http://2013.igem.org/wiki/index.php?title=Special:Contributions/Ariadni&feed=atom&limit=50&target=Ariadni&year=&month=2013.igem.org - User contributions [en]2024-03-28T08:48:16ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:DTU-Denmark/Notebook/6_June_2013Team:DTU-Denmark/Notebook/6 June 20132013-10-04T18:15:08Z<p>Ariadni: Created page with "{{:Team:DTU-Denmark/Templates/StartPage|6 June 2013}} No labwork was performed this date. Navigate to the Previous or the [[Team:DTU-D..."</p>
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/Notebook/8_June_2013Team:DTU-Denmark/Notebook/8 June 20132013-10-04T18:14:07Z<p>Ariadni: Created page with "{{:Team:DTU-Denmark/Templates/StartPage|8 June 2013}} No labwork was performed this date. Navigate to the Previous or the [[Team:DTU-D..."</p>
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<div>{{:Team:DTU-Denmark/Templates/StartPage|8 June 2013}}<br />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/Notebook/21_July_2013Team:DTU-Denmark/Notebook/21 July 20132013-10-04T18:12:52Z<p>Ariadni: Created page with "{{:Team:DTU-Denmark/Templates/StartPage|21 July 2013}} No labwork was performed this date. Navigate to the Previous or the [[Team:DTU..."</p>
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<div>{{:Team:DTU-Denmark/Templates/StartPage|21 July 2013}}<br />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/Notebook/20_July_2013Team:DTU-Denmark/Notebook/20 July 20132013-10-04T18:12:12Z<p>Ariadni: Created page with "{{:Team:DTU-Denmark/Templates/StartPage|20 July 2013}} No labwork was performed this date. Navigate to the Previous or the [[Team:DTU..."</p>
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<div>{{:Team:DTU-Denmark/Templates/StartPage|20 July 2013}}<br />
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No labwork was performed this date.<br />
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Navigate to the [[Team:DTU-Denmark/Notebook/19_July_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/21_July_2013|Next]] Entry.<br />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/Methods/Ammonium_colorimetric_measurementsTeam:DTU-Denmark/Methods/Ammonium colorimetric measurements2013-10-04T18:09:10Z<p>Ariadni: /* Ammonium colorimetric measurements */</p>
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<div>{{:Team:DTU-Denmark/Templates/StartPage|Ammonium colorimetric measurements}}<br />
<div class="overviewPage"><br />
==Ammonium colorimetric measurements==<br />
<br />
'''Measuring the concentration of NH<sub>4</sub> in an ''E. coli''/''P. aeruginosa'' growth experiment using Spectroquant kit<br />
'''<br />
<br />
Material:<br />
* Ammonium Test Kit (#00683)<br />
<br />
A liquid test solution should be performed to achieve a measurement between the ranges of 2-75 mg/L NH<sub>4</sub>-N.<br />
<br />
Ammonium solution<br />
<br />
Prepare solution with 39 mg NH<sub>4</sub>-N/L: <br />
* Weigh 220 mg Ammonium Bicarbonate into 100mL volumetric flask (10x concentrated)<br />
* Take 5 mL of the 10x solution in 45 mL water<br />
* Expected final concentration 39 mg N/L <br />
<br />
Performing Measurements:<br />
'''Ammonium'''<br />
<br />
''Measuring range of the kit: 2-75 mg/L NH<sub>4</sub>-N''<br />
# Pipette 5.0 ml of reagent 1 (NH<sub>4</sub>-1) into a test tube.<br />
# Add 0.20 mL of sample when it is among the range of measuring kit (otherwise make proper dilution)<br />
# Add 1 level spoon of reagent 2 (NH<sub>4</sub>-2).<br />
# Shake vigorously to dissolve.<br />
# Reaction time: 15 mins<br />
# Pour sample into the cuvette <br />
# Select method in the spectrometer by entering the AutoSelector (little black tube)<br />
# Note the result<br />
# If the read is above the linear range then put the sample back to the tube test, dilute it and try again.<br />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/Methods/rehydrating_primersTeam:DTU-Denmark/Methods/rehydrating primers2013-10-04T18:08:04Z<p>Ariadni: </p>
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<div>{{:Team:DTU-Denmark/Templates/StartPage|Rehydrating Primers}}<br />
<br />
We dilute the lyphelized pellet in 150uL of MilliQ water (diluted 150 times) and save this as stock at -80<sup>o</sup>C. From the stock we take 10uL and add to 90uL of MilliQ (diluted 10 times) to make working solution of primers kept in -20<sup>o</sup>C.<br />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/Methods/Visualizing_GFP_in_the_periplasmTeam:DTU-Denmark/Methods/Visualizing GFP in the periplasm2013-10-04T18:07:29Z<p>Ariadni: /* Guide on how to visualize GFP in the periplasm */</p>
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<div>{{:Team:DTU-Denmark/Templates/StartPage|Periplasmic GFP}}<br />
<div class="overviewPage"><br />
== Guide on how to visualize GFP in the periplasm ==<br />
<br />
This is a guide on how to use biobrick [http://parts.igem.org/Part:BBa_K1067009 BBa_K1067009] to acquire pictures of GFP exported into the periplasm. The guide can also be used if having GFP fused proteins directed to the periplasm with the Twin Arginine Translocation pathway (TAT). The guide is inspired by Skoog, Karl, et al.<br />
<br />
The produre is as follows:<br />
*TOP10 ''E. coli'' was transformed with the constructed plasmid and plated on LB agar plates.<br />
*Colonies was visually inspected the day after. All colonies should be white.<br />
*The plate was induced with arabinose by using an atomizer with a 1%w/vol sterile arabinose solution. <br />
*The day after colonies where visually inspected under brief exposure to UV light; a colony with a yellowish red where picked and cultured in LB containing 0.5%w/vol arabinose. The culture volume was 50mL and it was kept at 37C with shake until it reached saturation.<br />
*The culture was harvested by centrifugation at 5000g for 10 min at 4<sup>o</sup>C. Supernatant was discarded and the pellet was washed thoroughly by washing twice with fresh LB with no arabinose.<br />
*The pellet was resuspended in 25mL fresh LB with no arabinose and incubated additionally 3 hours and 40 min. to chase the cytoplasmic GFP to the periplasm. The incubation was done at 37C with shake.<br />
*Then the cells were harvested by taking 0.5mL into a centrifuge tube and adding a pinch of low melting point agarose. This was incubated at 50C for 10 min interrupted by vortexing middle ways. <br />
*The sample was vortexed and 20uL was loaded on a microscope slide and covered with a cover slip. This could then be stored at 4<sup>o</sup>C or immediately examined.<br />
*Pictures were acquired on fluorescents microscope with 100x lens. Both GFP and RFP filters was used. To get the clear difference between periplasm and cytoplasm 2 pictures with each filter was taken; one with short exposure time and one with long. The short exposure time was subtracted from the long and finally the GFP and RFP pictures were merged to get RFP as a cytoplasmic contrast to the periplasmic GFP.<br />
<br />
==References== <br />
<html><br />
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<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 />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/Methods/Determining_concentration_of_nitrogen_compounds/Experiment_4Team:DTU-Denmark/Methods/Determining concentration of nitrogen compounds/Experiment 42013-10-04T18:06:41Z<p>Ariadni: /* Procedure */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|Experiment 4}}<br />
<div class="overviewPage"><br />
==Procedure==<br />
<br />
In this aerobic experiment, we add increasing concentrations of ammonium NH<sub>4</sub><sup>+</sup> to transformed ''E. coli'' cells growing aerobically, in order to test whether our AMO transformant is converting ammonium to hydroxylamine (NH<sub>2</sub>OH) . The same protocol is used to measure whether our HAO transformant is converting hydroxylamine to nitrite NO<sub>2</sub><sup>-</sup>. <br />
<br />
The bottle is placed to a magnetic stirrer on 270 rpm and 37<sup>o</sup>C.<br />
<br />
The AMO experiment will need the following:<br />
*AMO '' E. coli'' transformant (duplicate)<br />
*Control: Untransformed ''E. coli''. <br />
*Control: ''Nitrosomonas'' at 26 <sup>o</sup>C<br />
*Control: Abiotic<br />
<br />
We expect the AMO transformant to use ammonium more quickly than the untransformed control, and ''Nitrosomonas'' to also use ammonia. <br />
<br />
The HAO experiment will need the following:<br />
*HAO ''E. coli'' transformant (duplicate)<br />
*Control: Untransformed ''E. coli''. <br />
*Control: ''Nitrosomonas'' at 26 <sup>o</sup>C<br />
*Control: Abiotic<br />
We expect the HAO transformant and ''Nitrosomonas'' to consume hydroxylamine and produce nitrite, and the untransformed ''E. coli'' will not.<br />
<br />
<br />
<br />
There will be 10 experimental flasks:<br />
<br />
# ''E. coli'' cultures will be grown at 37 <sup>o</sup>C in the incubator and<br />
# ''Nitrosomonas Europaea'' at 26 <sup>o</sup>C<br />
<br />
<br />
<br />
'''EQUIPMENT NEEDED'''<br />
<br />
*4 erlyenmeyer flasks for growing cultures<br />
*1 hot plate and magnet stirrer<br />
*Syringe filters with pore size 0.2μm<br />
*Stopwatch.<br />
*Ammonium Chloride (NH<sub>4</sub>Cl)<br />
*[[Team:DTU-Denmark/Methods/Modified_DM_minimal_medium|Modified DM minimal medium]]<br />
*''E. coli'' overnight culture<br />
*Nitrosomonas overnight culture<br />
*LB-broth medium<br />
*Flat bottom centrifuge tubes<br />
*50 mM ammonium chloride stock solution <br />
*2mL Eppendorf tubes<br />
*Colorimetric test kits for ammonium<br />
*Rack for the samples <br />
*Glass tubes for the colorimetric tests<br />
*10mL, 1mL, 200μL pipettes with tips<br />
*Single use plastic cuvettes<br />
*MilliQ water<br />
<br />
<br />
'''EXPERIMENTAL PROCEDURE''' <br />
<br />
First, prepare the solutions that will be used:<br />
# Prepare at least 2L DM minimal medium with ammonium chloride as a nitrogen source. Add 0.745 g NH<sub>4</sub>Cl to 1L of prepared DM medium. This is needed for:<br />
#* growing the AMO transformant (10 mL overnight culture + 400mL to resuspend)<br />
#* growing the HAO transformant (10 mL overnight culture + 400mL to resuspend)<br />
#* growing the ''E. coli'' control for both experiments (10 mL overnight culture + 400mL to resuspend x2)<br />
# Prepare at least 1L DM modified minimal medium (with no ammonium chloride). This is needed for:<br />
#* washing the ''E. coli'' cultures (5mL x 6 experiments)<br />
#* resuspending the ''E. coli'' cultures for the experiments (107.5 mL x6)<br />
#* media for the abiotic control experiments (100mL x2)<br />
# Prepare 1 L ''Nitrosomonas'' ATCC medium containing ammonia. This is needed for:<br />
#* overnight culture (10 mL) and resuspending (400 mL) x2<br />
# Prepare at least 500 mL Nitrosomonas ATCC medium with no ammonia source. This is needed for:<br />
#* washing (5mL) x2<br />
#* resuspending for the experiment (100mL) x2<br />
# Prepare 50mM ammonium chloride solution. <br />
#* Dissolve 2.4544 g NH4Cl in 50 mL DM modified minimal medium (no NH<sub>4</sub>Cl).<br />
# Prepare 80mM hydroxylamine solution.<br />
#* Dissolve 0.260 g hydroxylamine in 100 mL DM modified minimal medium (no NH<sub>4</sub>Cl).<br />
<br />
For the AMO and HAO experiments, repeat the following for both the transformed and untransformed ''E. coli'', and for ''N. europaea''.<br />
<br />
# Grow'' E. coli'' top10 overnight in 10mL of DM medium + NH<sub>4</sub>Cl prepared in step 1 at 37<sup>o</sup>C in an erlenmeyer flask.<br />
# Take 4mL of E. coli overnight culture and add to 400mL fresh DM medium + NH<sub>4</sub>Cl. <br />
# Grow the ''E. coli'' at 36.6<sup>o</sup>C in 210 RPM until OD=0.35 (about 5 hours). Grow the ''N. europaea'' at 26<sup>o</sup>C . <br />
# Pellet down the 400mL culture at 3000g for 4 min at room temperature.<br />
# Wash with 5 mL Modified DM minimal medium and centrifuge again.<br />
# Pour off the supernatant and resuspend the cell pellet. Pour samples together if they were made in more than one centrifuge tube. The OD should be around 0.3.<br />
# Remove one aliquot of 107.5 mL of the OD=0.3 suspension to a flask and keep at 37<sup>o</sup>C, but don't let it sit around for many hours. This is the experimental flask. <br />
For the abiotic control:<br />
# Remove one aliquot of 100mL of DM medium (without the added NH<sub>4</sub>Cl). <br />
Repeat the following steps for each experiment and for the control:<br />
# Put the experimental flask on the hot plate with magnet stirrer and stabilize the temperature at 37<sup>o</sup>C (26<sup>o</sup>C for ''Nitrosomonas'').<br />
# Remove 4 mL as the first sample t=0 colorimetric analysis. For the abiotic control, take samples only at the start and end of the experiment.<br />
# Use 2mL of the first sample to measure the OD.<br />
# Take a 2mL sample at 5 min and at 10 min for a baseline.<br />
# After the min 10 sample, quickly spike with <br />
#* 1.7 mL of 50mM ammonium stock solution. Then the ammonium concentration in 100mL is 0.85mM (equivalent to the Km of AMO) = 11.9mg/L Nitrogen.<br />
#* 50uL of 80mM hydroxylamine solution. Then the hydroxylamine concentration in 100 mL is 0.04mM (the Km of HAO) = 0.56 mg/L Nitrogen. <br />
# Then take another sample immediately after the spike.<br />
# Continue taking samples every 5 min until t=30 min.<br />
# Take samples every 10 min until t=60 min. <br />
At the end of the experiment:<br />
# Take an extra 2mL sample to measure the OD.<br />
# Measure the temperature. <br />
To finish gathering data:<br />
# Make the colorimetric measurements for:<br />
#* ammonium for each of the samples collected for the AMO experiment by using the protocol [[Team:DTU-Denmark/Methods/Ammonium_colorimetric_measurements#Ammonium_colorimetric_measurements|Ammonium measurement]].<br />
#* nitrite for each of the samples collected for the HAO experiment by using the protocol [[Team:DTU-Denmark/Methods/Nitrite_colorimetric_measurements|Nitrite measurement]]<br />
<br />
If none of the the added ammonia is converted to hydroxylamine, then the last sample will contain 11.9 mg N-Ammonia/L (the test kit measures between 0 and 70 mg/L approx). <br />
<br />
If all the added hydroxylamine is converted to nitrite, then the last sample will contain 0.56 mg N-Nitrite/L (the test kit measures between 0 and 1 mg/L approx).<br />
<br />
<br />
<br />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/Methods/Determining_concentration_of_nitrogen_compounds/Experiment_1aTeam:DTU-Denmark/Methods/Determining concentration of nitrogen compounds/Experiment 1a2013-10-04T18:03:52Z<p>Ariadni: /* Experiment 1a: Verify nitrite stability in anaerobic, untransformed E. coli */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|Determining concentration of nitrogen compounds}}<br />
<div class="overviewPage"><br />
<br />
==Experiment 1a: Verify nitrite stability in anaerobic, untransformed ''E. coli''==<br />
<br />
Characterization of the behavior of ''E. coli'' wild type while growing in the presence of nitrite in an anaerobic reaction chamber by measuring the conversion of nitrite to ammonium.<br />
<br />
In this anaerobic experiment three different concentrations of nitrite are added to a medium where a native strain of ''E. coli'' is growing, in order to test the rate at which nitrite is converted to ammonium. <br />
The hypothesis is that nitrite will be converted into ammonium. We want to determine how readily, and at what concentrations this happens.<br />
The anaerobic experiments will be performed in a sealed flask. <br />
The concentrations of NH<sub>4</sub><sup>+</sup> and NO<sub>2</sub><sup>-</sup> will be measured every 20 minutes and the experiment will last for 3 hours. The literature states that 10mmol of NO<sub>2</sub><sup>-</sup> is converted into NH<sub>4</sub><sup>+</sup> per mg cell biomass per hour.<br />
There will be also a magnetic stirrer on 250 rpm. Each sample with different concentrations of nitrite will be measured separately, as well as the controls. <br />
There will be three controls. One will be with only anaerobic biomass to check the behavior of cells without nitrite. A second one will be anaerobic abiotic with nitrite and medium to check that there is not any conversion of nitrite in the medium. A third anaerobic with killed biomass to confirm that reaction is catalyzed only by microbial activity. <br />
<br />
EQUIPMENT NEEDED<br />
* 6 bottle flasks of 100 ml with lids<br />
* 1 magnetic stirrer<br />
* Syringes with long needles and small needles <br />
* Syringe filters with pore size 0.2μm<br />
* Stopwatch.<br />
* Ammonium Bicarbonate (NH<sub>4</sub>)HCO<sub>3</sub> puriss., meets analytical specification of Ph.Eur., BP, E 503, 99-101% (Sigma-Aldrich)<br />
* Sodium nitrite – NaNO<sub>2</sub> , puriss. p.a., ACS reagent, reag. Ph. Eur., ≥99% (Sigma-Aldrich)<br />
* [[Team:DTU-Denmark/Methods/Modified DM minimal medium|Modified DM minimal medium]]<br />
* ''E. coli'' overnight culture<br />
* LB-broth medium<br />
* Flat bottom centrifuge tubes<br />
* 50 mM nitrite stock solution (MW NaNO<sub>2</sub> =69 g/mol; 345 mg in 100 mL water)<br />
* 2mL Eppendorf tubes<br />
* Colorimetric test kits for ammonium and nitrite.<br />
* Rack for the samples <br />
* Glass tubes for the colorimetric tests<br />
* 10mL, 1mL, 200μL pipettes with tips.<br />
* Single use plastic cuvettes.<br />
* MilliQ water. <br />
All preparations with cells will be done on ice so that the cells do not grow.<br />
<br />
EXPERIMENTAL PROCEDURE <br />
# Grow ''E. coli'' top10 overnight in 10mL of LB medium at 37◦C.<br />
# Take 5mL of overnight culture and add to 500mL fresh LB medium. <br />
# Grow the cells at 37<sup>o</sup>C in 210 RPM until OD=0.35 (about 2 hours).<br />
# Cool down the centrifuge for 30 min at 4<sup>o</sup>C.<br />
# Pellet down the 500mL culture, 3000g for 4 min at 4<sup>o</sup>C.<br />
# Discard supernatants and wash each aliquot with 10-20 ml cold Modified DM minimal medium and centrifuge again.<br />
# Pour off the supernatant once again and resuspend the cell pellets in 200mL Modified DM Minimal medium. <br />
# Measure OD of the 200 mL cell suspension and add cold Modified DM minimal medium until OD=0.3 (note the exact value). The final volume should be around 520-570 mL.<br />
# Killed biomass control: Add 100 mL of biomass in a flask, label accordingly and autoclave it.<br />
# 3 nitrite experiments plus control: Pour 100 mL of the OD=0.3 suspension into each flask and keep on ice. Label the flasks accordingly (nitrite1, nitrite 2, nitrite 3, no nitrite).<br />
# Abiotic control: Add 100 mL of DM medium to a flask and label accordingly.<br />
# Make experiments anaerobic by sparging N<sub>2</sub> in the solution by following the method for injecting N<sub>2</sub> (Appendix 1), for 3 min the nitrite stock solution and 5 min the cell suspensions and controls. Seal all bottles with rubber stoppers and aluminium caps.<br />
# Add 0.5 mL of 50mM nitrite stock solution to 99.5 ml of the cell suspension in flask 1. Then the nitrite concentration is about 0.25mM. <br />
# Add 1 mL of of 50mM nitrite stock solution to 99 ml of the cell suspension in a flask 2 and adjust the electrode. Then the nitrite concentration is about 0.5mM. <br />
# Add 2 mL of 50mM nitrite stock solution to 98 ml of the cell suspension in a flask 3. Then the nitrite concentration is about 1mM. <br />
# In the abiotic anaerobic control add 1 mL of nitrite stock.<br />
# Add 1mL of nitrite stock solution the anaerobic control with killed biomass.<br />
# Remove two 2.5mL samples as the t=0 samples from each of the reaction chambers.<br />
# Remove an initial 2mL sample to measure the initial Nitrate concentration.<br />
# Put the flask on the magnetic stirrer and start with 250 rpm at 37<sup>o</sup>C. <br />
# Every 20 min for 3 hours take two samples 2 ml for the colorimetric assay from each of the experiments.<br />
# Inject the sample using filter of pore size 0.2μm and fill into a 2mL Eppendorf tube. <br />
# Repeat the steps 22-23 for each flask.<br />
# After the end of the batches, take a final sample for colorimetric assay and measure OD+cell mass.<br />
# Take a final sample to measure the ending Nitrate concentration.<br />
# Make the colorimetric measurements for nitrite and ammonium by using the protocols [[Team:DTU-Denmark/Methods/Nitrite colorimetric measurements|Nitrite colorimetric measurements]] and [[Team:DTU-Denmark/Methods/Ammonium colorimetric measurements|Ammonium colorimetric measurements]], respectively.<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/Methods/dpni_treatmentTeam:DTU-Denmark/Methods/dpni treatment2013-10-04T18:03:13Z<p>Ariadni: </p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|DpnI treatment}}<br />
<br />
To digest methylated DNA, for example to clean up PCR products from template.<br />
<br />
<u>Reaction Mix</u>:<br />
* 44 uL sample<br />
* 5 uL NEB buffer 4<br />
* 1 uL DpnI<br />
<br />
Incubate 1h at 37<sup>o</sup>C and 20:00 at 80<sup>o</sup>C to inactivate the enzyme.</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/Methods/AutoclavingTeam:DTU-Denmark/Methods/Autoclaving2013-10-04T18:00:41Z<p>Ariadni: /* Autoclaving in the lab of DTU building 208 */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|Autoclaving}}<br />
<br />
<div class="overviewPage"><br />
<div class="overviewBox left"><br />
<br />
==Autoclaving in the lab of DTU building 208==<br />
*Close the lid<br />
*Check if it is locked (the orange pointer should be turned vertical)<br />
*Wait until the condensation goes out before closing the valve<br />
*Let temperature reach 125 <sup>o</sup>C and pressure 20 psi <br />
*Wait for 15 min before switching off the power and wait an hour.<br />
<br />
</div><br />
<div style="clear: both;"></div><br />
</div><br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/Methods/Determining_concentration_of_nitrogen_compounds/Experiment_1aTeam:DTU-Denmark/Methods/Determining concentration of nitrogen compounds/Experiment 1a2013-10-04T17:59:55Z<p>Ariadni: /* Experiment 1a: Verify nitrite stability in anaerobic, untransformed E. coli */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|Determining concentration of nitrogen compounds}}<br />
<div class="overviewPage"><br />
<br />
==Experiment 1a: Verify nitrite stability in anaerobic, untransformed ''E. coli''==<br />
<br />
Characterization of the behavior of ''E. coli'' wild type while growing in the presence of nitrite in an anaerobic reaction chamber by measuring the conversion of nitrite to ammonium.<br />
<br />
In this anaerobic experiment three different concentrations of nitrite are added to a medium where a native strain of ''E. coli'' is growing, in order to test the rate at which nitrite is converted to ammonium. <br />
The hypothesis is that nitrite will be converted into ammonium. We want to determine how readily, and at what concentrations this happens.<br />
The anaerobic experiments will be performed in a sealed flask. <br />
The concentrations of NH<sub>4</sub><sup>+</sup> and NO<sub>2</sub><sup>-</sup> will be measured every 20 minutes and the experiment will last for 3 hours. The literature states that 10mmol of NO<sub>2</sub><sup>-</sup> is converted into NH<sub>4</sub><sup>+</sup> per mg cell biomass per hour.<br />
There will be also a magnetic stirrer on 250 rpm. Each sample with different concentrations of nitrite will be measured separately, as well as the controls. <br />
There will be three controls. One will be with only anaerobic biomass to check the behavior of cells without nitrite. A second one will be anaerobic abiotic with nitrite and medium to check that there is not any conversion of nitrite in the medium. A third anaerobic with killed biomass to confirm that reaction is catalyzed only by microbial activity. <br />
<br />
EQUIPMENT NEEDED<br />
* 6 bottle flasks of 100 ml with lids<br />
* 1 magnetic stirrer<br />
* Syringes with long needles and small needles <br />
* Syringe filters with pore size 0.2μm<br />
* Stopwatch.<br />
* Ammonium Bicarbonate (NH<sub>4</sub>)HCO<sub>3</sub> puriss., meets analytical specification of Ph.Eur., BP, E 503, 99-101% (Sigma-Aldrich)<br />
* Sodium nitrite – NaNO<sub>2</sub> , puriss. p.a., ACS reagent, reag. Ph. Eur., ≥99% (Sigma-Aldrich)<br />
* [[Team:DTU-Denmark/Methods/Modified DM minimal medium|Modified DM minimal medium]]<br />
* ''E. coli'' overnight culture<br />
* LB-broth medium<br />
* Flat bottom centrifuge tubes<br />
* 50 mM nitrite stock solution (MW NaNO<sub>2</sub> =69 g/mol; 345 mg in 100 mL water)<br />
* 2mL Eppendorf tubes<br />
* Colorimetric test kits for ammonium and nitrite.<br />
* Rack for the samples <br />
* Glass tubes for the colorimetric tests<br />
* 10mL, 1mL, 200μL pipettes with tips.<br />
* Single use plastic cuvettes.<br />
* MilliQ water. <br />
All preparations with cells will be done on ice so that the cells do not grow.<br />
<br />
EXPERIMENTAL PROCEDURE <br />
# Grow ''E. coli'' top10 overnight in 10mL of LB medium at 37◦C.<br />
# Take 5mL of overnight culture and add to 500mL fresh LB medium. <br />
# Grow the cells at 37<sup>o</sup>C in 210 RPM until OD=0.35 (about 2 hours).<br />
# Cool down the centrifuge for 30 min at 4<sup>o</sup>C.<br />
# Pellet down the 500mL culture, 3000g for 4 min at 4<sup>o</sup>C.<br />
# Discard supernatants and wash each aliquot with 10-20 ml cold Modified DM minimal medium and centrifuge again.<br />
# Pour off the supernatant once again and resuspend the cell pellets in 200mL Modified DM Minimal medium. <br />
# Measure OD of the 200 mL cell suspension and add cold Modified DM minimal medium until OD=0.3 (note the exact value). The final volume should be around 520-570 mL.<br />
# Killed biomass control: Add 100 mL of biomass in a flask, label accordingly and autoclave it.<br />
# 3 nitrite experiments plus control: Pour 100 mL of the OD=0.3 suspension into each flask and keep on ice. Label the flasks accordingly (nitrite1, nitrite 2, nitrite 3, no nitrite).<br />
# Abiotic control: Add 100 mL of DM medium to a flask and label accordingly.<br />
# Make experiments anaerobic by sparging N<sub>2</sub> in the solution by following the method for injecting N<sub>2</sub> (Appendix 1), for 3 min the nitrite stock solution and 5 min the cell suspensions and controls. Seal all bottles with rubber stoppers and aluminium caps.<br />
# Add 0.5 mL of 50mM nitrite stock solution to 99.5 ml of the cell suspension in flask 1. Then the nitrite concentration is about 0.25mM. <br />
# Add 1 mL of of 50mM nitrite stock solution to 99 ml of the cell suspension in a flask 2 and adjust the electrode. Then the nitrite concentration is about 0.5mM. <br />
# Add 2 mL of 50mM nitrite stock solution to 98 ml of the cell suspension in a flask 3. Then the nitrite concentration is about 1mM. <br />
# In the abiotic anaerobic control add 1 mL of nitrite stock.<br />
# Add 1mL of nitrite stock solution the anaerobic control with killed biomass.<br />
# Remove two 2.5mL samples as the t=0 samples from each of the reaction chambers.<br />
# Remove an initial 2mL sample to measure the initial Nitrate concentration.<br />
# Put the flask on the magnetic stirrer and start with 250 rpm at 37<sup>o</sup>C. <br />
# Every 20 min for 3 hours take two samples 2 ml for the colorimetric assay from each of the experiments.<br />
# Inject the sample using filter of pore size 0.2μm and fill into a 2mL Eppendorf tube. <br />
# Repeat the steps 22-23 for each flask.<br />
# After the end of the batches, take a final sample for colorimetric assay and measure OD+cell mass.<br />
# Take a final sample to measure the ending Nitrate concentration.<br />
# Make the colorimetric measurements for nitrite and ammonium by using the appendices 3 and 4.<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-04T17:38:34Z<p>Ariadni: /* References */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
===What?===<br />
Our project removes ammonia (NH<sub>3</sub>) from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide (N<sub>2</sub>O).<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
<br />
===Why?===<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 [1]. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
In Europe the annual surplus of fixed nitrogen from crop production and livestock farming is 10 Tg (10 Teragrams = a million tonnes), 6 Tg of which leaches into ground and surface water [2]. If that was to be converted into N<sub>2</sub>O, it could be decomposed for 4.87 Billion kW h -- equivalent to the annual power consumption of 36,000 Avarage households.<br />
<br />
If released into the atmosphere, nitrous oxide is a potent greenhouse gas, and capable of degrading ozone, which is why it makes sense to convert it to N<sub>2</sub>.<br />
<br />
===How?===<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 [3]. We also provide [[Team:DTU-Denmark/Reactor_Model|a model 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 />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
[[File:Dtu-Mutant1.png|right]]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><br />
<br><br />
<br />
=== Mutant 2: Anaerobic ===<br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
[[File:dtu-Mutant2.png|right]]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, but we were able to [[Team:DTU-Denmark/Experiment4|verify that the AMO containing plasmid is working as designed]].<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 />
<br />
== USER Cloning ==<br />
To assemble our plasmids we employ uracil-specific excision reagent (USER) cloning [4]. 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 specially designed set of primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
## We are using the non-commercial polymerase X7, designed by Morten Nørholm [5].<br />
# Optional - If the method was used to insert genes into a backbone, with it's own resistance marker, the PCR product were treated with DpnI to digest the template DNA from the PCR reaction.<br />
# USER-reaction: Digesting the linearized PCR fragment with Uracil-Specific Excision Reagent (USER) enzyme removes the uracil-insertions. This will produce the required overhangs with the sequence and the length of your wish.<br />
# Transformation: The mix is immediately transformed into competent ''E. coli'' cells. The plasmid assembles itself by base pairing between the complementary overhangs and ligation is done by ''E.coli'' itself.<br />
<br />
'''Advantages to USER cloning'''<br />
*You only need one enzyme<br />
*You don't need any ligation reaction<br />
*Seamless assembly<br />
*Directional <br />
*Multiple fragment in one reaction - also in eukaryotes [6].<br />
*[https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play]<br />
<br />
[[File:Plug 'n' Play assembly samlet.png|thumb|upright=3|center|The [https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play] concept. Figure taken from DTU-2 iGEM team 2011]]<br />
<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 />
<br />
[1] Sutton, Mark A., et al. "Too much of a good thing." Nature 472.7342 (2011): 159-161.<br />
<br />
[2] Sutton, Mark A., Clare M. Howard, and Jan Willem Erisman, eds. The European nitrogen assessment: sources, effects and policy perspectives. Cambridge University Press, 2011.<br />
<br />
[3] Yaniv D. Scherson, George F. Wells, Sung-Geun Woo, Jangho Lee, Joonhong Park, Brian J. Cantwell and Craig S. Criddle; Nitrogen removal with energy recovery through N<sub>2</sub>O decomposition; Energy & Environmental Science. Issue 1 2013.<br />
<br />
[4] Nour-Eldin, HH et al (2010). USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories. Methods Mol Biol. 2010;643:185-200. doi: 10.1007/978-1-60761-723-5_13.<br />
<br />
[5] Nørholm, Morten HH. "A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering." BMC biotechnology 10.1 (2010): 21.<br />
<br />
[6] Frandsen, Rasmus, et al. "Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi." BMC molecular biology 9.1 (2008): 70.<br />
<br />
<br />
U.S. Energy Information Administration, Frequently Asked Questions: How much electricity does an American home use?<br />
<br />
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).<br />
<br />
Kanehisa, M. and Goto, S.; KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27-30 (2000).<br />
<br />
Zumft, Walter G.; Cell Biology and Molecular Basis of Denitrification. Microbiology and Molecular Biology Reviews, December 1997 p. 533–616<br />
<br />
Ingledew, W.J. and Poole, R.K.; The Respiratory Chains of Escherichia coli. Microbiological Reviews, Sept. 1984, p. 222-271<br />
<br />
<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-04T17:38:00Z<p>Ariadni: /* References */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
===What?===<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide (N<sub>2</sub>O).<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
<br />
===Why?===<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 [1]. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
In Europe the annual surplus of fixed nitrogen from crop production and livestock farming is 10 Tg (10 Teragrams = a million tonnes), 6 Tg of which leaches into ground and surface water [2]. If that was to be converted into N<sub>2</sub>O, it could be decomposed for 4.87 Billion kW h -- equivalent to the annual power consumption of 36,000 Avarage households.<br />
<br />
If released into the atmosphere, nitrous oxide is a potent greenhouse gas, and capable of degrading ozone, which is why it makes sense to convert it to N<sub>2</sub>.<br />
<br />
===How?===<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 [3]. We also provide [[Team:DTU-Denmark/Reactor_Model|a model 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 />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
[[File:Dtu-Mutant1.png|right]]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><br />
<br><br />
<br />
=== Mutant 2: Anaerobic ===<br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
[[File:dtu-Mutant2.png|right]]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, but we were able to [[Team:DTU-Denmark/Experiment4|verify that the AMO containing plasmid is working as designed]].<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 />
<br />
== USER Cloning ==<br />
To assemble our plasmids we employ uracil-specific excision reagent (USER) cloning [4]. 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 specially designed set of primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
## We are using the non-commercial polymerase X7, designed by Morten Nørholm [5].<br />
# Optional - If the method was used to insert genes into a backbone, with it's own resistance marker, the PCR product were treated with DpnI to digest the template DNA from the PCR reaction.<br />
# USER-reaction: Digesting the linearized PCR fragment with Uracil-Specific Excision Reagent (USER) enzyme removes the uracil-insertions. This will produce the required overhangs with the sequence and the length of your wish.<br />
# Transformation: The mix is immediately transformed into competent ''E. coli'' cells. The plasmid assembles itself by base pairing between the complementary overhangs and ligation is done by ''E.coli'' itself.<br />
<br />
'''Advantages to USER cloning'''<br />
*You only need one enzyme<br />
*You don't need any ligation reaction<br />
*Seamless assembly<br />
*Directional <br />
*Multiple fragment in one reaction - also in eukaryotes [6].<br />
*[https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play]<br />
<br />
[[File:Plug 'n' Play assembly samlet.png|thumb|upright=3|center|The [https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play] concept. Figure taken from DTU-2 iGEM team 2011]]<br />
<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 />
<br />
[1] Sutton, Mark A., et al. "Too much of a good thing." Nature 472.7342 (2011): 159-161.<br />
<br />
[2] Sutton, Mark A., Clare M. Howard, and Jan Willem Erisman, eds. The European nitrogen assessment: sources, effects and policy perspectives. Cambridge University Press, 2011.<br />
<br />
[3] Yaniv D. Scherson, George F. Wells, Sung-Geun Woo, Jangho Lee, Joonhong Park, Brian J. Cantwell and Craig S. Criddle; Nitrogen removal with energy recovery through N<sub>2</sub>O decomposition; Energy & Environmental Science. Issue 1 2013.<br />
<br />
[4] Nour-Eldin, HH et al (2010). USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories. Methods Mol Biol. 2010;643:185-200. doi: 10.1007/978-1-60761-723-5_13.<br />
<br />
[5] Nørholm, Morten HH. "A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering." BMC biotechnology 10.1 (2010): 21.<br />
<br />
[6] Frandsen, Rasmus, et al. "Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi." BMC molecular biology 9.1 (2008): 70.<br />
<br />
<br />
U.S. Energy Information Administration, Frequently Asked Questions: How much electricity does an American home use?<br />
<br />
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).<br />
<br />
Kanehisa, M. and Goto, S.; KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27-30 (2000).<br />
<br />
Zumft, Walter G.; Cell Biology and Molecular Basis of Denitrification. Microbiology and Molecular Biology Reviews, December 1997 p. 533–616<br />
<br />
Ingledew, WJ and Poole, RK (1984) The Respiratory Chains of Escherichia coli. Microbiological Reviews, Sept. 1984, p. 222-271<br />
<br />
<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/TeamPhotosTeam:DTU-Denmark/TeamPhotos2013-10-04T16:17:47Z<p>Ariadni: /* Additional Photos */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|Team Photos}}<br />
<div class="overviewPage"><br />
<div class="overviewBox"><br />
<br />
==Hackathon Timelapse==<br />
[[File:DTU-Hackathon.gif|thumb|center|600px|CLICK on the image to see our Hackathon Timelapse]]<br />
<br />
==Additional Photos==<br />
<br />
[[File:DTU_iGEM_workshop_marts_2013_004.jpg|thumb|left|200px|]][[File:Dtu IMAG0108.jpg|thumb|center|200px|]]<br />
<br />
[[File:IMG 4544.JPG|thumb|right|300px]][[File:DTU_iGEM_workshop_marts_2013_029.JPG|thumb|300px|]][[File:Dtu IMG 4546.JPG|thumb|300px|]][[File:Dtu IMG 4646.JPG|thumb|300px|]]<br />
<br />
<br />
[[File:Dtu IMG 4652.JPG|thumb|300px|]][[File:Dtu_P1010192.JPG|thumb|300px|]][[File:Dtu P1010248.JPG|thumb|300px|]][[File:Dtu P1010252.JPG|thumb|300px|]]<br />
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<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/TeamPhotosTeam:DTU-Denmark/TeamPhotos2013-10-04T16:17:11Z<p>Ariadni: /* Additional Photos */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|Team Photos}}<br />
<div class="overviewPage"><br />
<div class="overviewBox"><br />
<br />
==Hackathon Timelapse==<br />
[[File:DTU-Hackathon.gif|thumb|center|600px|CLICK on the image to see our Hackathon Timelapse]]<br />
<br />
==Additional Photos==<br />
<br />
[[File:DTU_iGEM_workshop_marts_2013_004.jpg|thumb|left|200px|]][[File:Dtu IMAG0108.jpg|thumb|center|200px|]]<br />
<br />
[[File:IMG 4544.JPG|thumb|right|300px]][[File:DTU_iGEM_workshop_marts_2013_029.JPG|thumb|300px|]][[File:Dtu IMG 4546.JPG|thumb|300px|]][[File:Dtu IMG 4646.JPG|thumb|300px|]]<br />
<br />
<br />
[[File:Dtu IMG 4652.JPG|thumb|300px|]][[File:Dtu_P1010192.JPG|thumb|300px|]][[File:Dtu P1010248.JPG|thumb|300px|]][[File:Dtu P1010252.JPG|thumb|300px|]]<br />
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<div style="clear: both;"></div><br />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/TeamPhotosTeam:DTU-Denmark/TeamPhotos2013-10-04T16:16:33Z<p>Ariadni: /* Additional Photos */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|Team Photos}}<br />
<div class="overviewPage"><br />
<div class="overviewBox"><br />
<br />
==Hackathon Timelapse==<br />
[[File:DTU-Hackathon.gif|thumb|center|600px|CLICK on the image to see our Hackathon Timelapse]]<br />
<br />
==Additional Photos==<br />
<br />
[[File:DTU_iGEM_workshop_marts_2013_004.jpg|thumb|left|200px|]][[File:Dtu IMAG0108.jpg|thumb|center|200px|]]<br />
<br />
[[File:IMG 4544.JPG|thumb|right|300px]][[File:DTU_iGEM_workshop_marts_2013_029.JPG|thumb|300px|]]<br />
<br />
<br />
[[File:Dtu IMG 4546.JPG|thumb|300px|]]<br />
<br />
<br />
[[File:Dtu IMG 4646.JPG|thumb|300px|]]<br />
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<br />
[[File:Dtu IMG 4652.JPG|thumb|300px|]]<br />
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<br />
[[File:Dtu_P1010192.JPG|thumb|300px|]]<br />
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[[File:Dtu P1010248.JPG|thumb|300px|]]<br />
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[[File:Dtu P1010252.JPG|thumb|300px|]]<br />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/TeamPhotosTeam:DTU-Denmark/TeamPhotos2013-10-04T16:15:05Z<p>Ariadni: /* Additional Photos */</p>
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==Hackathon Timelapse==<br />
[[File:DTU-Hackathon.gif|thumb|center|600px|CLICK on the image to see our Hackathon Timelapse]]<br />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/TeamPhotosTeam:DTU-Denmark/TeamPhotos2013-10-04T16:11:32Z<p>Ariadni: /* Additional Photos */</p>
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==Hackathon Timelapse==<br />
[[File:DTU-Hackathon.gif|thumb|center|600px|CLICK on the image to see our Hackathon Timelapse]]<br />
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==Additional Photos==<br />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/TeamPhotosTeam:DTU-Denmark/TeamPhotos2013-10-04T16:11:00Z<p>Ariadni: /* Additional Photos */</p>
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==Hackathon Timelapse==<br />
[[File:DTU-Hackathon.gif|thumb|center|600px|CLICK on the image to see our Hackathon Timelapse]]<br />
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==Additional Photos==<br />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/HelloWorldTeam:DTU-Denmark/HelloWorld2013-10-04T15:49:03Z<p>Ariadni: /* Construction */</p>
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<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 [1].<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. [2].<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, [3]. 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 [4].<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 />
<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] [5] 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 />
== Results ==<br />
We used different methods for testing that our construct actually did work and also for verifying the model we did before the test. <br />
<br />
<br />
===Periplasmic vs. cytoplasmic fraction===<br />
The first test we did was to purify the periplasmic fraction of the cells and then comparing with the cytoplasmic ([https://2013.igem.org/Team:DTU-Denmark/Notebook/6_July_2013 link to notebook]). We did this with the cold osmotic shock method and viewed the fraction under UV-light. <br />
[[File:Sucrose A+D+C.JPG|thumb|center|upright=3|Periplasmic fractions are clear green fluorescent while the pellet with the cytoplasmic fraction is red fluorescent]]<br />
<br />
<br />
===Fluorescent microscope images=== <br />
Next test was the "real thing". We wanted water-tight evidence that GFP SF was actually exported; and what better way to do this than getting pictures of it? After following our procedure for chasing GFP SF to the periplasm, we observed these images after background subtraction. Following is an image with no zoom:<br />
[[File:GFP in perimplasm RFP in cytoplasm.png|thumb|center|upright=3|Transformed ''E. coli'' taken with a Fluorescence microscope]]<br />
<br />
<br />
Next to test our model we picked a cell this good resolution and zoomed in on this:<br />
[[File:GFP in perimplasm RFP in cytoplasm close up.png|thumb|center|upright=3|Close up of several cells, showing GFP expression in the periplasm]]<br />
<br />
<br />
For this cell a linear profile was made that shows both the GFP and RFP signal across the cell:<br />
[[File:Graf.PNG|thumb|center|upright=3|Line profile of the cross section of the cell seen in the above close up]]<br />
These profiles closely resemble our model seen in next section.<br />
<br />
<br />
==Modeling==<br />
<br />
===Modeling Methods===<br />
To assess the ability to distinguish the periplasmic from the cytoplasmic space we made a model which is based on a perfect spherical cell with and even layer between inner and outer membrane. <br />
[[File:Cell model.png|300px|center|Conceptual drawing for the model]]<br />
<br />
To get a value of the RFP and GFP signal we used a cross section of the spherical cell and measured the length of RFP and GFP going across this section; see the measurement bars at the figure. The make a graph of this we set the middle of the cell to be 0 on the x-axis. On the y-axis we let two lines show the value of GFP and RFP in the cross section.<br />
<br />
The first graph can be seen below:<br />
[[File:First model.png|thumb|center|upright=2|The first model was done in Maple.]]<br />
<br />
<br />
===Model of "Hello World" construct===<br />
With the mathematical equations made for making the graph above we made a interactive model that enables the user to set their own settings with regard to GFP and RFP intensity and cytoplasmic vs. periplasmic fraction. If the parameters are set well the model reassembles the images we acquired with the fluorescence microscope. <br />
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<h4>Warning</h4><br />
<img src="/wiki/images/c/cb/Warning.png" height="70" width="70"><br />
<h3>Oh no! Your browser is too old to display this content</h3><br />
<p><b>Please install the newest version of any of these: </b></p><br />
<p><b><i>Chrome, Safari, Internet Explorer (Windows 7 or later only), Firefox, Opera</i></b></p><br />
<p><b>And please come back! It's worth it!</b></p><br />
</td><br />
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Cell membrane size:<br />
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<input type="range" name="red_intensity_slider" id="red_intensity_slider" min="0" max="255" step="1" value="255"><br />
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Green intensity:<br />
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<input type="range" name="green_intensity_slider" id="green_intensity_slider" min="0" max="255" step="1" value="255"><br />
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<script src="http://dworzynski.eu/visualization_logic.js"></script><br />
<script src="http://dworzynski.eu/visualization_main.js"></script><br />
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</html><br />
<br />
== Conclusions ==<br />
Biobrick [[Team:DTU-Denmark/Parts|BBa_K1067009]] successfully directs GFP SF to the periplasm of ''E. coli''. This experiment is a proof of concept that proteins can be exported to the periplasm. There have in previous iGEM project been a confusing whether or not it was possible to export GFP to the periplasm; this experiment verifies just that and gives a procedure on how to reproduce this. <br />
<br />
<br />
==References== <br />
<br />
[1] 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 />
<br />
[2] 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 />
<br />
[3] 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 />
<br />
[4] 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 />
<br />
[5] 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 />
<br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/AttributionsTeam:DTU-Denmark/Attributions2013-10-04T15:29:49Z<p>Ariadni: /* 30px Malgorzata Futyma */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|Attributions}}<br />
<br />
== Work done by the team ==<br />
<hr/><br />
===== [[File:Poland_Flag.gif|30px]] Katarzyna Chyzynska =====<br />
* Performed [[Team:DTU-Denmark/Experiments|characterization and verification experiments]]<br />
* Designed our poster<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Canada_Flag.jpg|30px]] Helen Cook =====<br />
* Performed and analyzed data from the [[Team:DTU-Denmark/Experiments|characterization and verification experiments]]<br />
* Created the diagrams describing our project<br />
* Participated in high school outreach, Biobrick workshop and the discussion on IP and synthetic biology.<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Kristian Davidsen =====<br />
* Lead user cloning work in the 208 lab; designed primers<br />
* Constructed the [[Team:DTU-Denmark/pBAD_SPL|pBAD_SPL]]<br />
* Found sponsors and funding<br />
* Starred in our [https://www.youtube.com/watch?v=7EiVttJpXH4 bricks of knowledge video on USER cloning]<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Greece_Flag.jpg|30px]] Ariadni Droumpali =====<br />
* Performed [[Team:DTU-Denmark/Experiments|characterization and verification experiments]]<br />
* Participated in high school outreach, Biobrick workshop and the discussion on IP and synthetic biology.<br />
<br />
===== [[File:Poland_Flag.gif|30px]] Piotr Dworzynski =====<br />
* Simulation of [[Team:DTU-Denmark/HelloWorld|GFP in the periplasm]] for Hello World project<br />
<br />
===== [[File:Poland_Flag.gif|30px]] Malgorzata Futyma =====<br />
* USER cloning and other work in 208 lab<br />
* Drew pictures for our banner and front page slideshow<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Jakob Berg Jespersen =====<br />
* Constructed [[Team:DTU-Denmark/Protein_Models| protein models]]<br />
* Designed our t-shirts and business cards<br />
* Found sponsors and funding<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Germany_Flag.gif|30px]] Vanessa Jurtz =====<br />
* Modeled [[Team:DTU-Denmark/Kinetic_Model|kinetics of our reactions]]<br />
* Modeled a [[Team:DTU-Denmark/Reactor_Model|continuous flow reactor]]<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Greece_Flag.jpg|30px]] Anastasia Mourka =====<br />
* Wiki design and implementation<br />
* Participated in the discussion on IP and synthetic biology.<br />
<br />
===== [[File:Greece_Flag.jpg|30px]] Natalia Papargyri =====<br />
* Responsible for our [[Team:DTU-Denmark/Safety|safety]] form and section<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Italy_Flag.png|30px]] Julia Villarroel =====<br />
* USER cloning in 208 lab<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Germany_Flag.gif|30px]] Henrike Zschach =====<br />
* USER cloning in the 208 lab<br />
* Participated in high school outreach, Biobrick workshop and the discussion on IP and synthetic biology.<br />
<br />
== Assistance provided by our supervisors ==<br />
<hr/><br />
===== [[File:USA_Flag.png|30px]] Chris Workman (PI)=====<br />
* Provided perl and R scripts to analyze raw biolector data<br />
* Provided lab space for us to work in (building 208)<br />
<br />
===== [[File:Flag_of_Belgium.svg.png|30px]] Barth Smets =====<br />
* Provided the idea for our project<br />
* Provided lab space for us to work in (building 115)<br />
<br />
== Assistance provided by our advisors ==<br />
<hr/><br />
===== [[File:Denmark_Flag.gif|30px]] Thomas Trolle =====<br />
* Provided early advice on the design of the pBAD SPL<br />
* Helped during the BioBrick workshop<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Anne Mathilde Lund =====<br />
* Generously provided us with X7 polymerase and assistance with debugging PCR reactions<br />
* Arranged all experiments under the BioBrick workshop <br />
* Great help with primer design for USER cloning as with introduction on how to use the gradient PCR-machine ([https://2013.igem.org/Team:DTU-Denmark/Notebook/13_August_2013 link to notebook])<br />
* Helped in "hard to amplify" PCR situations<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Andreas Porse =====<br />
* Reviewed our initial plan and provided insight into ''E. coli'' specific signal peptides<br />
* Assisted in primer design for USER cloning i the "Hello World project"<br />
* General questions about sequencing, PCR, lab techniques etc.<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Julie Rank =====<br />
* Viewed early versions of our presentation and provided excellent feedback on how we could improve<br />
* Helped during the BioBrick workshop<br />
<br />
===== [[File:Turkey_Flag.png|30px]] Ali Altıntaş =====<br />
* Provided assistance in the 208 lab with general lab technique<br />
* Help with debugging gel pictures and methods <br />
* Kindly gave us filter sterilization units<br />
<br />
===== [[File:Flag_of_France.png|30px]] Sébastien Muller =====<br />
* Provided assistance in the 208 lab with general lab technique<br />
* Helped debugging PCR-reactions<br />
* Kindly gave us loading dye and DNA ladder<br />
<br />
== Special Thanks to ==<br />
<hr/><br />
[http://www.env.dtu.dk/english/Research/RRE/RRE-Staff/Person?id=66486&cpid=86362&tab=2&qt=dtupublicationquery| Gizem Mutlu], PhD student DTU Environmental Engineering for her help with initial calibration of the N<sub>2</sub>O and NO sensors, and for preparing the NO standard solution.<br />
<br />
Tina Johansen, Laboratory Technician DTU Systems biology for her technical help with the bioreactor set up and to [http://www.dtu.dk/Service/Telefonbog/Person?id=7254&cpid=710&tab=2&qt=dtupublicationquery| Jette Thykær], Associate Professor DTU Systems Biology for her assistance scheduling time on the bioreactor.<br />
<br />
Natalia Skawińska, who participated in the high school outreach, and provided advice on cloning in ''E. coli''.<br />
<br />
Morten Nørholm, who developed the X7 polymerase [http://www.biomedcentral.com/1472-6750/10/21].<br />
<br />
[http://www.biotechacademy.dk/biotech%20academy/omos/hjg.aspx| Hans Jasper Genee], who introduced us to [http://www.cbs.dtu.dk/services/PHUSER/ PHUSER] during our BioBrick Workshop.<br />
<br />
[http://www.cbs.dtu.dk/ Center for Biological Sequence Analysis] for letting us use one of their offices and their coffee machine!<br />
<br />
[https://2011.igem.org/Team:DTU-Denmark/How_to_customize_an_iGEM_wiki DTU iGEM 2011 Team] for their Wiki Guide, which helped in the creation of this site.<br />
<br />
[https://2009.igem.org/Team:DTU_Denmark/USERprogram DTU iGEM 2009 Team] for their software, [http://www.cbs.dtu.dk/services/PHUSER/ PHUSER 2.0], that we have used and beta tested.<br />
<br />
[[Team:UNIK_Copenhagen|University of Copenhagen iGEM 2013 Team]] and [[Team:SDU-Denmark|University of Southern Denmark iGEM 2013 Team]] for joining us for the [[Team:DTU-Denmark/Biobrick_Workshop|Biobrick Workshop]] and indian food.<br />
<br />
[[Team:SDU-Denmark|University of Southern Denmark iGEM 2013 Team]] for arranging the DK-Meet-up.<br />
<br />
== Sponsors ==<br />
<hr/><br />
{|border="0" cellspacing="30"<br />
|[[File:DTU otto.jpg|450px|left|link=http://www.ottomoensted.dk/]] ||<br />
Otto Mønsted Fonden<br />
|-<br />
|[[File:DTU_Bruun.png|200px|left|link=http://ottobruunsfond.dk/index.html]] ||<br />
Otto Bruuns Fond<br />
|-<br />
|[[File:DTU_VFL_DK_Logo_singleline_RGB.png|200px|left|link=http://www.vfl.dk/]] ||<br />
Videncenteret for Landbrug<br />
|-<br />
|[[File:DTU Brenntag.jpg|250px|left|link=http://www.brenntag-nordic.com/]] ||<br />
Brenntag<br />
|-<br />
|[[File:DTU_kruger.png|250px|left|link=http://www.kruger.dk/en/]] ||<br />
Krüger<br />
|-<br />
|[[File:DTU Systembiologi.jpg|250px|left|link=http://www.bio.dtu.dk/]] ||<br />
DTU Systems Biology<br />
|-<br />
|[[File:DTU_Environment.jpg|250px|left|link=http://www.env.dtu.dk/]] ||<br />
DTU Environment<br />
|-<br />
|[[File:DTU logo2.jpg|200px|left|link=http://www.dtu.dk/]] ||<br />
Technical University of Denmark<br />
|-<br />
|[[File:DTU clcbio.jpg|200px|left|link=http://www.clcbio.com/]] ||<br />
CLC Bio<br />
|-<br />
|[[File:NEB_Header_iGem.jpg|200px|left|link=http://www.neb-online.de]] ||<br />
New England Biolabs GmbH<br />
|-<br />
|[[File:DTU_KAILOW_UK.png|200px|left|link=http://www.kailow.dk/index_en.html]] ||<br />
Kailow<br />
|-<br />
|}<br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}<br />
<!-- {{:Team:DTU-Denmark/Templates/Footer1}} --></div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/HelloWorldTeam:DTU-Denmark/HelloWorld2013-10-04T15:14:52Z<p>Ariadni: </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 [1].<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. [2].<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, [3]. 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 [4].<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] [5] 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 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 />
== Results ==<br />
We used different methods for testing that our construct actually did work and also for verifying the model we did before the test. <br />
<br />
<br />
===Periplasmic vs. cytoplasmic fraction===<br />
The first test we did was to purify the periplasmic fraction of the cells and then comparing with the cytoplasmic ([https://2013.igem.org/Team:DTU-Denmark/Notebook/6_July_2013 link to notebook]). We did this with the cold osmotic shock method and viewed the fraction under UV-light. <br />
[[File:Sucrose A+D+C.JPG|thumb|center|upright=3|Periplasmic fractions are clear green fluorescent while the pellet with the cytoplasmic fraction is red fluorescent]]<br />
<br />
<br />
===Fluorescent microscope images=== <br />
Next test was the "real thing". We wanted water-tight evidence that GFP SF was actually exported; and what better way to do this than getting pictures of it? After following our procedure for chasing GFP SF to the periplasm, we observed these images after background subtraction. Following is an image with no zoom:<br />
[[File:GFP in perimplasm RFP in cytoplasm.png|thumb|center|upright=3|Transformed ''E. coli'' taken with a Fluorescence microscope]]<br />
<br />
<br />
Next to test our model we picked a cell this good resolution and zoomed in on this:<br />
[[File:GFP in perimplasm RFP in cytoplasm close up.png|thumb|center|upright=3|Close up of several cells, showing GFP expression in the periplasm]]<br />
<br />
<br />
For this cell a linear profile was made that shows both the GFP and RFP signal across the cell:<br />
[[File:Graf.PNG|thumb|center|upright=3|Line profile of the cross section of the cell seen in the above close up]]<br />
These profiles closely resemble our model seen in next section.<br />
<br />
<br />
==Modeling==<br />
<br />
===Modeling Methods===<br />
To assess the ability to distinguish the periplasmic from the cytoplasmic space we made a model which is based on a perfect spherical cell with and even layer between inner and outer membrane. <br />
[[File:Cell model.png|300px|center|Conceptual drawing for the model]]<br />
<br />
To get a value of the RFP and GFP signal we used a cross section of the spherical cell and measured the length of RFP and GFP going across this section; see the measurement bars at the figure. The make a graph of this we set the middle of the cell to be 0 on the x-axis. On the y-axis we let two lines show the value of GFP and RFP in the cross section.<br />
<br />
The first graph can be seen below:<br />
[[File:First model.png|thumb|center|upright=2|The first model was done in Maple.]]<br />
<br />
<br />
===Model of "Hello World" construct===<br />
With the mathematical equations made for making the graph above we made a interactive model that enables the user to set their own settings with regard to GFP and RFP intensity and cytoplasmic vs. periplasmic fraction. If the parameters are set well the model reassembles the images we acquired with the fluorescence microscope. <br />
<br />
<html><br />
<br />
<br />
<script src="http://ajax.aspnetcdn.com/ajax/modernizr/modernizr-2.0.6-development-only.js"></script><br />
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<br />
<div id="visualization_complete" class="display:none"><br />
<br />
<table style="border: 0px"><br />
<tr><br />
<td><br />
Cell membrane size:<br />
</td><br />
<td><br />
<input type="range" name="inner_cell_prop_slider" id="inner_cell_prop_slider" min="0" max="1" step=".01" value="0.5"><br />
</td><br />
<td id="inner_cell_prop_num"><br />
</td><br />
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<tr><br />
<td><br />
Red intesity:<br />
</td><br />
<td><br />
<input type="range" name="red_intensity_slider" id="red_intensity_slider" min="0" max="255" step="1" value="255"><br />
</td><br />
<td id="red_intensity_num"><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
Green intensity:<br />
</td><br />
<td><br />
<input type="range" name="green_intensity_slider" id="green_intensity_slider" min="0" max="255" step="1" value="255"><br />
</td><br />
<td id="green_intensity_num"><br />
</td><br />
</tr><br />
</table><br />
<br />
<div id="visualization"><br />
</div><br />
<div id="cell_image"><br />
</div><br />
</div><br />
<!--End of visualization_complete --><br />
<br />
<br />
<script src="http://cdnjs.cloudflare.com/ajax/libs/d3/3.2.2/d3.v3.min.js" charset="utf-8"></script><br />
<br />
<script src="http://dworzynski.eu/visualization_logic.js"></script><br />
<script src="http://dworzynski.eu/visualization_main.js"></script><br />
<br />
<br />
</html><br />
<br />
== Conclusions ==<br />
Biobrick [[Team:DTU-Denmark/Parts|BBa_K1067009]] successfully directs GFP SF to the periplasm of ''E. coli''. This experiment is a proof of concept that proteins can be exported to the periplasm. There have in previous iGEM project been a confusing whether or not it was possible to export GFP to the periplasm; this experiment verifies just that and gives a procedure on how to reproduce this. <br />
<br />
<br />
==References== <br />
<br />
[1] 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 />
<br />
[2] 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 />
<br />
[3] 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 />
<br />
[4] 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 />
<br />
[5] 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 />
<br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/HelloWorldTeam:DTU-Denmark/HelloWorld2013-10-04T15:13:59Z<p>Ariadni: /* Methods */</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 [1].<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. [2].<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, [3]. 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 [4].<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] [5] 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 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 />
== Results ==<br />
We used different methods for testing that our construct actually did work and also for verifying the model we did before the test. <br />
<br />
<br />
===Periplasmic vs. cytoplasmic fraction===<br />
The first test we did was to purify the periplasmic fraction of the cells and then comparing with the cytoplasmic ([https://2013.igem.org/Team:DTU-Denmark/Notebook/6_July_2013 link to notebook]). We did this with the cold osmotic shock method and viewed the fraction under UV-light. <br />
[[File:Sucrose A+D+C.JPG|thumb|center|upright=3|Periplasmic fractions are clear green fluorescent while the pellet with the cytoplasmic fraction is red fluorescent]]<br />
<br />
<br />
===Fluorescent microscope images=== <br />
Next test was the "real thing". We wanted water-tight evidence that GFP SF was actually exported; and what better way to do this than getting pictures of it? After following our procedure for chasing GFP SF to the periplasm, we observed these images after background subtraction. Following is an image with no zoom:<br />
[[File:GFP in perimplasm RFP in cytoplasm.png|thumb|center|upright=3|Transformed ''E. coli'' taken with a Fluorescence microscope]]<br />
<br />
<br />
Next to test our model we picked a cell this good resolution and zoomed in on this:<br />
[[File:GFP in perimplasm RFP in cytoplasm close up.png|thumb|center|upright=3|Close up of several cells, showing GFP expression in the periplasm]]<br />
<br />
<br />
For this cell a linear profile was made that shows both the GFP and RFP signal across the cell:<br />
[[File:Graf.PNG|thumb|center|upright=3|Line profile of the cross section of the cell seen in the above close up]]<br />
These profiles closely resemble our model seen in next section.<br />
<br />
<br />
==Modeling==<br />
<br />
===Modeling Methods===<br />
To assess the ability to distinguish the periplasmic from the cytoplasmic space we made a model which is based on a perfect spherical cell with and even layer between inner and outer membrane. <br />
[[File:Cell model.png|300px|center|Conceptual drawing for the model]]<br />
<br />
To get a value of the RFP and GFP signal we used a cross section of the spherical cell and measured the length of RFP and GFP going across this section; see the measurement bars at the figure. The make a graph of this we set the middle of the cell to be 0 on the x-axis. On the y-axis we let two lines show the value of GFP and RFP in the cross section.<br />
<br />
The first graph can be seen below:<br />
[[File:First model.png|thumb|center|upright=2|The first model was done in Maple.]]<br />
<br />
<br />
===Model of "Hello World" construct===<br />
With the mathematical equations made for making the graph above we made a interactive model that enables the user to set their own settings with regard to GFP and RFP intensity and cytoplasmic vs. periplasmic fraction. If the parameters are set well the model reassembles the images we acquired with the fluorescence microscope. <br />
<br />
<html><br />
<br />
<br />
<script src="http://ajax.aspnetcdn.com/ajax/modernizr/modernizr-2.0.6-development-only.js"></script><br />
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<p><b>And please come back! It's worth it!</b></p><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div id="visualization_complete" class="display:none"><br />
<br />
<table style="border: 0px"><br />
<tr><br />
<td><br />
Cell membrane size:<br />
</td><br />
<td><br />
<input type="range" name="inner_cell_prop_slider" id="inner_cell_prop_slider" min="0" max="1" step=".01" value="0.5"><br />
</td><br />
<td id="inner_cell_prop_num"><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
Red intesity:<br />
</td><br />
<td><br />
<input type="range" name="red_intensity_slider" id="red_intensity_slider" min="0" max="255" step="1" value="255"><br />
</td><br />
<td id="red_intensity_num"><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
Green intensity:<br />
</td><br />
<td><br />
<input type="range" name="green_intensity_slider" id="green_intensity_slider" min="0" max="255" step="1" value="255"><br />
</td><br />
<td id="green_intensity_num"><br />
</td><br />
</tr><br />
</table><br />
<br />
<div id="visualization"><br />
</div><br />
<div id="cell_image"><br />
</div><br />
</div><br />
<!--End of visualization_complete --><br />
<br />
<br />
<script src="http://cdnjs.cloudflare.com/ajax/libs/d3/3.2.2/d3.v3.min.js" charset="utf-8"></script><br />
<br />
<script src="http://dworzynski.eu/visualization_logic.js"></script><br />
<script src="http://dworzynski.eu/visualization_main.js"></script><br />
<br />
<br />
</html><br />
<br />
== Conclusions ==<br />
Biobrick [[Team:DTU-Denmark/Parts|BBa_K1067009]] successfully directs GFP SF to the periplasm of ''E. coli''. This experiment is a proof of concept that proteins can be exported to the periplasm. There have in previous iGEM project been a confusing whether or not it was possible to export GFP to the periplasm; this experiment verifies just that and gives a procedure on how to reproduce this. <br />
<br />
<br />
<br />
<br />
==References== <br />
<br />
[1] 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 />
<br />
[2] 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 />
<br />
[3] 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 />
<br />
[4] 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 />
<br />
[5] 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 />
<br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/HelloWorldTeam:DTU-Denmark/HelloWorld2013-10-04T15:13:38Z<p>Ariadni: /* References */</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 [1].<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. [2].<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 />
<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 />
<br />
== Results ==<br />
We used different methods for testing that our construct actually did work and also for verifying the model we did before the test. <br />
<br />
<br />
===Periplasmic vs. cytoplasmic fraction===<br />
The first test we did was to purify the periplasmic fraction of the cells and then comparing with the cytoplasmic ([https://2013.igem.org/Team:DTU-Denmark/Notebook/6_July_2013 link to notebook]). We did this with the cold osmotic shock method and viewed the fraction under UV-light. <br />
[[File:Sucrose A+D+C.JPG|thumb|center|upright=3|Periplasmic fractions are clear green fluorescent while the pellet with the cytoplasmic fraction is red fluorescent]]<br />
<br />
<br />
===Fluorescent microscope images=== <br />
Next test was the "real thing". We wanted water-tight evidence that GFP SF was actually exported; and what better way to do this than getting pictures of it? After following our procedure for chasing GFP SF to the periplasm, we observed these images after background subtraction. Following is an image with no zoom:<br />
[[File:GFP in perimplasm RFP in cytoplasm.png|thumb|center|upright=3|Transformed ''E. coli'' taken with a Fluorescence microscope]]<br />
<br />
<br />
Next to test our model we picked a cell this good resolution and zoomed in on this:<br />
[[File:GFP in perimplasm RFP in cytoplasm close up.png|thumb|center|upright=3|Close up of several cells, showing GFP expression in the periplasm]]<br />
<br />
<br />
For this cell a linear profile was made that shows both the GFP and RFP signal across the cell:<br />
[[File:Graf.PNG|thumb|center|upright=3|Line profile of the cross section of the cell seen in the above close up]]<br />
These profiles closely resemble our model seen in next section.<br />
<br />
<br />
==Modeling==<br />
<br />
===Modeling Methods===<br />
To assess the ability to distinguish the periplasmic from the cytoplasmic space we made a model which is based on a perfect spherical cell with and even layer between inner and outer membrane. <br />
[[File:Cell model.png|300px|center|Conceptual drawing for the model]]<br />
<br />
To get a value of the RFP and GFP signal we used a cross section of the spherical cell and measured the length of RFP and GFP going across this section; see the measurement bars at the figure. The make a graph of this we set the middle of the cell to be 0 on the x-axis. On the y-axis we let two lines show the value of GFP and RFP in the cross section.<br />
<br />
The first graph can be seen below:<br />
[[File:First model.png|thumb|center|upright=2|The first model was done in Maple.]]<br />
<br />
<br />
===Model of "Hello World" construct===<br />
With the mathematical equations made for making the graph above we made a interactive model that enables the user to set their own settings with regard to GFP and RFP intensity and cytoplasmic vs. periplasmic fraction. If the parameters are set well the model reassembles the images we acquired with the fluorescence microscope. <br />
<br />
<html><br />
<br />
<br />
<script src="http://ajax.aspnetcdn.com/ajax/modernizr/modernizr-2.0.6-development-only.js"></script><br />
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Green intensity:<br />
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<br />
== Conclusions ==<br />
Biobrick [[Team:DTU-Denmark/Parts|BBa_K1067009]] successfully directs GFP SF to the periplasm of ''E. coli''. This experiment is a proof of concept that proteins can be exported to the periplasm. There have in previous iGEM project been a confusing whether or not it was possible to export GFP to the periplasm; this experiment verifies just that and gives a procedure on how to reproduce this. <br />
<br />
<br />
<br />
<br />
==References== <br />
<br />
[1] 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 />
<br />
[2] 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 />
<br />
[3] 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 />
<br />
[4] 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 />
<br />
[5] 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 />
<br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/HelloWorldTeam:DTU-Denmark/HelloWorld2013-10-04T15:06:41Z<p>Ariadni: /* Introduction */</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 [1].<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. [2].<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 />
<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 />
<br />
== Results ==<br />
We used different methods for testing that our construct actually did work and also for verifying the model we did before the test. <br />
<br />
<br />
===Periplasmic vs. cytoplasmic fraction===<br />
The first test we did was to purify the periplasmic fraction of the cells and then comparing with the cytoplasmic ([https://2013.igem.org/Team:DTU-Denmark/Notebook/6_July_2013 link to notebook]). We did this with the cold osmotic shock method and viewed the fraction under UV-light. <br />
[[File:Sucrose A+D+C.JPG|thumb|center|upright=3|Periplasmic fractions are clear green fluorescent while the pellet with the cytoplasmic fraction is red fluorescent]]<br />
<br />
<br />
===Fluorescent microscope images=== <br />
Next test was the "real thing". We wanted water-tight evidence that GFP SF was actually exported; and what better way to do this than getting pictures of it? After following our procedure for chasing GFP SF to the periplasm, we observed these images after background subtraction. Following is an image with no zoom:<br />
[[File:GFP in perimplasm RFP in cytoplasm.png|thumb|center|upright=3|Transformed ''E. coli'' taken with a Fluorescence microscope]]<br />
<br />
<br />
Next to test our model we picked a cell this good resolution and zoomed in on this:<br />
[[File:GFP in perimplasm RFP in cytoplasm close up.png|thumb|center|upright=3|Close up of several cells, showing GFP expression in the periplasm]]<br />
<br />
<br />
For this cell a linear profile was made that shows both the GFP and RFP signal across the cell:<br />
[[File:Graf.PNG|thumb|center|upright=3|Line profile of the cross section of the cell seen in the above close up]]<br />
These profiles closely resemble our model seen in next section.<br />
<br />
<br />
==Modeling==<br />
<br />
===Modeling Methods===<br />
To assess the ability to distinguish the periplasmic from the cytoplasmic space we made a model which is based on a perfect spherical cell with and even layer between inner and outer membrane. <br />
[[File:Cell model.png|300px|center|Conceptual drawing for the model]]<br />
<br />
To get a value of the RFP and GFP signal we used a cross section of the spherical cell and measured the length of RFP and GFP going across this section; see the measurement bars at the figure. The make a graph of this we set the middle of the cell to be 0 on the x-axis. On the y-axis we let two lines show the value of GFP and RFP in the cross section.<br />
<br />
The first graph can be seen below:<br />
[[File:First model.png|thumb|center|upright=2|The first model was done in Maple.]]<br />
<br />
<br />
===Model of "Hello World" construct===<br />
With the mathematical equations made for making the graph above we made a interactive model that enables the user to set their own settings with regard to GFP and RFP intensity and cytoplasmic vs. periplasmic fraction. If the parameters are set well the model reassembles the images we acquired with the fluorescence microscope. <br />
<br />
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<br />
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<br />
== Conclusions ==<br />
Biobrick [[Team:DTU-Denmark/Parts|BBa_K1067009]] successfully directs GFP SF to the periplasm of ''E. coli''. This experiment is a proof of concept that proteins can be exported to the periplasm. There have in previous iGEM project been a confusing whether or not it was possible to export GFP to the periplasm; this experiment verifies just that and gives a procedure on how to reproduce this. <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 />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/ToxicityExperimentTeam:DTU-Denmark/ToxicityExperiment2013-10-04T15:04:12Z<p>Ariadni: /* References */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|Toxicity Experiment}}<br />
<br />
== Description ==<br />
<br />
Determine whether the ions nitrite, nitrate and ammonium are toxic to ''E. coli'', and at what concentration. This experiment will use the Biolector to grow an ''E. coli'' strain that constitutively expresses RFP in the presence of these ions (at a range of concentrations) to determine the OD and fluorescence of the cells as a proxy for growth.<br />
<br />
== Results ==<br />
<br />
The graphs in the following sections show the response in biomass and fluorescence of GFP over time for varying concentrations of each ion. The data has been baseline adjusted by using a script from Chris Workman (unpublished). The horizontal facets show concentrations in mM.<br />
<br />
==== Ammonium ====<br />
<br />
[[File:DTU-Denmark-Toxicity-Ammonium-parsed.png|600px]]<br />
<br />
==== Nitrate ====<br />
<br />
[[File:DTU-Denmark-Toxicity-Nitrate-parsed.png|600px]]<br />
<br />
==== Nitrite ====<br />
<br />
[[File:DTU-Denmark-Toxicity-Nitrite-parsed.png|600px]]<br />
<br />
== Conclusions ==<br />
<br />
Very high concentrations of ammonium ≥500 mM were able to inhibit growth of ''E. coli''. Nitrate at concentrations of 25mM and greater also inhibits growth of ''E. coli'', whereas concentrations of nitrate at 5mM and greater show delayed expression of GFP and slower growth. The tested concentrations of nitrite were not sufficient to inhibit growth.<br />
<br />
== Discussion ==<br />
<br />
The average concentration of ammonium in wastewater treatment plants is 70 mg/L [1] which is much less than 500mM that we found to be toxic to ''E. coli''. If we assume the ammonia is stoichiometrically converted to nitrite via our mutants, the concentrations of nitrite that will be produced with this volume of ammonium will also not be toxic to the cell. We do not expect that a significant amount of nitrate will be produced during this process, since ''E. coli'' contains the Nap pathway that converts nitrate to nitrite. Therefore, we can expect that our transformants will survive in this environment.<br />
<br />
==References== <br />
[1] Sotirakou, E. ,Kladitis, G., Diamantis, N., Grigoropoulou, H.; Ammonia and Phosphorous Removal in Municipal Wastewater Treatment Plant With Extended Aeration. "Global Nest", Sept. 1999, Vol. 1, No 1, pp 47-53.<br />
<br />
<br />
<div style="clear: both;"></div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/ToxicityExperimentTeam:DTU-Denmark/ToxicityExperiment2013-10-04T15:03:48Z<p>Ariadni: /* Discussion */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|Toxicity Experiment}}<br />
<br />
== Description ==<br />
<br />
Determine whether the ions nitrite, nitrate and ammonium are toxic to ''E. coli'', and at what concentration. This experiment will use the Biolector to grow an ''E. coli'' strain that constitutively expresses RFP in the presence of these ions (at a range of concentrations) to determine the OD and fluorescence of the cells as a proxy for growth.<br />
<br />
== Results ==<br />
<br />
The graphs in the following sections show the response in biomass and fluorescence of GFP over time for varying concentrations of each ion. The data has been baseline adjusted by using a script from Chris Workman (unpublished). The horizontal facets show concentrations in mM.<br />
<br />
==== Ammonium ====<br />
<br />
[[File:DTU-Denmark-Toxicity-Ammonium-parsed.png|600px]]<br />
<br />
==== Nitrate ====<br />
<br />
[[File:DTU-Denmark-Toxicity-Nitrate-parsed.png|600px]]<br />
<br />
==== Nitrite ====<br />
<br />
[[File:DTU-Denmark-Toxicity-Nitrite-parsed.png|600px]]<br />
<br />
== Conclusions ==<br />
<br />
Very high concentrations of ammonium ≥500 mM were able to inhibit growth of ''E. coli''. Nitrate at concentrations of 25mM and greater also inhibits growth of ''E. coli'', whereas concentrations of nitrate at 5mM and greater show delayed expression of GFP and slower growth. The tested concentrations of nitrite were not sufficient to inhibit growth.<br />
<br />
== Discussion ==<br />
<br />
The average concentration of ammonium in wastewater treatment plants is 70 mg/L [1] which is much less than 500mM that we found to be toxic to ''E. coli''. If we assume the ammonia is stoichiometrically converted to nitrite via our mutants, the concentrations of nitrite that will be produced with this volume of ammonium will also not be toxic to the cell. We do not expect that a significant amount of nitrate will be produced during this process, since ''E. coli'' contains the Nap pathway that converts nitrate to nitrite. Therefore, we can expect that our transformants will survive in this environment.<br />
<br />
==References== <br />
<html><br />
<br />
<ul><br />
<li>Sotirakou, E. ,Kladitis, G., Diamantis, N., Grigoropoulou, H.; Ammonia and Phosphorous Removal in Municipal Wastewater Treatment Plant With Extended Aeration. "Global Nest", Sept. 1999, Vol. 1, No 1, pp 47-53.<br />
<br />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/pBAD_SPLTeam:DTU-Denmark/pBAD SPL2013-10-04T15:01:55Z<p>Ariadni: /* References */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|pBAD SPL}}<br />
<br />
== pBAD synthetic promoter library ==<br />
A synthetic promoter library (SPL) is a library of cells each having one promoter sequence that differs between them. This promoter is always the same and is usually upstream a fluorescent protein like GFP/RFP. The term was first coined in 1998 and used in ''Lactococcus lactis'' [1]. The method was adapted by [https://2010.igem.org/Team:DTU-Denmark/SPL 2010 DTU iGEM team] to make a SPL for ''E.coli'' that enabled the modulation of constitutive gene expression with great precision. They even made a new standard for this method with the use of biobricks ([http://dspace.mit.edu/handle/1721.1/60080 RFC 63]).<br />
<br />
We used the method to build a non-leaky arabinose inducible promoter as a tool for expressing lethal proteins in ''E. coli''. The reason was that many of the proteins we are working with are membrane bound or integral membrane proteins and will be lethal if expressed in to high quantities. <br />
<br />
For testing our pathways we needed to grow the transformed cell to a certain concentration and growth with the gene constitutively expressed was not working very well. We needed a inducible system but the standard arabinose inducible system was way too leaky and we got same stood with the same problem as before. Therefore we needed to either build or buy (if an possible) an inducible system with great tightness able to be induced easily. We choose to build such system.<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 />
# After inspection under UV-light the visually non RFP containing colonies where picked. <br />
# Plates were induced by spraying them with an 5% w/v aqueous arabinose solution.<br />
# The plates were again inspected under UV-light and this time the most red florescent cells were picked. <br />
# Colonies were grown in culture tubes and screened in parallel on BioLector. Wells on BioLector plate were loaded with culture by transferring a toothpick from each overnight culture selected and into the wells of the plate. 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 pBAD system [http://parts.igem.org/Part:BBa_K808000 BBa_K808000] was used as a reference.<br />
<br />
[[File:AraSPL primer.png|thumb|upright=3.5|center|This reverse primer sequence is incorporating randomized promoter sequences into the pBAD construct. The sequence is annotated with -10 and -35 consensus region. Note that I2-bindingsite is overlapping with the -35 region. Blue is the binding part of the primer, red is the USER made sticky ends. N=random, W=50% A and 50% T]]<br />
<br />
<br />
=== Data analysis ===<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.<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 />
<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. The induced activity of the arabinose reference promoter is shown as a band representing two replicates.<br />
<br />
[[File:Induced_vs_basal.png|600px]]<br />
<br />
=== Details ===<br />
<br />
Promoter strengths for two trials of each colony with and without arabinose. <br />
<br />
{|<br />
!Colony Number<br />
!With Arabinose 1<br />
!With Arabinose 2<br />
!Without Arabinose 1<br />
!Without Arabinose 2<br />
|-<br />
|Col2||14.4033||14.0052||0.2194||0.1869<br />
|-<br />
|Col3||16.2046||16.9829||0.4143||0.4376<br />
|-<br />
|Col4||13.7729||14.5287||0.3348||0.3548<br />
|-<br />
|Col5||17.2641||18.1032||0.4384||0.4111<br />
|-<br />
|Col12||13.0562||14.2424||0.3908||0.4378<br />
|-<br />
|Col10||17.4996||18.4075||0.4473||0.4774<br />
|-<br />
|Col9||15.1088||17.1372||0.5082||0.5523<br />
|-<br />
|Col8||13.2387||13.4653||0.2094||0.2121<br />
|-<br />
|Col13||10.8144||10.9013||0.1002||0.091<br />
|-<br />
|Col15||16.3058||14.6369||0.2307||0.2397<br />
|-<br />
|Col18||19.7533||19.8389||0.5438||0.5039<br />
|-<br />
|Col19||17.4378||18.422||0.3458||0.2838<br />
|-<br />
|Col31||3.6692||3.9245||0.1479||0.1741<br />
|-<br />
|Col29||13.6443||16.0181||0.3114||0.2914<br />
|-<br />
|Col26||13.8313||13.7574||0.2648||0.3195<br />
|-<br />
|Col22||17.5911||16.0539||0.4667||0.4657<br />
|-<br />
|Col33||14.4961||14.3026||0.8441||1.1722<br />
|-<br />
|Col34||12.6946||12.0078||0.1854||0.1894<br />
|-<br />
|Col35||18.9636||17.8541||0.8889||0.8904<br />
|-<br />
|ConRef||7.7632||7.9323||7.8893||7.9323<br />
|}<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-col5.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-col31.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 />
== Sequences ==<br />
<br />
We sequenced and aligned the promoters that are shown above. The sequences show conservation in the -10 and -35 regions by design. Within the -10 region, we allowed for two random weak (A or T) bases. We do not see a strong preference for A over T or vice versa. Within the regions where we allowed any base, we see a preference for C over the other bases. This could be due to bias during synthesis. <br />
<br />
[[File:dtu-spl-align.png]]<br />
<br />
<br />
== Example of use ==<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 close up.png|300px|thumbnail|upright=2|left|alt=Alt text|Zoomed in picture of "Hello World" transformants, showing a clear separation 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 fluorescence microscope and subjected to background subtraction]]<br />
[[File:Graf.PNG|290px|thumbnail|upright=2|left|alt=Alt text|Line profile through the cell. The profile shows both the green and red channel. It can be seen that the intensity of the red fluorescence is restricted to the cytoplasm while green fluorescence has it's peaks on the edges. A weak green signal is measured for the cytoplasmic region caused by the periplasm enveloping the cytoplasm.]]<br />
<br />
<br />
<br />
See also [https://2013.igem.org/Team:DTU-Denmark/HelloWorld "Hello World project"].<br />
<br />
<br />
==References== <br />
<br />
[1] Jensen, Peter Ruhdal, and Karin Hammer. "The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters." Applied and environmental microbiology 64.1 (1998): 82-87.<br />
<br />
<br />
<div style="clear: both;"></div><br />
<br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/pBAD_SPLTeam:DTU-Denmark/pBAD SPL2013-10-04T15:01:23Z<p>Ariadni: /* pBAD synthetic promoter library */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|pBAD SPL}}<br />
<br />
== pBAD synthetic promoter library ==<br />
A synthetic promoter library (SPL) is a library of cells each having one promoter sequence that differs between them. This promoter is always the same and is usually upstream a fluorescent protein like GFP/RFP. The term was first coined in 1998 and used in ''Lactococcus lactis'' [1]. The method was adapted by [https://2010.igem.org/Team:DTU-Denmark/SPL 2010 DTU iGEM team] to make a SPL for ''E.coli'' that enabled the modulation of constitutive gene expression with great precision. They even made a new standard for this method with the use of biobricks ([http://dspace.mit.edu/handle/1721.1/60080 RFC 63]).<br />
<br />
We used the method to build a non-leaky arabinose inducible promoter as a tool for expressing lethal proteins in ''E. coli''. The reason was that many of the proteins we are working with are membrane bound or integral membrane proteins and will be lethal if expressed in to high quantities. <br />
<br />
For testing our pathways we needed to grow the transformed cell to a certain concentration and growth with the gene constitutively expressed was not working very well. We needed a inducible system but the standard arabinose inducible system was way too leaky and we got same stood with the same problem as before. Therefore we needed to either build or buy (if an possible) an inducible system with great tightness able to be induced easily. We choose to build such system.<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 />
# After inspection under UV-light the visually non RFP containing colonies where picked. <br />
# Plates were induced by spraying them with an 5% w/v aqueous arabinose solution.<br />
# The plates were again inspected under UV-light and this time the most red florescent cells were picked. <br />
# Colonies were grown in culture tubes and screened in parallel on BioLector. Wells on BioLector plate were loaded with culture by transferring a toothpick from each overnight culture selected and into the wells of the plate. 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 pBAD system [http://parts.igem.org/Part:BBa_K808000 BBa_K808000] was used as a reference.<br />
<br />
[[File:AraSPL primer.png|thumb|upright=3.5|center|This reverse primer sequence is incorporating randomized promoter sequences into the pBAD construct. The sequence is annotated with -10 and -35 consensus region. Note that I2-bindingsite is overlapping with the -35 region. Blue is the binding part of the primer, red is the USER made sticky ends. N=random, W=50% A and 50% T]]<br />
<br />
<br />
=== Data analysis ===<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.<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 />
<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. The induced activity of the arabinose reference promoter is shown as a band representing two replicates.<br />
<br />
[[File:Induced_vs_basal.png|600px]]<br />
<br />
=== Details ===<br />
<br />
Promoter strengths for two trials of each colony with and without arabinose. <br />
<br />
{|<br />
!Colony Number<br />
!With Arabinose 1<br />
!With Arabinose 2<br />
!Without Arabinose 1<br />
!Without Arabinose 2<br />
|-<br />
|Col2||14.4033||14.0052||0.2194||0.1869<br />
|-<br />
|Col3||16.2046||16.9829||0.4143||0.4376<br />
|-<br />
|Col4||13.7729||14.5287||0.3348||0.3548<br />
|-<br />
|Col5||17.2641||18.1032||0.4384||0.4111<br />
|-<br />
|Col12||13.0562||14.2424||0.3908||0.4378<br />
|-<br />
|Col10||17.4996||18.4075||0.4473||0.4774<br />
|-<br />
|Col9||15.1088||17.1372||0.5082||0.5523<br />
|-<br />
|Col8||13.2387||13.4653||0.2094||0.2121<br />
|-<br />
|Col13||10.8144||10.9013||0.1002||0.091<br />
|-<br />
|Col15||16.3058||14.6369||0.2307||0.2397<br />
|-<br />
|Col18||19.7533||19.8389||0.5438||0.5039<br />
|-<br />
|Col19||17.4378||18.422||0.3458||0.2838<br />
|-<br />
|Col31||3.6692||3.9245||0.1479||0.1741<br />
|-<br />
|Col29||13.6443||16.0181||0.3114||0.2914<br />
|-<br />
|Col26||13.8313||13.7574||0.2648||0.3195<br />
|-<br />
|Col22||17.5911||16.0539||0.4667||0.4657<br />
|-<br />
|Col33||14.4961||14.3026||0.8441||1.1722<br />
|-<br />
|Col34||12.6946||12.0078||0.1854||0.1894<br />
|-<br />
|Col35||18.9636||17.8541||0.8889||0.8904<br />
|-<br />
|ConRef||7.7632||7.9323||7.8893||7.9323<br />
|}<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-col5.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-col31.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 />
== Sequences ==<br />
<br />
We sequenced and aligned the promoters that are shown above. The sequences show conservation in the -10 and -35 regions by design. Within the -10 region, we allowed for two random weak (A or T) bases. We do not see a strong preference for A over T or vice versa. Within the regions where we allowed any base, we see a preference for C over the other bases. This could be due to bias during synthesis. <br />
<br />
[[File:dtu-spl-align.png]]<br />
<br />
<br />
== Example of use ==<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 close up.png|300px|thumbnail|upright=2|left|alt=Alt text|Zoomed in picture of "Hello World" transformants, showing a clear separation 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 fluorescence microscope and subjected to background subtraction]]<br />
[[File:Graf.PNG|290px|thumbnail|upright=2|left|alt=Alt text|Line profile through the cell. The profile shows both the green and red channel. It can be seen that the intensity of the red fluorescence is restricted to the cytoplasm while green fluorescence has it's peaks on the edges. A weak green signal is measured for the cytoplasmic region caused by the periplasm enveloping the cytoplasm.]]<br />
<br />
<br />
<br />
See also [https://2013.igem.org/Team:DTU-Denmark/HelloWorld "Hello World project"].<br />
<br />
<br />
==References== <br />
<html><br />
<br />
<ul><br />
<li>Jensen, Peter Ruhdal, and Karin Hammer. "The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters." Applied and environmental microbiology 64.1 (1998): 82-87.<br />
<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 />
<br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-04T14:59:06Z<p>Ariadni: /* References */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
===What?===<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
===Why?===<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 [1]. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
In Europe the annual surplus of fixed nitrogen from crop production and livestock farming is 10 Tg (10 Teragrams = a million tonnes), 6 Tg leaches into ground and surface water [2]. If that was to be converted into N<sub>2</sub>O, it could be decomposed for 4.87 Billion kW h or equivalent to the annual power consumption of 36,000 Avarage households.<br />
<br />
===How?===<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 [3]. We also provide [https://2013.igem.org/Team:DTU-Denmark/Bioreactor 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 />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
[[File:Dtu-Mutant1.png|right]]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><br />
<br><br />
<br />
=== Mutant 2: Anaerobic ===<br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
[[File:dtu-Mutant2.png|right]]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, but we were able to [[Team:DTU-Denmark/Experiment4|verify that the AMO containing plasmid is working as designed]].<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 />
<br />
== USER Cloning ==<br />
To assemble our plasmids we employ uracil-specific excision reagent (USER) cloning [4]. 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 specially designed set of primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
## We are using the non-commercial polymerase X7, designed by Morten Nørholm [5].<br />
# Optional - If the method was used to insert genes into a backbone, with it's own resistance marker, the PCR product were treated with DpnI to digest the template DNA from the PCR reaction.<br />
# USER-reaction: Digesting the linearized PCR fragment with Uracil-Specific Excision Reagent (USER) enzyme removes the uracil-insertions. This will produce the required overhangs with the sequence and the length of your wish.<br />
# Transformation: The mix is immediately transformed into competent ''E. coli cells''. The plasmid assembles itself by base pairing between the complementary overhangs and ligation is done by ''E.coli'' itself.<br />
*You only need one enzyme<br />
*You don't need any ligation reaction<br />
*Seamless assembly<br />
*Directional <br />
*Multiple fragment in one reaction - also in eukaryotes [6].<br />
*[https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play]<br />
<br />
[[File:Plug 'n' Play assembly samlet.png|thumb|upright=3|center|The [https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play] concept. Figure taken from DTU-2 iGEM team 2011]]<br />
<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 />
<br />
[1] Sutton, Mark A., et al. "Too much of a good thing." Nature 472.7342 (2011): 159-161.<br />
<br />
[2] Sutton, Mark A., Clare M. Howard, and Jan Willem Erisman, eds. The European nitrogen assessment: sources, effects and policy perspectives. Cambridge University Press, 2011.<br />
<br />
[3] Yaniv D. Scherson, George F. Wells, Sung-Geun Woo, Jangho Lee, Joonhong Park, Brian J. Cantwell and Craig S. Criddle; Nitrogen removal with energy recovery through N<sub>2</sub>O decomposition; Energy & Environmental Science. Issue 1 2013.<br />
<br />
[4] Nour-Eldin, HH et al (2010). USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories. Methods Mol Biol. 2010;643:185-200. doi: 10.1007/978-1-60761-723-5_13.<br />
<br />
[5] Nørholm, Morten HH. "A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering." BMC biotechnology 10.1 (2010): 21.<br />
<br />
[6] Frandsen, Rasmus, et al. "Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi." BMC molecular biology 9.1 (2008): 70.<br />
<br />
<br />
U.S. Energy Information Administration, Frequently Asked Questions: How much electricity does an American home use?<br />
<br />
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).<br />
<br />
Kanehisa, M. and Goto, S.; KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27-30 (2000).<br />
<br />
Zumft, Walter G. (1997) Cell Biology and Molecular Basis of Denitrification. Microbiology and Molecular Biology Reviews, December 1997 p. 533–616<br />
<br />
Ingledew, WJ and Poole, RK (1984) The Respiratory Chains of Escherichia coli. Microbiological Reviews, Sept. 1984, p. 222-271<br />
<br />
<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-04T14:53:14Z<p>Ariadni: /* USER Cloning */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
===What?===<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
===Why?===<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 [1]. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
In Europe the annual surplus of fixed nitrogen from crop production and livestock farming is 10 Tg (10 Teragrams = a million tonnes), 6 Tg leaches into ground and surface water [2]. If that was to be converted into N<sub>2</sub>O, it could be decomposed for 4.87 Billion kW h or equivalent to the annual power consumption of 36,000 Avarage households.<br />
<br />
===How?===<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 [3]. We also provide [https://2013.igem.org/Team:DTU-Denmark/Bioreactor 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 />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
[[File:Dtu-Mutant1.png|right]]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><br />
<br><br />
<br />
=== Mutant 2: Anaerobic ===<br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
[[File:dtu-Mutant2.png|right]]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, but we were able to [[Team:DTU-Denmark/Experiment4|verify that the AMO containing plasmid is working as designed]].<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 />
<br />
== USER Cloning ==<br />
To assemble our plasmids we employ uracil-specific excision reagent (USER) cloning [4]. 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 specially designed set of primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
## We are using the non-commercial polymerase X7, designed by Morten Nørholm [5].<br />
# Optional - If the method was used to insert genes into a backbone, with it's own resistance marker, the PCR product were treated with DpnI to digest the template DNA from the PCR reaction.<br />
# USER-reaction: Digesting the linearized PCR fragment with Uracil-Specific Excision Reagent (USER) enzyme removes the uracil-insertions. This will produce the required overhangs with the sequence and the length of your wish.<br />
# Transformation: The mix is immediately transformed into competent ''E. coli cells''. The plasmid assembles itself by base pairing between the complementary overhangs and ligation is done by ''E.coli'' itself.<br />
*You only need one enzyme<br />
*You don't need any ligation reaction<br />
*Seamless assembly<br />
*Directional <br />
*Multiple fragment in one reaction - also in eukaryotes [6].<br />
*[https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play]<br />
<br />
[[File:Plug 'n' Play assembly samlet.png|thumb|upright=3|center|The [https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play] concept. Figure taken from DTU-2 iGEM team 2011]]<br />
<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 />
<br />
[1] Sutton, Mark A., et al. "Too much of a good thing." Nature 472.7342 (2011): 159-161.<br />
<br />
[2] Sutton, Mark A., Clare M. Howard, and Jan Willem Erisman, eds. The European nitrogen assessment: sources, effects and policy perspectives. Cambridge University Press, 2011.<br />
<br />
[3] Yaniv D. Scherson, George F. Wells, Sung-Geun Woo, Jangho Lee, Joonhong Park, Brian J. Cantwell and Craig S. Criddle; Nitrogen removal with energy recovery through N<sub>2</sub>O decomposition; Energy & Environmental Science. Issue 1 2013.<br />
<br />
[4] Nour-Eldin, HH et al (2010). USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories. Methods Mol Biol. 2010;643:185-200. doi: 10.1007/978-1-60761-723-5_13.<br />
<br />
[5] Nørholm, Morten HH. "A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering." BMC biotechnology 10.1 (2010): 21.<br />
<br />
[6] Frandsen, Rasmus, et al. "Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi." BMC molecular biology 9.1 (2008): 70.<br />
<br />
<br />
U.S. Energy Information Administration, Frequently Asked Questions: How much electricity does an American home use?</li><br />
<br />
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).<br />
<br />
Kanehisa, M. and Goto, S.; KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27-30 (2000).</li><br />
<br />
Zumft, Walter G. (1997) Cell Biology and Molecular Basis of Denitrification. Microbiology and Molecular Biology Reviews, December 1997 p. 533–616<br />
<br />
Ingledew, WJ and Poole, RK (1984) The Respiratory Chains of Escherichia coli. Microbiological Reviews, Sept. 1984, p. 222-271<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-04T14:53:09Z<p>Ariadni: /* References */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
===What?===<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
===Why?===<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 [1]. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
In Europe the annual surplus of fixed nitrogen from crop production and livestock farming is 10 Tg (10 Teragrams = a million tonnes), 6 Tg leaches into ground and surface water [2]. If that was to be converted into N<sub>2</sub>O, it could be decomposed for 4.87 Billion kW h or equivalent to the annual power consumption of 36,000 Avarage households.<br />
<br />
===How?===<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 [3]. We also provide [https://2013.igem.org/Team:DTU-Denmark/Bioreactor 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 />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
[[File:Dtu-Mutant1.png|right]]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><br />
<br><br />
<br />
=== Mutant 2: Anaerobic ===<br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
[[File:dtu-Mutant2.png|right]]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, but we were able to [[Team:DTU-Denmark/Experiment4|verify that the AMO containing plasmid is working as designed]].<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 />
<br />
== USER Cloning ==<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 specially designed set of primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
## We are using the non-commercial polymerase X7, designed by Morten Nørholm (Nørholm, Morten HH.).<br />
# Optional - If the method was used to insert genes into a backbone, with it's own resistance marker, the PCR product were treated with DpnI to digest the template DNA from the PCR reaction.<br />
# USER-reaction: Digesting the linearized PCR fragment with Uracil-Specific Excision Reagent (USER) enzyme removes the uracil-insertions. This will produce the required overhangs with the sequence and the length of your wish.<br />
# Transformation: The mix is immediately transformed into competent ''E. coli cells''. The plasmid assembles itself by base pairing between the complementary overhangs and ligation is done by ''E.coli'' itself.<br />
*You only need one enzyme<br />
*You don't need any ligation reaction<br />
*Seamless assembly<br />
*Directional <br />
*Multiple fragment in one reaction - also in eukaryotes (Frandsen, Rasmus, et al.)<br />
*[https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play]<br />
<br />
[[File:Plug 'n' Play assembly samlet.png|thumb|upright=3|center|The [https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play] concept. Figure taken from DTU-2 iGEM team 2011]]<br />
<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 />
<br />
[1] Sutton, Mark A., et al. "Too much of a good thing." Nature 472.7342 (2011): 159-161.<br />
<br />
[2] Sutton, Mark A., Clare M. Howard, and Jan Willem Erisman, eds. The European nitrogen assessment: sources, effects and policy perspectives. Cambridge University Press, 2011.<br />
<br />
[3] Yaniv D. Scherson, George F. Wells, Sung-Geun Woo, Jangho Lee, Joonhong Park, Brian J. Cantwell and Craig S. Criddle; Nitrogen removal with energy recovery through N<sub>2</sub>O decomposition; Energy & Environmental Science. Issue 1 2013.<br />
<br />
[4] Nour-Eldin, HH et al (2010). USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories. Methods Mol Biol. 2010;643:185-200. doi: 10.1007/978-1-60761-723-5_13.<br />
<br />
[5] Nørholm, Morten HH. "A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering." BMC biotechnology 10.1 (2010): 21.<br />
<br />
[6] Frandsen, Rasmus, et al. "Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi." BMC molecular biology 9.1 (2008): 70.<br />
<br />
<br />
U.S. Energy Information Administration, Frequently Asked Questions: How much electricity does an American home use?</li><br />
<br />
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).<br />
<br />
Kanehisa, M. and Goto, S.; KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27-30 (2000).</li><br />
<br />
Zumft, Walter G. (1997) Cell Biology and Molecular Basis of Denitrification. Microbiology and Molecular Biology Reviews, December 1997 p. 533–616<br />
<br />
Ingledew, WJ and Poole, RK (1984) The Respiratory Chains of Escherichia coli. Microbiological Reviews, Sept. 1984, p. 222-271<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-04T14:46:55Z<p>Ariadni: /* References */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
===What?===<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
===Why?===<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 [1]. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
In Europe the annual surplus of fixed nitrogen from crop production and livestock farming is 10 Tg (10 Teragrams = a million tonnes), 6 Tg leaches into ground and surface water [2]. If that was to be converted into N<sub>2</sub>O, it could be decomposed for 4.87 Billion kW h or equivalent to the annual power consumption of 36,000 Avarage households.<br />
<br />
===How?===<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 [3]. We also provide [https://2013.igem.org/Team:DTU-Denmark/Bioreactor 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 />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
[[File:Dtu-Mutant1.png|right]]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><br />
<br><br />
<br />
=== Mutant 2: Anaerobic ===<br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
[[File:dtu-Mutant2.png|right]]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, but we were able to [[Team:DTU-Denmark/Experiment4|verify that the AMO containing plasmid is working as designed]].<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 />
<br />
== USER Cloning ==<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 specially designed set of primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
## We are using the non-commercial polymerase X7, designed by Morten Nørholm (Nørholm, Morten HH.).<br />
# Optional - If the method was used to insert genes into a backbone, with it's own resistance marker, the PCR product were treated with DpnI to digest the template DNA from the PCR reaction.<br />
# USER-reaction: Digesting the linearized PCR fragment with Uracil-Specific Excision Reagent (USER) enzyme removes the uracil-insertions. This will produce the required overhangs with the sequence and the length of your wish.<br />
# Transformation: The mix is immediately transformed into competent ''E. coli cells''. The plasmid assembles itself by base pairing between the complementary overhangs and ligation is done by ''E.coli'' itself.<br />
*You only need one enzyme<br />
*You don't need any ligation reaction<br />
*Seamless assembly<br />
*Directional <br />
*Multiple fragment in one reaction - also in eukaryotes (Frandsen, Rasmus, et al.)<br />
*[https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play]<br />
<br />
[[File:Plug 'n' Play assembly samlet.png|thumb|upright=3|center|The [https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play] concept. Figure taken from DTU-2 iGEM team 2011]]<br />
<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 />
<br />
[1] Sutton, Mark A., et al. "Too much of a good thing." Nature 472.7342 (2011): 159-161.<br />
<br />
[2] Sutton, Mark A., Clare M. Howard, and Jan Willem Erisman, eds. The European nitrogen assessment: sources, effects and policy perspectives. Cambridge University Press, 2011.<br />
<br />
Mark A. Sutton, Gilles Billen, Albert Bleeker, Jan Willem Erisman, Peringe Grennfelt, Hans van Grinsven, Bruna Grizzetti, Clare M. Howard and Adrian Leip; Technical summary, European Nitrogen Assessment<br />
<br />
[3] Yaniv D. Scherson, George F. Wells, Sung-Geun Woo, Jangho Lee, Joonhong Park, Brian J. Cantwell and Craig S. Criddle; Nitrogen removal with energy recovery through N<sub>2</sub>O decomposition; Energy & Environmental Science. Issue 1 2013.<br />
<br />
U.S. Energy Information Administration, Frequently Asked Questions: How much electricity does an American home use?</li><br />
<br />
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).<br />
<br />
Kanehisa, M. and Goto, S.; KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27-30 (2000).</li><br />
<br />
Zumft, Walter G. (1997) Cell Biology and Molecular Basis of Denitrification. Microbiology and Molecular Biology Reviews, December 1997 p. 533–616<br />
<br />
Ingledew, WJ and Poole, RK (1984) The Respiratory Chains of Escherichia coli. Microbiological Reviews, Sept. 1984, p. 222-271<br />
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 />
<br />
Nørholm, Morten HH. "A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering." BMC biotechnology 10.1 (2010): 21.<br />
<br />
Frandsen, Rasmus, et al. "Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi." BMC molecular biology 9.1 (2008): 70.<br />
<br />
Scherson, Yaniv D., et al. "Nitrogen removal with energy recovery through N<sub>2</sub>O decomposition." Energy & Environmental Science 6.1 (2013): 241-248.<br />
<br />
<br />
<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-04T14:45:52Z<p>Ariadni: /* How? */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
===What?===<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
===Why?===<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 [1]. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
In Europe the annual surplus of fixed nitrogen from crop production and livestock farming is 10 Tg (10 Teragrams = a million tonnes), 6 Tg leaches into ground and surface water [2]. If that was to be converted into N<sub>2</sub>O, it could be decomposed for 4.87 Billion kW h or equivalent to the annual power consumption of 36,000 Avarage households.<br />
<br />
===How?===<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 [3]. We also provide [https://2013.igem.org/Team:DTU-Denmark/Bioreactor 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 />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
[[File:Dtu-Mutant1.png|right]]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><br />
<br><br />
<br />
=== Mutant 2: Anaerobic ===<br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
[[File:dtu-Mutant2.png|right]]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, but we were able to [[Team:DTU-Denmark/Experiment4|verify that the AMO containing plasmid is working as designed]].<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 />
<br />
== USER Cloning ==<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 specially designed set of primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
## We are using the non-commercial polymerase X7, designed by Morten Nørholm (Nørholm, Morten HH.).<br />
# Optional - If the method was used to insert genes into a backbone, with it's own resistance marker, the PCR product were treated with DpnI to digest the template DNA from the PCR reaction.<br />
# USER-reaction: Digesting the linearized PCR fragment with Uracil-Specific Excision Reagent (USER) enzyme removes the uracil-insertions. This will produce the required overhangs with the sequence and the length of your wish.<br />
# Transformation: The mix is immediately transformed into competent ''E. coli cells''. The plasmid assembles itself by base pairing between the complementary overhangs and ligation is done by ''E.coli'' itself.<br />
*You only need one enzyme<br />
*You don't need any ligation reaction<br />
*Seamless assembly<br />
*Directional <br />
*Multiple fragment in one reaction - also in eukaryotes (Frandsen, Rasmus, et al.)<br />
*[https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play]<br />
<br />
[[File:Plug 'n' Play assembly samlet.png|thumb|upright=3|center|The [https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play] concept. Figure taken from DTU-2 iGEM team 2011]]<br />
<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 />
<br />
[1] Sutton, Mark A., et al. "Too much of a good thing." Nature 472.7342 (2011): 159-161.<br />
<br />
[2] Sutton, Mark A., Clare M. Howard, and Jan Willem Erisman, eds. The European nitrogen assessment: sources, effects and policy perspectives. Cambridge University Press, 2011.<br />
<br />
Mark A. Sutton, Gilles Billen, Albert Bleeker, Jan Willem Erisman, Peringe Grennfelt, Hans van Grinsven, Bruna Grizzetti, Clare M. Howard and Adrian Leip; Technical summary, European Nitrogen Assessment<br />
<br />
Yaniv D. Scherson, George F. Wells, Sung-Geun Woo, Jangho Lee, Joonhong Park, Brian J. Cantwell and Craig S. Criddle; Nitrogen removal with energy recovery through N<sub>2</sub>O decomposition; Energy & Environmental Science. Issue 1 2013.<br />
<br />
U.S. Energy Information Administration, Frequently Asked Questions: How much electricity does an American home use?</li><br />
<br />
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).<br />
<br />
Kanehisa, M. and Goto, S.; KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27-30 (2000).</li><br />
<br />
Zumft, Walter G. (1997) Cell Biology and Molecular Basis of Denitrification. Microbiology and Molecular Biology Reviews, December 1997 p. 533–616<br />
<br />
Ingledew, WJ and Poole, RK (1984) The Respiratory Chains of Escherichia coli. Microbiological Reviews, Sept. 1984, p. 222-271<br />
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 />
<br />
Nørholm, Morten HH. "A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering." BMC biotechnology 10.1 (2010): 21.<br />
<br />
Frandsen, Rasmus, et al. "Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi." BMC molecular biology 9.1 (2008): 70.<br />
<br />
Scherson, Yaniv D., et al. "Nitrogen removal with energy recovery through N<sub>2</sub>O decomposition." Energy & Environmental Science 6.1 (2013): 241-248.<br />
<br />
<br />
<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-04T14:45:18Z<p>Ariadni: /* References */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
===What?===<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
===Why?===<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 [1]. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
In Europe the annual surplus of fixed nitrogen from crop production and livestock farming is 10 Tg (10 Teragrams = a million tonnes), 6 Tg leaches into ground and surface water [2]. If that was to be converted into N<sub>2</sub>O, it could be decomposed for 4.87 Billion kW h or equivalent to the annual power consumption of 36,000 Avarage households.<br />
<br />
===How?===<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 (Scherson, Yaniv D., et al.). We also provide [https://2013.igem.org/Team:DTU-Denmark/Bioreactor 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 />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
[[File:Dtu-Mutant1.png|right]]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><br />
<br><br />
<br />
=== Mutant 2: Anaerobic ===<br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
[[File:dtu-Mutant2.png|right]]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, but we were able to [[Team:DTU-Denmark/Experiment4|verify that the AMO containing plasmid is working as designed]].<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 />
<br />
== USER Cloning ==<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 specially designed set of primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
## We are using the non-commercial polymerase X7, designed by Morten Nørholm (Nørholm, Morten HH.).<br />
# Optional - If the method was used to insert genes into a backbone, with it's own resistance marker, the PCR product were treated with DpnI to digest the template DNA from the PCR reaction.<br />
# USER-reaction: Digesting the linearized PCR fragment with Uracil-Specific Excision Reagent (USER) enzyme removes the uracil-insertions. This will produce the required overhangs with the sequence and the length of your wish.<br />
# Transformation: The mix is immediately transformed into competent ''E. coli cells''. The plasmid assembles itself by base pairing between the complementary overhangs and ligation is done by ''E.coli'' itself.<br />
*You only need one enzyme<br />
*You don't need any ligation reaction<br />
*Seamless assembly<br />
*Directional <br />
*Multiple fragment in one reaction - also in eukaryotes (Frandsen, Rasmus, et al.)<br />
*[https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play]<br />
<br />
[[File:Plug 'n' Play assembly samlet.png|thumb|upright=3|center|The [https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play] concept. Figure taken from DTU-2 iGEM team 2011]]<br />
<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 />
<br />
[1] Sutton, Mark A., et al. "Too much of a good thing." Nature 472.7342 (2011): 159-161.<br />
<br />
[2] Sutton, Mark A., Clare M. Howard, and Jan Willem Erisman, eds. The European nitrogen assessment: sources, effects and policy perspectives. Cambridge University Press, 2011.<br />
<br />
Mark A. Sutton, Gilles Billen, Albert Bleeker, Jan Willem Erisman, Peringe Grennfelt, Hans van Grinsven, Bruna Grizzetti, Clare M. Howard and Adrian Leip; Technical summary, European Nitrogen Assessment<br />
<br />
Yaniv D. Scherson, George F. Wells, Sung-Geun Woo, Jangho Lee, Joonhong Park, Brian J. Cantwell and Craig S. Criddle; Nitrogen removal with energy recovery through N<sub>2</sub>O decomposition; Energy & Environmental Science. Issue 1 2013.<br />
<br />
U.S. Energy Information Administration, Frequently Asked Questions: How much electricity does an American home use?</li><br />
<br />
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).<br />
<br />
Kanehisa, M. and Goto, S.; KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27-30 (2000).</li><br />
<br />
Zumft, Walter G. (1997) Cell Biology and Molecular Basis of Denitrification. Microbiology and Molecular Biology Reviews, December 1997 p. 533–616<br />
<br />
Ingledew, WJ and Poole, RK (1984) The Respiratory Chains of Escherichia coli. Microbiological Reviews, Sept. 1984, p. 222-271<br />
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 />
<br />
Nørholm, Morten HH. "A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering." BMC biotechnology 10.1 (2010): 21.<br />
<br />
Frandsen, Rasmus, et al. "Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi." BMC molecular biology 9.1 (2008): 70.<br />
<br />
Scherson, Yaniv D., et al. "Nitrogen removal with energy recovery through N<sub>2</sub>O decomposition." Energy & Environmental Science 6.1 (2013): 241-248.<br />
<br />
<br />
<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-04T14:44:48Z<p>Ariadni: /* References */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
===What?===<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
===Why?===<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 [1]. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
In Europe the annual surplus of fixed nitrogen from crop production and livestock farming is 10 Tg (10 Teragrams = a million tonnes), 6 Tg leaches into ground and surface water [2]. If that was to be converted into N<sub>2</sub>O, it could be decomposed for 4.87 Billion kW h or equivalent to the annual power consumption of 36,000 Avarage households.<br />
<br />
===How?===<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 (Scherson, Yaniv D., et al.). We also provide [https://2013.igem.org/Team:DTU-Denmark/Bioreactor 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 />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
[[File:Dtu-Mutant1.png|right]]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><br />
<br><br />
<br />
=== Mutant 2: Anaerobic ===<br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
[[File:dtu-Mutant2.png|right]]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, but we were able to [[Team:DTU-Denmark/Experiment4|verify that the AMO containing plasmid is working as designed]].<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 />
<br />
== USER Cloning ==<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 specially designed set of primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
## We are using the non-commercial polymerase X7, designed by Morten Nørholm (Nørholm, Morten HH.).<br />
# Optional - If the method was used to insert genes into a backbone, with it's own resistance marker, the PCR product were treated with DpnI to digest the template DNA from the PCR reaction.<br />
# USER-reaction: Digesting the linearized PCR fragment with Uracil-Specific Excision Reagent (USER) enzyme removes the uracil-insertions. This will produce the required overhangs with the sequence and the length of your wish.<br />
# Transformation: The mix is immediately transformed into competent ''E. coli cells''. The plasmid assembles itself by base pairing between the complementary overhangs and ligation is done by ''E.coli'' itself.<br />
*You only need one enzyme<br />
*You don't need any ligation reaction<br />
*Seamless assembly<br />
*Directional <br />
*Multiple fragment in one reaction - also in eukaryotes (Frandsen, Rasmus, et al.)<br />
*[https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play]<br />
<br />
[[File:Plug 'n' Play assembly samlet.png|thumb|upright=3|center|The [https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play] concept. Figure taken from DTU-2 iGEM team 2011]]<br />
<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 />
<br />
[1] Sutton, Mark A., et al. "Too much of a good thing." Nature 472.7342 (2011): 159-161.<br />
<br />
[2] Sutton, Mark A., Clare M. Howard, and Jan Willem Erisman, eds. The European nitrogen assessment: sources, effects and policy perspectives. Cambridge University Press, 2011.<br />
<br />
Mark A. Sutton, Gilles Billen, Albert Bleeker, Jan Willem Erisman, Peringe Grennfelt, Hans van Grinsven, Bruna Grizzetti, Clare M. Howard and Adrian Leip; Technical summary, European Nitrogen Assessment<br />
<br />
Yaniv D. Scherson, George F. Wells, Sung-Geun Woo, Jangho Lee, Joonhong Park, Brian J. Cantwell and Craig S. Criddle; Nitrogen removal with energy recovery through N<sub>2</sub>O decomposition; Energy & Environmental Science. Issue 1 2013.<br />
<br />
U.S. Energy Information Administration, Frequently Asked Questions: How much electricity does an American home use?</li><br />
<br />
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).<br />
<br />
Kanehisa, M. and Goto, S.; KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27-30 (2000).</li><br />
<br />
Zumft, Walter G. (1997) Cell Biology and Molecular Basis of Denitrification. Microbiology and Molecular Biology Reviews, December 1997 p. 533–616<br />
<br />
Ingledew, WJ and Poole, RK (1984) The Respiratory Chains of Escherichia coli. Microbiological Reviews, Sept. 1984, p. 222-271<br />
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 />
<br />
Nørholm, Morten HH. "A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering." BMC biotechnology 10.1 (2010): 21.<br />
<br />
Frandsen, Rasmus, et al. "Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi." BMC molecular biology 9.1 (2008): 70.<br />
<br />
Scherson, Yaniv D., et al. "Nitrogen removal with energy recovery through N<sub2</sub>O decomposition." Energy & Environmental Science 6.1 (2013): 241-248.<br />
<br />
<br />
<br />
<br />
<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-04T14:44:25Z<p>Ariadni: /* References */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
===What?===<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
===Why?===<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 [1]. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
In Europe the annual surplus of fixed nitrogen from crop production and livestock farming is 10 Tg (10 Teragrams = a million tonnes), 6 Tg leaches into ground and surface water [2]. If that was to be converted into N<sub>2</sub>O, it could be decomposed for 4.87 Billion kW h or equivalent to the annual power consumption of 36,000 Avarage households.<br />
<br />
===How?===<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 (Scherson, Yaniv D., et al.). We also provide [https://2013.igem.org/Team:DTU-Denmark/Bioreactor 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 />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
[[File:Dtu-Mutant1.png|right]]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><br />
<br><br />
<br />
=== Mutant 2: Anaerobic ===<br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
[[File:dtu-Mutant2.png|right]]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, but we were able to [[Team:DTU-Denmark/Experiment4|verify that the AMO containing plasmid is working as designed]].<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 />
<br />
== USER Cloning ==<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 specially designed set of primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
## We are using the non-commercial polymerase X7, designed by Morten Nørholm (Nørholm, Morten HH.).<br />
# Optional - If the method was used to insert genes into a backbone, with it's own resistance marker, the PCR product were treated with DpnI to digest the template DNA from the PCR reaction.<br />
# USER-reaction: Digesting the linearized PCR fragment with Uracil-Specific Excision Reagent (USER) enzyme removes the uracil-insertions. This will produce the required overhangs with the sequence and the length of your wish.<br />
# Transformation: The mix is immediately transformed into competent ''E. coli cells''. The plasmid assembles itself by base pairing between the complementary overhangs and ligation is done by ''E.coli'' itself.<br />
*You only need one enzyme<br />
*You don't need any ligation reaction<br />
*Seamless assembly<br />
*Directional <br />
*Multiple fragment in one reaction - also in eukaryotes (Frandsen, Rasmus, et al.)<br />
*[https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play]<br />
<br />
[[File:Plug 'n' Play assembly samlet.png|thumb|upright=3|center|The [https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play] concept. Figure taken from DTU-2 iGEM team 2011]]<br />
<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 />
<br />
[1] Sutton, Mark A., et al. "Too much of a good thing." Nature 472.7342 (2011): 159-161.<br />
<br />
[2] Sutton, Mark A., Clare M. Howard, and Jan Willem Erisman, eds. The European nitrogen assessment: sources, effects and policy perspectives. Cambridge University Press, 2011.<br />
<br />
Mark A. Sutton, Gilles Billen, Albert Bleeker, Jan Willem Erisman, Peringe Grennfelt, Hans van Grinsven, Bruna Grizzetti, Clare M. Howard and Adrian Leip; Technical summary, European Nitrogen Assessment<br />
<br />
Yaniv D. Scherson, George F. Wells, Sung-Geun Woo, Jangho Lee, Joonhong Park, Brian J. Cantwell and Craig S. Criddle; Nitrogen removal with energy recovery through N<sub>2</sub>O decomposition; Energy & Environmental Science. Issue 1 2013.<br />
<br />
U.S. Energy Information Administration, Frequently Asked Questions: How much electricity does an American home use?</li><br />
<br />
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).<br />
<br />
Kanehisa, M. and Goto, S.; KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27-30 (2000).</li><br />
<br />
Zumft, Walter G. (1997) Cell Biology and Molecular Basis of Denitrification. Microbiology and Molecular Biology Reviews, December 1997 p. 533–616<br />
<br />
Ingledew, WJ and Poole, RK (1984) The Respiratory Chains of Escherichia coli. Microbiological Reviews, Sept. 1984, p. 222-271<br />
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 />
<br />
Nørholm, Morten HH. "A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering." BMC biotechnology 10.1 (2010): 21.<br />
<br />
Frandsen, Rasmus, et al. "Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi." BMC molecular biology 9.1 (2008): 70.<br />
<br />
Scherson, Yaniv D., et al. "Nitrogen removal with energy recovery through N<sub2</sub>O decomposition." Energy & Environmental Science 6.1 (2013): 241-248.<br />
<br />
</ul><br />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-04T14:42:57Z<p>Ariadni: /* References */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
===What?===<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
===Why?===<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 [1]. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
In Europe the annual surplus of fixed nitrogen from crop production and livestock farming is 10 Tg (10 Teragrams = a million tonnes), 6 Tg leaches into ground and surface water [2]. If that was to be converted into N<sub>2</sub>O, it could be decomposed for 4.87 Billion kW h or equivalent to the annual power consumption of 36,000 Avarage households.<br />
<br />
===How?===<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 (Scherson, Yaniv D., et al.). We also provide [https://2013.igem.org/Team:DTU-Denmark/Bioreactor 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 />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
[[File:Dtu-Mutant1.png|right]]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><br />
<br><br />
<br />
=== Mutant 2: Anaerobic ===<br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
[[File:dtu-Mutant2.png|right]]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, but we were able to [[Team:DTU-Denmark/Experiment4|verify that the AMO containing plasmid is working as designed]].<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 />
<br />
== USER Cloning ==<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 specially designed set of primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
## We are using the non-commercial polymerase X7, designed by Morten Nørholm (Nørholm, Morten HH.).<br />
# Optional - If the method was used to insert genes into a backbone, with it's own resistance marker, the PCR product were treated with DpnI to digest the template DNA from the PCR reaction.<br />
# USER-reaction: Digesting the linearized PCR fragment with Uracil-Specific Excision Reagent (USER) enzyme removes the uracil-insertions. This will produce the required overhangs with the sequence and the length of your wish.<br />
# Transformation: The mix is immediately transformed into competent ''E. coli cells''. The plasmid assembles itself by base pairing between the complementary overhangs and ligation is done by ''E.coli'' itself.<br />
*You only need one enzyme<br />
*You don't need any ligation reaction<br />
*Seamless assembly<br />
*Directional <br />
*Multiple fragment in one reaction - also in eukaryotes (Frandsen, Rasmus, et al.)<br />
*[https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play]<br />
<br />
[[File:Plug 'n' Play assembly samlet.png|thumb|upright=3|center|The [https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play] concept. Figure taken from DTU-2 iGEM team 2011]]<br />
<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 />
<br />
[1] Sutton, Mark A., et al. "Too much of a good thing." Nature 472.7342 (2011): 159-161.<br />
<br />
[2] Sutton, Mark A., Clare M. Howard, and Jan Willem Erisman, eds. The European nitrogen assessment: sources, effects and policy perspectives. Cambridge University Press, 2011.<br />
<li>Mark A. Sutton, Gilles Billen, Albert Bleeker, Jan Willem Erisman, Peringe Grennfelt, Hans van Grinsven, Bruna Grizzetti, Clare M. Howard and Adrian Leip; Technical summary, European Nitrogen Assessment</li><br />
<li> Yaniv D. Scherson, George F. Wells, Sung-Geun Woo, Jangho Lee, Joonhong Park, Brian J. Cantwell and Craig S. Criddle; Nitrogen removal with energy recovery through N<sub>2</sub>O decomposition; Energy & Environmental Science. Issue 1 2013.</li><br />
<li>U.S. Energy Information Administration, Frequently Asked Questions: How much electricity does an American home use?</li><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 />
<li>Nørholm, Morten HH. "A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering." BMC biotechnology 10.1 (2010): 21.<br />
<li>Frandsen, Rasmus, et al. "Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi." BMC molecular biology 9.1 (2008): 70.<br />
<li>Scherson, Yaniv D., et al. "Nitrogen removal with energy recovery through N<sub2</sub>O decomposition." Energy & Environmental Science 6.1 (2013): 241-248.<br />
<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 />
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<div style="clear: both;"></div><br />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-04T14:42:23Z<p>Ariadni: /* References */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
===What?===<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
===Why?===<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 [1]. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
In Europe the annual surplus of fixed nitrogen from crop production and livestock farming is 10 Tg (10 Teragrams = a million tonnes), 6 Tg leaches into ground and surface water [2]. If that was to be converted into N<sub>2</sub>O, it could be decomposed for 4.87 Billion kW h or equivalent to the annual power consumption of 36,000 Avarage households.<br />
<br />
===How?===<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 (Scherson, Yaniv D., et al.). We also provide [https://2013.igem.org/Team:DTU-Denmark/Bioreactor 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 />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
[[File:Dtu-Mutant1.png|right]]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><br />
<br><br />
<br />
=== Mutant 2: Anaerobic ===<br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
[[File:dtu-Mutant2.png|right]]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, but we were able to [[Team:DTU-Denmark/Experiment4|verify that the AMO containing plasmid is working as designed]].<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 />
<br />
== USER Cloning ==<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 specially designed set of primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
## We are using the non-commercial polymerase X7, designed by Morten Nørholm (Nørholm, Morten HH.).<br />
# Optional - If the method was used to insert genes into a backbone, with it's own resistance marker, the PCR product were treated with DpnI to digest the template DNA from the PCR reaction.<br />
# USER-reaction: Digesting the linearized PCR fragment with Uracil-Specific Excision Reagent (USER) enzyme removes the uracil-insertions. This will produce the required overhangs with the sequence and the length of your wish.<br />
# Transformation: The mix is immediately transformed into competent ''E. coli cells''. The plasmid assembles itself by base pairing between the complementary overhangs and ligation is done by ''E.coli'' itself.<br />
*You only need one enzyme<br />
*You don't need any ligation reaction<br />
*Seamless assembly<br />
*Directional <br />
*Multiple fragment in one reaction - also in eukaryotes (Frandsen, Rasmus, et al.)<br />
*[https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play]<br />
<br />
[[File:Plug 'n' Play assembly samlet.png|thumb|upright=3|center|The [https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play] concept. Figure taken from DTU-2 iGEM team 2011]]<br />
<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 />
[1] Sutton, Mark A., et al. "Too much of a good thing." Nature 472.7342 (2011): 159-161.<br />
<br />
<br />
[2] Sutton, Mark A., Clare M. Howard, and Jan Willem Erisman, eds. The European nitrogen assessment: sources, effects and policy perspectives. Cambridge University Press, 2011.<br />
<li>Mark A. Sutton, Gilles Billen, Albert Bleeker, Jan Willem Erisman, Peringe Grennfelt, Hans van Grinsven, Bruna Grizzetti, Clare M. Howard and Adrian Leip; Technical summary, European Nitrogen Assessment</li><br />
<li> Yaniv D. Scherson, George F. Wells, Sung-Geun Woo, Jangho Lee, Joonhong Park, Brian J. Cantwell and Craig S. Criddle; Nitrogen removal with energy recovery through N<sub>2</sub>O decomposition; Energy & Environmental Science. Issue 1 2013.</li><br />
<li>U.S. Energy Information Administration, Frequently Asked Questions: How much electricity does an American home use?</li><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 />
<li>Nørholm, Morten HH. "A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering." BMC biotechnology 10.1 (2010): 21.<br />
<li>Frandsen, Rasmus, et al. "Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi." BMC molecular biology 9.1 (2008): 70.<br />
<li>Scherson, Yaniv D., et al. "Nitrogen removal with energy recovery through N<sub2</sub>O decomposition." Energy & Environmental Science 6.1 (2013): 241-248.<br />
<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>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-04T14:41:59Z<p>Ariadni: /* References */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
===What?===<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
===Why?===<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 [1]. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
In Europe the annual surplus of fixed nitrogen from crop production and livestock farming is 10 Tg (10 Teragrams = a million tonnes), 6 Tg leaches into ground and surface water [2]. If that was to be converted into N<sub>2</sub>O, it could be decomposed for 4.87 Billion kW h or equivalent to the annual power consumption of 36,000 Avarage households.<br />
<br />
===How?===<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 (Scherson, Yaniv D., et al.). We also provide [https://2013.igem.org/Team:DTU-Denmark/Bioreactor 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 />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
[[File:Dtu-Mutant1.png|right]]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><br />
<br><br />
<br />
=== Mutant 2: Anaerobic ===<br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
[[File:dtu-Mutant2.png|right]]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, but we were able to [[Team:DTU-Denmark/Experiment4|verify that the AMO containing plasmid is working as designed]].<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 />
<br />
== USER Cloning ==<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 specially designed set of primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
## We are using the non-commercial polymerase X7, designed by Morten Nørholm (Nørholm, Morten HH.).<br />
# Optional - If the method was used to insert genes into a backbone, with it's own resistance marker, the PCR product were treated with DpnI to digest the template DNA from the PCR reaction.<br />
# USER-reaction: Digesting the linearized PCR fragment with Uracil-Specific Excision Reagent (USER) enzyme removes the uracil-insertions. This will produce the required overhangs with the sequence and the length of your wish.<br />
# Transformation: The mix is immediately transformed into competent ''E. coli cells''. The plasmid assembles itself by base pairing between the complementary overhangs and ligation is done by ''E.coli'' itself.<br />
*You only need one enzyme<br />
*You don't need any ligation reaction<br />
*Seamless assembly<br />
*Directional <br />
*Multiple fragment in one reaction - also in eukaryotes (Frandsen, Rasmus, et al.)<br />
*[https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play]<br />
<br />
[[File:Plug 'n' Play assembly samlet.png|thumb|upright=3|center|The [https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play] concept. Figure taken from DTU-2 iGEM team 2011]]<br />
<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 />
[1] Sutton, Mark A., et al. "Too much of a good thing." Nature 472.7342 (2011): 159-161.<br />
<br />
<br />
[2] Sutton, Mark A., Clare M. Howard, and Jan Willem Erisman, eds. The European nitrogen assessment: sources, effects and policy perspectives. Cambridge University Press, 2011.<br />
<li>Mark A. Sutton, Gilles Billen, Albert Bleeker, Jan Willem Erisman, Peringe Grennfelt, Hans van Grinsven, Bruna Grizzetti, Clare M. Howard and Adrian Leip; Technical summary, European Nitrogen Assessment</li><br />
<li> Yaniv D. Scherson, George F. Wells, Sung-Geun Woo, Jangho Lee, Joonhong Park, Brian J. Cantwell and Craig S. Criddle; Nitrogen removal with energy recovery through N<sub>2</sub>O decomposition; Energy & Environmental Science. Issue 1 2013.</li><br />
<li>U.S. Energy Information Administration, Frequently Asked Questions: How much electricity does an American home use?</li><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 />
<li>Nørholm, Morten HH. "A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering." BMC biotechnology 10.1 (2010): 21.<br />
<li>Frandsen, Rasmus, et al. "Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi." BMC molecular biology 9.1 (2008): 70.<br />
<li>Scherson, Yaniv D., et al. "Nitrogen removal with energy recovery through N<sub2</sub>O decomposition." Energy & Environmental Science 6.1 (2013): 241-248.<br />
<br />
</ul><br />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-04T14:41:31Z<p>Ariadni: /* References */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
===What?===<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
===Why?===<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 [1]. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
In Europe the annual surplus of fixed nitrogen from crop production and livestock farming is 10 Tg (10 Teragrams = a million tonnes), 6 Tg leaches into ground and surface water [2]. If that was to be converted into N<sub>2</sub>O, it could be decomposed for 4.87 Billion kW h or equivalent to the annual power consumption of 36,000 Avarage households.<br />
<br />
===How?===<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 (Scherson, Yaniv D., et al.). We also provide [https://2013.igem.org/Team:DTU-Denmark/Bioreactor 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 />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
[[File:Dtu-Mutant1.png|right]]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><br />
<br><br />
<br />
=== Mutant 2: Anaerobic ===<br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
[[File:dtu-Mutant2.png|right]]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, but we were able to [[Team:DTU-Denmark/Experiment4|verify that the AMO containing plasmid is working as designed]].<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 />
<br />
== USER Cloning ==<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 specially designed set of primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
## We are using the non-commercial polymerase X7, designed by Morten Nørholm (Nørholm, Morten HH.).<br />
# Optional - If the method was used to insert genes into a backbone, with it's own resistance marker, the PCR product were treated with DpnI to digest the template DNA from the PCR reaction.<br />
# USER-reaction: Digesting the linearized PCR fragment with Uracil-Specific Excision Reagent (USER) enzyme removes the uracil-insertions. This will produce the required overhangs with the sequence and the length of your wish.<br />
# Transformation: The mix is immediately transformed into competent ''E. coli cells''. The plasmid assembles itself by base pairing between the complementary overhangs and ligation is done by ''E.coli'' itself.<br />
*You only need one enzyme<br />
*You don't need any ligation reaction<br />
*Seamless assembly<br />
*Directional <br />
*Multiple fragment in one reaction - also in eukaryotes (Frandsen, Rasmus, et al.)<br />
*[https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play]<br />
<br />
[[File:Plug 'n' Play assembly samlet.png|thumb|upright=3|center|The [https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play] concept. Figure taken from DTU-2 iGEM team 2011]]<br />
<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 />
[1] Sutton, Mark A., et al. "Too much of a good thing." Nature 472.7342 (2011): 159-161.<br />
<br />
[2] Sutton, Mark A., Clare M. Howard, and Jan Willem Erisman, eds. The European nitrogen assessment: sources, effects and policy perspectives. Cambridge University Press, 2011.<br />
<li>Mark A. Sutton, Gilles Billen, Albert Bleeker, Jan Willem Erisman, Peringe Grennfelt, Hans van Grinsven, Bruna Grizzetti, Clare M. Howard and Adrian Leip; Technical summary, European Nitrogen Assessment</li><br />
<li> Yaniv D. Scherson, George F. Wells, Sung-Geun Woo, Jangho Lee, Joonhong Park, Brian J. Cantwell and Craig S. Criddle; Nitrogen removal with energy recovery through N<sub>2</sub>O decomposition; Energy & Environmental Science. Issue 1 2013.</li><br />
<li>U.S. Energy Information Administration, Frequently Asked Questions: How much electricity does an American home use?</li><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 />
<li>Nørholm, Morten HH. "A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering." BMC biotechnology 10.1 (2010): 21.<br />
<li>Frandsen, Rasmus, et al. "Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi." BMC molecular biology 9.1 (2008): 70.<br />
<li>Scherson, Yaniv D., et al. "Nitrogen removal with energy recovery through N<sub2</sub>O decomposition." Energy & Environmental Science 6.1 (2013): 241-248.<br />
<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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<!-- Main content area --><br />
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</body><br />
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</html><br />
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<div style="clear: both;"></div><br />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-04T14:41:19Z<p>Ariadni: /* References */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
===What?===<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
===Why?===<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 [1]. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
In Europe the annual surplus of fixed nitrogen from crop production and livestock farming is 10 Tg (10 Teragrams = a million tonnes), 6 Tg leaches into ground and surface water [2]. If that was to be converted into N<sub>2</sub>O, it could be decomposed for 4.87 Billion kW h or equivalent to the annual power consumption of 36,000 Avarage households.<br />
<br />
===How?===<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 (Scherson, Yaniv D., et al.). We also provide [https://2013.igem.org/Team:DTU-Denmark/Bioreactor 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 />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
[[File:Dtu-Mutant1.png|right]]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><br />
<br><br />
<br />
=== Mutant 2: Anaerobic ===<br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
[[File:dtu-Mutant2.png|right]]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, but we were able to [[Team:DTU-Denmark/Experiment4|verify that the AMO containing plasmid is working as designed]].<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 />
<br />
== USER Cloning ==<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 specially designed set of primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
## We are using the non-commercial polymerase X7, designed by Morten Nørholm (Nørholm, Morten HH.).<br />
# Optional - If the method was used to insert genes into a backbone, with it's own resistance marker, the PCR product were treated with DpnI to digest the template DNA from the PCR reaction.<br />
# USER-reaction: Digesting the linearized PCR fragment with Uracil-Specific Excision Reagent (USER) enzyme removes the uracil-insertions. This will produce the required overhangs with the sequence and the length of your wish.<br />
# Transformation: The mix is immediately transformed into competent ''E. coli cells''. The plasmid assembles itself by base pairing between the complementary overhangs and ligation is done by ''E.coli'' itself.<br />
*You only need one enzyme<br />
*You don't need any ligation reaction<br />
*Seamless assembly<br />
*Directional <br />
*Multiple fragment in one reaction - also in eukaryotes (Frandsen, Rasmus, et al.)<br />
*[https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play]<br />
<br />
[[File:Plug 'n' Play assembly samlet.png|thumb|upright=3|center|The [https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play] concept. Figure taken from DTU-2 iGEM team 2011]]<br />
<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 />
[1] Sutton, Mark A., et al. "Too much of a good thing." Nature 472.7342 (2011): 159-161.<br />
<br />
[2] Sutton, Mark A., Clare M. Howard, and Jan Willem Erisman, eds. The European nitrogen assessment: sources, effects and policy perspectives. Cambridge University Press, 2011.<br />
<li>Mark A. Sutton, Gilles Billen, Albert Bleeker, Jan Willem Erisman, Peringe Grennfelt, Hans van Grinsven, Bruna Grizzetti, Clare M. Howard and Adrian Leip; Technical summary, European Nitrogen Assessment</li><br />
<li> Yaniv D. Scherson, George F. Wells, Sung-Geun Woo, Jangho Lee, Joonhong Park, Brian J. Cantwell and Craig S. Criddle; Nitrogen removal with energy recovery through N<sub>2</sub>O decomposition; Energy & Environmental Science. Issue 1 2013.</li><br />
<li>U.S. Energy Information Administration, Frequently Asked Questions: How much electricity does an American home use?</li><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 />
<li>Nørholm, Morten HH. "A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering." BMC biotechnology 10.1 (2010): 21.<br />
<li>Frandsen, Rasmus, et al. "Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi." BMC molecular biology 9.1 (2008): 70.<br />
<li>Scherson, Yaniv D., et al. "Nitrogen removal with energy recovery through N<sub2</sub>O decomposition." Energy & Environmental Science 6.1 (2013): 241-248.<br />
<br />
</ul><br />
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</td><br />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-04T14:40:28Z<p>Ariadni: /* References */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
===What?===<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
===Why?===<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 [1]. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
In Europe the annual surplus of fixed nitrogen from crop production and livestock farming is 10 Tg (10 Teragrams = a million tonnes), 6 Tg leaches into ground and surface water [2]. If that was to be converted into N<sub>2</sub>O, it could be decomposed for 4.87 Billion kW h or equivalent to the annual power consumption of 36,000 Avarage households.<br />
<br />
===How?===<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 (Scherson, Yaniv D., et al.). We also provide [https://2013.igem.org/Team:DTU-Denmark/Bioreactor 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 />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
[[File:Dtu-Mutant1.png|right]]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><br />
<br><br />
<br />
=== Mutant 2: Anaerobic ===<br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
[[File:dtu-Mutant2.png|right]]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, but we were able to [[Team:DTU-Denmark/Experiment4|verify that the AMO containing plasmid is working as designed]].<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 />
<br />
== USER Cloning ==<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 specially designed set of primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
## We are using the non-commercial polymerase X7, designed by Morten Nørholm (Nørholm, Morten HH.).<br />
# Optional - If the method was used to insert genes into a backbone, with it's own resistance marker, the PCR product were treated with DpnI to digest the template DNA from the PCR reaction.<br />
# USER-reaction: Digesting the linearized PCR fragment with Uracil-Specific Excision Reagent (USER) enzyme removes the uracil-insertions. This will produce the required overhangs with the sequence and the length of your wish.<br />
# Transformation: The mix is immediately transformed into competent ''E. coli cells''. The plasmid assembles itself by base pairing between the complementary overhangs and ligation is done by ''E.coli'' itself.<br />
*You only need one enzyme<br />
*You don't need any ligation reaction<br />
*Seamless assembly<br />
*Directional <br />
*Multiple fragment in one reaction - also in eukaryotes (Frandsen, Rasmus, et al.)<br />
*[https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play]<br />
<br />
[[File:Plug 'n' Play assembly samlet.png|thumb|upright=3|center|The [https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play] concept. Figure taken from DTU-2 iGEM team 2011]]<br />
<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 />
[1]Sutton, Mark A., et al. "Too much of a good thing." Nature 472.7342 (2011): 159-161.<br />
<br />
[2]Sutton, Mark A., Clare M. Howard, and Jan Willem Erisman, eds. The European nitrogen assessment: sources, effects and policy perspectives. Cambridge University Press, 2011.<br />
<li>Mark A. Sutton, Gilles Billen, Albert Bleeker, Jan Willem Erisman, Peringe Grennfelt, Hans van Grinsven, Bruna Grizzetti, Clare M. Howard and Adrian Leip; Technical summary, European Nitrogen Assessment</li><br />
<li> Yaniv D. Scherson, George F. Wells, Sung-Geun Woo, Jangho Lee, Joonhong Park, Brian J. Cantwell and Craig S. Criddle; Nitrogen removal with energy recovery through N<sub>2</sub>O decomposition; Energy & Environmental Science. Issue 1 2013.</li><br />
<li>U.S. Energy Information Administration, Frequently Asked Questions: How much electricity does an American home use?</li><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 />
<li>Nørholm, Morten HH. "A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering." BMC biotechnology 10.1 (2010): 21.<br />
<li>Frandsen, Rasmus, et al. "Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi." BMC molecular biology 9.1 (2008): 70.<br />
<li>Scherson, Yaniv D., et al. "Nitrogen removal with energy recovery through N<sub2</sub>O decomposition." Energy & Environmental Science 6.1 (2013): 241-248.<br />
<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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<!-- Main content area --><br />
<br />
</body><br />
<br />
</html><br />
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<div style="clear: both;"></div><br />
</div><br />
{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-04T14:40:02Z<p>Ariadni: </p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
===What?===<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
===Why?===<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 [1]. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
In Europe the annual surplus of fixed nitrogen from crop production and livestock farming is 10 Tg (10 Teragrams = a million tonnes), 6 Tg leaches into ground and surface water [2]. If that was to be converted into N<sub>2</sub>O, it could be decomposed for 4.87 Billion kW h or equivalent to the annual power consumption of 36,000 Avarage households.<br />
<br />
===How?===<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 (Scherson, Yaniv D., et al.). We also provide [https://2013.igem.org/Team:DTU-Denmark/Bioreactor 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 />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
[[File:Dtu-Mutant1.png|right]]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><br />
<br><br />
<br />
=== Mutant 2: Anaerobic ===<br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
[[File:dtu-Mutant2.png|right]]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, but we were able to [[Team:DTU-Denmark/Experiment4|verify that the AMO containing plasmid is working as designed]].<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 />
<br />
== USER Cloning ==<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 specially designed set of primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
## We are using the non-commercial polymerase X7, designed by Morten Nørholm (Nørholm, Morten HH.).<br />
# Optional - If the method was used to insert genes into a backbone, with it's own resistance marker, the PCR product were treated with DpnI to digest the template DNA from the PCR reaction.<br />
# USER-reaction: Digesting the linearized PCR fragment with Uracil-Specific Excision Reagent (USER) enzyme removes the uracil-insertions. This will produce the required overhangs with the sequence and the length of your wish.<br />
# Transformation: The mix is immediately transformed into competent ''E. coli cells''. The plasmid assembles itself by base pairing between the complementary overhangs and ligation is done by ''E.coli'' itself.<br />
*You only need one enzyme<br />
*You don't need any ligation reaction<br />
*Seamless assembly<br />
*Directional <br />
*Multiple fragment in one reaction - also in eukaryotes (Frandsen, Rasmus, et al.)<br />
*[https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play]<br />
<br />
[[File:Plug 'n' Play assembly samlet.png|thumb|upright=3|center|The [https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play] concept. Figure taken from DTU-2 iGEM team 2011]]<br />
<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 />
[1]Sutton, Mark A., et al. "Too much of a good thing." Nature 472.7342 (2011): 159-161.<br />
[2]Sutton, Mark A., Clare M. Howard, and Jan Willem Erisman, eds. The European nitrogen assessment: sources, effects and policy perspectives. Cambridge University Press, 2011.<br />
<li>Mark A. Sutton, Gilles Billen, Albert Bleeker, Jan Willem Erisman, Peringe Grennfelt, Hans van Grinsven, Bruna Grizzetti, Clare M. Howard and Adrian Leip; Technical summary, European Nitrogen Assessment</li><br />
<li> Yaniv D. Scherson, George F. Wells, Sung-Geun Woo, Jangho Lee, Joonhong Park, Brian J. Cantwell and Craig S. Criddle; Nitrogen removal with energy recovery through N<sub>2</sub>O decomposition; Energy & Environmental Science. Issue 1 2013.</li><br />
<li>U.S. Energy Information Administration, Frequently Asked Questions: How much electricity does an American home use?</li><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 />
<li>Nørholm, Morten HH. "A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering." BMC biotechnology 10.1 (2010): 21.<br />
<li>Frandsen, Rasmus, et al. "Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi." BMC molecular biology 9.1 (2008): 70.<br />
<li>Scherson, Yaniv D., et al. "Nitrogen removal with energy recovery through N<sub2</sub>O decomposition." Energy & Environmental Science 6.1 (2013): 241-248.<br />
<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>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/ProjectTeam:DTU-Denmark/Project2013-10-04T14:39:11Z<p>Ariadni: /* Why? */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|}}<br />
<div class="overviewPage"><br />
__NOTOC__<br />
==Project Description==<br />
===What?===<br />
Our project removes ammonia from waste water, and via two ''E. coli'' mutants, turns it into nitrous oxide.<br />
<br />
[[File:Dtu overview.png|400px|center]]<br />
===Why?===<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 [1]. This results in decreased biodiversity throughout the watershed. Within Europe, 53% of lakes are eutrophic. <br />
<br />
In Europe the annual surplus of fixed nitrogen from crop production and livestock farming is 10 Tg (10 Teragrams = a million tonnes), 6 Tg leaches into ground and surface water [2]. If that was to be converted into N<sub>2</sub>O, it could be decomposed for 4.87 Billion kW h or equivalent to the annual power consumption of 36,000 Avarage households.<br />
<br />
===How?===<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 (Scherson, Yaniv D., et al.). We also provide [https://2013.igem.org/Team:DTU-Denmark/Bioreactor 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 />
<br />
[[File:Dtu-pathway-mutant1.png]]<br />
<br />
[[File:Dtu-Mutant1.png|right]]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><br />
<br><br />
<br />
=== Mutant 2: Anaerobic ===<br />
<br />
[[File:Dtu-pathway-mutant2.png]]<br />
<br />
[[File:dtu-Mutant2.png|right]]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, but we were able to [[Team:DTU-Denmark/Experiment4|verify that the AMO containing plasmid is working as designed]].<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 />
<br />
== USER Cloning ==<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 specially designed set of primers with a tail that will become the overhang and a polymerase that is able to perform uracil-insertion.<br />
## We are using the non-commercial polymerase X7, designed by Morten Nørholm (Nørholm, Morten HH.).<br />
# Optional - If the method was used to insert genes into a backbone, with it's own resistance marker, the PCR product were treated with DpnI to digest the template DNA from the PCR reaction.<br />
# USER-reaction: Digesting the linearized PCR fragment with Uracil-Specific Excision Reagent (USER) enzyme removes the uracil-insertions. This will produce the required overhangs with the sequence and the length of your wish.<br />
# Transformation: The mix is immediately transformed into competent ''E. coli cells''. The plasmid assembles itself by base pairing between the complementary overhangs and ligation is done by ''E.coli'' itself.<br />
*You only need one enzyme<br />
*You don't need any ligation reaction<br />
*Seamless assembly<br />
*Directional <br />
*Multiple fragment in one reaction - also in eukaryotes (Frandsen, Rasmus, et al.)<br />
*[https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play]<br />
<br />
[[File:Plug 'n' Play assembly samlet.png|thumb|upright=3|center|The [https://2011.igem.org/Team:DTU-Denmark-2 Plug 'n' Play] concept. Figure taken from DTU-2 iGEM team 2011]]<br />
<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>Mark A. Sutton, Gilles Billen, Albert Bleeker, Jan Willem Erisman, Peringe Grennfelt, Hans van Grinsven, Bruna Grizzetti, Clare M. Howard and Adrian Leip; Technical summary, European Nitrogen Assessment</li><br />
<li> Yaniv D. Scherson, George F. Wells, Sung-Geun Woo, Jangho Lee, Joonhong Park, Brian J. Cantwell and Craig S. Criddle; Nitrogen removal with energy recovery through N<sub>2</sub>O decomposition; Energy & Environmental Science. Issue 1 2013.</li><br />
<li>U.S. Energy Information Administration, Frequently Asked Questions: How much electricity does an American home use?</li><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 />
<li>Nørholm, Morten HH. "A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering." BMC biotechnology 10.1 (2010): 21.<br />
<li>Frandsen, Rasmus, et al. "Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi." BMC molecular biology 9.1 (2008): 70.<br />
<li>Scherson, Yaniv D., et al. "Nitrogen removal with energy recovery through N<sub2</sub>O decomposition." Energy & Environmental Science 6.1 (2013): 241-248.<br />
<li>Sutton, Mark A., et al. "Too much of a good thing." Nature 472.7342 (2011): 159-161.<br />
<li>Sutton, Mark A., Clare M. Howard, and Jan Willem Erisman, eds. The European nitrogen assessment: sources, effects and policy perspectives. Cambridge University Press, 2011.<br />
</ul><br />
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{{:Team:DTU-Denmark/Templates/EndPage}}</div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/AttributionsTeam:DTU-Denmark/Attributions2013-10-04T14:20:27Z<p>Ariadni: /* Special Thanks to */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|Attributions}}<br />
<br />
== Work done by the team ==<br />
<hr/><br />
===== [[File:Poland_Flag.gif|30px]] Katarzyna Chyzynska =====<br />
* Performed [[Team:DTU-Denmark/Experiments|characterization and verification experiments]]<br />
* Designed our poster<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Canada_Flag.jpg|30px]] Helen Cook =====<br />
* Performed and analyzed data from the [[Team:DTU-Denmark/Experiments|characterization and verification experiments]]<br />
* Created the diagrams describing our project<br />
* Participated in high school outreach, Biobrick workshop and the discussion on IP and synthetic biology.<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Kristian Davidsen =====<br />
* Lead user cloning work in the 208 lab; designed primers<br />
* Constructed the [[Team:DTU-Denmark/pBAD_SPL|pBAD_SPL]]<br />
* Found sponsors and funding<br />
* Starred in our [https://www.youtube.com/watch?v=7EiVttJpXH4 bricks of knowledge video on USER cloning]<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Greece_Flag.jpg|30px]] Ariadni Droumpali =====<br />
* Performed [[Team:DTU-Denmark/Experiments|characterization and verification experiments]]<br />
* Participated in high school outreach, Biobrick workshop and the discussion on IP and synthetic biology.<br />
<br />
===== [[File:Poland_Flag.gif|30px]] Piotr Dworzynski =====<br />
* Simulation of [[Team:DTU-Denmark/HelloWorld|GFP in the periplasm]] for Hello World project<br />
<br />
===== [[File:Poland_Flag.gif|30px]] Malgorzata Futyma =====<br />
* USER cloning and other work in 208 lab<br />
* Drew pictures for the [[Team:project video|project video]]<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Jakob Berg Jespersen =====<br />
* Constructed [[Team:DTU-Denmark/Protein_Models| protein models]]<br />
* Designed our t-shirts and business cards<br />
* Found sponsors and funding<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Germany_Flag.gif|30px]] Vanessa Jurtz =====<br />
* Modeled [[Team:DTU-Denmark/Kinetic_Model|kinetics of our reactions]]<br />
* Modeled a [[Team:DTU-Denmark/Reactor_Model|continuous flow reactor]]<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Greece_Flag.jpg|30px]] Anastasia Mourka =====<br />
* Wiki design and implementation<br />
* Participated in the discussion on IP and synthetic biology.<br />
<br />
===== [[File:Greece_Flag.jpg|30px]] Natalia Papargyri =====<br />
* Responsible for our [[Team:DTU-Denmark/Safety|safety]] form and section<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Italy_Flag.png|30px]] Julia Villarroel =====<br />
* USER cloning in 208 lab<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Germany_Flag.gif|30px]] Henrike Zschach =====<br />
* USER cloning in the 208 lab<br />
* Participated in high school outreach, Biobrick workshop and the discussion on IP and synthetic biology.<br />
<br />
== Assistance provided by our supervisors ==<br />
<hr/><br />
===== [[File:USA_Flag.png|30px]] Chris Workman (PI)=====<br />
* Provided perl and R scripts to analyze raw biolector data<br />
* Provided lab space for us to work in (building 208)<br />
<br />
===== [[File:Flag_of_Belgium.svg.png|30px]] Barth Smets =====<br />
* Provided the idea for our project<br />
* Provided lab space for us to work in (building 115)<br />
<br />
== Assistance provided by our advisors ==<br />
<hr/><br />
===== [[File:Denmark_Flag.gif|30px]] Thomas Trolle =====<br />
* Provided early advice on the design of the pBAD SPL<br />
* Helped during the BioBrick workshop<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Anne Mathilde Lund =====<br />
* Generously provided us with X7 polymerase and assistance with debugging PCR reactions<br />
* Arranged all experiments under the BioBrick workshop <br />
* Great help with primer design for USER cloning as with introduction on how to use the gradient PCR-machine ([https://2013.igem.org/Team:DTU-Denmark/Notebook/13_August_2013 link to notebook])<br />
* Helped in "hard to amplify" PCR situations<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Andreas Porse =====<br />
* Reviewed our initial plan and provided insight into ''E. coli'' specific signal peptides<br />
* Assisted in primer design for USER cloning i the "Hello World project"<br />
* General questions about sequencing, PCR, lab techniques etc.<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Julie Rank =====<br />
* Viewed early versions of our presentation and provided excellent feedback on how we could improve<br />
* Helped during the BioBrick workshop<br />
<br />
===== [[File:Turkey_Flag.png|30px]] Ali Altıntaş =====<br />
* Provided assistance in the 208 lab with general lab technique<br />
* Help with debugging gel pictures and methods <br />
* Kindly gave us filter sterilization units<br />
<br />
===== [[File:Flag_of_France.png|30px]] Sébastien Muller =====<br />
* Provided assistance in the 208 lab with general lab technique<br />
* Helped debugging PCR-reactions<br />
* Kindly gave us loading dye and DNA ladder<br />
<br />
== Special Thanks to ==<br />
<hr/><br />
[http://www.env.dtu.dk/english/Research/RRE/RRE-Staff/Person?id=66486&cpid=86362&tab=2&qt=dtupublicationquery| Gizem Mutlu], PhD student DTU Environmental Engineering for her help with initial calibration of the N<sub>2</sub>O and NO sensors, and for preparing the NO standard solution.<br />
<br />
Tina Johansen, Laboratory Technician DTU Systems biology for her technical help with the bioreactor set up and to [http://www.dtu.dk/Service/Telefonbog/Person?id=7254&cpid=710&tab=2&qt=dtupublicationquery| Jette Thykær], Associate Professor DTU Systems Biology for her assistance scheduling time on the bioreactor.<br />
<br />
Natalia Skawińska, who participated in the high school outreach, and provided advice on cloning in ''E. coli''.<br />
<br />
Morten Nørholm, who developed the X7 polymerase [http://www.biomedcentral.com/1472-6750/10/21].<br />
<br />
[http://www.biotechacademy.dk/biotech%20academy/omos/hjg.aspx| Hans Jasper Genee], who introduced us to [http://www.cbs.dtu.dk/services/PHUSER/ PHUSER] during our BioBrick Workshop.<br />
<br />
[http://www.cbs.dtu.dk/ Center for Biological Sequence Analysis] for letting us use one of their offices and their coffee machine!<br />
<br />
[https://2011.igem.org/Team:DTU-Denmark/How_to_customize_an_iGEM_wiki DTU iGEM 2011 Team] for their Wiki Guide, which helped in the creation of this site.<br />
<br />
[https://2009.igem.org/Team:DTU_Denmark/USERprogram DTU iGEM 2009 Team] for their software, [http://www.cbs.dtu.dk/services/PHUSER/ PHUSER 2.0], that we have used and beta tested.<br />
<br />
[[Team:UNIK_Copenhagen|University of Copenhagen iGEM 2013 Team]] and [[Team:SDU-Denmark|University of Southern Denmark iGEM 2013 Team]] for joining us for the [[Team:DTU-Denmark/Biobrick_Workshop|Biobrick Workshop]] and indian food.<br />
<br />
[[Team:SDU-Denmark|University of Southern Denmark iGEM 2013 Team]] for arranging the DK-Meet-up.<br />
<br />
== Sponsors ==<br />
<hr/><br />
{|border="0" cellspacing="30"<br />
|[[File:DTU otto.jpg|450px|left|link=http://www.ottomoensted.dk/]] ||<br />
Otto Mønsted Fonden<br />
|-<br />
|[[File:DTU_Bruun.png|200px|left|link=http://ottobruunsfond.dk/index.html]] ||<br />
Otto Bruuns Fond<br />
|-<br />
|[[File:DTU_VFL_DK_Logo_singleline_RGB.png|200px|left|link=http://www.vfl.dk/]] ||<br />
Videncenteret for Landbrug<br />
|-<br />
|[[File:DTU Brenntag.jpg|250px|left|link=http://www.brenntag-nordic.com/]] ||<br />
Brenntag<br />
|-<br />
|[[File:DTU_kruger.png|250px|left|link=http://www.kruger.dk/en/]] ||<br />
Krüger<br />
|-<br />
|[[File:DTU Systembiologi.jpg|250px|left|link=http://www.bio.dtu.dk/]] ||<br />
DTU Systems Biology<br />
|-<br />
|[[File:DTU_Environment.jpg|250px|left|link=http://www.env.dtu.dk/]] ||<br />
DTU Environment<br />
|-<br />
|[[File:DTU logo2.jpg|200px|left|link=http://www.dtu.dk/]] ||<br />
Technical University of Denmark<br />
|-<br />
|[[File:DTU clcbio.jpg|200px|left|link=http://www.clcbio.com/]] ||<br />
CLC Bio<br />
|-<br />
|[[File:NEB_Header_iGem.jpg|200px|left|link=http://www.neb-online.de]] ||<br />
New England Biolabs GmbH<br />
|-<br />
|[[File:DTU_KAILOW_UK.png|200px|left|link=http://www.kailow.dk/index_en.html]] ||<br />
Kailow<br />
|-<br />
|}<br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}<br />
<!-- {{:Team:DTU-Denmark/Templates/Footer1}} --></div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/AttributionsTeam:DTU-Denmark/Attributions2013-10-04T14:16:19Z<p>Ariadni: /* Special Thanks to */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|Attributions}}<br />
<br />
== Work done by the team ==<br />
<hr/><br />
===== [[File:Poland_Flag.gif|30px]] Katarzyna Chyzynska =====<br />
* Performed [[Team:DTU-Denmark/Experiments|characterization and verification experiments]]<br />
* Designed our poster<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Canada_Flag.jpg|30px]] Helen Cook =====<br />
* Performed and analyzed data from the [[Team:DTU-Denmark/Experiments|characterization and verification experiments]]<br />
* Created the diagrams describing our project<br />
* Participated in high school outreach, Biobrick workshop and the discussion on IP and synthetic biology.<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Kristian Davidsen =====<br />
* Lead user cloning work in the 208 lab; designed primers<br />
* Constructed the [[Team:DTU-Denmark/pBAD_SPL|pBAD_SPL]]<br />
* Found sponsors and funding<br />
* Starred in our [https://www.youtube.com/watch?v=7EiVttJpXH4 bricks of knowledge video on USER cloning]<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Greece_Flag.jpg|30px]] Ariadni Droumpali =====<br />
* Performed [[Team:DTU-Denmark/Experiments|characterization and verification experiments]]<br />
* Participated in high school outreach, Biobrick workshop and the discussion on IP and synthetic biology.<br />
<br />
===== [[File:Poland_Flag.gif|30px]] Piotr Dworzynski =====<br />
* Simulation of [[Team:DTU-Denmark/HelloWorld|GFP in the periplasm]] for Hello World project<br />
<br />
===== [[File:Poland_Flag.gif|30px]] Malgorzata Futyma =====<br />
* USER cloning and other work in 208 lab<br />
* Drew pictures for the [[Team:project video|project video]]<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Jakob Berg Jespersen =====<br />
* Constructed [[Team:DTU-Denmark/Protein_Models| protein models]]<br />
* Designed our t-shirts and business cards<br />
* Found sponsors and funding<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Germany_Flag.gif|30px]] Vanessa Jurtz =====<br />
* Modeled [[Team:DTU-Denmark/Kinetic_Model|kinetics of our reactions]]<br />
* Modeled a [[Team:DTU-Denmark/Reactor_Model|continuous flow reactor]]<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Greece_Flag.jpg|30px]] Anastasia Mourka =====<br />
* Wiki design and implementation<br />
* Participated in the discussion on IP and synthetic biology.<br />
<br />
===== [[File:Greece_Flag.jpg|30px]] Natalia Papargyri =====<br />
* Responsible for our [[Team:DTU-Denmark/Safety|safety]] form and section<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Italy_Flag.png|30px]] Julia Villarroel =====<br />
* USER cloning in 208 lab<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Germany_Flag.gif|30px]] Henrike Zschach =====<br />
* USER cloning in the 208 lab<br />
* Participated in high school outreach, Biobrick workshop and the discussion on IP and synthetic biology.<br />
<br />
== Assistance provided by our supervisors ==<br />
<hr/><br />
===== [[File:USA_Flag.png|30px]] Chris Workman (PI)=====<br />
* Provided perl and R scripts to analyze raw biolector data<br />
* Provided lab space for us to work in (building 208)<br />
<br />
===== [[File:Flag_of_Belgium.svg.png|30px]] Barth Smets =====<br />
* Provided the idea for our project<br />
* Provided lab space for us to work in (building 115)<br />
<br />
== Assistance provided by our advisors ==<br />
<hr/><br />
===== [[File:Denmark_Flag.gif|30px]] Thomas Trolle =====<br />
* Provided early advice on the design of the pBAD SPL<br />
* Helped during the BioBrick workshop<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Anne Mathilde Lund =====<br />
* Generously provided us with X7 polymerase and assistance with debugging PCR reactions<br />
* Arranged all experiments under the BioBrick workshop <br />
* Great help with primer design for USER cloning as with introduction on how to use the gradient PCR-machine ([https://2013.igem.org/Team:DTU-Denmark/Notebook/13_August_2013 link to notebook])<br />
* Helped in "hard to amplify" PCR situations<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Andreas Porse =====<br />
* Reviewed our initial plan and provided insight into ''E. coli'' specific signal peptides<br />
* Assisted in primer design for USER cloning i the "Hello World project"<br />
* General questions about sequencing, PCR, lab techniques etc.<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Julie Rank =====<br />
* Viewed early versions of our presentation and provided excellent feedback on how we could improve<br />
* Helped during the BioBrick workshop<br />
<br />
===== [[File:Turkey_Flag.png|30px]] Ali Altıntaş =====<br />
* Provided assistance in the 208 lab with general lab technique<br />
* Help with debugging gel pictures and methods <br />
* Kindly gave us filter sterilization units<br />
<br />
===== [[File:Flag_of_France.png|30px]] Sébastien Muller =====<br />
* Provided assistance in the 208 lab with general lab technique<br />
* Helped debugging PCR-reactions<br />
* Kindly gave us loading dye and DNA ladder<br />
<br />
== Special Thanks to ==<br />
<hr/><br />
[http://www.env.dtu.dk/english/Research/RRE/RRE-Staff/Person?id=66486&cpid=86362&tab=2&qt=dtupublicationquery| Gizem Mutlu], PhD student DTU Environmental Engineering for her help with initial calibration of the N<sub>2</sub>O and NO sensors, and for preparing the NO standard solution.<br />
<br />
Tina Johansen, Laboratory Technician DTU Systems biology for her technical help with the bioreactor set up and to [http://www.dtu.dk/Service/Telefonbog/Person?id=7254&cpid=710&tab=2&qt=dtupublicationquery| Jette Thykær], Associate Professor DTU Systems Biology for her assistance scheduling time on the bioreactor.<br />
<br />
Natalia Skawińska, who participated in the high school outreach, and provided advice on cloning in ''E. coli''.<br />
<br />
Morten Nørholm, who developed the X7 polymerase [http://www.biomedcentral.com/1472-6750/10/21].<br />
<br />
Hans Jasper Genee, who introduced us to [http://www.cbs.dtu.dk/services/PHUSER/ PHUSER] during our BioBrick Workshop.<br />
<br />
[http://www.cbs.dtu.dk/ Center for Biological Sequence Analysis] for letting us use one of their offices and their coffee machine!<br />
<br />
[https://2011.igem.org/Team:DTU-Denmark/How_to_customize_an_iGEM_wiki DTU iGEM 2011 Team] for their Wiki Guide, which helped in the creation of this site.<br />
<br />
[https://2009.igem.org/Team:DTU_Denmark/USERprogram DTU iGEM 2009 Team] for their software, [http://www.cbs.dtu.dk/services/PHUSER/ PHUSER 2.0], that we have used and beta tested.<br />
<br />
[[Team:UNIK_Copenhagen|University of Copenhagen iGEM 2013 Team]] and [[Team:SDU-Denmark|University of Southern Denmark iGEM 2013 Team]] for joining us for the [[Team:DTU-Denmark/Biobrick_Workshop|Biobrick Workshop]] and indian food.<br />
<br />
[[Team:SDU-Denmark|University of Southern Denmark iGEM 2013 Team]] for arranging the DK-Meet-up.<br />
<br />
== Sponsors ==<br />
<hr/><br />
{|border="0" cellspacing="30"<br />
|[[File:DTU otto.jpg|450px|left|link=http://www.ottomoensted.dk/]] ||<br />
Otto Mønsted Fonden<br />
|-<br />
|[[File:DTU_Bruun.png|200px|left|link=http://ottobruunsfond.dk/index.html]] ||<br />
Otto Bruuns Fond<br />
|-<br />
|[[File:DTU_VFL_DK_Logo_singleline_RGB.png|200px|left|link=http://www.vfl.dk/]] ||<br />
Videncenteret for Landbrug<br />
|-<br />
|[[File:DTU Brenntag.jpg|250px|left|link=http://www.brenntag-nordic.com/]] ||<br />
Brenntag<br />
|-<br />
|[[File:DTU_kruger.png|250px|left|link=http://www.kruger.dk/en/]] ||<br />
Krüger<br />
|-<br />
|[[File:DTU Systembiologi.jpg|250px|left|link=http://www.bio.dtu.dk/]] ||<br />
DTU Systems Biology<br />
|-<br />
|[[File:DTU_Environment.jpg|250px|left|link=http://www.env.dtu.dk/]] ||<br />
DTU Environment<br />
|-<br />
|[[File:DTU logo2.jpg|200px|left|link=http://www.dtu.dk/]] ||<br />
Technical University of Denmark<br />
|-<br />
|[[File:DTU clcbio.jpg|200px|left|link=http://www.clcbio.com/]] ||<br />
CLC Bio<br />
|-<br />
|[[File:NEB_Header_iGem.jpg|200px|left|link=http://www.neb-online.de]] ||<br />
New England Biolabs GmbH<br />
|-<br />
|[[File:DTU_KAILOW_UK.png|200px|left|link=http://www.kailow.dk/index_en.html]] ||<br />
Kailow<br />
|-<br />
|}<br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}<br />
<!-- {{:Team:DTU-Denmark/Templates/Footer1}} --></div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/AttributionsTeam:DTU-Denmark/Attributions2013-10-04T14:15:57Z<p>Ariadni: /* Special Thanks to */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|Attributions}}<br />
<br />
== Work done by the team ==<br />
<hr/><br />
===== [[File:Poland_Flag.gif|30px]] Katarzyna Chyzynska =====<br />
* Performed [[Team:DTU-Denmark/Experiments|characterization and verification experiments]]<br />
* Designed our poster<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Canada_Flag.jpg|30px]] Helen Cook =====<br />
* Performed and analyzed data from the [[Team:DTU-Denmark/Experiments|characterization and verification experiments]]<br />
* Created the diagrams describing our project<br />
* Participated in high school outreach, Biobrick workshop and the discussion on IP and synthetic biology.<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Kristian Davidsen =====<br />
* Lead user cloning work in the 208 lab; designed primers<br />
* Constructed the [[Team:DTU-Denmark/pBAD_SPL|pBAD_SPL]]<br />
* Found sponsors and funding<br />
* Starred in our [https://www.youtube.com/watch?v=7EiVttJpXH4 bricks of knowledge video on USER cloning]<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Greece_Flag.jpg|30px]] Ariadni Droumpali =====<br />
* Performed [[Team:DTU-Denmark/Experiments|characterization and verification experiments]]<br />
* Participated in high school outreach, Biobrick workshop and the discussion on IP and synthetic biology.<br />
<br />
===== [[File:Poland_Flag.gif|30px]] Piotr Dworzynski =====<br />
* Simulation of [[Team:DTU-Denmark/HelloWorld|GFP in the periplasm]] for Hello World project<br />
<br />
===== [[File:Poland_Flag.gif|30px]] Malgorzata Futyma =====<br />
* USER cloning and other work in 208 lab<br />
* Drew pictures for the [[Team:project video|project video]]<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Jakob Berg Jespersen =====<br />
* Constructed [[Team:DTU-Denmark/Protein_Models| protein models]]<br />
* Designed our t-shirts and business cards<br />
* Found sponsors and funding<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Germany_Flag.gif|30px]] Vanessa Jurtz =====<br />
* Modeled [[Team:DTU-Denmark/Kinetic_Model|kinetics of our reactions]]<br />
* Modeled a [[Team:DTU-Denmark/Reactor_Model|continuous flow reactor]]<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Greece_Flag.jpg|30px]] Anastasia Mourka =====<br />
* Wiki design and implementation<br />
* Participated in the discussion on IP and synthetic biology.<br />
<br />
===== [[File:Greece_Flag.jpg|30px]] Natalia Papargyri =====<br />
* Responsible for our [[Team:DTU-Denmark/Safety|safety]] form and section<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Italy_Flag.png|30px]] Julia Villarroel =====<br />
* USER cloning in 208 lab<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Germany_Flag.gif|30px]] Henrike Zschach =====<br />
* USER cloning in the 208 lab<br />
* Participated in high school outreach, Biobrick workshop and the discussion on IP and synthetic biology.<br />
<br />
== Assistance provided by our supervisors ==<br />
<hr/><br />
===== [[File:USA_Flag.png|30px]] Chris Workman (PI)=====<br />
* Provided perl and R scripts to analyze raw biolector data<br />
* Provided lab space for us to work in (building 208)<br />
<br />
===== [[File:Flag_of_Belgium.svg.png|30px]] Barth Smets =====<br />
* Provided the idea for our project<br />
* Provided lab space for us to work in (building 115)<br />
<br />
== Assistance provided by our advisors ==<br />
<hr/><br />
===== [[File:Denmark_Flag.gif|30px]] Thomas Trolle =====<br />
* Provided early advice on the design of the pBAD SPL<br />
* Helped during the BioBrick workshop<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Anne Mathilde Lund =====<br />
* Generously provided us with X7 polymerase and assistance with debugging PCR reactions<br />
* Arranged all experiments under the BioBrick workshop <br />
* Great help with primer design for USER cloning as with introduction on how to use the gradient PCR-machine ([https://2013.igem.org/Team:DTU-Denmark/Notebook/13_August_2013 link to notebook])<br />
* Helped in "hard to amplify" PCR situations<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Andreas Porse =====<br />
* Reviewed our initial plan and provided insight into ''E. coli'' specific signal peptides<br />
* Assisted in primer design for USER cloning i the "Hello World project"<br />
* General questions about sequencing, PCR, lab techniques etc.<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Julie Rank =====<br />
* Viewed early versions of our presentation and provided excellent feedback on how we could improve<br />
* Helped during the BioBrick workshop<br />
<br />
===== [[File:Turkey_Flag.png|30px]] Ali Altıntaş =====<br />
* Provided assistance in the 208 lab with general lab technique<br />
* Help with debugging gel pictures and methods <br />
* Kindly gave us filter sterilization units<br />
<br />
===== [[File:Flag_of_France.png|30px]] Sébastien Muller =====<br />
* Provided assistance in the 208 lab with general lab technique<br />
* Helped debugging PCR-reactions<br />
* Kindly gave us loading dye and DNA ladder<br />
<br />
== Special Thanks to ==<br />
<hr/><br />
[http://www.env.dtu.dk/english/Research/RRE/RRE-Staff/Person?id=66486&cpid=86362&tab=2&qt=dtupublicationquery| Gizem Mutlu], PhD student DTU Environmental Engineering for her help with initial calibration of the N<sub>2</sub>O and NO sensors, and for preparing the NO standard solution.<br />
<br />
Tina Johansen, Laboratory Technician DTU Systems biology for her technical help with the bioreactor set up and to [http://www.dtu.dk/Service/Telefonbog/Person?id=7254&cpid=710&tab=2&qt=dtupublicationquery|Jette Thykær], Associate Professor DTU Systems Biology for her assistance scheduling time on the bioreactor.<br />
<br />
Natalia Skawińska, who participated in the high school outreach, and provided advice on cloning in ''E. coli''.<br />
<br />
Morten Nørholm, who developed the X7 polymerase [http://www.biomedcentral.com/1472-6750/10/21].<br />
<br />
Hans Jasper Genee, who introduced us to [http://www.cbs.dtu.dk/services/PHUSER/ PHUSER] during our BioBrick Workshop.<br />
<br />
[http://www.cbs.dtu.dk/ Center for Biological Sequence Analysis] for letting us use one of their offices and their coffee machine!<br />
<br />
[https://2011.igem.org/Team:DTU-Denmark/How_to_customize_an_iGEM_wiki DTU iGEM 2011 Team] for their Wiki Guide, which helped in the creation of this site.<br />
<br />
[https://2009.igem.org/Team:DTU_Denmark/USERprogram DTU iGEM 2009 Team] for their software, [http://www.cbs.dtu.dk/services/PHUSER/ PHUSER 2.0], that we have used and beta tested.<br />
<br />
[[Team:UNIK_Copenhagen|University of Copenhagen iGEM 2013 Team]] and [[Team:SDU-Denmark|University of Southern Denmark iGEM 2013 Team]] for joining us for the [[Team:DTU-Denmark/Biobrick_Workshop|Biobrick Workshop]] and indian food.<br />
<br />
[[Team:SDU-Denmark|University of Southern Denmark iGEM 2013 Team]] for arranging the DK-Meet-up.<br />
<br />
== Sponsors ==<br />
<hr/><br />
{|border="0" cellspacing="30"<br />
|[[File:DTU otto.jpg|450px|left|link=http://www.ottomoensted.dk/]] ||<br />
Otto Mønsted Fonden<br />
|-<br />
|[[File:DTU_Bruun.png|200px|left|link=http://ottobruunsfond.dk/index.html]] ||<br />
Otto Bruuns Fond<br />
|-<br />
|[[File:DTU_VFL_DK_Logo_singleline_RGB.png|200px|left|link=http://www.vfl.dk/]] ||<br />
Videncenteret for Landbrug<br />
|-<br />
|[[File:DTU Brenntag.jpg|250px|left|link=http://www.brenntag-nordic.com/]] ||<br />
Brenntag<br />
|-<br />
|[[File:DTU_kruger.png|250px|left|link=http://www.kruger.dk/en/]] ||<br />
Krüger<br />
|-<br />
|[[File:DTU Systembiologi.jpg|250px|left|link=http://www.bio.dtu.dk/]] ||<br />
DTU Systems Biology<br />
|-<br />
|[[File:DTU_Environment.jpg|250px|left|link=http://www.env.dtu.dk/]] ||<br />
DTU Environment<br />
|-<br />
|[[File:DTU logo2.jpg|200px|left|link=http://www.dtu.dk/]] ||<br />
Technical University of Denmark<br />
|-<br />
|[[File:DTU clcbio.jpg|200px|left|link=http://www.clcbio.com/]] ||<br />
CLC Bio<br />
|-<br />
|[[File:NEB_Header_iGem.jpg|200px|left|link=http://www.neb-online.de]] ||<br />
New England Biolabs GmbH<br />
|-<br />
|[[File:DTU_KAILOW_UK.png|200px|left|link=http://www.kailow.dk/index_en.html]] ||<br />
Kailow<br />
|-<br />
|}<br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}<br />
<!-- {{:Team:DTU-Denmark/Templates/Footer1}} --></div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/AttributionsTeam:DTU-Denmark/Attributions2013-10-04T14:14:52Z<p>Ariadni: /* Special Thanks to */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|Attributions}}<br />
<br />
== Work done by the team ==<br />
<hr/><br />
===== [[File:Poland_Flag.gif|30px]] Katarzyna Chyzynska =====<br />
* Performed [[Team:DTU-Denmark/Experiments|characterization and verification experiments]]<br />
* Designed our poster<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Canada_Flag.jpg|30px]] Helen Cook =====<br />
* Performed and analyzed data from the [[Team:DTU-Denmark/Experiments|characterization and verification experiments]]<br />
* Created the diagrams describing our project<br />
* Participated in high school outreach, Biobrick workshop and the discussion on IP and synthetic biology.<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Kristian Davidsen =====<br />
* Lead user cloning work in the 208 lab; designed primers<br />
* Constructed the [[Team:DTU-Denmark/pBAD_SPL|pBAD_SPL]]<br />
* Found sponsors and funding<br />
* Starred in our [https://www.youtube.com/watch?v=7EiVttJpXH4 bricks of knowledge video on USER cloning]<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Greece_Flag.jpg|30px]] Ariadni Droumpali =====<br />
* Performed [[Team:DTU-Denmark/Experiments|characterization and verification experiments]]<br />
* Participated in high school outreach, Biobrick workshop and the discussion on IP and synthetic biology.<br />
<br />
===== [[File:Poland_Flag.gif|30px]] Piotr Dworzynski =====<br />
* Simulation of [[Team:DTU-Denmark/HelloWorld|GFP in the periplasm]] for Hello World project<br />
<br />
===== [[File:Poland_Flag.gif|30px]] Malgorzata Futyma =====<br />
* USER cloning and other work in 208 lab<br />
* Drew pictures for the [[Team:project video|project video]]<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Jakob Berg Jespersen =====<br />
* Constructed [[Team:DTU-Denmark/Protein_Models| protein models]]<br />
* Designed our t-shirts and business cards<br />
* Found sponsors and funding<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Germany_Flag.gif|30px]] Vanessa Jurtz =====<br />
* Modeled [[Team:DTU-Denmark/Kinetic_Model|kinetics of our reactions]]<br />
* Modeled a [[Team:DTU-Denmark/Reactor_Model|continuous flow reactor]]<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Greece_Flag.jpg|30px]] Anastasia Mourka =====<br />
* Wiki design and implementation<br />
* Participated in the discussion on IP and synthetic biology.<br />
<br />
===== [[File:Greece_Flag.jpg|30px]] Natalia Papargyri =====<br />
* Responsible for our [[Team:DTU-Denmark/Safety|safety]] form and section<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Italy_Flag.png|30px]] Julia Villarroel =====<br />
* USER cloning in 208 lab<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Germany_Flag.gif|30px]] Henrike Zschach =====<br />
* USER cloning in the 208 lab<br />
* Participated in high school outreach, Biobrick workshop and the discussion on IP and synthetic biology.<br />
<br />
== Assistance provided by our supervisors ==<br />
<hr/><br />
===== [[File:USA_Flag.png|30px]] Chris Workman (PI)=====<br />
* Provided perl and R scripts to analyze raw biolector data<br />
* Provided lab space for us to work in (building 208)<br />
<br />
===== [[File:Flag_of_Belgium.svg.png|30px]] Barth Smets =====<br />
* Provided the idea for our project<br />
* Provided lab space for us to work in (building 115)<br />
<br />
== Assistance provided by our advisors ==<br />
<hr/><br />
===== [[File:Denmark_Flag.gif|30px]] Thomas Trolle =====<br />
* Provided early advice on the design of the pBAD SPL<br />
* Helped during the BioBrick workshop<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Anne Mathilde Lund =====<br />
* Generously provided us with X7 polymerase and assistance with debugging PCR reactions<br />
* Arranged all experiments under the BioBrick workshop <br />
* Great help with primer design for USER cloning as with introduction on how to use the gradient PCR-machine ([https://2013.igem.org/Team:DTU-Denmark/Notebook/13_August_2013 link to notebook])<br />
* Helped in "hard to amplify" PCR situations<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Andreas Porse =====<br />
* Reviewed our initial plan and provided insight into ''E. coli'' specific signal peptides<br />
* Assisted in primer design for USER cloning i the "Hello World project"<br />
* General questions about sequencing, PCR, lab techniques etc.<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Julie Rank =====<br />
* Viewed early versions of our presentation and provided excellent feedback on how we could improve<br />
* Helped during the BioBrick workshop<br />
<br />
===== [[File:Turkey_Flag.png|30px]] Ali Altıntaş =====<br />
* Provided assistance in the 208 lab with general lab technique<br />
* Help with debugging gel pictures and methods <br />
* Kindly gave us filter sterilization units<br />
<br />
===== [[File:Flag_of_France.png|30px]] Sébastien Muller =====<br />
* Provided assistance in the 208 lab with general lab technique<br />
* Helped debugging PCR-reactions<br />
* Kindly gave us loading dye and DNA ladder<br />
<br />
== Special Thanks to ==<br />
<hr/><br />
[http://www.env.dtu.dk/english/Research/RRE/RRE-Staff/Person?id=66486&cpid=86362&tab=2&qt=dtupublicationquery| Gizem Mutlu], PhD student DTU Environmental Engineering for her help with initial calibration of the N<sub>2</sub>O and NO sensors, and for preparing the NO standard solution.<br />
<br />
Tina Johansen, Laboratory Technician DTU Systems biology for her technical help with the bioreactor set up and to Jette Thykær, Associate Professor DTU Systems Biology for her assistance scheduling time on the bioreactor.<br />
<br />
Natalia Skawińska, who participated in the high school outreach, and provided advice on cloning in ''E. coli''.<br />
<br />
Morten Nørholm, who developed the X7 polymerase [http://www.biomedcentral.com/1472-6750/10/21].<br />
<br />
Hans Jasper Genee, who introduced us to [http://www.cbs.dtu.dk/services/PHUSER/ PHUSER] during our BioBrick Workshop.<br />
<br />
[http://www.cbs.dtu.dk/ Center for Biological Sequence Analysis] for letting us use one of their offices and their coffee machine!<br />
<br />
[https://2011.igem.org/Team:DTU-Denmark/How_to_customize_an_iGEM_wiki DTU iGEM 2011 Team] for their Wiki Guide, which helped in the creation of this site.<br />
<br />
[https://2009.igem.org/Team:DTU_Denmark/USERprogram DTU iGEM 2009 Team] for their software, [http://www.cbs.dtu.dk/services/PHUSER/ PHUSER 2.0], that we have used and beta tested.<br />
<br />
[[Team:UNIK_Copenhagen|University of Copenhagen iGEM 2013 Team]] and [[Team:SDU-Denmark|University of Southern Denmark iGEM 2013 Team]] for joining us for the [[Team:DTU-Denmark/Biobrick_Workshop|Biobrick Workshop]] and indian food.<br />
<br />
[[Team:SDU-Denmark|University of Southern Denmark iGEM 2013 Team]] for arranging the DK-Meet-up.<br />
<br />
== Sponsors ==<br />
<hr/><br />
{|border="0" cellspacing="30"<br />
|[[File:DTU otto.jpg|450px|left|link=http://www.ottomoensted.dk/]] ||<br />
Otto Mønsted Fonden<br />
|-<br />
|[[File:DTU_Bruun.png|200px|left|link=http://ottobruunsfond.dk/index.html]] ||<br />
Otto Bruuns Fond<br />
|-<br />
|[[File:DTU_VFL_DK_Logo_singleline_RGB.png|200px|left|link=http://www.vfl.dk/]] ||<br />
Videncenteret for Landbrug<br />
|-<br />
|[[File:DTU Brenntag.jpg|250px|left|link=http://www.brenntag-nordic.com/]] ||<br />
Brenntag<br />
|-<br />
|[[File:DTU_kruger.png|250px|left|link=http://www.kruger.dk/en/]] ||<br />
Krüger<br />
|-<br />
|[[File:DTU Systembiologi.jpg|250px|left|link=http://www.bio.dtu.dk/]] ||<br />
DTU Systems Biology<br />
|-<br />
|[[File:DTU_Environment.jpg|250px|left|link=http://www.env.dtu.dk/]] ||<br />
DTU Environment<br />
|-<br />
|[[File:DTU logo2.jpg|200px|left|link=http://www.dtu.dk/]] ||<br />
Technical University of Denmark<br />
|-<br />
|[[File:DTU clcbio.jpg|200px|left|link=http://www.clcbio.com/]] ||<br />
CLC Bio<br />
|-<br />
|[[File:NEB_Header_iGem.jpg|200px|left|link=http://www.neb-online.de]] ||<br />
New England Biolabs GmbH<br />
|-<br />
|[[File:DTU_KAILOW_UK.png|200px|left|link=http://www.kailow.dk/index_en.html]] ||<br />
Kailow<br />
|-<br />
|}<br />
<br />
<br />
{{:Team:DTU-Denmark/Templates/EndPage}}<br />
<!-- {{:Team:DTU-Denmark/Templates/Footer1}} --></div>Ariadnihttp://2013.igem.org/Team:DTU-Denmark/AttributionsTeam:DTU-Denmark/Attributions2013-10-04T14:13:17Z<p>Ariadni: /* Special Thanks to */</p>
<hr />
<div>{{:Team:DTU-Denmark/Templates/StartPage|Attributions}}<br />
<br />
== Work done by the team ==<br />
<hr/><br />
===== [[File:Poland_Flag.gif|30px]] Katarzyna Chyzynska =====<br />
* Performed [[Team:DTU-Denmark/Experiments|characterization and verification experiments]]<br />
* Designed our poster<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Canada_Flag.jpg|30px]] Helen Cook =====<br />
* Performed and analyzed data from the [[Team:DTU-Denmark/Experiments|characterization and verification experiments]]<br />
* Created the diagrams describing our project<br />
* Participated in high school outreach, Biobrick workshop and the discussion on IP and synthetic biology.<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Kristian Davidsen =====<br />
* Lead user cloning work in the 208 lab; designed primers<br />
* Constructed the [[Team:DTU-Denmark/pBAD_SPL|pBAD_SPL]]<br />
* Found sponsors and funding<br />
* Starred in our [https://www.youtube.com/watch?v=7EiVttJpXH4 bricks of knowledge video on USER cloning]<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Greece_Flag.jpg|30px]] Ariadni Droumpali =====<br />
* Performed [[Team:DTU-Denmark/Experiments|characterization and verification experiments]]<br />
* Participated in high school outreach, Biobrick workshop and the discussion on IP and synthetic biology.<br />
<br />
===== [[File:Poland_Flag.gif|30px]] Piotr Dworzynski =====<br />
* Simulation of [[Team:DTU-Denmark/HelloWorld|GFP in the periplasm]] for Hello World project<br />
<br />
===== [[File:Poland_Flag.gif|30px]] Malgorzata Futyma =====<br />
* USER cloning and other work in 208 lab<br />
* Drew pictures for the [[Team:project video|project video]]<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Jakob Berg Jespersen =====<br />
* Constructed [[Team:DTU-Denmark/Protein_Models| protein models]]<br />
* Designed our t-shirts and business cards<br />
* Found sponsors and funding<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Germany_Flag.gif|30px]] Vanessa Jurtz =====<br />
* Modeled [[Team:DTU-Denmark/Kinetic_Model|kinetics of our reactions]]<br />
* Modeled a [[Team:DTU-Denmark/Reactor_Model|continuous flow reactor]]<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Greece_Flag.jpg|30px]] Anastasia Mourka =====<br />
* Wiki design and implementation<br />
* Participated in the discussion on IP and synthetic biology.<br />
<br />
===== [[File:Greece_Flag.jpg|30px]] Natalia Papargyri =====<br />
* Responsible for our [[Team:DTU-Denmark/Safety|safety]] form and section<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Italy_Flag.png|30px]] Julia Villarroel =====<br />
* USER cloning in 208 lab<br />
* Participated in high school outreach and the Biobrick workshop<br />
<br />
===== [[File:Germany_Flag.gif|30px]] Henrike Zschach =====<br />
* USER cloning in the 208 lab<br />
* Participated in high school outreach, Biobrick workshop and the discussion on IP and synthetic biology.<br />
<br />
== Assistance provided by our supervisors ==<br />
<hr/><br />
===== [[File:USA_Flag.png|30px]] Chris Workman (PI)=====<br />
* Provided perl and R scripts to analyze raw biolector data<br />
* Provided lab space for us to work in (building 208)<br />
<br />
===== [[File:Flag_of_Belgium.svg.png|30px]] Barth Smets =====<br />
* Provided the idea for our project<br />
* Provided lab space for us to work in (building 115)<br />
<br />
== Assistance provided by our advisors ==<br />
<hr/><br />
===== [[File:Denmark_Flag.gif|30px]] Thomas Trolle =====<br />
* Provided early advice on the design of the pBAD SPL<br />
* Helped during the BioBrick workshop<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Anne Mathilde Lund =====<br />
* Generously provided us with X7 polymerase and assistance with debugging PCR reactions<br />
* Arranged all experiments under the BioBrick workshop <br />
* Great help with primer design for USER cloning as with introduction on how to use the gradient PCR-machine ([https://2013.igem.org/Team:DTU-Denmark/Notebook/13_August_2013 link to notebook])<br />
* Helped in "hard to amplify" PCR situations<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Andreas Porse =====<br />
* Reviewed our initial plan and provided insight into ''E. coli'' specific signal peptides<br />
* Assisted in primer design for USER cloning i the "Hello World project"<br />
* General questions about sequencing, PCR, lab techniques etc.<br />
<br />
===== [[File:Denmark_Flag.gif|30px]] Julie Rank =====<br />
* Viewed early versions of our presentation and provided excellent feedback on how we could improve<br />
* Helped during the BioBrick workshop<br />
<br />
===== [[File:Turkey_Flag.png|30px]] Ali Altıntaş =====<br />
* Provided assistance in the 208 lab with general lab technique<br />
* Help with debugging gel pictures and methods <br />
* Kindly gave us filter sterilization units<br />
<br />
===== [[File:Flag_of_France.png|30px]] Sébastien Muller =====<br />
* Provided assistance in the 208 lab with general lab technique<br />
* Helped debugging PCR-reactions<br />
* Kindly gave us loading dye and DNA ladder<br />
<br />
== Special Thanks to ==<br />
<hr/><br />
[http://www.env.dtu.dk/english/Research/RRE/RRE-Staff/Person?id=66486&cpid=86362&tab=2&qt=dtupublicationquery| Gizem Mutlu] , PhD student DTU Environmental Engineering for her help with initial calibration of the N<sub>2</sub>O and NO sensors, and for preparing the NO standard solution.<br />
<br />
Tina Johansen, Laboratory Technician DTU Systems biology for her technical help with the bioreactor set up and to Jette Thykær, Associate Professor DTU Systems Biology for her assistance scheduling time on the bioreactor.<br />
<br />
Natalia Skawińska, who participated in the high school outreach, and provided advice on cloning in ''E. coli''.<br />
<br />
Morten Nørholm, who developed the X7 polymerase [http://www.biomedcentral.com/1472-6750/10/21].<br />
<br />
Hans Jasper Genee, who introduced us to [http://www.cbs.dtu.dk/services/PHUSER/ PHUSER] during our BioBrick Workshop.<br />
<br />
[http://www.cbs.dtu.dk/ Center for Biological Sequence Analysis] for letting us use one of their offices and their coffee machine!<br />
<br />
[https://2011.igem.org/Team:DTU-Denmark/How_to_customize_an_iGEM_wiki DTU iGEM 2011 Team] for their Wiki Guide, which helped in the creation of this site.<br />
<br />
[https://2009.igem.org/Team:DTU_Denmark/USERprogram DTU iGEM 2009 Team] for their software, [http://www.cbs.dtu.dk/services/PHUSER/ PHUSER 2.0], that we have used and beta tested.<br />
<br />
[[Team:UNIK_Copenhagen|University of Copenhagen iGEM 2013 Team]] and [[Team:SDU-Denmark|University of Southern Denmark iGEM 2013 Team]] for joining us for the [[Team:DTU-Denmark/Biobrick_Workshop|Biobrick Workshop]] and indian food.<br />
<br />
[[Team:SDU-Denmark|University of Southern Denmark iGEM 2013 Team]] for arranging the DK-Meet-up.<br />
<br />
== Sponsors ==<br />
<hr/><br />
{|border="0" cellspacing="30"<br />
|[[File:DTU otto.jpg|450px|left|link=http://www.ottomoensted.dk/]] ||<br />
Otto Mønsted Fonden<br />
|-<br />
|[[File:DTU_Bruun.png|200px|left|link=http://ottobruunsfond.dk/index.html]] ||<br />
Otto Bruuns Fond<br />
|-<br />
|[[File:DTU_VFL_DK_Logo_singleline_RGB.png|200px|left|link=http://www.vfl.dk/]] ||<br />
Videncenteret for Landbrug<br />
|-<br />
|[[File:DTU Brenntag.jpg|250px|left|link=http://www.brenntag-nordic.com/]] ||<br />
Brenntag<br />
|-<br />
|[[File:DTU_kruger.png|250px|left|link=http://www.kruger.dk/en/]] ||<br />
Krüger<br />
|-<br />
|[[File:DTU Systembiologi.jpg|250px|left|link=http://www.bio.dtu.dk/]] ||<br />
DTU Systems Biology<br />
|-<br />
|[[File:DTU_Environment.jpg|250px|left|link=http://www.env.dtu.dk/]] ||<br />
DTU Environment<br />
|-<br />
|[[File:DTU logo2.jpg|200px|left|link=http://www.dtu.dk/]] ||<br />
Technical University of Denmark<br />
|-<br />
|[[File:DTU clcbio.jpg|200px|left|link=http://www.clcbio.com/]] ||<br />
CLC Bio<br />
|-<br />
|[[File:NEB_Header_iGem.jpg|200px|left|link=http://www.neb-online.de]] ||<br />
New England Biolabs GmbH<br />
|-<br />
|[[File:DTU_KAILOW_UK.png|200px|left|link=http://www.kailow.dk/index_en.html]] ||<br />
Kailow<br />
|-<br />
|}<br />
<br />
<br />
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