Team:Bielefeld-Germany/Results/Summary

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<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>TTHL</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>BHAL</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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<li><a href="#11"><strong>Shuttle vector</strong></a></li>
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                 <li><a href="#12"><strong>Collaboration with UCL</strong></a></li>
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<h1>Summary</h1>
<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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|-
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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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|-
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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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|-
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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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|-
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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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<img src="https://static.igem.org/mediawiki/2012/3/3e/Bielefeld2012_Overview.jpg" />
<h1>Datapage</h1>
<h1>Datapage</h1>
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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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==How our system works==
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==Data for our favorite new parts==
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# <partinfo>K863000</partinfo> - '''bpul (laccase from ''Bacillus pumilus'') with T7 promoter, RBS and HIS tag''':
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# <partinfo>K863005</partinfo> - '''ecol (laccase from ''E. coli'') with T7 promoter, RBS and HIS tag''':
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==Data for pre-existing parts==
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==We have also characterized the following parts==
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# <partinfo>BBa_K863012</partinfo> - '''tthl laccase ( from T. thermophilus) with constitutive promoter J23100, RBS and HIS tag''':
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# <partinfo>BBa_K863022</partinfo> - '''bhal laccase (from Bacillus halodurans) with constitutive promoter J23100, RBS and HIS tag''':
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<h1>Laccase from Bacillus pumilus DSM 27 (ATCC7061)</h1>
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<img src="https://static.igem.org/mediawiki/2012/6/6c/Bielefeld2012_Plan.jpg" />
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First some trails of shaking flask cultivations were made with different parameters to define the best conditions for the production of the His-tagged BPUL. Because of no activity in the cell lysate a purification method was established (using Ni-NTA-Histag resin). The purified BPUL could be detected by SDS-PAGE (molecular weight of 58,6&nbsp;kDa) as well as MALDI-TOF. To improve the purification strategies the length of the elution gradient was increased. The fractionated samples were also tested concerning their activity. A maximal activity of X was reached. After measuring activity of BPUL a scale up was made up to 3&nbsp;L and also up to 6&nbsp;L.
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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 to 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 Cu<sub>2</sub>Cl. Due to the screening experiments we identified the best conditions for expression of BPUL:
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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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==3&nbsp;L Fermentation ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo> ==
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[[File:Bielefeld2012_BPUL3LFermentation.jpg|450px|thumb|left|'''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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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&nbsp;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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== 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 chromatogram of the BPUL-elution is shown in figure 2:
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[[File:Bielefeld2012_BPUL3LChromatogramm.jpg|450px|thumb|left|'''Figure 2:''' Chromatogram 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 chromatogram 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 analyzed 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 peaks (up to a UV-detection-signal of 980&nbsp;mAU) detectable. These results are caused by an accidental detachment in front of the UV-detector. Just to be on the safe side, the corresponding fractions were analyzed by SDS-PAGE analysis. The results of the SDS-PAGE are shown in the following pictures.
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==SDS-PAGES of purified BPUL==
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[[File:Bielefeld2012_SDS_BPUL3L.jpg|widths={320}|heights{120}|thumb|left|'''Figure 3:''' SDS-Pages of purified E. coli KRX containing BBa_K863000 lysate (fermented in 3 L Biostat Braun B). The elution 1 to 3 are shown before buffer exchange and elution 1 and 2 also after buffer exchange. The arrow marks the BPUL band with a molecular weight of 58,6 kDa. !MARKER ANPASSEN!]]
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Figure 3 shows the purified culture of ''E. coli'' KRX with <partinfo>BBa_K863000</partinfo> lysates (fermented in 3 L Braun Biostat B). For this SDS-PAGE two of three samples were treated before. The cell disrupting buffer was exchanged to MilliQ, because of its usability for the activity test. It shows the elution fractions 1 and 2 before and after the buffer exchange and elution fraction 3 only before buffer exchange. The arrow marks the BPUL band with a molecular weight of 58,6 kDa. This PAGES shows, that BPUL could be expressed and eluted (first 2 fractions) successfully and that the exchange of buffers leads to a loss of the amount of proteins.
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The appearing bands were analysed by MALDI-TOF and could be identified as CotA (BPUL).
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==6&nbsp;L Fermentation of ''E.&nbsp;coli'' KRX with <partinfo>BBa_K863000</partinfo>==
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[[File:Bielefeld2012_BPUL6LFermentation.jpg|450px|thumb|left|'''Figure 4:''' 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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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. Agitation speed was increased up to 425&nbsp;rpm after one hour due to problems caused by the control panel. The pO<sub>2</sub> decreased until a cultivation time of 4,75&nbsp;hours. The increasing pO<sub>2</sub>-Level indicates the beginning of the deceleration phase. There is no visible break in cell growth caused by an induction of protein expression. A maximal OD<sub>600</sub> of 3,68 was reached after 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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==Purification of BPUL==
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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>. 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 chromatogram of the BPUL-elution is shown in figure 5.
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[[File:Bielefeld2012_BPUL6LChromatogramm.jpg|450px|thumb|left|'''Figure 5:''' Chromatogram 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.]]
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The chromatogram shows a 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 imidazol) . The corresponding fractions were analyzed 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 a 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 analyzed by SDS-PAGE analysis to detect BPUL. The results of the SDS-PAGE are shown in the following pictures.
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==SDS-PAGES of purified BPUL==
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[[File:Bielefeld2012 0914.jpg|450px|thumb|left|'''Figure 6:''' SDS-PAGE of purified ''E.&nbsp;coli'' with <partinfo>BBa_K863000</partinfo> lysate (fermented in Bioengineering, 6 L). The flow-through, wash and elution fraction 1 to 9 are shown. The arrow marks the BPUL band with a molecular weight of 58,6 kDa. ]]
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In figure 6 the SDS-PAGE of the Ni-NTA-Histag purification of the lysed culture of ''E. coli'' KRX containing <partinfo>BBa_K863000</partinfo> is illustrated. It shows the flow-through, wash and elution fractions 1-9. BPUL has a molecular weight of 58,6 kDA and was marked with a red arrow. The band appears in all fractions from 2 to 9 with a variing strength, with the strongest ones in fractions 7 to 9. There are also some other non-specific bands, which could not be identified. Therefore the purification method has to be improved for the next time.
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Furthermore the bands were analysed by MALDI-TOF and identified as CotA (BPUL).
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<h1>Laccases</h1>
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<h1>Laccase from Escherichia coli BL21 (DE3)</h1>
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<p class="more"> First some trails of shaking flask cultivations were made with changing parameters to identify the best conditions for the production of the laccase ECOL fused to a Histag. Because of no activity in the cell lysate a purification method was established (using Ni-NTA-Histag resin). The purified ECOL could be identified by SDS-PAGE (molecular weight of 53,4 kDa) as well as MALDI-TOF. The fractionated samples were also tested concerning their activity. A maximal activity of X was reached. After measuring activity of ECOL a scale up was made up to 3 L and then also up to 6 L.
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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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==Shaking Flask Cultivations==
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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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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 ([[https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium autoinduction autoinduction] and manual induction), several cultivation times (6 - 24&nbsp;h) and in absence or presence of 0,25&nbsp;mM Cu<sub>2</sub>Cl. Due to the screening experiments we identified the best conditions under which ECOL was expressed:
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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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* flask design: shaking flask without baffles
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:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/comparison Comparison of the different laccases]
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* medium: [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium autoinduction medium]
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:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/trametis Purchased positive control ''Trametes versicolor'' laccase TVEL0]
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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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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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The reproducibility and repeatability of the measured data and results were investigated for the shaking flask and bioreactor cultivation with n ≤ 3.
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==3&nbsp;L Fermentation ''E. coli'' KRX with <partinfo>BBa_K863005</partinfo>==
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[[File:Bielefeld2012_ECOL3LFermentation.jpg|450px|thumb|left|'''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.]]
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<p align="justify">
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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 lower. In the following hours, the OD<sub>600,max</sub> and the agitation speed decreased and the pO<sub>2</sub> increased, which indicates the death phase of the cells. This is caused by 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.
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</p>
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<br style="clear: both" />
 
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==Purification of ECOL==
 
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<p align="justify">
 
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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] step elution from 5&nbsp;% (equates to 25&nbsp;mM imidazol) with a length of 60&nbsp;mL, to 50&nbsp;% (equates to 250&nbsp;mM Imidazol) with a length of 60&nbsp;mL, to 80&nbsp;% (equates to 400&nbsp;mM imidazol) with a length of  40&nbsp;mL and finally to 100&nbsp;% (equates to 500&nbsp;mM imidazol) with a length 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 chromatogram of the BPUL-elution is shown in figure 2:
 
</p>
</p>
-
[[File:Bielefeld2012_ECOL3LChromatogramm.jpg|450px|thumb|left|'''Figure 2:''' Chromatogram 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. ]]
 
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<p align="justify">
 
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The chromatogram 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 to 25&nbsp;mM 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 peak up to a UV-detection signal of 292&nbsp;mAU was measured. The area 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 figure 3.
 
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<br style="clear: both" />
 
-
 
-
==SDS-PAGE of ECOL purification==
 
-
 
-
[[File:Bielefeld2012_SDS_ECOL3L.jpg|450px|thumb|left|'''Figure 3:''' SDS-Pages of purified  ''E.&nbsp;coli'' KRX containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005] lysate (fermented in 3&nbsp;L Infors Labfors). The flow-through, wash and elution fraction 2-9 are shown. The arrow marks the ECOL band with a molecular weight of 53,4&nbsp;kDa.]]
 
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In figure 3 the SDS-PAGE of the Ni-NTA-Histag purification of the lysed culture ( ''E.&nbsp;coli'' KRX containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005]) is illustrated including the flow-through and the fractions 2 to 9. The red arrow indicates the band of ECOL with a molecular weight of 53,4&nbsp;kDa, which appears in all fractions. The strongest bands appear in fractions 6 and 7. Another small band with a lower molecular weight appears, but was not identified.
 
-
 
-
Furthermore the bands were analysed by [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Analytics#MALDI MALDI-TOF] and identified as CueO (ECOL).
 
-
<br style="clear: both" />
 
-
 
-
==6&nbsp;L Fermentation ''E. coli'' KRX with <partinfo>BBa_K863005</partinfo>==
 
-
 
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[[File:Bielefeld2012_ECOL6LFermentation.jpg|450px|thumb|left|'''Figure 4:''' 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.]]
 
-
 
-
<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 a 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. The death phase started, which is indicated by an increasing pO<sub>2</sub> and a decreasing OD<sub>600</sub>. 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 lower. Cells were harvested after 12&nbsp;hours.
 
-
</p>
 
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<br style="clear: both" />
 
-
 
-
==Purification of ECOL==
 
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<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>. 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 length of 200&nbsp;mL and the elution was collected in 10&nbsp;mL fractions. The chromatogram of the ECOL-elution is shown in figure&nbsp;4:
 
-
</p>
 
-
 
-
[[File:Bielefeld2012_ECOL6LChromatogramm.jpg|450px|thumb|left|'''Figure 5:''' Chromatogram 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.]]
 
-
 
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<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 chromatogram shows a remarkable 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.
 
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</p>
 
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<br style="clear: both" />
 
-
 
-
==SDS-PAGES of ECOL purification==
 
-
[[File:Bielefeld2012_coli0910.jpg|450px|thumb|left|'''Figure 6:''' SDS-Pages of lysed culture of ''E.&nbsp;coli'' KRX containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005] (fermented in 6&nbsp;L Bioengineering NFL22) after purification. The flow-through, wash and elution fraction 1-15 are shown (except from fraction 11/12). The arrow marks the ECOL band with a molecular weight of 53,4&nbsp;kDa.]]
 
-
 
-
 
-
In figure 6 the result of the Ni-NTA-Histag purification of the lysed culture ''E.&nbsp;coli'' KRX containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005] (6&nbsp;L fermentation) including the flow-through, wash and the fractions 1 to 15 (except from fraction 11/12) are shown. The red arrow indicates the band of ECOL with a molecular weight of 53,4&nbsp;kDa, which appears in all fractions. The strongest bands appear from fractions 3 and 8 with a decreasing amount of other non-specific bands.
 
-
 
-
Furthermore the bands were analysed by [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Analytics#MALDI MALDI-TOF] and identified as CueO (ECOL).
 
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<br style="clear: both" />
 
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<img src="https://static.igem.org/mediawiki/2012/2/25/Bielefeld2012_Immo.jpeg" />
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<h1>Laccase from Thermus thermophilus HB27</h1>
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<h1>Immobilization</h1>
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<p class="more">
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<p class="more">
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First some trails of shaking flask cultivations were made with different parameters to define the best conditions for the production of the His-tagged TTHL. Because of no activity in the cell lysate a purification method was established (using Ni-NTA-Histag resin). The purified TTHL could not be detected by SDS-PAGE (theoretical molecular weight of 53&nbsp;kDa) by using the '' E. coli'' KRX as expression system. Due to this results we decided to change the expression to ''E. coli'' Rossetta-Gami&nbsp;2. With this expression system the TTHL could be detected by SDS-PAGE as well as MALDI-TOF analysis. To prove the activity of the produced laccase we used a small scale Ni-NTA-column to purify our laccase. The fractionated samples were also tested concerning their activity. A maximal activity of X was reached. After measuring activity of TTHL a scale up was made up to 6&nbsp;L
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</html>
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__NOTOC__
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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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<html>
-
==Shaking Flask Cultivation==
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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
+
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* medium: [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials LB-Medium]
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* 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:
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-
SDS PAGES!!!!!
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ACTIVITÄTSMESSUNGEN VOM ERSTEN AKTIVEN B PUMI!!!!!!
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+
-
==Fermentation of ''E. coli'' KRX with <partinfo>BBa_K863012</partinfo>==
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+
-
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.
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<p align="justify">
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[[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.]]
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<br style="clear: both" />
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+
:* [http://openwetware.org/wiki/E._coli_genotypes#BL21.28DE3.29 ''E. coli'' BL21 (DE3)] (named ECOL)
-
 
+
:* [http://www.dsmz.de/catalogues/details/culture/DSM-27.html ''Bacillus pumilus'' DSM 27 (ATCC7061)] (named BPUL)
-
==Purification of TTHL==
+
:* [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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<p align="justify">
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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:
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</p>
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==SDS-PAGE of purification 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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<h1>Laccase from Bacillus halodurans C-125</h1>
 
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First some trails of shaking flask cultivations were made with various parameters to identify the best conditions for the production of the His-tagged BHAL. Because of no activity in the cell lysate a purification method was established (using Ni-NTA-Histag resin). The BHAL could not be detected by SDS-PAGE (theoretical molecular weight of 56&nbsp;kDa) by using the '' E. coli'' KRX as expression system. Due to this results we decided to change the expression to ''E. coli'' Rossetta-Gami&nbsp;2. With this expression system the BHAL could be found by SDS-PAGE as well as MALDI-TOF analysis. To prove the activity of the produced laccase we used a small scale Ni-NTA-column to purify our laccase. The fractionated samples were also tested concerning their activity. A maximal activity of X was reached. A scale up could not be implemented yet.
 
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</p>
 
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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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<h1>Laccase from Trametes versicolor </h1>
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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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<img src="https://static.igem.org/mediawiki/2012/6/6d/Bielefeld2012_GFP.jpg" />
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<h1>Cellulose binding domain</h1>
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<p class="more">
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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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<h1>Subtrate Analytics</h1>
 
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<img src="https://static.igem.org/mediawiki/2012/1/17/Bielefeld2012_PECPP11JS.JPG" />
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<h1>Shuttle vector</h1>
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__NOTOC__
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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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==Dilutioin series==
+
</p>
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[[File:Bielefeld2012_detectionlimit.JPG|200px|thumb|right|'''Figure 1.1:'''The detection range of ethinyl estradiol. Concentrations between 0.1 µg ml <sup>-1</sup> and 3 µg ml <sup>-1</sup> are detecable.]]
+
-
 
+
-
At first we mesured dilution series of all different substrates.
+
-
* estrogens
+
-
** estradiol
+
-
** estrone
+
-
** ethinyl estradiol
+
-
* analgesics
+
-
** diclofenac
+
-
** naproxen
+
-
** ibuprofen
+
-
* PAH´s
+
-
** naphthalene
+
-
** acenaphthene
+
-
** phenantrene
+
-
** anthracen
+
-
 
+
-
==Negative controls==
+
-
[[File:Bielefeld2012_neg_pahs.png|700px|thumb|center|'''Figure 2.1:'''The PAH´s naphthalene,
+
-
acenaphthene and phenantrene in BR-buffer at 30 °C. The startconcentration was 1 µg mL <sup>-1</sup> for all PAH´s. After 1 houre nearly all PAH´s decayed completely.]]
+
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[[File:Bielefeld2012_naphneg.png|700px|thumb|center|'''Figure 2.2:'''Naphthalene decay in four different conditions. Dissolved in MeOH, dissolved in acetonitrile, with BR-buffer and with BR-buffer together with ABTS ]]
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==Degradation==
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[[File:Bielefeld2012_EBT_ABTS.png|800px|thumb|center|'''Figure 3.1:'''Comparison between two of our Laccases BPUL and ECOL with the purchased Laccase TVEL0 under ABTS influence.]]
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<h1>Cellulose binding domain</h1>
 
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__NOTOC__
 
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==Introduction==
 
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<p align="justify">
 
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In the field of cheap protein-extraction cellulose binding domains (CBD) have made themselfes a name. A lot of publications have been made, concerning a cheap strategy to capture a protein from the cell-lysate with a CBD-tag. Also enhanced segregation with CBD-tagged proteins have been observed. Here the idea is different, we want to take advantage of the binding capacity of binding domains not only for purification reasons (it is still a benefit), but also as an immobilizing-protocol for our laccases.
 
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[[Image:Bielefeld2012 Osaka3.jpg|500px|thumb|right|Figure 1:Graphical sequence-alignment of <partinfo>K392014</partinfo> and the [http://www.ncbi.nlm.nih.gov/nuccore/AB041993 Xyn10A gene of C.josui]]]
 
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To make a purification and immobilization-tag out of a protein domain, there are a lot of decisions and characterizations you have to get through.
 
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Starting with the choice of the binding domain, the first limitation is accessibility. Our first place to look was, of course the [http://partsregistry.org Partsregistry]. We found a promising Cellulose binding motif of the ''C. josui'' Xyn10A gene (<partinfo>BBa_K392014</partinfo>) there and ordered it right from the spot for our project. After some research later concerning the sequence of that BioBrick it turned out that the part is not the CBD of the Xylanase as it should be, but the glycosyl hydrolase domain of the protein (Figure 1). This result made the part useless for our project ([http://partsregistry.org/Part:BBa_K392014:Experience complete review]) and it was the only binding domain in the [http://partsregistry.org Partsregistry] that fitted to our project.
 
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So we started to search the accessible organisms we had via NCBI for binding-domains, -proteins and -motifs and asked in work-groups if they could help us out. The results of our database research were only two chitin/carbohydate binding modules within the ''Bacillus halodurans'' genome (we ordered that stain for it's laccase <partinfo>BBa_K863020</partinfo>). One is in the [http://www.ncbi.nlm.nih.gov/nucleotide/289656506?report=genbank&log$=nuclalign&blast_rank=1&RID=0JPT9WMS01N Cochin chitinase gene] and the other in a [http://www.ncbi.nlm.nih.gov/protein/BAB05022.1 chitin binding protein].
 
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[[Image:Bielefeld2012_p714.jpg|200px|thumb|left|Figure 2: Plasmid with CBDcex]][[Image:Bielefeld2012_p570.jpg|220px|thumb|left|Figure 3: Plasmid with CBDclos]]
 
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Meanwhile the Fermentation of our university group offered use two plasmids (p570 & p671), containing two different cellulose binding domains. The Cellulose binding domain of the [http://www.ncbi.nlm.nih.gov/nucleotide/327179207?report=genbank&log$=nucltop&blast_rank=3&RID=152ZCN0E01N ''Cellulomonas fimi'' ATCC 484 exoglucanase gene] (CBDcex) and the Cellulose binding domain of [http://www.ncbi.nlm.nih.gov/nuccore/M73817 ''Clostridium cellulovorans'' cellulose binding protein gene (cbp A)] (CBDclos). At that point we decided to use these two domains. Staying within the cellulose binding domain-family and leave other protein domains like carbohydrate binding domains aside will keep the results comparable. Like changing to a different binding material would change the binding capacities of both domains in the same way. Also we would stay within bacterial CBD and didn't have to spend time thinking about post-translational modification and glycosylation.
 
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-
To get to know more about these two domains, their properties and their proteins we consulted NCBI and used the [http://blast.ncbi.nlm.nih.gov/ BLAST]-tool to identify the cellulose binding domains and ExPASy-tools for further measurements.
 
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[[Image:Bielefeld2012_pfam00553.jpg|130px|thumb|left|Figure 5: Crystal structure of the CBM_2-family]]
 
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[[Image:Bielefeld2012_cfimiexo.jpg|500px|thumb|right|Figure 6: Protein-BLAST of [http://www.ncbi.nlm.nih.gov/nucleotide/327179207?report=genbank&log$=nucltop&blast_rank=3&RID=152ZCN0E01N ''Cellulomonas fimi'' ATCC 484 exoglucanase gene]]]
 
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The CBD of the Cellulomonas fimi ATCC 484 exoglucanase gene (Figure 6) is a 100 amino acid long domain, close to the C-terminal ending of the protein with a theoretical pI of 8.07 and a molecular weight of 10.3 kDa. It is classified to be stable and belongs to the Cellulose Binding Modul family 2 (pfam00553/cl02709). This means two tryptophan residues are involved in cellulose binding, this type of CBD is only found in bacteria. Also a CBM49 Carbohydrate binding domain is found within the protein domain, where [http://www.ncbi.nlm.nih.gov/pubmed/17322304?dopt=Abstract binding studies] have shown, that it binds to crystalline cellulose, which could be a possible target for immobilization.
 
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[[Image:Bielefeld2012_ccellubp.jpg|450px|thumb|left|Figure 7: Protein-BLAST of [http://www.ncbi.nlm.nih.gov/nuccore/M73817 ''Clostridium cellulovorans'' cellulose binding protein gene (cbp A)]]]
 
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[[Image:Bielefeld2012_pfam00942.jpg|130px|thumb|right|Figure 8: Crystal structure of the CBM_3-family]]
 
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The CBD of the [http://www.ncbi.nlm.nih.gov/nuccore/M73817 ''Clostridium cellulovorans'' cellulose binding protein gene (cbp A)] on the other hand is a N-terminal domain with 92 amino acids, theoretical pI of 4.56 and ist also classified as stable. It belongs to the Cellulose Binding Modul family 3 (pfam00942/cl03026) and is part of a very large cellulose binding protein with four other carbohydrate binding moduls and a lot of docking interfaces for the proteins in its amino acid sequence (Figure 7).
 
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==The Binding Assay==
 
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[[image:Bielefeld2012_GFP.jpg|300px|thumb|right|Aequorea victoria green fluorescent protein in action]]
 
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To measure the capacity and strength of the bonding between the cellulose binding domains and different types of cellulose many different assays have been made. One of the simplest and most often used is the fusion of the CBD to a reporter-protein, especially [http://www.ncbi.nlm.nih.gov/pubmed/22305911a green or red fluorescent protein (GFP/RFP)] is very common. The place of the CBD is measured through the fluorescence of the fused GFP and quantification can easily been done.
 
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[http://www.ncbi.nlm.nih.gov/pubmed/18573384 Protocol]:
 
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* Harvest the ''E coli''-cells producing the fusion-protein of CBD and GFP and centifuge 10 minutes at top speed.
 
-
* Re-suspend the cell-pellet in 50 mM Tris-HCl-Buffer (pH 8.0).
 
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* Break down cells via sonication.
 
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* Centrifuge at top speed for 20 minutes to get rid of the cell-debris.
 
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* Take the supernatant and measure the emission at 511 nm (excitation at 501 nm) (<partinfo>E0040</partinfo>)
 
-
* Mix a definite volume of lysate with a definite volume or mass of e.g. crystalline cellulose (CC) or reactivated amorphous cellulose (RAC)
 
-
* Wait 15 (RAC) to 30 (CC) minutes
 
-
* Take supernatant and measure the emission at 511 nm again.
 
-
* The difference between the first an the second measurement is the relative quantity of what has bound to the cellulose.
 
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==Cloning of the Cellulose Binding Domains==
 
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The cloning of the CBDs should fit to the cloning of our laccases, so we designed the BioBricks with a T7-promoter and the B0034 RBS to have a similar method of cultivation. After we investigated the restriction-sites it showed, that at least for the characterization of the CBDs a quick in-frame assembly of the CBDs and a GFP would be possible, because nether the CBDs nor the GFP (<partinfo>I13522</partinfo>) of the partsregistry inherits a ''Age''I- or ''Ngo''MIV-site, which makes Freiburg-assembly possible. To do so, we designed [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Primers primers] for the constructs <partinfo>BBa_K863101</partinfo> (CBDcex(T7)), carrying the CBDcex domain from the ''C. fimi'' exoglucanase and <partinfo>BBa_K863111</partinfo> (CBDclos(T7)), carrying the CBDclos domain from the ''C.cellulovorans'' binding protein. The protein-BLAST of the two CBDs gave a exact picture of which bases are count to the binding domains and which are not. To be sure not to disturb the folding anyway we kept 6 to 12 bases up and downstream of the domains as conserved sequences. Even if the [http://partsregistry.org/Part:BBa_K863101 CBDcex]is a N-terminal domain we chose to make the domains N-terminal, so the BioBrick already carries all regulatory parts and the linked protein can easily be exchanged. This also would be nice for other people using this part.
 
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<center>
 
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{|class="wikitable"
 
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| CBDcex_T7RBS|| 80 || TGAATTCGCGGCCGCTTCTAGAGTAATACGACTCACTATAGGGAAAGAGGAGAAATAATGGGT<br />CCGGCCGGGTGCCAGGT
 
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|-
 
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| CBDcex_2AS-Link_compl || 56 || CTGCAGCGGCCGCTACTAGTATTAACCGGTGCTGCCGCCGACCGTGCAGGGCGTGC
 
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| CBDclos_T7RBS || 73 || CCGCTTCTAGAGTAATACGACTCACTATAGGGAAAGAGGAGAAATAATGTCAGTTGAATTTTACAACTCTAAC
 
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| CBDclos_2ASlink_compl|| 63 || CTGCAGCGGCCGCTACTAGTATTAACCGGTGCTGCCTGCAAATCCAAATTCAACATATGTATC
 
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The listed complementary primers added, besides the Freiburg-suffix, a two amino acid Glycine-Serine-linker to the end of the CBDs. This is a very short linker, but as GFP-experts and [http://www.ncbi.nlm.nih.gov/pubmed/17394253 publications] described GFP and CBDs are very stable proteins and should cope with a very short linker. The benefit of a short linker is that protease activity is kept minimal.
 
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The GFP <partinfo>K863121</partinfo> we wanted to use for the assay was an alternated version of the <partinfo>BBa_I13522</partinfo>. We made [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Primers primers] that added a Freiburg-pre- and suffix to the GFP coding sequence and a His-tag to the C-terminus to get it purified for the measurements. This part <partinfo>BBa_K863121</partinfo> (GFP_His) should be easily added to the CBDs and assemble to the fusion-proteins <partinfo>BBa_K863103</partinfo> CBDcex(T7)+GFP_His] and <partinfo>BBa_K863113</partinfo> CBDclos(T7)+GFP_His]).
 
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<center>
 
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{|class="wikitable"
 
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| GFP_Frei || 54 || TACGGAATTCGCGGCCGCTTCTAGATGGCCGGCATGCGTAAAGGAGAAGAACTT
 
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|-
 
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| GFP_His6_compl || 74 || CTGCAGCGGCCGCTACTAGTATTAACCGGTGTGATGGTGATGGTGATGTTTGTATAGTTCATCCATGCCATGTG
 
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|}
 
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</center>
 
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Since we added a His-tag to the end of the GFP, we had to make a alternated version of the <partinfo>BBa_I13522</partinfo> to compare binding of GFP with and without CBD. Therefore we made a forward primer, to amplify the whole <partinfo>BBa_I13522</partinfo> and used the GFP_His_compl-primer to add the His-tag to the C-terminus.
 
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<center>
 
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{|class="wikitable"
 
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| GFP_FW_SV || 39 || acgtcacctgcgtgtagctCGTAAAGGAGAAGAACTTTT
 
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Due to bad cleavage efficiency at the ''Pst''I restiction-site in nearly all PCR-products the cloning of the CBDs ([http://partsregistry.org/Part:BBa_K863102 CBDcex(T7)] and [http://partsregistry.org/Part:BBa_K863112 CBDclos(T7)])and especially the insertion of [http://partsregistry.org/Part:BBa_K863121 GFP_His] to the CBDs took a lot more time as expected. This was because, while designing the primers, we missed to add additional base pairs from the cleavage-site to the termini to increase the efficiency to standard. When we got aware of the problem, cloning got a lot quicker and more successful.
 
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As the project went on and the T7-constructs didn't seem to work one suspected reason was that a stop codon (TAA) that accidentally resulted between the RBS and ATG could be the reason for that. To solve the problem we started to change to a constitutive promoter (<partinfo>BBa_J61101</partinfo>) using the Freiburg-assembly. Therefore Freiburg forward primers for the CBDs were made.
 
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<center>
 
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{|class="wikitable"
 
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| CBDcex_Freiburg-Prefix|| 54 || GCTAGAATTCGCGGCCGCTTCTAGATGGCCGGCGGTCCGGCCGGGTGCCAGGTG
 
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| CBDclos_Freiburg-Prefix || 57 || GCTAGAATTCGCGGCCGCTTCTAGATGGCCGGCTCATCAATGTCAGTTGAATTTTAC
 
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|}
 
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The second advantage of these primers were, that the order of the fusion proteins could easily been changed, when a Freiburg suffix-primer for the GFP would be available, so we ordered that.
 
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<center>
 
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{|class="wikitable"
 
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| GFP_Freiburg_compl || 61 || ACGTCTGCAGCGGCCGCTACTAGTATTAACCGGTTTTGTATAGTTCATCCATGCCATGTGT
 
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|}
 
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[[File:Bielefeld2012_S3N10linker.jpg|500px|thumb|right|PCR-product of BBa_K863104 with S3N10-primers]]
 
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Sadly the primers arrived just a few days before wiki freeze and we had no time to test that. The switching to the constitutive promoter had no obvious effect (no green colonies or culture). Changing the expression strain of ''E. coli'' BL21 didn't do no positive effect ether. Which led us to the conclusion, that besides further information the problem has to be the space in between the two proteins and the CBD and GFP must hamper each other from folding correctly. To test and solve this a very long linker with three serines followed by ten asparagines should be assembled in the already existing parts via a blunt end cloning. This also is right at hand, just wasn't successful in the time which was left.
 
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<center>
 
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{|class="wikitable"
 
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| S3N10_Cex_compl || 40 || TTGTTGTTGTTCGAGCTCGAGCCGACCGTGCAGGGCGTGC
 
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|-
 
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| S3N10_Clos_compl || 40 || TTGTTGTTGTTCGAGCTCGAGCTGCCGCCGACCGTGCAGG
 
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|-
 
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| S3N10_GFP || 40 || CAATAACAATAACAACAACCGTAAAGGAGAAGAACTTTTC
 
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|}
 
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== Literature ==
 
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Biotechnol Bioeng. 2007 Oct 15;98(3):599-610.
 
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Strategy for selecting and characterizing linker peptides for CBM9-tagged fusion proteins expressed in Escherichia coli.
 
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Kavoosi M, Creagh AL, Kilburn DG, Haynes CA.
 
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J Biol Chem. 2007 Apr 20;282(16):12066-74. Epub 2007 Feb 23.
 
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A tomato endo-beta-1,4-glucanase, SlCel9C1, represents a distinct subclass with a new family of carbohydrate binding modules (CBM49).
 
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Urbanowicz BR, Catalá C, Irwin D, Wilson DB, Ripoll DR, Rose JK.
 
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Protein Expr Purif. 2012 Apr;82(2):290-6. Epub 2012 Jan 28.
 
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Cellulose affinity purification of fusion proteins tagged with fungal family 1 cellulose-binding domain.
 
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Sugimoto N, Igarashi K, Samejima M.
 
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Anal Chim Acta. 2008 Jul 28;621(2):193-9. Epub 2008 May 27.
 
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Bioseparation of recombinant cellulose-binding module-proteins by affinity adsorption on an ultra-high-capacity cellulosic adsorbent.
 
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Hong J, Ye X, Wang Y, Zhang YH.
 
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<h1>Shuttle vector</h1>
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<h1>Collaboration with UCL</h1>
<h1>Collaboration with UCL</h1>
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The BioBrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_K729002 BBa_K729002] from the University&nbsp;College&nbsp;London was characterized by us using our Chassi (''E. coli'' KRX and our acitvity assay. We cultivcate a ''E. coli'' KRX containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K729002 BBa_K729002] in 50&nbsp;mL shaking flask for 12 h. The harvested cells were lysed and the supernatant was purified from substances with a low molecular weight. The purified supernatant analyzed by SDS-PAGE analysis, our etablished activity assay as well as MALDI-TOF. A maximal activity of X was reached.
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To compare the laccase the University&nbsp;College&nbsp;London we cultivated the ''E. coli'' KRX and ''E. coli'' KRX  containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K7863005].
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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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__NOTOC__
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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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== PCIL - Laccase from ''Pycnoporus cinnabarinus'' ==
 
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== TVEL5 - Laccase from ''Trametes versicolor '' ==
 
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== TVEL10 - Laccase from ''Trametes versicolor '' ==
 
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== TVEL13 - Laccase from ''Trametes versicolor'' ==
 
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== 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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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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