen has increased to the point that half the human population now relies on chemical fertilizer to grow their food. While fertilizer is a requirement for modern life, runoff from overfertilized farmland can cause eutrophication. In the presence of abundant ammonia, algae overgrow and consume the much of the available oxygen in the water. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
Using two ''E. coli'' mutants built with genes from ''Nitrosomonas europaea'' and ''Pseudomonas aeruginosa'', we provide a system to reverse nitrogen fixation. Our mutants consume ammonia and produce nitrous oxide, releases a sustainable source of energy when decomposed into nitrogen and oxygen. We also provide a prototype of a bioreactor that could be scaled up and deployed in the field to simultaneously clean the water and produce energy.<br />
<br />
==Details==<br />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
The first mutant incorporates AMO, HAO and two cytochromes c554 and cm552 from ''Nitrosomonas europaea'' (shown in blue). AMO is a 3 subunit protein which converts ammonia (NH<sub>4</sub> to an intermediate called hydroxylamine (NH<sub>2</sub>OH). HAO then converts the hydroxylamine to nitrite (NO<sub>2</sub><sup>-</sup>. During this conversion process, cytochrome c554 accepts an electron from HAO, and then passes this electron on to ccm552. The terminal electron acceptor in this chain is quinone. AMO and ccm552 are embedded in the cytoplasmic membrane, and all other proteins in this process are found in the periplasm. <br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
The second mutant incorporates the Nir region from ''Pseudomonas aeruginosa'' (shown in yellow). NirS converts nitrite to nitric oxide (NO), while removing an electron from NirM. The remainder of the Nir region is necessary for the synthesis of NirM, and so we have included these genes as well. NOR, which is present natively in ''E. coli'', converts nitric oxide to nitrous oxide (N<sub>2</sub>O). In contrast to ''N. europaea'', ''E. coli'' is not a nitrifying bacterium and so does not convert nitrous oxide into nitrogen to complete the denitrification process.<br />
<br />
== Plasmids ==<br />
<br />
=== Mutant 1 ===<br />
<br />
The final plasmid for Mutant 1 should be the following:<br />
<br />
[[File:dtu_Plasmid_mutant_1-01.png|200px]]<br />
<br />
In order to simplify and to be able to debug each part of this pathway separately, we implemented the following.<br />
<br />
Unfortunately, we did not have time to combine all the genes into the final plasmid.<br />
<br />
[[File:Dtu Plasmids amo.png|100px]] [[File:Dtu Plasmids hao-01.png|150px]][[File:Dtu_Plasmids_cyt-01.png|150px]]<br />
<br />
=== Mutant 2 ===<br />
<br />
The final plasmid for Mutant 2 should be the following:<br />
<br />
[[File:Dtu Plasmid mutant 2-01.png|200px]]<br />
<br />
Since we had difficulty extracting Nir from ''Pseudomonas aeruginosa'' in one piece, we have implemented TODO<br />
<br />
== User Cloning ==<br />
<br />
See our "Bricks of Knowledge" [https://www.youtube.com/watch?v=7EiVttJpXH4 video on USER cloning] for more information.<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-01T14:17:25Z<p>Hezscha: /* Plasmids */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
Global demand for fixed nitrogen has increased to the point that half the human population now relies on chemical fertilizer to grow their food. While fertilizer is a requirement for modern life, runoff from overfertilized farmland can cause eutrophication. In the presence of abundant ammonia, algae overgrow and consume the much of the available oxygen in the water. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
Using two ''E. coli'' mutants built with genes from ''Nitrosomonas europaea'' and ''Pseudomonas aeruginosa'', we provide a system to reverse nitrogen fixation. Our mutants consume ammonia and produce nitrous oxide, releases a sustainable source of energy when decomposed into nitrogen and oxygen. We also provide a prototype of a bioreactor that could be scaled up and deployed in the field to simultaneously clean the water and produce energy.<br />
<br />
==Details==<br />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
The first mutant incorporates AMO, HAO and two cytochromes c554 and cm552 from ''Nitrosomonas europaea'' (shown in blue). AMO is a 3 subunit protein which converts ammonia (NH<sub>4</sub> to an intermediate called hydroxylamine (NH<sub>2</sub>OH). HAO then converts the hydroxylamine to nitrite (NO<sub>2</sub><sup>-</sup>. During this conversion process, cytochrome c554 accepts an electron from HAO, and then passes this electron on to ccm552. The terminal electron acceptor in this chain is quinone. AMO and ccm552 are embedded in the cytoplasmic membrane, and all other proteins in this process are found in the periplasm. <br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
The second mutant incorporates the Nir region from ''Pseudomonas aeruginosa'' (shown in yellow). NirS converts nitrite to nitric oxide (NO), while removing an electron from NirM. The remainder of the Nir region is necessary for the synthesis of NirM, and so we have included these genes as well. NOR, which is present natively in ''E. coli'', converts nitric oxide to nitrous oxide (N<sub>2</sub>O). In contrast to ''N. europaea'', ''E. coli'' is not a nitrifying bacterium and so does not convert nitrous oxide into nitrogen to complete the denitrification process.<br />
<br />
== Plasmids ==<br />
<br />
To assemble our plasmids we employ USER cloning[there should be a ref here]. This technique enables us to speed up the cloning process, to clone seamless and to assemble many fragments in one reaction. The steps are:<br />
<br />
# PCR: The backbone and all fragments are amplified as linear products containing a uracil instead of a thymine at a specific position. Requires special uracil-containing primers and a polymerase that is able to perform uracil-insertion.<br />
# Optional - If circular DNA was used as PCR template a DpnI digestion can be performed to eliminate this DNA.<br />
# USER-reaction: Digesting the linearized PCR fragment with the USER enzyme removes the uracil-insertions, which will produce the required overhangs.<br />
# Ligation: Ligation is performed in chemically compentent E. coli, making use of this bacterium's repair system to assemble the plasmid.<br />
<br />
=== Mutant 1 ===<br />
<br />
The final plasmid for Mutant 1 should be the following:<br />
<br />
[[File:dtu_Plasmid_mutant_1-01.png|200px]]<br />
<br />
In order to simplify and to be able to debug each part of this pathway separately, we implemented the following.<br />
<br />
Unfortunately, we did not have time to combine all the genes into the final plasmid.<br />
<br />
[[File:Dtu Plasmids amo.png|100px]] [[File:Dtu Plasmids hao-01.png|150px]][[File:Dtu_Plasmids_cyt-01.png|150px]]<br />
<br />
=== Mutant 2 ===<br />
<br />
The final plasmid for Mutant 2 should be the following:<br />
<br />
[[File:Dtu Plasmid mutant 2-01.png|200px]]<br />
<br />
Since we had difficulty extracting Nir from ''Pseudomonas aeruginosa'' in one piece, we have implemented TODO<br />
<br />
== User Cloning ==<br />
<br />
See our "Bricks of Knowledge" [https://www.youtube.com/watch?v=7EiVttJpXH4 video on USER cloning] for more information.<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/pBAD_SPLTeam:DTU-Denmark/pBAD SPL2013-10-01T13:56:53Z<p>Hezscha: /* Experimental procedure */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|pBAD SPL}}<br />
<br />
== pBAD synthetic promoter library ==<br />
<br />
As a tool for expressing lethal proteins in ''E. coli'' we made a synthetic promoter library (SPL, [http://dspace.mit.edu/handle/1721.1/60080 RFC 63]) with the pBAD arabinose inducible promoter. The concept was taken from the [https://2010.igem.org/Team:DTU-Denmark/SPL DTU iGEM team from 2010].<br />
<br />
== Methods ==<br />
<br />
=== Experimental procedure ===<br />
<br />
# Random promoter sequences were ordered matching the sequence CTGACGNNNNNNNNNNNNNNNNNNTAWWATNNNNA.<br />
# USER cloning to add RFP downstream of promoter.<br />
# Colonies were plated.<br />
# Plates were induced by spraying them with an aqueous arabinose solution.<br />
# Colonies that were not red prior to induction with arabinose but that did turn red after induction with arabinose were selected and re-innoculated as liquid cultures.<br />
# Biolector: Wells were inoculated from overnight cultures of each of the selected colonies. All wells were run in duplicate.<br />
# All duplicate colonies were run twice -- once with arabinose added at t=0, and again without arabinose. <br />
# The constitutive promoter TODO:which was used as a reference.<br />
<br />
=== Data analysis ===<br />
<br />
# Data was collected from the Biolector, and analyzed using a series of R scripts written by Chris Workman (unpublished). <br />
#* The maturation and degradation times for mCherry were both assumed to be 40 min. TODOref<br />
#* The growth rate, mu, was estimated to be 1.28 (from an average of all wells on all plates) since we expect each strain to grow at the same rate.<br />
#* A time window representing exponential growth was selected (between 1 and 4.5 hours).<br />
# The RFP measurement for a constitutively expressed strain was used as a standard measure of growth. This is plotted on the x-axis in the detailed plots per colony below. <br />
# Figures were plotted using R.<br />
<br />
== Results ==<br />
<br />
=== Summary ===<br />
<br />
Promoter activity when induced (with arabinose added) plotted vs basal activity (without arabinose; ie leakiness of the promoter). The colonies that we selected all show less activity than the the constitutive promoter, and when induced, show higher activity than the constitutive promoter. <br />
[[File:Induced_vs_basal.png]]<br />
<br />
=== Details ===<br />
<br />
Promoter strengths for two trials of each colony with and without arabinose. <br />
{| class="wikitable"<br />
!Colony Number<br />
!With arabinose 1<br />
!With arabinose 2<br />
!Without arabinose 1<br />
!Without arabinose 2<br />
|-<br />
|Col2||8.9025||7.8699||0.727||0.9552<br />
|-<br />
|Col3||9.2724||12.1142||0.5248||0.6982<br />
|-<br />
|Col4||9.4571||11.4522||0.5231||0.2508<br />
|-<br />
|Col12||6.3641||10.5389||0.5897||0.6869<br />
|-<br />
|Col10||7.9697||9.7949||0.3392||0.733<br />
|-<br />
|Col9||7.8563||20.1094||0.6995||0.7432<br />
|-<br />
|Col8||12.2318||15.4548||0.4203||0.4538<br />
|-<br />
|Col13||11.0377||7.3343||0.482||0.4641<br />
|-<br />
|Col15||15.6817||8.2707||0.8169||0.1343<br />
|-<br />
|Col18||14.7916||15.5736||0.6674||0.6745<br />
|-<br />
|Col19||14.2126||16.4898||0.4545||0.3566<br />
|-<br />
|Col29||7.1853||16.3467||0.5445||0.5013<br />
|-<br />
|Col26||9.7724||9.6269||0.7118||0.7865<br />
|-<br />
|Col22||8.4168||5.5958||0.6049||0.5645<br />
|-<br />
|Col33||9.1982||8.9987||0.6508||1.374<br />
|-<br />
|Col34||10.6987||7.883||0.5067||0.5031<br />
|-<br />
|Col35||13.8427||7.5469||0.4363||0.6281<br />
|-<br />
|ConRef||6.506||7.9323||8.7811||7.9323<br />
|}<br />
<br />
[[File:dtu-Fss-plot-col2.png|300px]]<br />
[[File:dtu-Fss-plot-col3.png|300px]]<br />
[[File:dtu-Fss-plot-col4.png|300px]]<br />
[[File:dtu-Fss-plot-col12.png|300px]]<br />
[[File:dtu-Fss-plot-col10.png|300px]]<br />
[[File:dtu-Fss-plot-col9.png|300px]]<br />
[[File:dtu-Fss-plot-col8.png|300px]]<br />
[[File:dtu-Fss-plot-col13.png|300px]]<br />
[[File:dtu-Fss-plot-col15.png|300px]]<br />
[[File:dtu-Fss-plot-col18.png|300px]]<br />
[[File:dtu-Fss-plot-col19.png|300px]]<br />
[[File:dtu-Fss-plot-col29.png|300px]]<br />
[[File:dut-Fss-plot-col26.png|300px]]<br />
[[File:dtu-Fss-plot-col22.png|300px]]<br />
[[File:dtu-Fss-plot-col33.png|300px]]<br />
[[File:dtu-Fss-plot-col34.png|300px]]<br />
[[File:dtu-Fss-plot-col25.png|300px]]<br />
[[File:dtu-Fss-plot-conref.png|300px]]<br />
<br />
== Example of use ==<br />
[[File:GFP in perimplasm RFP in cytoplasm.png|650px|thumbnail|upright=4|left|alt=Alt text|Caption text]]<br />
[[File:GFP in perimplasm RFP in cytoplasm close up.png|300px|thumbnail|upright=2|left|alt=Alt text|Caption text]]<br />
[[File:Graf.PNG|290px|thumbnail|upright=2|left|alt=Alt text|Caption text]]<br />
<br />
<br />
<br />
See also [https://2013.igem.org/Team:DTU-Denmark/HelloWorld "Hello World project"].<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/NotebookTeam:DTU-Denmark/Notebook2013-10-01T13:39:30Z<p>Hezscha: </p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|Notebook}}<br />
<br />
Our team decided to keep an online notebook for our lab work. Click in the calendar to see the entry for a specific day.<br />
<br />
<div class="overviewPage"><br />
==Lab Book Entries==<br />
<br />
{| style="width: 60%; height: 200px;" border="1" <br />
|-<br />
| <center> {{#calendar: title=Team:DTU-Denmark/Notebook |year=2013 | month=06}} </center><br />
| <center> {{#calendar: title=Team:DTU-Denmark/Notebook |year=2013 | month=07}} </center><br />
| <center> {{#calendar: title=Team:DTU-Denmark/Notebook |year=2013 | month=08}} </center><br />
| <center> {{#calendar: title=Team:DTU-Denmark/Notebook |year=2013 | month=09}} </center><br />
|}<br />
<br />
<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/Notebook/25_August_2013Team:DTU-Denmark/Notebook/25 August 20132013-09-28T18:43:25Z<p>Hezscha: /* Who was in the lab */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|25 August 2013}}<br />
Navigate to the [[Team:DTU-Denmark/Notebook/24_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/26_August_2013|Next]] Entry<br />
=Lab 208=<br />
<hr/><br />
==Main purpose==<br />
<hr/><br />
*colony PCR<br />
<br />
==Who was in the lab==<br />
<hr/><br />
Kristian, Henrike<br />
<br />
==Procedure==<br />
<hr/><br />
<br />
===Colony PCR===<br />
<br />
Performed colony PCR to confirm insert for HAO, AMO, cycAX and Nir transformants. Used Q5 premix with the following reaction mix:<br />
<br />
{| class="wikitable" style="text-align: right"<br />
! compound !! amount<br />
|-<br />
| Q5 mix || 25 uL<br />
|-<br />
| FW primer || 3 uL<br />
|-<br />
| RV primer || 3 uL<br />
|-<br />
| template || 1 uL<br />
|-<br />
| MilliQ || 18 uL<br />
|-<br />
|}<br />
<br />
Template was made by resuspending 1 culture in 100uL MilliQ.<br />
<br />
program:<br />
{| class="wikitable" style="text-align: right"<br />
! temperature !! time !! cycles<br />
|-<br />
| 98C || 10:00 || - <br />
|-<br />
| 98C || 0:10 || 36 <br />
|-<br />
| annealing temperature || 0:30 || 36 <br />
|-<br />
| 72C || 0:20 || 36<br />
|-<br />
| 72C || 5:00 || - <br />
|-<br />
| 10C || hold || -<br />
|-<br />
|}<br />
<br />
details (primers, temp, expected fragment length):<br />
* cycAX - FW_1, RV_2, 61C, 749bp<br />
* HAO - FW_2, RV_3, 64C, 751bp<br />
* AMO - FW_2, RV_3, 63C, 750bp<br />
* Nir - FW_2, RV_3, 64C, 733bp<br />
* Nir - FW_5, RV_6, 71C, 737bp<br />
<br />
==Results==<br />
<hr/><br />
<br />
==Conclusion==<br />
<hr/><br />
<br />
Navigate to the [[Team:DTU-Denmark/Notebook/24_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/26_August_2013|Next]] Entry<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/Notebook/30_August_2013Team:DTU-Denmark/Notebook/30 August 20132013-09-28T18:05:59Z<p>Hezscha: /* lab 208 */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|30 August 2013}}<br />
Navigate to the [[Team:DTU-Denmark/Notebook/29_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/31_August_2013|Next]] Entry<br />
=lab 208=<br />
<hr/><br />
==Main purpose==<br />
<hr/><br />
*User reaction<br />
*PCR to amplify HAO and cycAX USER fragments<br />
<br />
==Who was in the lab==<br />
<hr/><br />
<br />
==Procedure==<br />
<hr/><br />
<br />
===USER reaction===<br />
<br />
Redid USER reaction for ligating our inserts into the Biobrick backbone. There was no cycAX fragment left.<br />
<br />
HAO in pSB1C3<br />
AMO in pSB1C3<br />
<br />
===PCR to amplify HAO and cycAX USER fragments===<br />
<br />
Made 6 reactions for each, using either the extraction PCR fragment or the glycerol stock as template<br />
<br />
{| class="wikitable" style="text-align: right"<br />
! label !! cyc gly D !! cyc gly % !! cyc gly 1uL D !! cyc ext D !! cyc ext % !! cyc ext 1uL D <br />
|-<br />
| template || glycerol stock || glycerol stock || glycerol stock || extraction PCR fragment || extraction PCR fragment || extraction PCR fragment <br />
|-<br />
| DMSO || 1,5 || - || 1,5 || 1,5 || - || 1,5 <br />
|-<br />
| MgCl2 || - || - || 1 || - || - || 1<br />
|-<br />
| MQ || 1 || 2,5 || - || 1 || 2,5 || - <br />
|-<br />
|}<br />
<br />
{| class="wikitable" style="text-align: right"<br />
! label !! HAO gly D !! HAO gly % !! HAO gly 1uL D !! HAO ext D !! HAO ext % !! HAO ext 1uL D <br />
|-<br />
| template || glycerol stock || glycerol stock || glycerol stock || extraction PCR fragment || extraction PCR fragment || extraction PCR fragment <br />
|-<br />
| DMSO || 1,5 || - || 1,5 || 1,5 || - || 1,5 <br />
|-<br />
| MgCl2 || - || - || 1 || - || - || 1<br />
|-<br />
| MQ || 1 || 2,5 || - || 1 || 2,5 || - <br />
|-<br />
|}<br />
<br />
Program:<br />
{| class="wikitable" style="text-align: right"<br />
! temperature !! time !! cycles<br />
|-<br />
| 98C || 2:00 || - <br />
|-<br />
| 98C || 0:20 || 36 <br />
|-<br />
| annealing temperature || 1:00 || 36 <br />
|-<br />
| 72C || extension time || 36<br />
|-<br />
| 72C || 5:00 || - <br />
|-<br />
| 10C || hold || -<br />
|-<br />
|}<br />
<br />
HAO - 59C, 3:00<br />
<br />
cycAX - 55C, 1:30<br />
<br />
==Results==<br />
<hr/><br />
<br />
===Gel===<br />
<br />
* 1 kb ladder<br />
* psB1C3 for Nir, 2% DMSO<br />
* psB1C3 for Nir, 2% DMSO, 1 uL 50mM MgCl2<br />
* psB1C3 for Nir, 4% DMSO<br />
* psB1C3 for Nir, 4% DMSO, 1 uL 50mM MgCl2<br />
* psB1C3 for Nir, 1M Betaine<br />
* purification of psB1C3 USER fragment to make Biobricks (escaped from well -> Ethanol?)<br />
* purification of HAO fragment (escaped from well -> Ethanol?)<br />
* purification of AMO fragment (escaped from well -> Ethanol?)<br />
* purification of psB1C3 USER fragment to make Biobricks (used more loading buffer)<br />
* purification of HAO fragment (used more loading buffer)<br />
* purification of AMO fragment (used more loading buffer)<br />
* purification of pZA21::ara(tight)<br />
* purification of pZA21::ara(tight) with endings for Nir<br />
* 1 kb ladder<br />
<br />
[[File:2013-08-30 psbnir puri verification2.jpg|600px]]<br />
<br />
==Conclusion==<br />
<hr/><br />
Navigate to the [[Team:DTU-Denmark/Notebook/29_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/31_August_2013|Next]] Entry<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/Notebook/28_August_2013Team:DTU-Denmark/Notebook/28 August 20132013-09-28T18:04:33Z<p>Hezscha: /* Main purpose */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|28 August 2013}}<br />
Navigate to the [[Team:DTU-Denmark/Notebook/27_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/29_August_2013|Next]] Entry<br />
=Lab 208=<br />
<hr/><br />
==Main purpose==<br />
<hr/><br />
PCR to amplify pSB1C3 and pZA21::ara for Nir<br />
<br />
==Who was in the lab==<br />
<hr/><br />
Henrike, Kristian, Julia<br />
==Procedure==<br />
<hr/><br />
A gradient PCR was made for pSB1C3 and pZA21::ara for Nir.<br />
<br />
The [[Team:DTU-Denmark/Methods/PCR-mix|master mix]] contains:<br />
*CG buffer, <br />
*X7 polymerase, <br />
*2% of DMSO (1uL of 100%DMSO) per reaction <br />
*1mM MgCl2 (1uL of 50mM MgCl2) per reaction<br />
{| class="wikitable" style="text-align: right"<br />
! !! 1 !! 2<br />
|-<br />
| Template || pSB1C3 || pZA21::ara<br />
|-<br />
| Primers || 57a + 57b || 58a + 58b<br />
|-<br />
| Temperature range || 58C-78C || 50C-65C<br />
|-<br />
| Elongation time per cycle || 1:30 min || 2 min<br />
|-<br />
|}<br />
<br />
Each reaction was run in one of the 12 different annealing temperatures within the range indicated in the table.<br />
We followed the [[Team:DTU-Denmark/Methods/PCR#Generel_example_on_a_normal_PCR-program|standard PCR program]].<br />
<br />
==Results==<br />
<hr/><br />
<br />
==Conclusion==<br />
<hr/><br />
<br />
<br />
=lab 115=<br />
<hr/><br />
<br />
==Main purpose==<br />
<hr/><br />
Run [[Team:DTU-Denmark/Experiment2|Experiment 4]] in two different samples aerobically in order to characterize the behavior of ''HAO transformants'' and the ''Nitrosomonas europaea'' strain.<br />
<br />
==Who was in the lab==<br />
<hr/><br />
Ariadni, Helen, Kashia<br />
<br />
==Procedure==<br />
<hr/><br />
<br />
Adjusting the temperature at 36 degrees and calibrating the probes as described in [[Team:DTU-Denmark/Methods/Calibrating_Electrodes|Calibration protocol]].<br />
<br />
<br />
Following the protocol [[Team:DTU-Denmark/Methods/Determining_concentration_of_nitrogen_compounds/Experiment_4|Experiment 4]]<br />
<br />
Growing:<br />
*two replicates of 4 ml from HAO overnight culture in 50 ml of DM minimal medium with NH<sub>4</sub>Cl and 25 ul of hydroxylamine solution<br />
* 4 ml from ''Nitrosomonas Europaea'' in 42 ml of ATCC media with 20 ul of hydroxylamine solution<br />
* 4 ml from ''Nitrosomonas Europaea'' in 44 ml of ATCC media with 0.7 ml of ammonium solution<br />
<br />
<br />
OD was measured OD=0.4017 and 0.3864 for the HAO mutant. 0.0822 and 0.0947. For ''Nitrosomonas Europaea'' was 0.0095 and 0.0084.<br />
<br />
==Results==<br />
<hr/><br />
<br />
===Colorimetric results===<br />
<br />
'''Ranges'''<br />
* ''Measuring range 5-150 mg/L NH<sub>4</sub>-N''<br />
* ''Measuring range 0.02-1 mg/L NO<sub>2</sub>-N''<br />
<br />
<br />
Standard solutions <br />
* Ammonium - 66.6 mg/L (expected 39 mg/L)<br />
* Nitrite - 0.72 mg/L (expected 0.5 mg/L)<br />
<br />
<br />
{| class="wikitable" style="text-align: right"<br />
! time (min)!!nitrite HAO1(mg/L)!! nitrite HAO2(mg/L)!! Ammonium ''N.Europaea''(mg/L) !! nitrite ''N.Europaea''<br />
|-<br />
| 0 || <0.02 || <0.02 || 0 || <0.02<br />
|-<br />
| 6 || <0.02 || <0.02 || 0 || <0.02<br />
|-<br />
| 13 || <0.02 || <0.02 || 0 || <0.02<br />
|-<br />
| spike || <0.02 || <0.02 || 246 || <0.02<br />
|-<br />
| 25 || <0.02 || <0.02 || 250 || <0.02<br />
|-<br />
| 39 || <0.02 || <0.02 || 278 || <0.02<br />
|-<br />
| 105 || <0.02 || <0.02 || 266 || <0.02<br />
|-<br />
| 19 hours|| <0.02 || <0.02 || 220 || <0.02<br />
|-<br />
|}<br />
<br />
Note: there was some more nitrite for ''N.Europaea'' but it was still less than <0.02 mg/L at the end of the experiment<br />
<br />
===OD at the end of the experiment===<br />
<br />
OD( HAO1)=0.28 and OD(HAO2)=0.278. For the ''N.Europaea'' culture the OD was 0.1348 and 0.0114 for the solution with ammonium and for the one with hydroxylamine, respectively.<br />
<br />
==Conclusion==<br />
<hr/><br />
<br />
Navigate to the [[Team:DTU-Denmark/Notebook/27_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/29_August_2013|Next]] Entry<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/Notebook/27_August_2013Team:DTU-Denmark/Notebook/27 August 20132013-09-28T18:03:03Z<p>Hezscha: /* Main purpose */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|27 August 2013}}<br />
Navigate to the [[Team:DTU-Denmark/Notebook/26_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/28_August_2013|Next]] Entry<br />
=lab 208=<br />
<hr/><br />
==Main purpose==<br />
<hr/><br />
various PCR reactions<br />
<br />
==Who was in the lab==<br />
<hr/><br />
Kristian, Henrike<br />
<br />
==Procedure==<br />
<hr/><br />
<br />
===PCR reactions===<br />
<br />
Set up PCR reactions for:<br />
* Biobrick backbone pSB1C3 with USER endings fitting for our inserts<br />
* constitutive reference promoter in pZA21 (for SPL)<br />
* pZA21::ara vector with USER endings fitting for Nir1 and Nir2 inserts<br />
<br />
<u>first round of PCRs</u>:<br />
<br />
pSB1C3:<br />
{| class="wikitable" style="text-align: right"<br />
! temperature !! time !! cycles<br />
|-<br />
| 98C || 2:00 || - <br />
|-<br />
| 98C || 0:10 || 36 <br />
|-<br />
| 70C || 0:45 || 36 <br />
|-<br />
| 72C || 1:30 || 36<br />
|-<br />
| 72C || 5:00 || - <br />
|-<br />
| 10C || hold || -<br />
|-<br />
|}<br />
Four different reaction mixes: HF, HF+5%DMSO, GC, GC+5%DMSO<br />
<br />
pZA21::ara for Nir:<br />
{| class="wikitable" style="text-align: right"<br />
! temperature !! time !! cycles<br />
|-<br />
| 98C || 2:00 || - <br />
|-<br />
| 98C || 0:10 || 36 <br />
|-<br />
| 60C || 1:00 || 36 <br />
|-<br />
| 72C || 2:00 || 36<br />
|-<br />
| 72C || 5:00 || - <br />
|-<br />
| 10C || hold || -<br />
|-<br />
|}<br />
<br />
Standard reaction mix.<br />
<br />
constitutive reference promoter:<br />
{| class="wikitable" style="text-align: right"<br />
! temperature !! time !! cycles<br />
|-<br />
| 98C || 2:00 || - <br />
|-<br />
| 98C || 0:20 || 36 <br />
|-<br />
| 58.1C || 1:00 || 36 <br />
|-<br />
| 72C || 3:00 || 36<br />
|-<br />
| 72C || 5:00 || - <br />
|-<br />
| 10C || hold || -<br />
|-<br />
|}<br />
Mix with GC, 5%DMSO and 2uL MgCl2.<br />
<br />
pSB1C3 and pZA21::ara for Nir were without results (see gel picture). New PCRs were set up with one standard reaction and one reaction with additives. The annealing temperature was lowered.<br />
<br />
<u>second round of PCRs</u>:<br />
<br />
{| class="wikitable" style="text-align: right"<br />
! compound !! amount (in uL)<br />
|-<br />
| || standard mix || additive mix<br />
|-<br />
| dNTPs || 1 || 1<br />
|-<br />
| X7 ploymerase || 0.5 || 0.5<br />
|-<br />
| HF buffer || 10 || 10<br />
|-<br />
| MilliQ || 31.5 || 27<br />
|-<br />
| FW primer || 3 || 3<br />
|-<br />
| RV primer || 3 || 3<br />
|-<br />
| template || 1 || 1<br />
|-<br />
| DMSO || - || 2.5<br />
|-<br />
| 50 mM MgCl2 || - || 2<br />
|-<br />
|}<br />
<br />
<br />
pSB1C3:<br />
{| class="wikitable" style="text-align: right"<br />
! temperature !! time !! cycles<br />
|-<br />
| 98C || 2:00 || - <br />
|-<br />
| 98C || 0:10 || 36 <br />
|-<br />
| 60C || 1:00 || 36 <br />
|-<br />
| 72C || 1:30 || 36<br />
|-<br />
| 72C || 5:00 || - <br />
|-<br />
| 10C || hold || -<br />
|-<br />
|}<br />
<br />
<br />
pZA21::ara for Nir:<br />
{| class="wikitable" style="text-align: right"<br />
! temperature !! time !! cycles<br />
|-<br />
| 98C || 2:00 || - <br />
|-<br />
| 98C || 0:10 || 36 <br />
|-<br />
| 55C || 1:00 || 36 <br />
|-<br />
| 72C || 2:00 || 36<br />
|-<br />
| 72C || 5:00 || - <br />
|-<br />
| 10C || hold || -<br />
|-<br />
|}<br />
<br />
<br />
Additionally set up a PCR for cycAX with USER endings since the amount of fragment is running low.<br />
<br />
cycAX for USER:<br />
{| class="wikitable" style="text-align: right"<br />
! temperature !! time !! cycles<br />
|-<br />
| 98C || 2:00 || - <br />
|-<br />
| 98C || 0:20 || 36 <br />
|-<br />
| 57C || 0:45 || 36 <br />
|-<br />
| 72C || 1:30 || 36<br />
|-<br />
| 72C || 5:00 || - <br />
|-<br />
| 10C || hold || -<br />
|-<br />
|}<br />
Standard reaction mix.<br />
<br />
==Results==<br />
<hr/><br />
<br />
===Gel===<br />
<br />
* 1 kb ladder<br />
* pSB1C3 for Biobricks, HF buffer<br />
* pSB1C3 for Biobricks, HF buffer, 5%DMSO<br />
* pSB1C3 for Biobricks, GC buffer<br />
* pSB1C3 for Biobricks, GC buffer, 5%DMSO<br />
* negative pSB1C3 for Biobricks<br />
* pZA21::ara for USER ligation with Nir<br />
* pZA21::ara for USER ligation with Nir (duplicate)<br />
* 1 kb ladder<br />
<br />
Note: It seems the ladder overflew.<br />
<br />
[[File:2013-08-27 bb pzaarafornir.jpg|600px]]<br />
<br />
==Conclusion==<br />
<hr/><br />
<br />
<br />
=lab 115=<br />
<hr/><br />
<br />
==Main purpose==<br />
<hr/><br />
Run [[Team:DTU-Denmark/Experiment2|Experiment 4]] in two different samples aerobically in order to characterize the behavior of ''AMO transformants'' and the '''E.coli''' strain.<br />
<br />
==Who was in the lab==<br />
<hr/><br />
Ariadni, Helen, Kashia<br />
<br />
==Procedure==<br />
<hr/><br />
<br />
Adjusting the temperature at 36 degrees and calibrating the probes as described in [[Team:DTU-Denmark/Methods/Calibrating_Electrodes|Calibration protocol]].<br />
<br />
<br />
Following the protocol [[Team:DTU-Denmark/Methods/Determining_concentration_of_nitrogen_compounds/Experiment_4|Experiment 4]]<br />
<br />
Changing the steps :<br />
<br />
step 2 . 4 ml of the overnight culture growing in DM minimal medium with NH<sub>4</sub>Cl<br />
<br />
and step 3 from the experimental procedure where the OD was measured OD=0.0761 and 0.0720 for the AMO mutant. For the ''E.coli'' culture the OD was 0.0822 and 0.0947.The abiotic controls have OD= 0.002 and 0.0006.<br />
<br />
==Results==<br />
<hr/><br />
<br />
===Colorimetric results===<br />
<br />
'''Ranges'''<br />
* ''Measuring range 5-150 mg/L NH<sub>4</sub>-N''<br />
* ''Measuring range 0.02-1 mg/L NO<sub>2</sub>-N''<br />
<br />
<br />
Standard solutions <br />
* Ammonium - 66.6 mg/L (expected 39 mg/L)<br />
* Nitrite - 0.72 mg/L (expected 0.5 mg/L)<br />
<br />
<br />
{| class="wikitable" style="text-align: right"<br />
! time (min)!!ammonium AMO1(mg/L)!! ammonium AMO2(mg/L)!! Ammonium ''E.coli''(mg/L) !! nitrite ''E.coli''!! ammonium abiotic (mg/L) !!nitrite abiotic(mg/L)<br />
|-<br />
| 0 || <5 || <5 || <5 || <0.02 || 5 || <0.02<br />
|-<br />
| 13 || <5 || <5 || 5 || <0.02 || - || -<br />
|-<br />
| 19 || <5 || <5 || 5 || <0.02 || - || -<br />
|-<br />
| spike || 146|| 140 || <5 || <0.02 || - || -<br />
|-<br />
| 35 || 158 || 244|| 258 || <0.02 || - || -<br />
|-<br />
| 41 || 169.5 || 171 || 178 || <0.02 || - || -<br />
|-<br />
| 52 || 226 || 280 || 250 || <0.02 || - || -<br />
|-<br />
| 64 || 232 || 254 || 124 || <0.02 || - || -<br />
|-<br />
| 90|| 238 || 272 || 246 || <0.02 || - || -<br />
|-<br />
| 10 hours|| 172|| 218 || 16 || <0.02 || 154 || <0.02<br />
|-<br />
|}<br />
<br />
===OD at the end of the experiment===<br />
<br />
OD=0.2083 and 0.2232 for the AMO mutants. For the ''E.coli'' culture the OD was 2.39 and 0.1173.The abiotic controls have OD=0.00144 and 0.0004.<br />
<br />
==Conclusion==<br />
<hr/><br />
<br />
<br />
<br />
Navigate to the [[Team:DTU-Denmark/Notebook/26_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/28_August_2013|Next]] Entry<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/Notebook/27_August_2013Team:DTU-Denmark/Notebook/27 August 20132013-09-28T18:01:52Z<p>Hezscha: /* Who was in the lab */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|27 August 2013}}<br />
Navigate to the [[Team:DTU-Denmark/Notebook/26_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/28_August_2013|Next]] Entry<br />
=lab 208=<br />
<hr/><br />
==Main purpose==<br />
<hr/><br />
<br />
==Who was in the lab==<br />
<hr/><br />
Kristian, Henrike<br />
<br />
==Procedure==<br />
<hr/><br />
<br />
===PCR reactions===<br />
<br />
Set up PCR reactions for:<br />
* Biobrick backbone pSB1C3 with USER endings fitting for our inserts<br />
* constitutive reference promoter in pZA21 (for SPL)<br />
* pZA21::ara vector with USER endings fitting for Nir1 and Nir2 inserts<br />
<br />
<u>first round of PCRs</u>:<br />
<br />
pSB1C3:<br />
{| class="wikitable" style="text-align: right"<br />
! temperature !! time !! cycles<br />
|-<br />
| 98C || 2:00 || - <br />
|-<br />
| 98C || 0:10 || 36 <br />
|-<br />
| 70C || 0:45 || 36 <br />
|-<br />
| 72C || 1:30 || 36<br />
|-<br />
| 72C || 5:00 || - <br />
|-<br />
| 10C || hold || -<br />
|-<br />
|}<br />
Four different reaction mixes: HF, HF+5%DMSO, GC, GC+5%DMSO<br />
<br />
pZA21::ara for Nir:<br />
{| class="wikitable" style="text-align: right"<br />
! temperature !! time !! cycles<br />
|-<br />
| 98C || 2:00 || - <br />
|-<br />
| 98C || 0:10 || 36 <br />
|-<br />
| 60C || 1:00 || 36 <br />
|-<br />
| 72C || 2:00 || 36<br />
|-<br />
| 72C || 5:00 || - <br />
|-<br />
| 10C || hold || -<br />
|-<br />
|}<br />
<br />
Standard reaction mix.<br />
<br />
constitutive reference promoter:<br />
{| class="wikitable" style="text-align: right"<br />
! temperature !! time !! cycles<br />
|-<br />
| 98C || 2:00 || - <br />
|-<br />
| 98C || 0:20 || 36 <br />
|-<br />
| 58.1C || 1:00 || 36 <br />
|-<br />
| 72C || 3:00 || 36<br />
|-<br />
| 72C || 5:00 || - <br />
|-<br />
| 10C || hold || -<br />
|-<br />
|}<br />
Mix with GC, 5%DMSO and 2uL MgCl2.<br />
<br />
pSB1C3 and pZA21::ara for Nir were without results (see gel picture). New PCRs were set up with one standard reaction and one reaction with additives. The annealing temperature was lowered.<br />
<br />
<u>second round of PCRs</u>:<br />
<br />
{| class="wikitable" style="text-align: right"<br />
! compound !! amount (in uL)<br />
|-<br />
| || standard mix || additive mix<br />
|-<br />
| dNTPs || 1 || 1<br />
|-<br />
| X7 ploymerase || 0.5 || 0.5<br />
|-<br />
| HF buffer || 10 || 10<br />
|-<br />
| MilliQ || 31.5 || 27<br />
|-<br />
| FW primer || 3 || 3<br />
|-<br />
| RV primer || 3 || 3<br />
|-<br />
| template || 1 || 1<br />
|-<br />
| DMSO || - || 2.5<br />
|-<br />
| 50 mM MgCl2 || - || 2<br />
|-<br />
|}<br />
<br />
<br />
pSB1C3:<br />
{| class="wikitable" style="text-align: right"<br />
! temperature !! time !! cycles<br />
|-<br />
| 98C || 2:00 || - <br />
|-<br />
| 98C || 0:10 || 36 <br />
|-<br />
| 60C || 1:00 || 36 <br />
|-<br />
| 72C || 1:30 || 36<br />
|-<br />
| 72C || 5:00 || - <br />
|-<br />
| 10C || hold || -<br />
|-<br />
|}<br />
<br />
<br />
pZA21::ara for Nir:<br />
{| class="wikitable" style="text-align: right"<br />
! temperature !! time !! cycles<br />
|-<br />
| 98C || 2:00 || - <br />
|-<br />
| 98C || 0:10 || 36 <br />
|-<br />
| 55C || 1:00 || 36 <br />
|-<br />
| 72C || 2:00 || 36<br />
|-<br />
| 72C || 5:00 || - <br />
|-<br />
| 10C || hold || -<br />
|-<br />
|}<br />
<br />
<br />
Additionally set up a PCR for cycAX with USER endings since the amount of fragment is running low.<br />
<br />
cycAX for USER:<br />
{| class="wikitable" style="text-align: right"<br />
! temperature !! time !! cycles<br />
|-<br />
| 98C || 2:00 || - <br />
|-<br />
| 98C || 0:20 || 36 <br />
|-<br />
| 57C || 0:45 || 36 <br />
|-<br />
| 72C || 1:30 || 36<br />
|-<br />
| 72C || 5:00 || - <br />
|-<br />
| 10C || hold || -<br />
|-<br />
|}<br />
Standard reaction mix.<br />
<br />
==Results==<br />
<hr/><br />
<br />
===Gel===<br />
<br />
* 1 kb ladder<br />
* pSB1C3 for Biobricks, HF buffer<br />
* pSB1C3 for Biobricks, HF buffer, 5%DMSO<br />
* pSB1C3 for Biobricks, GC buffer<br />
* pSB1C3 for Biobricks, GC buffer, 5%DMSO<br />
* negative pSB1C3 for Biobricks<br />
* pZA21::ara for USER ligation with Nir<br />
* pZA21::ara for USER ligation with Nir (duplicate)<br />
* 1 kb ladder<br />
<br />
Note: It seems the ladder overflew.<br />
<br />
[[File:2013-08-27 bb pzaarafornir.jpg|600px]]<br />
<br />
==Conclusion==<br />
<hr/><br />
<br />
<br />
=lab 115=<br />
<hr/><br />
<br />
==Main purpose==<br />
<hr/><br />
Run [[Team:DTU-Denmark/Experiment2|Experiment 4]] in two different samples aerobically in order to characterize the behavior of ''AMO transformants'' and the '''E.coli''' strain.<br />
<br />
==Who was in the lab==<br />
<hr/><br />
Ariadni, Helen, Kashia<br />
<br />
==Procedure==<br />
<hr/><br />
<br />
Adjusting the temperature at 36 degrees and calibrating the probes as described in [[Team:DTU-Denmark/Methods/Calibrating_Electrodes|Calibration protocol]].<br />
<br />
<br />
Following the protocol [[Team:DTU-Denmark/Methods/Determining_concentration_of_nitrogen_compounds/Experiment_4|Experiment 4]]<br />
<br />
Changing the steps :<br />
<br />
step 2 . 4 ml of the overnight culture growing in DM minimal medium with NH<sub>4</sub>Cl<br />
<br />
and step 3 from the experimental procedure where the OD was measured OD=0.0761 and 0.0720 for the AMO mutant. For the ''E.coli'' culture the OD was 0.0822 and 0.0947.The abiotic controls have OD= 0.002 and 0.0006.<br />
<br />
==Results==<br />
<hr/><br />
<br />
===Colorimetric results===<br />
<br />
'''Ranges'''<br />
* ''Measuring range 5-150 mg/L NH<sub>4</sub>-N''<br />
* ''Measuring range 0.02-1 mg/L NO<sub>2</sub>-N''<br />
<br />
<br />
Standard solutions <br />
* Ammonium - 66.6 mg/L (expected 39 mg/L)<br />
* Nitrite - 0.72 mg/L (expected 0.5 mg/L)<br />
<br />
<br />
{| class="wikitable" style="text-align: right"<br />
! time (min)!!ammonium AMO1(mg/L)!! ammonium AMO2(mg/L)!! Ammonium ''E.coli''(mg/L) !! nitrite ''E.coli''!! ammonium abiotic (mg/L) !!nitrite abiotic(mg/L)<br />
|-<br />
| 0 || <5 || <5 || <5 || <0.02 || 5 || <0.02<br />
|-<br />
| 13 || <5 || <5 || 5 || <0.02 || - || -<br />
|-<br />
| 19 || <5 || <5 || 5 || <0.02 || - || -<br />
|-<br />
| spike || 146|| 140 || <5 || <0.02 || - || -<br />
|-<br />
| 35 || 158 || 244|| 258 || <0.02 || - || -<br />
|-<br />
| 41 || 169.5 || 171 || 178 || <0.02 || - || -<br />
|-<br />
| 52 || 226 || 280 || 250 || <0.02 || - || -<br />
|-<br />
| 64 || 232 || 254 || 124 || <0.02 || - || -<br />
|-<br />
| 90|| 238 || 272 || 246 || <0.02 || - || -<br />
|-<br />
| 10 hours|| 172|| 218 || 16 || <0.02 || 154 || <0.02<br />
|-<br />
|}<br />
<br />
===OD at the end of the experiment===<br />
<br />
OD=0.2083 and 0.2232 for the AMO mutants. For the ''E.coli'' culture the OD was 2.39 and 0.1173.The abiotic controls have OD=0.00144 and 0.0004.<br />
<br />
==Conclusion==<br />
<hr/><br />
<br />
<br />
<br />
Navigate to the [[Team:DTU-Denmark/Notebook/26_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/28_August_2013|Next]] Entry<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/Notebook/25_August_2013Team:DTU-Denmark/Notebook/25 August 20132013-09-28T17:59:45Z<p>Hezscha: /* Main purpose */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|25 August 2013}}<br />
Navigate to the [[Team:DTU-Denmark/Notebook/24_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/26_August_2013|Next]] Entry<br />
=Lab 208=<br />
<hr/><br />
==Main purpose==<br />
<hr/><br />
*colony PCR<br />
<br />
==Who was in the lab==<br />
<hr/><br />
<br />
==Procedure==<br />
<hr/><br />
<br />
===Colony PCR===<br />
<br />
Performed colony PCR to confirm insert for HAO, AMO, cycAX and Nir transformants. Used Q5 premix with the following reaction mix:<br />
<br />
{| class="wikitable" style="text-align: right"<br />
! compound !! amount<br />
|-<br />
| Q5 mix || 25 uL<br />
|-<br />
| FW primer || 3 uL<br />
|-<br />
| RV primer || 3 uL<br />
|-<br />
| template || 1 uL<br />
|-<br />
| MilliQ || 18 uL<br />
|-<br />
|}<br />
<br />
Template was made by resuspending 1 culture in 100uL MilliQ.<br />
<br />
program:<br />
{| class="wikitable" style="text-align: right"<br />
! temperature !! time !! cycles<br />
|-<br />
| 98C || 10:00 || - <br />
|-<br />
| 98C || 0:10 || 36 <br />
|-<br />
| annealing temperature || 0:30 || 36 <br />
|-<br />
| 72C || 0:20 || 36<br />
|-<br />
| 72C || 5:00 || - <br />
|-<br />
| 10C || hold || -<br />
|-<br />
|}<br />
<br />
details (primers, temp, expected fragment length):<br />
* cycAX - FW_1, RV_2, 61C, 749bp<br />
* HAO - FW_2, RV_3, 64C, 751bp<br />
* AMO - FW_2, RV_3, 63C, 750bp<br />
* Nir - FW_2, RV_3, 64C, 733bp<br />
* Nir - FW_5, RV_6, 71C, 737bp<br />
<br />
==Results==<br />
<hr/><br />
<br />
==Conclusion==<br />
<hr/><br />
<br />
Navigate to the [[Team:DTU-Denmark/Notebook/24_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/26_August_2013|Next]] Entry<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/Notebook/25_August_2013Team:DTU-Denmark/Notebook/25 August 20132013-09-28T17:59:27Z<p>Hezscha: /* Main purpose */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|25 August 2013}}<br />
Navigate to the [[Team:DTU-Denmark/Notebook/24_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/26_August_2013|Next]] Entry<br />
=Lab 208=<br />
<hr/><br />
==Main purpose==<br />
<hr/><br />
*PCR<br />
<br />
==Who was in the lab==<br />
<hr/><br />
<br />
==Procedure==<br />
<hr/><br />
<br />
===Colony PCR===<br />
<br />
Performed colony PCR to confirm insert for HAO, AMO, cycAX and Nir transformants. Used Q5 premix with the following reaction mix:<br />
<br />
{| class="wikitable" style="text-align: right"<br />
! compound !! amount<br />
|-<br />
| Q5 mix || 25 uL<br />
|-<br />
| FW primer || 3 uL<br />
|-<br />
| RV primer || 3 uL<br />
|-<br />
| template || 1 uL<br />
|-<br />
| MilliQ || 18 uL<br />
|-<br />
|}<br />
<br />
Template was made by resuspending 1 culture in 100uL MilliQ.<br />
<br />
program:<br />
{| class="wikitable" style="text-align: right"<br />
! temperature !! time !! cycles<br />
|-<br />
| 98C || 10:00 || - <br />
|-<br />
| 98C || 0:10 || 36 <br />
|-<br />
| annealing temperature || 0:30 || 36 <br />
|-<br />
| 72C || 0:20 || 36<br />
|-<br />
| 72C || 5:00 || - <br />
|-<br />
| 10C || hold || -<br />
|-<br />
|}<br />
<br />
details (primers, temp, expected fragment length):<br />
* cycAX - FW_1, RV_2, 61C, 749bp<br />
* HAO - FW_2, RV_3, 64C, 751bp<br />
* AMO - FW_2, RV_3, 63C, 750bp<br />
* Nir - FW_2, RV_3, 64C, 733bp<br />
* Nir - FW_5, RV_6, 71C, 737bp<br />
<br />
==Results==<br />
<hr/><br />
<br />
==Conclusion==<br />
<hr/><br />
<br />
Navigate to the [[Team:DTU-Denmark/Notebook/24_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/26_August_2013|Next]] Entry<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/Notebook/24_August_2013Team:DTU-Denmark/Notebook/24 August 20132013-09-28T17:58:41Z<p>Hezscha: /* Conclusion */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|24 August 2013}}<br />
Navigate to the [[Team:DTU-Denmark/Notebook/23_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/25_August_2013|Next]] Entry<br />
=Lab 208=<br />
<hr/><br />
==Main purpose==<br />
<hr/><br />
*fix problem with gels<br />
*run gels<br />
<br />
==Who was in the lab==<br />
<hr/><br />
Kristian, Henrike<br />
<br />
==Procedure==<br />
<hr/><br />
Running and loading gels.<br />
<br />
==Results==<br />
<hr/><br />
<br />
===Gels===<br />
<br />
* 1 kb<br />
* colony PCR NirG<br />
* constitutive reference promoter, GC buffer<br />
* constitutive reference promoter, GC buffer + 3% DMSO<br />
* constitutive reference promoter, HF buffer<br />
* constitutive reference promoter, HF buffer + 3% DMSO<br />
* 1 kb<br />
<br />
[[File:2013-08-24 nircol ref const.jpg|600px]]<br />
<br />
Gel could not be read and will be redone.<br />
<br />
* 1 kb<br />
* colony PCR cycAX, colony 2<br />
* colony PCR cycAX, colony 3<br />
* colony PCR cycAX, colony 4<br />
* colony PCR cycAX, colony 6<br />
* colony PCR cycAX, colony 7<br />
* colony PCR cycAX, colony 8<br />
* colony PCR cycAX, colony 9<br />
* constitutive reference promoter, GC buffer<br />
* constitutive reference promoter, GC buffer + 3% DMSO<br />
* constitutive reference promoter, HF buffer<br />
* constitutive reference promoter, HF buffer + 3% DMSO<br />
* 1 kb<br />
<br />
[[File:2013-08-24 cyccol refconst.jpg|600px]]<br />
<br />
The insert is confirmed for the colonies 3, 4 and 8.<br />
<br />
===Gel optimization test===<br />
<br />
Made two gels with TAE buffer to run on the two different power supplies and used two different loading dyes to try and resolve the problem with the bad gels we've been having.<br />
<br />
Loading dyes: Agarose Blue was made by us, GelPilot is supplied by Qiagen. The const ref samples were mixed with agarose blue before, so we cannot test the other loading dye on those.<br />
<br />
Run on the old power supply (the grey one):<br />
<br />
* 1 kb<br />
* cycAX col 3, agarose blue<br />
* cycAX col 4, GelPilot<br />
* cycAX col 8, GelPilot<br />
* cycAX col 2, agarose blue<br />
* cycAX col 6, agarose blue<br />
* cycAX col 7, GelPilot<br />
* cycAX col 9, GelPilot<br />
* constitutive reference promoter, GC buffer<br />
* constitutive reference promoter, GC buffer + 3% DMSO<br />
* constitutive reference promoter, HF buffer<br />
* constitutive reference promoter, HF buffer + 3% DMSO<br />
* NirG col, agarose blue<br />
* NirG col, GelPilot<br />
* HAO col, agarose blue<br />
* HAO col, GelPilot<br />
* 1 kb<br />
<br />
[[File:2013-08-24 check big.jpg|600px]]<br />
<br />
Run on the new power supply (the green one):<br />
<br />
* 1 kb<br />
* cycAX col 3, agarose blue<br />
* cycAX col 4, GelPilot<br />
* cycAX col 8, GelPilot<br />
* cycAX col 2, agarose blue<br />
* cycAX col 7, GelPilot<br />
* constitutive reference promoter, GC buffer<br />
* constitutive reference promoter, GC buffer + 3% DMSO<br />
* constitutive reference promoter, HF buffer<br />
* cycAX col 3, GelPilot<br />
* NirG col, agarose blue<br />
* NirG col, GelPilot<br />
* HAO col, agarose blue<br />
* HAO col, GelPilot<br />
* 1 kb<br />
<br />
[[File:2013-08-24 check small.jpg|600px]]<br />
<br />
==Conclusion==<br />
<hr/><br />
There is no visible difference between the two power-supplies and loading dyes. Probably the TBE we made from powder was not good, we will use TAE from now on.<br />
<br />
Navigate to the [[Team:DTU-Denmark/Notebook/23_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/25_August_2013|Next]] Entry<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/Notebook/24_August_2013Team:DTU-Denmark/Notebook/24 August 20132013-09-28T17:56:58Z<p>Hezscha: /* Procedure */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|24 August 2013}}<br />
Navigate to the [[Team:DTU-Denmark/Notebook/23_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/25_August_2013|Next]] Entry<br />
=Lab 208=<br />
<hr/><br />
==Main purpose==<br />
<hr/><br />
*fix problem with gels<br />
*run gels<br />
<br />
==Who was in the lab==<br />
<hr/><br />
Kristian, Henrike<br />
<br />
==Procedure==<br />
<hr/><br />
Running and loading gels.<br />
<br />
==Results==<br />
<hr/><br />
<br />
===Gels===<br />
<br />
* 1 kb<br />
* colony PCR NirG<br />
* constitutive reference promoter, GC buffer<br />
* constitutive reference promoter, GC buffer + 3% DMSO<br />
* constitutive reference promoter, HF buffer<br />
* constitutive reference promoter, HF buffer + 3% DMSO<br />
* 1 kb<br />
<br />
[[File:2013-08-24 nircol ref const.jpg|600px]]<br />
<br />
Gel could not be read and will be redone.<br />
<br />
* 1 kb<br />
* colony PCR cycAX, colony 2<br />
* colony PCR cycAX, colony 3<br />
* colony PCR cycAX, colony 4<br />
* colony PCR cycAX, colony 6<br />
* colony PCR cycAX, colony 7<br />
* colony PCR cycAX, colony 8<br />
* colony PCR cycAX, colony 9<br />
* constitutive reference promoter, GC buffer<br />
* constitutive reference promoter, GC buffer + 3% DMSO<br />
* constitutive reference promoter, HF buffer<br />
* constitutive reference promoter, HF buffer + 3% DMSO<br />
* 1 kb<br />
<br />
[[File:2013-08-24 cyccol refconst.jpg|600px]]<br />
<br />
The insert is confirmed for the colonies 3, 4 and 8.<br />
<br />
===Gel optimization test===<br />
<br />
Made two gels with TAE buffer to run on the two different power supplies and used two different loading dyes to try and resolve the problem with the bad gels we've been having.<br />
<br />
Loading dyes: Agarose Blue was made by us, GelPilot is supplied by Qiagen. The const ref samples were mixed with agarose blue before, so we cannot test the other loading dye on those.<br />
<br />
Run on the old power supply (the grey one):<br />
<br />
* 1 kb<br />
* cycAX col 3, agarose blue<br />
* cycAX col 4, GelPilot<br />
* cycAX col 8, GelPilot<br />
* cycAX col 2, agarose blue<br />
* cycAX col 6, agarose blue<br />
* cycAX col 7, GelPilot<br />
* cycAX col 9, GelPilot<br />
* constitutive reference promoter, GC buffer<br />
* constitutive reference promoter, GC buffer + 3% DMSO<br />
* constitutive reference promoter, HF buffer<br />
* constitutive reference promoter, HF buffer + 3% DMSO<br />
* NirG col, agarose blue<br />
* NirG col, GelPilot<br />
* HAO col, agarose blue<br />
* HAO col, GelPilot<br />
* 1 kb<br />
<br />
[[File:2013-08-24 check big.jpg|600px]]<br />
<br />
Run on the new power supply (the green one):<br />
<br />
* 1 kb<br />
* cycAX col 3, agarose blue<br />
* cycAX col 4, GelPilot<br />
* cycAX col 8, GelPilot<br />
* cycAX col 2, agarose blue<br />
* cycAX col 7, GelPilot<br />
* constitutive reference promoter, GC buffer<br />
* constitutive reference promoter, GC buffer + 3% DMSO<br />
* constitutive reference promoter, HF buffer<br />
* cycAX col 3, GelPilot<br />
* NirG col, agarose blue<br />
* NirG col, GelPilot<br />
* HAO col, agarose blue<br />
* HAO col, GelPilot<br />
* 1 kb<br />
<br />
[[File:2013-08-24 check small.jpg|600px]]<br />
<br />
==Conclusion==<br />
<hr/><br />
<br />
Navigate to the [[Team:DTU-Denmark/Notebook/23_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/25_August_2013|Next]] Entry<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/Notebook/24_August_2013Team:DTU-Denmark/Notebook/24 August 20132013-09-28T17:56:31Z<p>Hezscha: /* Main purpose */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|24 August 2013}}<br />
Navigate to the [[Team:DTU-Denmark/Notebook/23_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/25_August_2013|Next]] Entry<br />
=Lab 208=<br />
<hr/><br />
==Main purpose==<br />
<hr/><br />
*fix problem with gels<br />
*run gels<br />
<br />
==Who was in the lab==<br />
<hr/><br />
Kristian, Henrike<br />
<br />
==Procedure==<br />
<hr/><br />
<br />
==Results==<br />
<hr/><br />
<br />
===Gels===<br />
<br />
* 1 kb<br />
* colony PCR NirG<br />
* constitutive reference promoter, GC buffer<br />
* constitutive reference promoter, GC buffer + 3% DMSO<br />
* constitutive reference promoter, HF buffer<br />
* constitutive reference promoter, HF buffer + 3% DMSO<br />
* 1 kb<br />
<br />
[[File:2013-08-24 nircol ref const.jpg|600px]]<br />
<br />
Gel could not be read and will be redone.<br />
<br />
* 1 kb<br />
* colony PCR cycAX, colony 2<br />
* colony PCR cycAX, colony 3<br />
* colony PCR cycAX, colony 4<br />
* colony PCR cycAX, colony 6<br />
* colony PCR cycAX, colony 7<br />
* colony PCR cycAX, colony 8<br />
* colony PCR cycAX, colony 9<br />
* constitutive reference promoter, GC buffer<br />
* constitutive reference promoter, GC buffer + 3% DMSO<br />
* constitutive reference promoter, HF buffer<br />
* constitutive reference promoter, HF buffer + 3% DMSO<br />
* 1 kb<br />
<br />
[[File:2013-08-24 cyccol refconst.jpg|600px]]<br />
<br />
The insert is confirmed for the colonies 3, 4 and 8.<br />
<br />
===Gel optimization test===<br />
<br />
Made two gels with TAE buffer to run on the two different power supplies and used two different loading dyes to try and resolve the problem with the bad gels we've been having.<br />
<br />
Loading dyes: Agarose Blue was made by us, GelPilot is supplied by Qiagen. The const ref samples were mixed with agarose blue before, so we cannot test the other loading dye on those.<br />
<br />
Run on the old power supply (the grey one):<br />
<br />
* 1 kb<br />
* cycAX col 3, agarose blue<br />
* cycAX col 4, GelPilot<br />
* cycAX col 8, GelPilot<br />
* cycAX col 2, agarose blue<br />
* cycAX col 6, agarose blue<br />
* cycAX col 7, GelPilot<br />
* cycAX col 9, GelPilot<br />
* constitutive reference promoter, GC buffer<br />
* constitutive reference promoter, GC buffer + 3% DMSO<br />
* constitutive reference promoter, HF buffer<br />
* constitutive reference promoter, HF buffer + 3% DMSO<br />
* NirG col, agarose blue<br />
* NirG col, GelPilot<br />
* HAO col, agarose blue<br />
* HAO col, GelPilot<br />
* 1 kb<br />
<br />
[[File:2013-08-24 check big.jpg|600px]]<br />
<br />
Run on the new power supply (the green one):<br />
<br />
* 1 kb<br />
* cycAX col 3, agarose blue<br />
* cycAX col 4, GelPilot<br />
* cycAX col 8, GelPilot<br />
* cycAX col 2, agarose blue<br />
* cycAX col 7, GelPilot<br />
* constitutive reference promoter, GC buffer<br />
* constitutive reference promoter, GC buffer + 3% DMSO<br />
* constitutive reference promoter, HF buffer<br />
* cycAX col 3, GelPilot<br />
* NirG col, agarose blue<br />
* NirG col, GelPilot<br />
* HAO col, agarose blue<br />
* HAO col, GelPilot<br />
* 1 kb<br />
<br />
[[File:2013-08-24 check small.jpg|600px]]<br />
<br />
==Conclusion==<br />
<hr/><br />
<br />
Navigate to the [[Team:DTU-Denmark/Notebook/23_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/25_August_2013|Next]] Entry<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezschahttp://2013.igem.org/Team:DTU-Denmark/Notebook/23_August_2013Team:DTU-Denmark/Notebook/23 August 20132013-09-28T17:55:23Z<p>Hezscha: /* Lab 208 */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|23 August 2013}}<br />
Navigate to the [[Team:DTU-Denmark/Notebook/22_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/24_August_2013|Next]] Entry<br />
=Lab 208=<br />
<hr/><br />
==Main purpose==<br />
<hr/><br />
*PCR to isolate Biobricks<br />
*PCR to make the constitutive reference promoter<br />
*colony PCR on Nir transformants<br />
<br />
==Who was in the lab==<br />
<hr/><br />
Kristian, Henrike<br />
<br />
==Procedure==<br />
<hr/><br />
<br />
===PCR Biobrick Isolation===<br />
<br />
Used new primers to isolate Hello World constructs. Used HF buffer and tested three different concentrations of DMSO: 0%, 2% and 5%.<br />
<br />
Primers: 56a, 56b<br />
<br />
template: TAT2-1 (miniprep)<br />
<br />
program:<br />
{| class="wikitable" style="text-align: right"<br />
! temperature !! time !! cycles<br />
|-<br />
| 98C || 2:00 || - <br />
|-<br />
| 98C || 0:20 || 36 <br />
|-<br />
| 56C || 0:30 || 36 <br />
|-<br />
| 72C || 0:45 || 36<br />
|-<br />
| 72C || 5:00 || - <br />
|-<br />
| 10C || hold || -<br />
|-<br />
|}<br />
<br />
===PCR constitutive reference promoter===<br />
<br />
Got new primers for the constitutive reference promoter since the old reverse primer (52b2) had a mistake. Repeated reaction set-up and program from the 13.08. Used GC buffer, 5% DMSO and 2uL 50 mM MgCl2. <br />
<br />
primers: 52a, 52bn<br />
<br />
template: pZA21::RFP<br />
<br />
<br />
program:<br />
{| class="wikitable" style="text-align: right"<br />
! temperature !! time !! cycles<br />
|-<br />
| 98C || 2:00 || - <br />
|-<br />
| 98C || 0:20 || 36 <br />
|-<br />
| 58.1C || 1:00 || 36 <br />
|-<br />
| 72C || 3:00 || 36<br />
|-<br />
| 72C || 5:00 || - <br />
|-<br />
| 10C || hold || -<br />
|-<br />
|}<br />
<br />
===Colony PCR on Nir transformants===<br />
<br />
Used Q5 premix from NEB.<br />
<br />
{| class="wikitable" style="text-align: right"<br />
! Reagent !! volume (in uL)<br />
|-<br />
| Q5 master mix || 12,5 <br />
|-<br />
| FW primer || 3<br />
|-<br />
| RV primer || 3<br />
|-<br />
| template || 1<br />
|-<br />
| MilliQ || 5,5<br />
|-<br />
|}<br />
<br />
primers: 45a, 45b<br />
<br />
program:<br />
{| class="wikitable" style="text-align: right"<br />
! temperature !! time !! cycles<br />
|-<br />
| 98C || 10:00 || - <br />
|-<br />
| 98C || 0:10 || 36 <br />
|-<br />
| 56C || 0:30 || 36 <br />
|-<br />
| 72C || 0:45 || 36<br />
|-<br />
| 72C || 5:00 || - <br />
|-<br />
| 10C || hold || -<br />
|-<br />
|}<br />
<br />
==Results==<br />
<hr/><br />
<br />
===Gel electrophoresis===<br />
<br />
* 1 kb ladder<br />
* Sec2 Biobrick <br />
* Sec2 Biobrick<br />
* Sec2 neg<br />
* col AMO 1<br />
* col AMO 2<br />
* col AMO 3<br />
* col AMO 4<br />
* col AMO 5<br />
* col AMO 6<br />
* col AMO 7<br />
* col AMO 8<br />
* col AMO 9<br />
* col AMO 10<br />
* 1 kb ladder<br />
<br />
[[File:2013-08-23 bb sec amo colony.jpg|600px]]<br />
<br />
* 1 kb ladder <br />
* col Nir<br />
* constitutive reference promoter<br />
* constitutive reference promoter<br />
* Biobrick Isolation TAT2-1 negative<br />
* Biobrick Isolation TAT2-1 2% DMSO<br />
* Biobrick Isolation TAT2-1 5% DMSO<br />
* Biobrick Isolation TAT2-1 w/o DMSO<br />
* 1 kb ladder<br />
<br />
==Conclusion==<br />
<hr/><br />
The transformation of AMO into pZA21::ara was successful, all tested colonies have the insert.<br />
<br />
Navigate to the [[Team:DTU-Denmark/Notebook/22_August_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/24_August_2013|Next]] Entry<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Hezscha