Team:Bielefeld-Germany/Test

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<a href="#1"><strong>Chemical Waste Water</strong>
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<a href="#2"><strong>Our Focus</strong>
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<a href="#3"><strong>Laccase - Our ??</strong>
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<a href="#4"><strong>Laccase-Donators</strong>
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<li><a href="#1"><strong>Woche 2</strong></a></li>
 
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<li><a href="#3"><strong>Woche 3</strong></a></li>
 
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<li><a href="#4"><strong>Woche 4</strong></a></li>
 
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<li><a href="#5"><strong>Woche 5</strong></a></li>
 
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<li><a href="#1"><strong>Woche 6</strong></a></li>
 
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<li><a href="#2"><strong>Woche 7</strong></a></li>
 
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<li><a href="#3"><strong>Woche 8</strong></a></li>
 
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<li><a href="#4"><strong>Woche 9</strong></a></li>
 
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                <li><a href="#4"><strong>Woche 10</strong></a></li>
 
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<div><h3 style="text-decoration:none; color:black;">Summary</h3>
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Beginning in January and February members of the former iGEM team from Bielefeld started seminars to inform interested students about synthetic biology, iGEM and the past Bielefeld projects. In March the final 2012 iGEM Bielefeld team was formed of 15 students and weekly meetings began. Our team was established and it was time to find a suitable project. The first weekly meeting were more like big group brainstorming and we discussed idea, which in some cases were totally different from each other. Everyone had to inform about ideas of others so that, in the end, we all could discuss together. First project ideas were:
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<h3>Chemical waste in water?!</h3>
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  <li>the detection of multiresistent pathogens</li>
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  <li>communication between bacteria and fungi using quorum sensing</li>
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  <li>a bacterial hand warmer</li>
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  <li>a possibility to detect and destroy mold fungus</li>
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  <li>something about spontaneous combustion of hay bale</li>
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  <li>an enzyme dispenser</li>
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After some reports in media and press about the environmental effects of steroid hormones, we decided to go for hormones. From the beginning our aim was not to detect but to degrade hormones. We found several possible ways for degradation as there are the hydrolysis of estradiol-derivates with sufatases and glucoronidases. But we thought the best way to degrade steroid hormones would be with the use of laccases. Laccases have the ability to radicalize aromatic rings and can therefore be used to degrade or polymerize a broad range of substances, such as steroid hormones, special insecticides, polycyclic aromatic carbohydrates and aromatic acids. In nature laccases are often used for degradation or polymerization of lignin or pigments.
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=== Monday April 30th ===
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Water is the source of all life and covers 71% of the Earth’s surface.  Healthy drinking water is an important aspect and essential  for mankind. However, the growing industrialization, the production of chemical agents and the increasing consumption of pharmaceuticals is among the causes of the ever increasing pressure on the aquatic environment and on the availability and quality of safe and clean water.  The continuous release of pharmaceuticals into the environment and the proven effects on biological systems. The measured environmental concentrations cause that water pollution is one of the main environmental worries of our society. According to a survey of March 2012 of the Public Opinion Analysis sector of the [http://www.europarl.europa.eu/meetdocs/2009_2014/documents/envi/pr/909/909091/909091en.pdf European Commission],
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* '''Team Student Academy:''' We got the chance to organize one part of the first school academy “synthetic biology/ biotechnology” at the CeBiTec of University Bielefeld by arranging experiments for the pupils and by presenting us and the iGEM competition. For the experimental part our general idea was to give them an understanding of principle methods in biotechnology / synthetic biology by using fluorescent proteins. We planned the following experiments:
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* 68 % of Europeans think that water quantity and quality problems are serious.
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** Plasmid isolation of RFP/GFP from a liquid culture.
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* 80 % believe that chemical pollution is a threat to the water environment.
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** Transformation of a plasmid mixture consisting of two different fluorescent proteins (e.g. RFP and GFP) and different antibiotic resistances into ''E.coli'' KRX. It will be plated out on LB agar plates without antibiotics and on plates containing one of the two antibiotics, which are present on the plasmids. This way we can demonstrate the effect of antibiotics as selective pressure.  
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* 62 % feel that they are not sufficiently informed about problems facing groundwater, lakes, rivers and coastal waters in their countries.
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But which chemical and pharmaceutical agents are detected in the surface water?
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</p>
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<p align="justify">
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The birth control pill is a widespread contraception method. However, large amounts of these modified estrogens leave the body again in urine. According to the Federal Environment Agency in Germany (Umwelt Bundes Amt-UBA) several hundred tons of analgetics, antibiotics, beta blockers, X-ray contrast agents, anti-epileptic drugs, poly aromatic hydrocarbons pesticides ‘’etc.’’  get into the waste water through various ways, too, and in the end finds its way into rivers, lakes and in the drinking water.  
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* '''Team Bacterial Laccases:''' Before our lab time started we send requests for different plasmids to working groups, which have already worked with laccases we are interested in. Sadly just one working group responded to us. We got answer for a vector with the laccase-ORF [http://www.ncbi.nlm.nih.gov/protein/194015788 CotA] from ''Bacillus pumilus ATCC7061'' and a ampicillin resistance from the Swiss Federal Laboratories for Materials Science and Technology, Laboratory for Biomaterials in Switzerland. They promised to send us the plasmid pBpL6. [http://www.biomedcentral.com/1472-6750/11/9 More information...]
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[[File:Bielefeld2012_Wastewater.jpg|400px|left|thumb|Different sources of micro-contaminants]]
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:In a paper we found the research group worked with a laccase [http://www.ncbi.nlm.nih.gov/protein/21230052 CopA] from ''Xanthomonas pv. campestris ATCC33913''. Luckily the sequence of this laccase is the same in ''Xanthomonas campestris pv. campestris B100'' which we got from a working group at our university. The same thing with a laccase from ''E. coli''. We found papers which described the laccase [http://www.ncbi.nlm.nih.gov/protein/85674340 CueO] from ''E. coli W3110''. After blasting this laccase we found out that ''E. coli BL21(DE3)'' has this laccase, too. We decided to isolate the laccase from ''E. coli BL21(DE3)''.
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:* Generating new competent ''E.coli KRX cells''.
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</p>
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:* Cultivation of ''Xanthomonas campestris B100'' and ''E. coli BL21(DE3)''. The bacterial strains we got from a working group at our University. After cultivation we isolated the genomic DNA. The DNA was needed as template for PCRs to purify the wanted laccase ORFs.
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<p align="justify">
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:* Primer design for isolation of laccases from genomic DNA of ''Xanthomonas campestris B100'' and ''E. coli BL21(DE3)'' and for isolation of CotA from ''Bacillus pumilus ATCC7061'' from plasmid. The forward primers were designed with T7 promotor, RBS  and the first 20 bases of the wanted gene after prefix. The reverse primers were designed with the last 20 bases of the wanted gene without the stop codon, a HIS-Tag, two stop codons and suffix sequence. [https://2012.igem.org/Team:Bielefeld-Germany/Protocols#Primers Primers]: Xcc_LAC_FW_T7, Xcc_LAC_RV_HIS, E.coli_LAC_FW_T7, E.coli_LAC_RV_HIS, B.pumi_LAC_FW_T7 and B. pumi_LAC_RV_HIS
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The most substances which can be detected in the surface water possess one or more aromatic ring structure. Due to the aromatic structures the sewage treatment plants are not able to effectively degrade these substances with conventional methods. This means that a high proportion of these substances is being released into the environment. According to the IWW Rhine Westphalian Institute of Water Research gGmbH (IWW) environmental concentrations of [http://www.umweltbundesamt.de/chemikalien/veranstaltungen/ws-monitoring-arzneimittel/11_vortrag-abstract_bergmann.pdf 247] human and veterinary drugs can be measured in the environmental sewage effluent, the surface water, the groundwater, the drinking water and the sewage sludge. These substances include:
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</p>
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=== Tuesday May 1th ===
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'''Sweeteners'''
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* '''Team Student Academy:''' Searching for two plasmids with different fluorescent proteins behind and antibiotic resistance in parts registry. Found [http://partsregistry.org/Part:BBa_J04450 BBa_J04450], a Plasmid with RFP and chloramphenicol resistance (but lacI and CAP sensitive), [http://partsregistry.org/Part:BBa_J23100 BBa_J23100], a plasmid with RFP and ampicillin resistance and [http://partsregistry.org/wiki/index.php?title=Part:BBa_I13522 BBa_I13522], a Plasmid with GFP and ampicillin resistance in Kit Plate 2011.
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Acesulfam
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Sucralose
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=== Wednesday May 2th ===
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'''Antibiotics'''
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* '''Team Activity Test''': Good morning everybody and welcome to the labjournal of Team Activity Tests. Today we started our work with some literature research about enzyme activity tests, laccases and its substrates. So today was filled with online research, reading papers and collecting information about the laccases our team decided to use.
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Clarithromycin
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Sulfamethoxazol
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N4-Acetylsulfamethoxazol
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Carbamazepin
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=== Thursday May 3th ===
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'''Analgetics'''
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* '''Team Bacterial Laccases''':
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Diclofenac
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** After the vector with the laccase gene CotA from ''Bacillus pumilus'' arrived, we transformed it into the competent ''E.coli KRX'' which we have already made competent to have a greater amount of vector. The protocol we used was as followed:
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Ibuprofen
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*** The electroporation setup: U= 2,5kV C= 25 µF and R= 400 <math>\omega</math>
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*** Since we did not know the efficient of our competent KRX we used two different ''E.coli'' volumes for the transformation, 50µL and 100µL. We gave 50µL 10% Glycerol to the reaction tubes with 1µL of the vector DNA (''Bacillus pumilus''). After the transformation we plated them into ampicillin plates.
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** PCR with the ''Xanthomonas campestris B100'' and ''E. coli BL21(DE3)'' genomic DNA to isolate the laccases. Therefore we used the primers Xcc_LAC_FW_T7, Xcc_LAC_RV_HIS, E.coli_LAC_FW_T7 and E.coli_LAC_RV_HIS which are listed under Materials.
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** '''PCR table'''
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{| class="wikitable"
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! Material !! Volume
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| Buffer (10x Phusion) || 10µL
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| Phusion Polymerase || 0,5µL
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|-
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| dNTPs || 1µL
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|-
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| Primer Mix || 1µL
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| Template DNA || 1µL
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| DMSO || 1,5µL
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| Water || 35µL
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** ''' PCR program'''
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{| class="wikitable"
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! Temperature !! Time
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| 1) 98°C || 30 sec
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| 2) 98°C || 15 sec
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|-
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| 3) 62°C || 45 sec
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|-
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| 4) 72°C || 1 min
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|-
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| 5) 72°C || 3 min
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|-
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| 6) 12°C ||
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|-
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|}
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Cycle between step 2 and 4 35 times.
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=== Friday May 4th ===
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'''Benzotriazole'''
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'''Team Bacterial Laccases''': We did Colony PCR on the transformed the ''Bacillus pumilus'' CotA plasmid. Unfortunately the control with colony PCR didn't work. So we just picked some colonies for plasmid isolation in the hope that on the AMP plate were no false positives colonies.
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Benzotriazol
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4-Methylbenzotriazol
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5-Methylbenzotriazol
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'''Beta-Blocker'''
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Metoprolol
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Sotalol
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'''X-Ray contrast agents'''
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Amidotrizoesäure
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Iomeprol
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Iopamidol
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Iopromid
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<p align="justify">
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The concentrations of these substances and its metabolites are under their therapeutically effective concentration after the wastewater treatment, so that these agents are grouped under the concepts micro-contaminants. But the  impacts of the micro-contaminants on the environment are already evident. [http://toxsci.oxfordjournals.org/content/106/1/93.short N.Shved ''et al.''] e.g. has shown that 17α-Ethinylestradiol at environmentally relevant concentration is able to influence fish growth and reproductive functions of bony fishes.
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<p align="justify">
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The long-term consequences of increasing estrogen concentration for human beings are still largely unknown. Nonetheless, declining sperm counts and thereby increasing infertility in men living in industrial nations may well relate to this hormonal pollution. In addition, [http://bmjopen.bmj.com/content/1/2/e000311.full testicular and prostate cancers] as well as osteoporosis could be a consequence of overly high concentrations of estrogen in the human body. At the  moment only the acute, short term effects are obvious, but the long term effects of continuous exposure of ecosystems, nor the effects that occur even below therapeutic levels in non-target organisms are not [http://www.umweltbundesamt.de/chemikalien/veranstaltungen/ws-monitoring-arzneimittel/16_vortrag-abstract_knacker.pdf predictable].
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<p align="justify">
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Only for 70 of the [http://www.umweltbundesamt.de/chemikalien/veranstaltungen/ws-monitoring-arzneimittel/11_vortrag-abstract_bergmann.pdf 247] substances sufficient information exist for an ecotoxicological assesment. To assess the risk, the Federal Environment Agency (UBA) published a recommendation for not or only partially assessable micro-contaminants,  defined as “Gesundheitlicher Orientierungswert" [http://www.umweltbundesamt.de/chemikalien/veranstaltungen/ws-monitoring-arzneimittel/3_vortrag-abstract_vietoris.pdf  GOW]. Some of these recommended values already have been exceeded by several agents, like Diclofenac (up to 10,5µg mL-1) and Iboprofen up to 9,79 µg mL-1).
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<p align="justify">
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Detailed concentrations in sewage effluent and surface water of Diclofenac and some other substances can be found [https://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Project/Background/Concentrations here].
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The increasing amounts of some micro-contaminants, prompted the European Commission to adds [http://www.europarl.europa.eu/meetdocs/2009_2014/documents/envi/pr/909/909091/909091en.pdf 15 new priority substances] to the list of priority hazardous substances:
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* six incredients of pesticides (Aclonifen, Bifenox, Cypermethrin, Dicofol, Heptachlor und Quinoxyfen),
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* six incredients biocides (Cybtryn, Dichlorvos und Terbutryn),
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* two industrial Chemicals (Perfluoroctansulfonat (PFOS) und Hexabromcyclododecan (HBCDD)),
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* three pharmaceutical agents (Diclofenac, 17α-Ethinylestradiol (EE2) und Estradiol (E2)) and
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* Dioxin and dioxin like polychlorinated Biphenyle (dl-PCB).
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</p>
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<p align="justify">
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This list defines priority substances in the field of water policy, namely chemicals presenting a significant risk to or via  the aquatic environment.
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Besides these 15 substances  the European commission plans to [http://www.europarl.europa.eu/meetdocs/2009_2014/documents/envi/pr/909/909091/909091en.pdf increase the priorization of other substances] such as Anthracen, brominated diphenylether, Naphtalin and Polycyclic Aromatic Hydrocarbons.
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<p align="justify">
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But ultimately, currently there are no legally binding limits for concentrations of pharmaceutically active compounds in surface water, groundwater or drinking water. Consequently the increasing consumption of pharmaceuticals leads to an increasing pressure on the aquatic environment and on the availability and quality of healthy and clean water.  
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This is the problem the iGEM Team wants to solve. The Bielefeld iGEM team is to developing a biological filter using immobilized [https://2012.igem.org/Team:Bielefeld-Germany/Project/Background#3 laccases] to purify municipal and industrial wastewater from synthetic estrogens and other micro-contaminants.  
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<div><h3 style="text-decoration:none; color:black;">Woche 2</h3>
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<h3>Our Focus</h3>
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==Week 2 (05/07 - 05/13/12)==
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As soon as possible we are going to present the chemicals which we want to degrade on this site. Don't miss it.
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''' weekly seminar:'''
 
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* Found our first sponsors: [http://corporate.evonik.com/en/Pages/default.aspx Evonik], [http://www.biocircle.com/en-ca/ BioCircle] and [http://www.merckgroup.com/en/index.html Merck], now treaties have to be created and signed
 
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* Julia is working on the database
 
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* Decision to organize waver sell to fill up our petty cash
 
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* Gabi and Isabel are designing a poster for the waver sell
 
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* For our human practices we wanted to find a sociology student, willing to think about bioethics, but failed
 
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* Our video is nearly done, is cutted and only needs be underlain with music
 
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=== Monday May 7th ===
 
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* '''Team Student Academy:'''
 
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**First transformation of [http://partsregistry.org/Part:BBa_J04450 BBa_J04450] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_I13522 BBa_I13522] and plating on selective agar. Result: We got little colonies. There weren’t any green colonies and only some pale red fluorescent colonies.
 
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<div><h3 style="text-decoration:none; color:black;">Woche 3</h3>
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hier eine Zusammenfassung und ein <a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week3">read more</a>
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<h3>Our Partner - Laccase</h3>
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In the last few years a lot attention has been drawn to Laccases due to their ability to oxidise both phenolic and nonphenolic lignin related compounds as well as highly recalcitrant environmental pollutants. This makes them very useful for applications concernig several biotechnological processes. This includes the detoxification of industrial effluents, for example the paper and pulp, textile and petrochemical industries, the useage as a tool for medical diagnostics and as a bioremediation agent to clean up herbicides, pesticides and certain explosives in soil.Laccases are also used as catalysts for the manufacture of anti-cancer drugs and even as ingredients in cosmetics<sup>[1]</sup>.
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In our project Laccases are used as cleaning agents for a water purification systems. Their capacity to remove xenobiotic substances and produce polymeric products makes them a useful tool for bioremediation purposes."
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Laccases are copper-containing polyphenol oxidase enzymes '''(EC 1.10.3.2)''' that are found in many plants, insects, microorganisms and mainly in fungi. These enzymes are used in different metabolic pathways and fulfill several functions. E.g. these Enzymes are necessary on the one hand to degrade Lignin in ''Basidiomycetes'' and on the other hand to synthesize complex polymers like Melanin in ''Ascomycentes''.In General, laccases are extracellular enzyms and consists usually of 15-20 % carbon-hydrogen. The molecular weight of the deglycated protein is 60 to 80 kDa (about 480-650 aminoacids). These enzymes can occur as monomers, dimers, trimers and tetramers. The first crystal structure of a laccase from the organism ''Trametes versicolor''was published in 2002.
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Laccases are able to oxidize a broad range of substrates due to the contained copper-cluster, by reducing oxygen to water. The active site of the enzym includes a four-copper-ion-cluster, which can be differed by spectroscopically analyses. This Cluster consists of one blue copper-ion (type 1), one type 2 copper ions and two type 3 copper-ions.  Because of the blue copper-ion, the laccases belongs to the big family of the blue copper proteins. This specific blue copper ion is essential for the radical oxidation of the phenolic group. In the enzyme-reaction the electron from the oxidation is transferred to the other three copper ions. These ions are forming a trinuclearic cluster, which transfers electrons to the terminal electron acceptor oxygen. The molecular oxygen is reduced by four electrons to water.   
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<sup>[1]</sup> Susana Rodríguez Couto & José Luis Toca Herrera;<i>Industrial and biotechnological applications of laccases: A review</i>;  2006; Biotechnology Advances 24 500–513
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<h3>Laccase-donators</h3>
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Here you can find out witch donators we used.
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* [https://2012.igem.org/Team:Bielefeld-Germany/Project/Background/thaliana#Arabidopsis_thaliana ''Arabidopsis thaliana''] 
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* [https://2012.igem.org/Team:Bielefeld-Germany/Project/Background/thaliana#Bacillus_pumilus ''Bacillus pumilus''] 
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* [https://2012.igem.org/Team:Bielefeld-Germany/Project/Background/thaliana#Bacillus_halodurans ''Bacillus halodurans'']
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* [https://2012.igem.org/Team:Bielefeld-Germany/Project/Background/thaliana#Escherichia_coli ''Escherichia coli'']
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* [https://2012.igem.org/Team:Bielefeld-Germany/Project/Background/thaliana#Thermus_thermophilus ''Thermus thermophilus'']
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Revision as of 13:48, 23 September 2012

Chemical waste in water?!

Water is the source of all life and covers 71% of the Earth’s surface. Healthy drinking water is an important aspect and essential for mankind. However, the growing industrialization, the production of chemical agents and the increasing consumption of pharmaceuticals is among the causes of the ever increasing pressure on the aquatic environment and on the availability and quality of safe and clean water. The continuous release of pharmaceuticals into the environment and the proven effects on biological systems. The measured environmental concentrations cause that water pollution is one of the main environmental worries of our society. According to a survey of March 2012 of the Public Opinion Analysis sector of the European Commission,

  • 68 % of Europeans think that water quantity and quality problems are serious.
  • 80 % believe that chemical pollution is a threat to the water environment.
  • 62 % feel that they are not sufficiently informed about problems facing groundwater, lakes, rivers and coastal waters in their countries.
But which chemical and pharmaceutical agents are detected in the surface water?

The birth control pill is a widespread contraception method. However, large amounts of these modified estrogens leave the body again in urine. According to the Federal Environment Agency in Germany (Umwelt Bundes Amt-UBA) several hundred tons of analgetics, antibiotics, beta blockers, X-ray contrast agents, anti-epileptic drugs, poly aromatic hydrocarbons pesticides ‘’etc.’’ get into the waste water through various ways, too, and in the end finds its way into rivers, lakes and in the drinking water.

Different sources of micro-contaminants

The most substances which can be detected in the surface water possess one or more aromatic ring structure. Due to the aromatic structures the sewage treatment plants are not able to effectively degrade these substances with conventional methods. This means that a high proportion of these substances is being released into the environment. According to the IWW Rhine Westphalian Institute of Water Research gGmbH (IWW) environmental concentrations of 247 human and veterinary drugs can be measured in the environmental sewage effluent, the surface water, the groundwater, the drinking water and the sewage sludge. These substances include:

Sweeteners Acesulfam Sucralose

Antibiotics Clarithromycin Sulfamethoxazol N4-Acetylsulfamethoxazol Carbamazepin

Analgetics Diclofenac Ibuprofen

Benzotriazole Benzotriazol 4-Methylbenzotriazol 5-Methylbenzotriazol

Beta-Blocker Metoprolol Sotalol

X-Ray contrast agents Amidotrizoesäure Iomeprol Iopamidol Iopromid

The concentrations of these substances and its metabolites are under their therapeutically effective concentration after the wastewater treatment, so that these agents are grouped under the concepts micro-contaminants. But the impacts of the micro-contaminants on the environment are already evident. N.Shved et al. e.g. has shown that 17α-Ethinylestradiol at environmentally relevant concentration is able to influence fish growth and reproductive functions of bony fishes.

The long-term consequences of increasing estrogen concentration for human beings are still largely unknown. Nonetheless, declining sperm counts and thereby increasing infertility in men living in industrial nations may well relate to this hormonal pollution. In addition, testicular and prostate cancers as well as osteoporosis could be a consequence of overly high concentrations of estrogen in the human body. At the moment only the acute, short term effects are obvious, but the long term effects of continuous exposure of ecosystems, nor the effects that occur even below therapeutic levels in non-target organisms are not predictable.

Only for 70 of the 247 substances sufficient information exist for an ecotoxicological assesment. To assess the risk, the Federal Environment Agency (UBA) published a recommendation for not or only partially assessable micro-contaminants, defined as “Gesundheitlicher Orientierungswert" GOW. Some of these recommended values already have been exceeded by several agents, like Diclofenac (up to 10,5µg mL-1) and Iboprofen up to 9,79 µg mL-1).

Detailed concentrations in sewage effluent and surface water of Diclofenac and some other substances can be found here. The increasing amounts of some micro-contaminants, prompted the European Commission to adds 15 new priority substances to the list of priority hazardous substances:

  • six incredients of pesticides (Aclonifen, Bifenox, Cypermethrin, Dicofol, Heptachlor und Quinoxyfen),
  • six incredients biocides (Cybtryn, Dichlorvos und Terbutryn),
  • two industrial Chemicals (Perfluoroctansulfonat (PFOS) und Hexabromcyclododecan (HBCDD)),
  • three pharmaceutical agents (Diclofenac, 17α-Ethinylestradiol (EE2) und Estradiol (E2)) and
  • Dioxin and dioxin like polychlorinated Biphenyle (dl-PCB).

This list defines priority substances in the field of water policy, namely chemicals presenting a significant risk to or via the aquatic environment. Besides these 15 substances the European commission plans to increase the priorization of other substances such as Anthracen, brominated diphenylether, Naphtalin and Polycyclic Aromatic Hydrocarbons.

But ultimately, currently there are no legally binding limits for concentrations of pharmaceutically active compounds in surface water, groundwater or drinking water. Consequently the increasing consumption of pharmaceuticals leads to an increasing pressure on the aquatic environment and on the availability and quality of healthy and clean water. This is the problem the iGEM Team wants to solve. The Bielefeld iGEM team is to developing a biological filter using immobilized laccases to purify municipal and industrial wastewater from synthetic estrogens and other micro-contaminants.

Our Focus

As soon as possible we are going to present the chemicals which we want to degrade on this site. Don't miss it.


Our Partner - Laccase

In the last few years a lot attention has been drawn to Laccases due to their ability to oxidise both phenolic and nonphenolic lignin related compounds as well as highly recalcitrant environmental pollutants. This makes them very useful for applications concernig several biotechnological processes. This includes the detoxification of industrial effluents, for example the paper and pulp, textile and petrochemical industries, the useage as a tool for medical diagnostics and as a bioremediation agent to clean up herbicides, pesticides and certain explosives in soil.Laccases are also used as catalysts for the manufacture of anti-cancer drugs and even as ingredients in cosmetics[1]. In our project Laccases are used as cleaning agents for a water purification systems. Their capacity to remove xenobiotic substances and produce polymeric products makes them a useful tool for bioremediation purposes."


Laccases are copper-containing polyphenol oxidase enzymes (EC 1.10.3.2) that are found in many plants, insects, microorganisms and mainly in fungi. These enzymes are used in different metabolic pathways and fulfill several functions. E.g. these Enzymes are necessary on the one hand to degrade Lignin in Basidiomycetes and on the other hand to synthesize complex polymers like Melanin in Ascomycentes.In General, laccases are extracellular enzyms and consists usually of 15-20 % carbon-hydrogen. The molecular weight of the deglycated protein is 60 to 80 kDa (about 480-650 aminoacids). These enzymes can occur as monomers, dimers, trimers and tetramers. The first crystal structure of a laccase from the organism Trametes versicolorwas published in 2002.


Laccases are able to oxidize a broad range of substrates due to the contained copper-cluster, by reducing oxygen to water. The active site of the enzym includes a four-copper-ion-cluster, which can be differed by spectroscopically analyses. This Cluster consists of one blue copper-ion (type 1), one type 2 copper ions and two type 3 copper-ions. Because of the blue copper-ion, the laccases belongs to the big family of the blue copper proteins. This specific blue copper ion is essential for the radical oxidation of the phenolic group. In the enzyme-reaction the electron from the oxidation is transferred to the other three copper ions. These ions are forming a trinuclearic cluster, which transfers electrons to the terminal electron acceptor oxygen. The molecular oxygen is reduced by four electrons to water.


[1] Susana Rodríguez Couto & José Luis Toca Herrera;Industrial and biotechnological applications of laccases: A review; 2006; Biotechnology Advances 24 500–513

Laccase-donators

Here you can find out witch donators we used.


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About our Wiki

Fifteen students from different fields of study from the Bielefeld University aspired to complete a project called TOXIC COMPOUNDS IN NATURAL WATER - A CASE FOR LACCASE. In his Wiki we document our achievements and our approach.

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Universität Bielefeld
Universitätsstraße 25
D-33615 Bielefeld

info(at)igem-bielefeld.de

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