Team:Bielefeld-Germany/Results/Summary

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<ul style="list-style-type:none">
<ul style="list-style-type:none">
<li><a href="#1"><strong>Summary</strong></a></li>
<li><a href="#1"><strong>Summary</strong></a></li>
<li><a href="#2"><strong>Datapage</strong></a></li>
<li><a href="#2"><strong>Datapage</strong></a></li>
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<li><a href="#3"><strong>BPUL</strong></a></li>
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<li><a href="#3"><strong>Laccases</strong></a></li>
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<li><a href="#4"><strong>ECOL</strong></a></li>
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<li><a href="#4"><strong>Immobilization</strong></a></li>
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<li><a href="#5"><strong>BHAL</strong></a></li>
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<li><a href="#5"><strong>Substrate Analysis</strong></a></li>
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                <li><a href="#6"><strong>TTHL</strong></a></li>
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<li><a href="#6"><strong>CBD</strong></a></li>
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<li><a href="#7"><strong>TVE??</strong></a></li>
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<li><a href="#7"><strong>Shuttle vector</strong></a></li>
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<li><a href="#8"><strong>Immobilization</strong></a></li>
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                <li><a href="#8"><strong>Collaboration with UCL</strong></a></li>
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<li><a href="#9"><strong>Substrate Analytics</strong></a></li>
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<li><a href="#10"><strong>CBD</strong></a></li>
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<h1>Summary</h1>
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All BioBricks of the iGEM Team Bielefeld were screened to identify the best conditions for protein expression. The first trials were made by shaking flask cultivations with different parameters. These parameters were various shaking flask designs, different temperatures, different concentrations of chloramphenicol, various induction strategies, several cultivation times and some cultivations in absence or presence of CuCl<sub>2</sub>. To detect the produced laccases, different analysis methods were performed like SDS-PAGE analysis as well as MALDI-TOF. The iGEM Team successfully produced four active bacterial laccases and succeeded to purify four of them. Besides the successfully scale-up fermentation, these laccases could be purified in a high amount to characterize the optimal activity conditions regarding pH, temperature, buffer solutions and organic solvent resistance. Furthermore, the iGEM Team Bielefeld demonstrated that the produced laccases can be immobilized maintaining their activity and the degradation capacity was screened for several micro-contaminants. These tests indicate that all of our produced laccases are able to degrade estradiol and the two laccases TTHL and BPUL are able to degrade ethinyl-estradiol in combination with a mediator. At this moment, the self-designed shuttle-vector for the production of eukaryotic laccases in yeast is ready to go. This vector was tested to integrate by courtesy of homologous recombination genes of eukaryotic laccases into Pichia Pastoris and produce them in an active form. First experiments show a successful production of one laccase of ''Trametes versicolor''. A cheap alternative purification and immobilization method via a cellulose binding tag is also close at hand. During our research, we cultivated the following BioBricks and produced several laccase. To simplify the presentation of our results we named the produced laccase like the corresponding system.
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{| class="wikitable"
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!colspan="5"|Produced and generated BioBricks with the source strain of the DNA-sequence, promoter, protein name and the names given by the iGEM Team Bielefeld
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|BioBrick code
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|strain
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|promoter
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|name  of protein
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|name given by the iGEM Team
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|<partinfo>K863000</partinfo>
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|''Bacillus pumilus'' DSM 27
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|T7 promoter
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|align="center"|CotA
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|align="center"|'''BPUL'''
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|-
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|<partinfo>K863005</partinfo>
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|''E. coli'' BL21(DE3)
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| T7 promoter
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|align="center"|CueO
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|align="center"|'''ECOL'''
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|-
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| <partinfo>K863010</partinfo>
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|''Thermus thermophilus'' HB27
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| T7 promoter
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|align="center"|tthL
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|align="center"|'''TTHL'''
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|-
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| <partinfo>K863012</partinfo>
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|''Thermus thermophilus'' HB27
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| constitutive promoter  (<partinfo>BBa_J23100</partinfo>)
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|align="center"|tthL
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|align="center"|'''TTHL'''
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| <partinfo>K863015</partinfo>
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| ''Xanthomonas campestris pv. campestris'' B100
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|T7
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|align="center"|CopA
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|align="center"|'''XCCL'''
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|<partinfo>K863020</partinfo>
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|''Bacillus halodurans'' C-125
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|T7
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|align="center"|Lbh1
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|align="center"|'''BHAL'''
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|<partinfo>K863022</partinfo>
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|''Bacillus halodurans''  C-125
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| constitutive promoter  (<partinfo>BBa_J23100</partinfo>)
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|align="center"|Lbh1
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|align="center"|'''BHAL'''
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| <partinfo>K863030</partinfo>
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|''Trametes versicolor ''
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| AOX1 promoter
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|align="center"|TVL5
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|align="center"|'''TVEL5'''
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iGEM Team Bielefeld is developing a biological filter using immobilized laccases, enzymes able to radicalize and break down a broad range of aromatic substances. For the production of laccases from different bacteria, fungi and plants, two expression systems are used: ''Escherichia coli'' and the yeast ''Pichia pastoris''. Immobilization is carried out either by using CPC-silica beads or by fusing the enzymes to cellulose binding domains. The concept could be extended to other toxic pollutants in drinking and wastewater, as well as to industrial applications in paper and textile industries or even for bioremediation of contaminated soil.
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[https://2012.igem.org/Team:Bielefeld-Germany/Results/Datapage Read more.]
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<h3>Laccase from Bacillus pumilus DSM 27 (ATCC7061)</h3>
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<h1>Laccases</h1>
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The iGEM Team successfully produced four active bacterial laccases and an eukaryotic laccase (click for the results):
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:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/coli ''Escherichia coli'' laccase ECOL]
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:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/pumi''Bacillus pumilus'' laccase BPUL]
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:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/halo''Bacillus halodurans'' laccase BHAL]
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:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/thermo ''Thermus thermophilus'' laccase TTHL]
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:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/tvel5 ''Trametes versicolor'' laccase TVEL5]
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:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/comparison Comparison of the different laccases]
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:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/trametis Purchased positive control ''Trametes versicolor'' laccase TVEL0]
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All bacterial laccases (ECOL, BHAL, TTHL and BPUL) we accomplished to purify. Besides the successfully scale-up fermentation these laccases could be purified in a high amount to characterize the optimal activity conditions regarding  pH, temperature, buffer solutions  and  organic solvent resistance. Furthermore the iGEM Team Bielefeld demonstrated that the produced laccases can be immobilized maintaining their activity and the degradation capacity was screened for several micro-contaminants. These tests indicate that they are able to degrade estradiol and ethinyl-estradiol.
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<h3>Laccase from Escherichia coli BL21 (DE3)</h3>
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<img src="https://static.igem.org/mediawiki/2012/2/25/Bielefeld2012_Immo.jpeg" />
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<h1>Immobilization</h1>
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'''Using commercially acquired laccases from ''Trametes versicolor'' (named TVEL0) as a standard, it was possible to optimize an immobilization method of the purified laccases from
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:* [http://openwetware.org/wiki/E._coli_genotypes#BL21.28DE3.29 ''E. coli'' BL21 (DE3)] (named ECOL)
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:* [http://www.dsmz.de/catalogues/details/culture/DSM-27.html ''Bacillus pumilus'' DSM 27 (ATCC7061)] (named BPUL)
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:* [http://www.dsmz.de/catalogues/details/culture/DSM-18197.html?tx_dsmzresources_pi5 ''Bacillus halodurans'' C-125 ] (named BHAL) and from
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:* [http://www.dsmz.de/catalogues/details/culture/DSM-7039.html?tx_dsmzreso ''Thermus thermophilus'' HB27] (named TTHL)
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on CPC-silica beads. All four purified laccases were successfully immobilized, with ECOL and BPUL showing the highest binding ability to beads. Moreover, all four immobilized laccases showed activity. Whereas immobilized BPUL showed a relatively high activity, the results couldn't be compared to BHAL und TTHL due to the low concentration of the latters. ''' For immobilization results see [https://2012.igem.org/Team:Bielefeld-Germany/Results/immo here]
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<h3>Laccase from Thermus thermophilus HB27</h3>
 
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<img src="https://static.igem.org/mediawiki/2012/a/a7/Bielefeld2012-estradiol-control-spectroflurophotometer.JPG" />
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<h1>Substrate Analysis</h1>
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<h3>Laccase from Trametes versicolor </h3>
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To establish the methods for degradation analysis of different substrates TVEL0 was used as positive control.
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After that the four produced bacterial laccases were analyzed. The HPLC results showed that estradiol and ethinyl estradiol ( with addition of ABTS) are degradable with our laccases.To determine degradation products of estradiol and ethinyl estradiol after laccase treatment LCMS-MS analysis were done. For more informations [https://2012.igem.org/Team:Bielefeld-Germany/Results/substrate click here].
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A cheap alternative purification method combined with a powerful immobilization tool could be the solution to prevail over other more expensive water cleaning methods like oxidization with ozone or using tons of activated carbon which just capture microcontaminates, but does not dismantle them. A promising solution to this could be cellulose binding domains (CBDs). Cellulose is ubiquitous and sustainable. Following this idea fusion-protein-constructs with cellulose binding domains have been made. To characterize a GFP has been introduced as a C or N-terminal domain of the cellulose binding protein. After delays in cloning the constructs for two fusion proteins with a T7-promoter could be finished, but did not express the protein in ''E. coli'' KRX and BL21. An alternative construct with a constitutive promoter could also be finished, but gave the same results. Changing the order of CBD and GFP was carried out, but was hampered by a base deletion in the GFP gene causing a frame shift and could not be redone in time.
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<a href="https://2012.igem.org/Team:Bielefeld-Germany/Results/cbc">Read more</a>
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A shuttle vector for site-directed recombination into the yeast ''P. pastoris'' does not exist in the parts registry and could be developed by our team. With this system it is possible to recombine a protein of interest with a N-terminal mating factor alpha 1 for secretion the protein into the media. This protein of interest could be cloned in frame with one restriction ligate cloning step. The selection depends not on an antibiotic resistance like zeocine, but on a complementation of histidine auxotrophy. This system is for us important because some of our laccases can not be expressed in the prokaryotic expression system ''E. coli'', because the protein needs glycosylation.
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[https://2012.igem.org/Team:Bielefeld-Germany/Results/vector Read more.]
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The BioBrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_K729006 BBa_K729006] from the [https://2012.igem.org/Team:University_College_London University&nbsp;College&nbsp;London] was characterized by us. Therefore ''E. coli'' KRX containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K729006 BBa_K729006] and ''E. coli'' KRX as a negative control were cultivated in shaking flasks and a growth kinetic was determined. The harvested cells were lysed via sonication and substances with a low molecular weight were seperated out of the supernatant. After purification the sample was analyzed by SDS-PAGE and MALDI-TOF.
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For a comparison ''E.&nbsp;coli'' KRX containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K7863005] was cultivated and analysed by SDS-PAGE as well as tested with a laccase activity assay. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K729006 BBa_K729006] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K7863005] showed a similar behaviour in oxidizing ABTS.
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[https://2012.igem.org/Team:Bielefeld-Germany/Results/london Read more.]
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== Summary ==
 
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== BPUL - Laccase from ''Bacillus pumilus  DSM 27 (ATCC7061)'' ==
 
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===Shaking Flask Cultivation===
 
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The first trails to produce the BPUL were produced in shaking flask with various designs (from 100&nbsp;mL<sup>-1</sup> to 1&nbsp;L flasks, with and without baffles) and under different conditions. The parameters we have changed during our screening experiments were the temperature (27&nbsp;°C,30&nbsp;°C and 37&nbsp;°C), different concentrations of chloramphenicol (20-170&nbsp;µg&nbsp;mL<sup>-1</sup>), various induction strategies (autoinduction and manual induction), several cultivation times (6 - 24&nbsp;h) and in absence or presence of 0,25&nbsp;mM CuCl<sub>2</sub>. Due to the screening experiments we identified the best conditions under which BPUL was expressed:
 
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* flask design: shaking flask without baffles
 
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* medium: [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium autoinduction medium]
 
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* antibiotics: 60&nbsp;µg&nbsp;mL<sup>-1</sup> chloramphenicol
 
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* temperature: 37&nbsp;°C
 
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* cultivation time: 12&nbsp;h
 
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The reproducibility and repeatability of the measured data and results were investigated for the shaking flask and bioreactor cultivation with n&le;3.
 
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The Results of the SDS-PAGE analysis are shown in the following images:
 
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SDS PAGES!!!!!
 
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ACTIVITÄTSMESSUNGEN VOM ERSTEN AKTIVEN B PUMI!!!!!!
 
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===3&nbsp;L Fermentation ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> ===
 
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After the measurement of activity of BPUL we made a scale-up and fermented ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> in Braun Biostat B with a total volume of 3&nbsp;L. Agitation speed, pO<sub>2</sub> and OD<sub>600</sub> were determined and illustrated in Figure 1. We got a long lag phase of 2&nbsp;hours due to a relativly old preculture. The cell growth caused a decrease in pO<sub>2</sub> and after 3&nbsp;hours the value fell below 50&nbsp;%, so that the agitation speed increased automatically. After 8,5&nbsp;hours the deceleration phase started and therefore the agitation speed was decreased. There is no visible break in cell growth through induction of protein expression. It is probably that we did not produce such a great amount of BPUL that it had any influence on cell growth or that it is not active so far. The maximal OD<sub>600</sub> of 3,53 was reached after 10&nbsp;hours, which means a decrease of 28&nbsp;% in comparison to the fermentation of ''E.&nbsp;coli'' KRX under the same conditions(OD<sub>600,max</sub> =4,86 after 8,5&nbsp;hours, time shift due to long lag phase). The cells were harvested after 11&nbsp;hours.
 
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[[File:Bielefeld2012_BPUL3LFermentation.jpg|600px|thumb|center|Figure 1: Fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> (BPUL) in Braun Biostat B, scale: 3&nbsp;L, [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium autoinduction medium] + 60&nbsp;µg/mL chloramphenicol, 37&nbsp;°C, pH&nbsp;7, agitation on cascade to hold a pO<sub>2</sub> of 50&nbsp;%, OD<sub>600</sub> taken every 30&nbsp;minutes.]]
 
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=== Purification of BPUL ===
 
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The harvested cells were resuspended in [https://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-equilibrationbuffer], mechanically lysed by [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Production#Mechanical_lysis_of_the_.28bio-reactor.29_cultivation homogenization] and centrifuged. The supernatant of the lysed cell paste was loaded on the Ni-NTA-column (15&nbsp;mL Ni-NTA resin) with a flowrate of 1&nbsp;mL min<sup>-1</sup> cm<sup>-2</sup>. Then the column was washed by 10&nbsp;column&nbsp;volumes (CV) with [https://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-equilibrationbuffer]. The bound proteins were eluted by an increasing [https://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-elutionbuffer] gradient from 0&nbsp;% to 100&nbsp;% with a total volume of 100&nbsp;mL and the elution was collected in 10&nbsp;mL fractions. The chromatogramm of the BPUL-elution is shown in figure 2:
 
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[[File:Bielefeld2012_BPUL3LChromatogramm.jpg|600px|thumb|center|Figure 2: Chromatogramm of wash and elution from FLPC Ni-NTA-Histag Purification of BPUL produced by 3&nbsp;L fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo>. BPUL was eluted between a process volume of 460&nbsp;mL to 480&nbsp;mL with a maximal UV-detection signal of 69 mAU]]
 
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The chromatogramm shows a remarkable widespread peak between the process volume of 460&nbsp;mL to 480&nbsp;mL with the highest UV-detection signal of 69 mAU , which can be explained by the elution of bound proteins. The corresponding fractions were analysed by SDS-PAGE analysis. Afterwards the UV-signal increased caused by the changing imidazol concentration during the elution gradient. Between the process volume of 550 and 580&nbsp;mL there are several strong peaks (up to a UV-detection-signal of 980&nbsp;mAU) detectable. These results are caused by an accidental detachement in front of the UV-detector. Just to be on the safe side, the corresponding fractions were analysed by SDS-PAGE analysis. The results of the SDS-PAGE are shown in the following pictures.
 
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===6&nbsp;L Fermentation of ''E.&nbsp;coli'' with <partinfo>BBa_K863000</partinfo>===
 
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Another scale-up of the fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> was made up to a final working volume of 6&nbsp;L in Bioengineering NFL22. Agitation speed, pO<sub>2</sub> and OD<sub>600</sub> were determined and illustrated in Figure 3. There was no noticeable lag phase and the cells immediatly began to grow. Agitation speed was increased up to 425&nbsp;rpm after one hour due to control problems. Then the pO<sub>2</sub> sank until a cultivation time of 4,75&nbsp;hours, when the deceleration phase started. Then it increased again. There is no visible break through induction of protein expression. A maximal OD<sub>600</sub> of 3,68 was reached after 7/8&nbsp;hours of cultivation, which is similar to the 3&nbsp;L fermentation (OD<sub>600</sub> = 3,58 after 10 hours, time shift due to long lag phase). The cells were harvested after 12&nbsp;hours.
 
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[[File:Bielefeld2012_BPUL6LFermentation.jpg|600px|thumb|center|Figure 3: Fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> (BPUL) in Bioengineering NFL22, scale: 6&nbsp;L, [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium autoinduction medium] + 60&nbsp;µg/mL chloramphenicol, 37&nbsp;°C, pH&nbsp;7, agitation increased when pO<sub>2</sub> was below 30&nbsp;%, OD<sub>600</sub> taken every hour. ]]
 
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===Purification of BPUL===
 
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</center>
 
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<p align="justify">
 
-
The harvested cells were prepared in [https://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-equilibrationbuffer], mechanically lysed by [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Production#Mechanical_lysis_of_the_.28bio-reactor.29_cultivation homogenization] and centrifuged. The supernatant of the lysed cell paste was loaded on the Ni-NTA-column (15&nbsp;mL Ni-NTA resin) with a flowrate of 1&nbsp;mL min<sup>-1</sup> cm<sup>-2</sup>. Then the column was washed by 5&nbsp;column&nbsp;volumes (CV) with [https://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-equilibrationbuffer].The bound proteins were eluted by an increasing elutionbuffer gradient from 0&nbsp;% (equates to 20&nbsp;mM Imidazol) to 100&nbsp;% (equates to 500&nbsp;mM imidazol) with a total volume of 200&nbsp;mL. This strategy was chosen to improve of purification by a slower increase of [https://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-elutionbuffer] concentration. The elution was collected in 10&nbsp;mL fractions. The chromatogramm of the BPUL-elution is shown in figure 2:
 
-
</p>
 
-
 
-
<br style="clear: both" />
 
-
[[File:Bielefeld2012_BPUL6LChromatogramm.jpg|600px|thumb|center|Figure 4: Chromatogramm of wash and elution from FLPC Ni-NTA-Histag Purification of BPUL produced by 6&nbsp;L fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo>. BPUL was eluted between a process volume of 832&nbsp;mL and 900&nbsp;mL with a maximal UV-detection signal of 115&nbsp;mAU.]]
 
-
<br style="clear: both" />
 
-
 
-
<p align="justify">
 
-
The chromatogramm shows a strong peak at the beginning of the elution. This can be explained by pressure fluctuations upon starting the elution procedure. Between a process volume of 832&nbsp;mL and 900&nbsp;mL there is remarkable widespread peak with a UV-detection signal of 115&nbsp;mAU. This peak corresponds to an elution of bound proteins at a [https://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-elutionbuffer] concentration between 10&nbsp;% and 20&nbsp;% (equates to 50-100&nbsp;mM imidazole) . The corresponding fractions were analysed by SDS-PAGE analysis. The ensuing upwards trend of the UV-signal is caused by the increasing imidazol concentration during the elution gradient. Towards the end of the elution procedure there is constant UV-detection signal, which shows, that the most of the bound proteins was already eluted. Just to be on the safe side, all fractions were analysed by SDS-PAGE analysis to detect BPUL. The results of the SDS-PAGE are shown in the following pictures.
 
-
</p>
 
-
 
-
== ECOL - Laccase from ''Escherichia coli BL21 (DE3)'' ==
 
-
<center>
 
-
===Shaking Flask Cultivation===
 
-
</center>
 
-
The first trails to produce ECOL were produced in shaking flask with various designs (from 100&nbsp;mL<sup>-1</sup> to 1&nbsp;L flasks, with and without baffles) and under different conditions. The parameters we have changed during our screening experiments were the temperature (27&nbsp;°C,30&nbsp;°C and 37&nbsp;°C), different concentrations of chloramphenicol (20-170&nbsp;µg&nbsp;mL<sup>-1</sup>), various induction strategies (autoinduction and manual induction), several cultivation times (6 - 24&nbsp;h) and in absence or presence of 0,25&nbsp;mM CuCl<sub>2</sub>. Due to the screening experiments we identified the best conditions under which ECOL was expressed:
 
-
 
-
* flask design: shaking flask without baffles
 
-
* medium: [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium autoinduction medium]
 
-
* antibiotics: 60&nbsp;µg&nbsp;mL<sup>-1</sup> chloramphenicol
 
-
* temperature: 37&nbsp;°C
 
-
* cultivation time: 12&nbsp;h
 
-
 
-
The Results of the SDS-PAGE analysis are shown in the following images:
 
-
 
-
SDS PAGES!!!!!
 
-
 
-
 
-
ACTIVITÄTSMESSUNGEN VOM ERSTEN AKTIVEN ECOL!!!!!!
 
-
 
-
<center>
 
-
 
-
<center>
 
-
 
-
===3&nbsp;L Fermentation ''E. coli'' KRX with <partinfo>BBa_K863005</partinfo>===
 
-
</center>
 
-
 
-
<p align="justify">
 
-
After the measurement of activity of ECOL we made a scale-up and fermented ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863005</partinfo> in Infors Labfors with a total volume of 3&nbsp;L. Agitation speed, pO<sub>2</sub> and OD<sub>600</sub> were determined and illustrated in Figure 1. The exponential phase started after 1,5&nbsp;hours of cultivation. The cell growth caused a decrease in pO<sub>2</sub>. After 2&nbsp;hours of cultivation the agitation speed increased up to 629&nbsp;rmp (5,9&nbsp;hours) to hold the minimal pO<sub>2</sub> level of 50&nbsp;%. Then, after 4&nbsp;hours there was a break in cell growth due to induction of protein expression. The maximal OD<sub>600</sub> of 2,78 was reached after 5&nbsp;hours. In comparison to ''E.&nbsp;coli'' KRX (OD<sub>600,max</sub> =4,86 after 8,5 hours) and to ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> (OD<sub>600,max</sub> =3,53 after 10 hours, time shift due to long lag phase) the OD<sub>600,max</sub> is 22&nbsp;% or rather 43&nbsp;% lower. In the following hours the cells began to die because of the celltoxicity of ECOL (reference: [http://www.dbu.de/OPAC/ab/DBU-Abschlussbericht-AZ-13191.pdf  DBU final report]). Therefore they were harvested after 12&nbsp;hours.
 
-
</p>
 
-
 
-
[[File:Bielefeld2012_ECOL3LFermentation.jpg|600px|thumb|center|Figure 1: Fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863005</partinfo> (ECOL) in Infors Labfors Bioreactor, scale: 3&nbsp;L, [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium autoinduction medium] + 60&nbsp;µg/mL chloramphenicol, 37&nbsp;°C, pH&nbsp;7, agitation on cascade to hold a pO<sub>2</sub> of 50&nbsp;%, OD<sub>600</sub> taken every 30&nbsp;minutes.]]
 
-
<br style="clear: both" />
 
-
 
-
<center>
 
-
 
-
===Purification of ECOL===
 
-
</center>
 
-
<p align="justify">
 
-
The harvested cells were resuspended in [https://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-equilibrationbuffer], mechanically lysed by [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Production#Mechanical_lysis_of_the_.28bio-reactor.29_cultivation homogenization] and centrifuged. The supernatant of the lysed cell paste was loaded on the Ni-NTA-column (15&nbsp;mL Ni-NTA resin) with a flowrate of 1&nbsp;mL min<sup>-1</sup> cm<sup>-2</sup>. Then the column was washed by 10&nbsp;column&nbsp;volumes (CV) with [https://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-equilibrationbuffer]. The bound proteins were eluted by an increasing [https://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-elutionbuffer] step elution from 5&nbsp;% (equates to 25&nbsp;mM imidazol) with a total volume of 60&nbsp;mL, to 50&nbsp;% (equates to 250&nbsp;mM Imidazol) with a total volume of 60&nbsp;mL, to 80&nbsp;% (equates to 400&nbsp;mM imidazol) with a total volume of  40&nbsp;mL and finaly to 100&nbsp;% (equates to 500&nbsp;mM imidazol) with a total volume of 80&nbsp;mL. This strategies was chosen to improve the purification caused by a step by step increasing [https://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-elutionbuffer] concentration. The elution was collected in 10&nbsp;mL fractions. The chromatogramm of the BPUL-elution is shown in figure 2:
 
-
</p>
 
-
 
-
[[File:Bielefeld2012_ECOL3LChromatogramm.jpg|600px|thumb|center|Figure 2: Chromatogramm of Wash and Elution from FLPC Ni-NTA-Histag Purification of ECOL produced by 3&nbsp;L fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863005</partinfo>. ECOL was eluted by a concentration of 50&nbsp;% (equates to 250&nbsp;mM imidazol) with a maximal UV-detection signal of 292&nbsp;mAU. ]]
 
-
<br style="clear: both" />
 
-
 
-
<p align="justify">
 
-
The chromatogramm shows two remarkable peaks. The first peak was detected by a [https://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-equilibrationbuffer] concentration of 5&nbsp;% (equates 25mM Imidazol) and resulted from the elution of weakly bound proteins. After increasing the [https://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-elutionbuffer] concentration to 50&nbsp;% (equates to 250&nbsp;mM imidazol) a large peak up to a UV-detection signal of 292&nbsp;mAU was measured. The strength of this peak indicates that a high amount of protein was eluted. The corresponding fractions were analysed by SDS-PAGE analysis to detect ECOL. There were no further peaks detectable. The following increasing UV-dectection-signals equates to the imidazol concentration of the [https://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-elutionbuffer]. The corresponding SDS-PAGES are shown in the following images.
 
-
</p>
 
-
<center>
 
-
 
-
===SDS-Pages===
 
-
</center>
 
-
 
-
[[File:Bielefeld2012_SDSPAGES_ECOL6L_0910_1.jpg|600px|thumbs|center|Figure 5: x]]
 
-
<center>
 
-
 
-
===6&nbsp;L Fermentation ''E. coli'' KRX with <partinfo>BBa_K863005</partinfo>===
 
-
</center>
 
-
 
-
<p align="justify">
 
-
Another scale-up of the fermentation of E.&nbsp;coli KRX with <partinfo>BBa_K863005</partinfo> was made up to a final working volume of 6&nbsp;L in Bioengineering NFL22. Agitation speed, pO<sub>2</sub> and OD<sub>600</sub> were determined and illustrated in Figure&nbsp;3. There was no noticeable lag phase and the cells immediatly began to grow. The cells were in an exponential phase between 2 and 4&nbsp;hours of cultivation, which results in an decrease of pO<sub>2</sub> value and therefore in an increase of agitation speed. After 4&nbsp;hours of cultivation the maximal OD<sub>600</sub> of 2,76 was reached, which is comparable to the 3&nbsp;L fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863005</partinfo>. Due to induction of protein expression there is a break in cell growth then and the cells began to die. This demonstrates the cytotoxity of the laccases for ''E. coli'', which was reported by the [http://www.dbu.de/OPAC/ab/DBU-Abschlussbericht-AZ-13191.pdf DBU]. In comparison to the fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> under the same conditions (OD<sub>600,max</sub>= 3,53), the OD<sub>600,max</sub> was 22&nbsp;%. Cells were harvested after 12&nbsp;hours.
 
-
</p>
 
-
 
-
 
-
[[File:Bielefeld2012_ECOL6LFermentation.jpg|600px|thumb|center|Figure 3: Fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863005</partinfo> (ECOL) in Bioengineering NFL22, scale: 6&nbsp;L, [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium autoinduction medium] + 60&nbsp;µg/mL chloramphenicol, 37&nbsp;°C, pH&nbsp;7, agitation increased when pO<sub>2</sub> was below 30&nbsp;%, OD<sub>600</sub> taken every hour.]]
 
-
<br style="clear: both" />
 
-
 
-
<center>
 
-
 
-
===Purification of ECOL===
 
-
</center>
 
-
<p align="justify">
 
-
The harvested cells were resuspended in [https://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-equilibrationbuffer], mechanically lysed by [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Production#Mechanical_lysis_of_the_.28bio-reactor.29_cultivation homogenization] and centrifuged. The supernatant of the lysed cell paste was loaded on the Ni-NTA-column (15&nbsp;mL Ni-NTA resin) with a flowrate of 1&nbsp;mL min<sup>-1</sup> cm<sup>-2</sup>. Then the column was washed by 10&nbsp;column&nbsp;volumes (CV) with [https://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-equilibrationbuffer]. The bound proteins were eluted by an increasing [https://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-equilibrationbuffer] gradient from 0&nbsp;% to 100&nbsp;% with a total volume of 200&nbsp;mL and the elution was collected in 10&nbsp;mL fractions. The chromatogramm of the BPUL-elution is shown in figure 4:
 
-
</p>
 
-
 
-
[[File:Bielefeld2012_ECOL6LChromatogramm.jpg|600px|thumb|center|Figure 4: Chromatogramm of Wash and Elution from FLPC Ni-NTA-Histag Purification of ECOL produced by 3&nbsp;L fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863005</partinfo>. ECOL was eluted between a process volume 670&nbsp;mL to 750&nbsp;mL with a maximal UV-detection signal of 189&nbsp;mAU.]]
 
-
<br style="clear: both" />
 
-
 
-
<p align="justify">
 
-
After washing the column with 10 CV [https://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-elutionbuffer] the elution process was started. At a process volume of 670&nbsp;mL to 750&nbsp;mL the chromatogramm shows a strong widespread peak (UV-detection signal 189&nbsp;mAU) caused by the elution of a high amount of proteins. The run of the curve show a fronting. This can be explained by the elution of weakly bound proteins, which elutes at low imidazol concentrations. A better result could be achieved with a step elution strategy ([https://2012.igem.org/Team:Bielefeld-Germany/Results/Summary#Purification_of_ECOL see purification of the 3 L Fermentation above]). To detect ECOL the corresponding fractions were analysed by SDS-PAGE analysis.
 
-
</p>
 
-
 
-
::::::::::: '''!!SDS-Page!!'''
 
-
 
-
== XCCL - Laccase from ''Xanthomonas campestris pv. campestris B100'' ==
 
-
 
-
== BHAL - Laccase from ''Bacillus halodurans C-125'' ==
 
-
 
-
== TTHL - Laccase from ''Thermus thermophilus HB27'' ==
 
-
<center>
 
-
 
-
===Shaking Flask Cultivation===
 
-
</center>
 
-
The first trails to produce the ''Thermo thermophilus''-laccase (TTHL) were produced in shaking flask with various designs (from 100&nbsp;mL<sup>-1</sup> to 1&nbsp;L flasks, with and without baffles) and under different conditions. The parameters we have changed during our screening experiments were the temperature (27&nbsp;°C,30&nbsp;°C and 37&nbsp;°C), different concentrations of chloramphenicol (20-170&nbsp;µg&nbsp;mL<sup>-1</sup>), various induction strategies (autoinduction and manual induction), several cultivation times (6 - 24&nbsp;h) and in absence or presence of 0,25&nbsp;mM CuCl<sub>2</sub>. Due to the screening experiments we wasn't able to detect the best conditions for the production with the  ''E. coli'' KRX chassi.
 
-
Due to the failed screening results we decided to produce the TTHL in an other ''E. coli'' strain called ''E. coli '' Rosetta-Gami 2 containing <partinfo>BBa_K863012</partinfo>. We decided to use ''E. coli '' Rosetta-Gami 2 becaus of his skill to translate rare codons. We produced our TTHL with the following conditions:
 
-
 
-
* flask design: shaking flask without baffles
 
-
* medium: [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials LB-Medium]
 
-
* antibiotics: 60&nbsp;µg&nbsp;mL<sup>-1</sup> chloramphenicol and 300&nbsp;µg&nbsp;mL<sup>-1</sup> ampicillin
 
-
* temperature: 37&nbsp;°C
 
-
* cultivation time: 24&nbsp;h
 
-
 
-
The reproducibility and repeatability of the measured data and results were investigated for the shaking flask and bioreactor cultivation with n&le;3.
 
-
 
-
The Results of the SDS-PAGE analysis are shown in the following images:
 
-
 
-
SDS PAGES!!!!!
 
-
 
-
 
-
ACTIVITÄTSMESSUNGEN VOM ERSTEN AKTIVEN B PUMI!!!!!!
 
-
 
-
===Fermentation of ''E. coli'' KRX with <partinfo>BBa_K863012</partinfo>===
 
-
</center>
 
-
After measuring activity of TTHL we made a scale-up and fermented ''E.&nbsp;coli'' Rosetta-Gami 2 with <partinfo>BBa_K863000</partinfo> in Bioengineering NFL22 with a total volume of 6&nbsp;L. Agitation speed, pO<sub>2</sub> and OD<sub>600</sub> were determined and illustrated in Figure 1. The cells immediatly began to grow and therefore the pO<sub>2</sub> decreased up to a value of 0&nbsp;%, because the breakdown of the control unit. After a cultivation time of 9&nbsp;hours the agitation speed was increased up to a 500&nbsp;rpm, which resulted in a pO<sub>2</sub> value of more than 100&nbsp;% for the rest of the cultivation. During the whole process the OD<sub>600</sub> increased slowly in comparison to the fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> or <partinfo>BBa_K863005</partinfo>. The maximal OD<sub>600</sub> was reached after 19 hours of cultivation, when the cells were harvested.
 
-
 
-
<p align="justify">
 
-
[[File:Bielefeld2012_TTHL6LFermentation.jpg|600px|thumb|center|Figure 1: Fermentation of ''E.&nbsp;coli'' Rosetta-Gami 2 with <partinfo>BBa_K863012</partinfo> (TTHL) in Bioengineering NFL22, scale: 6&nbsp;L, [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium autoinduction medium] + 60&nbsp;µg/mL chloramphenicol, 37&nbsp;°C, pH&nbsp;7, agitation increased when pO<sub>2</sub> was below 30&nbsp;%, OD<sub>600</sub> taken every hour.]]
 
-
<br style="clear: both" />
 
-
</p>
 
-
 
-
<center>
 
-
 
-
===Purification of ECOL===
 
-
</center>
 
-
 
-
<p align="justify">
 
-
The cells were harvested and resuspended in [https://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-equilibrationbuffer], mechanically lysed by [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Production#Mechanical_lysis_of_the_.28bio-reactor.29_cultivation homogenization] and centrifuged. After preparing the cell paste we did not have the possibility to purificate the TTHL with the 15&nbsp;mL. To detect and to analyse our produced TTHL we implement a small scale purification of  6&nbsp;mL of the supernatant with a 1&nbsp;mL Ni-NTA-column. The results of the SDS-PAGE analysis are shown the following images:
 
-
</p>
 
-
 
-
::::::::::: '''!!SDS-Page!!'''
 
-
 
-
== PCIL - Laccase from ''Pycnoporus cinnabarinus'' ==
 
-
 
-
== TVEL5 - Laccase from ''Trametes versicolor '' ==
 
-
 
-
== TVEL10 - Laccase from ''Trametes versicolor '' ==
 
-
 
-
== TVEL13 - Laccase from ''Trametes versicolor'' ==
 
-
 
-
== TVEL20 - Laccase from ''Trametes versicolor'' ==
 
{{Team:Bielefeld/Sponsoren}}
{{Team:Bielefeld/Sponsoren}}

Latest revision as of 00:15, 3 December 2012

Results

Summary

All BioBricks of the iGEM Team Bielefeld were screened to identify the best conditions for protein expression. The first trials were made by shaking flask cultivations with different parameters. These parameters were various shaking flask designs, different temperatures, different concentrations of chloramphenicol, various induction strategies, several cultivation times and some cultivations in absence or presence of CuCl2. To detect the produced laccases, different analysis methods were performed like SDS-PAGE analysis as well as MALDI-TOF. The iGEM Team successfully produced four active bacterial laccases and succeeded to purify four of them. Besides the successfully scale-up fermentation, these laccases could be purified in a high amount to characterize the optimal activity conditions regarding pH, temperature, buffer solutions and organic solvent resistance. Furthermore, the iGEM Team Bielefeld demonstrated that the produced laccases can be immobilized maintaining their activity and the degradation capacity was screened for several micro-contaminants. These tests indicate that all of our produced laccases are able to degrade estradiol and the two laccases TTHL and BPUL are able to degrade ethinyl-estradiol in combination with a mediator. At this moment, the self-designed shuttle-vector for the production of eukaryotic laccases in yeast is ready to go. This vector was tested to integrate by courtesy of homologous recombination genes of eukaryotic laccases into Pichia Pastoris and produce them in an active form. First experiments show a successful production of one laccase of Trametes versicolor. A cheap alternative purification and immobilization method via a cellulose binding tag is also close at hand. During our research, we cultivated the following BioBricks and produced several laccase. To simplify the presentation of our results we named the produced laccase like the corresponding system.

Produced and generated BioBricks with the source strain of the DNA-sequence, promoter, protein name and the names given by the iGEM Team Bielefeld
BioBrick code strain promoter name of protein name given by the iGEM Team
<partinfo>K863000</partinfo> Bacillus pumilus DSM 27 T7 promoter CotA BPUL
<partinfo>K863005</partinfo> E. coli BL21(DE3) T7 promoter CueO ECOL
<partinfo>K863010</partinfo> Thermus thermophilus HB27 T7 promoter tthL TTHL
<partinfo>K863012</partinfo> Thermus thermophilus HB27 constitutive promoter (<partinfo>BBa_J23100</partinfo>) tthL TTHL
<partinfo>K863015</partinfo> Xanthomonas campestris pv. campestris B100 T7 CopA XCCL
<partinfo>K863020</partinfo> Bacillus halodurans C-125 T7 Lbh1 BHAL
<partinfo>K863022</partinfo> Bacillus halodurans C-125 constitutive promoter (<partinfo>BBa_J23100</partinfo>) Lbh1 BHAL
<partinfo>K863030</partinfo> Trametes versicolor AOX1 promoter TVL5 TVEL5

Datapage

iGEM Team Bielefeld is developing a biological filter using immobilized laccases, enzymes able to radicalize and break down a broad range of aromatic substances. For the production of laccases from different bacteria, fungi and plants, two expression systems are used: Escherichia coli and the yeast Pichia pastoris. Immobilization is carried out either by using CPC-silica beads or by fusing the enzymes to cellulose binding domains. The concept could be extended to other toxic pollutants in drinking and wastewater, as well as to industrial applications in paper and textile industries or even for bioremediation of contaminated soil.

Read more.

Laccases

The iGEM Team successfully produced four active bacterial laccases and an eukaryotic laccase (click for the results):

All bacterial laccases (ECOL, BHAL, TTHL and BPUL) we accomplished to purify. Besides the successfully scale-up fermentation these laccases could be purified in a high amount to characterize the optimal activity conditions regarding pH, temperature, buffer solutions and organic solvent resistance. Furthermore the iGEM Team Bielefeld demonstrated that the produced laccases can be immobilized maintaining their activity and the degradation capacity was screened for several micro-contaminants. These tests indicate that they are able to degrade estradiol and ethinyl-estradiol.

Immobilization

Using commercially acquired laccases from Trametes versicolor (named TVEL0) as a standard, it was possible to optimize an immobilization method of the purified laccases from

on CPC-silica beads. All four purified laccases were successfully immobilized, with ECOL and BPUL showing the highest binding ability to beads. Moreover, all four immobilized laccases showed activity. Whereas immobilized BPUL showed a relatively high activity, the results couldn't be compared to BHAL und TTHL due to the low concentration of the latters. For immobilization results see here

Substrate Analysis

To establish the methods for degradation analysis of different substrates TVEL0 was used as positive control. After that the four produced bacterial laccases were analyzed. The HPLC results showed that estradiol and ethinyl estradiol ( with addition of ABTS) are degradable with our laccases.To determine degradation products of estradiol and ethinyl estradiol after laccase treatment LCMS-MS analysis were done. For more informations click here.

Cellulose binding domain

A cheap alternative purification method combined with a powerful immobilization tool could be the solution to prevail over other more expensive water cleaning methods like oxidization with ozone or using tons of activated carbon which just capture microcontaminates, but does not dismantle them. A promising solution to this could be cellulose binding domains (CBDs). Cellulose is ubiquitous and sustainable. Following this idea fusion-protein-constructs with cellulose binding domains have been made. To characterize a GFP has been introduced as a C or N-terminal domain of the cellulose binding protein. After delays in cloning the constructs for two fusion proteins with a T7-promoter could be finished, but did not express the protein in E. coli KRX and BL21. An alternative construct with a constitutive promoter could also be finished, but gave the same results. Changing the order of CBD and GFP was carried out, but was hampered by a base deletion in the GFP gene causing a frame shift and could not be redone in time.

Read more

Shuttle vector

A shuttle vector for site-directed recombination into the yeast P. pastoris does not exist in the parts registry and could be developed by our team. With this system it is possible to recombine a protein of interest with a N-terminal mating factor alpha 1 for secretion the protein into the media. This protein of interest could be cloned in frame with one restriction ligate cloning step. The selection depends not on an antibiotic resistance like zeocine, but on a complementation of histidine auxotrophy. This system is for us important because some of our laccases can not be expressed in the prokaryotic expression system E. coli, because the protein needs glycosylation. Read more.

Collaboration with UCL

The BioBrick BBa_K729006 from the University College London was characterized by us. Therefore E. coli KRX containing BBa_K729006 and E. coli KRX as a negative control were cultivated in shaking flasks and a growth kinetic was determined. The harvested cells were lysed via sonication and substances with a low molecular weight were seperated out of the supernatant. After purification the sample was analyzed by SDS-PAGE and MALDI-TOF. For a comparison E. coli KRX containing BBa_K7863005 was cultivated and analysed by SDS-PAGE as well as tested with a laccase activity assay. BBa_K729006 and BBa_K7863005 showed a similar behaviour in oxidizing ABTS. Read more.


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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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