Team:Bielefeld-Germany/Results/Summary

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                Results
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<li><a href="#1"><strong>Summary</strong></a></li>
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<li><a href="#2"><strong>Datapage</strong></a></li>
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<a href="#1"><strong>BioBricks</strong>
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<li><a href="#3"><strong>Laccases</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>Substrate Analysis</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>Shuttle vector</strong></a></li>
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                <li><a href="#8"><strong>Collaboration with UCL</strong></a></li>
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<a href="#3">Judging Criteria<strong></strong>
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<h1>Summary</h1>
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<h3>BioBricks</h3>
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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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|<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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| <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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| <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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coming soon. <a href="https://2012.igem.org/Team:Bielefeld-Germany/BioBricks">read more</a>
 
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<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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<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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<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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== Summary ==
 
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== BPUL - Laccase from ''Bacillus pumilus  DSM 27 (ATCC7061)'' ==
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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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<h1>Substrate Analysis</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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=== 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>. After loading the supernatant on the column, the column was washed by 10&nbsp;column&nbsp;volumes (CV) with equilibrationbuffer.The bounded Protein's were eluted by an increasing elutionbuffer gradient from 0% to 100% with a length of 100&nbsp;mL and the elution was collected in 10&nbsp;mL Fraction. The chromatogramm of the BPUL-elution is shown in the following graph:
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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>.]]
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The chromatogramm shows a remarkable widespread peak at the beginning of the elution. This peak can be explained by the elution of bounded proteins. The corresponding fractions were analysed by SDS-PAGE. Afterwads there is an upwards trend of the UV-signal. This trend is caused by the increasing Imidazol concentration during the elution gradient. Towards the end of the elution procedure there are different and strong peaks detectable. These peaks results from a problem with the tube between the conductivity sensor and the UV-detector. During the elution the connector of the tube has become detached in front of the UV-detector. The Connection were not screwed corretly which leaded to a lost of volume of the corresponding fractions and most probably the measured peaks caused by air bubbles. 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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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>Shuttle vector</h1>
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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 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>. After loading the supernatant on the column, the column was washed by 5&nbsp;column&nbsp;volumes (CV) with equilibrationbuffer.The bounded Protein's were eluted by an increasing elutionbuffer gradient from 0% (equates 20mM Imidazol) to 100% (equates 500mM Imidazol) with a length of 200&nbsp;mL. This strategies was chosen to get a better result of purification caused by a slowly increasing elutionbuffer concentration.The elution was collected in 10&nbsp;mL Fraction. The chromatogramm of the BPUL-elution is shown in the following graph:
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[[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>.]]
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<h1>Collaboration with UCL</h1>
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The chromatogramm shows a strong peak the beginning of the elution. This peak can be explained by pressure fluctuations upon starting the elution  procedure. In the following part ot the chromatogramm there is remarkable widespread peak which results in the elution of bounded proteins.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 bounded proteins elute at low imidazol concentration. Just to be on the safe side, all fractions were analysed by SDS-PAGE analysis to detect the B.&nsbp;pumi-Laccase. The results of the SDS-PAGE are shown in the following pictures.
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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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== ECOL - Laccase from ''Escherichia coli BL21 (DE3)'' ==
 
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===3&nbsp;L Fermentation ''E. coli'' KRX with <partinfo>BBa_K863005</partinfo>===
 
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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;%. 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.
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[[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.]]
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===Purification of ECOL===
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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>. After loading the supernatant on the column, the column was washed by 10&nbsp;column&nbsp;volumes (CV) with equilibrationbuffer.The bounded Protein's were eluted by an increasing elutionbuffer gradient step-by-step from 5&nbsp;% (equates 25&nbsp;mM Imidazol)with a length of 60&nbsp;mL, to 50&nbsp;% (equates 250mM Imidazol) with a length of 60&nbsp;mL, to 80&nbsp;% (equates 400mM Imidazol) with a length of  40mL and finaly to 100&nbsp;% (equates 500mM Imidazol) with a length of 80&nbsp;mL. This strategies was chosen to get a better result of purification caused by a step-by-step increasing elutionbuffer concentration.The elution was collected in 10&nbsp;mL Fraction. The chromatogramm of the BPUL-elution is shown in the following graph:
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[[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>.]]
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The chromatogramm shows two peaks. The first peak was detected by an elutionbuffer concentration of 5&nbsp;% and can result from the elution of weakly bounded proteins. After increasing the elutionbuffer concentration to 50&nbsp;% (equates 250mM Imidazol) a large peak was measured. The strength of this peak indicates that a high amount of protein eluates at this moment. The corresponding fractions were analysed by SDS-PAGE analysis to detect our produced laccase. During the following elution process there were no further peaks detectable. The following increasing UV-dectection-signals equates to the imidazol concentration of the elutionsbuffer. The output of the SDS-PAGES are shown in the following images.
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===6&nbsp;L Fermentation ''E. coli'' KRX with <partinfo>BBa_K863005</partinfo>===
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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, 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 reportet 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.
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[[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.]]
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===Purification of ECOL===
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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>. After loading the supernatant on the column, the column was washed by 10&nbsp;column&nbsp;volumes (CV) with equilibrationbuffer.The bounded Protein's were eluted by an increasing elutionbuffer gradient from 0% to 100% with a length of 200&nbsp;mL and the elution was collected in 10&nbsp;mL Fraction. The chromatogramm of the BPUL-elution is shown in the following graph:
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[[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>.]]
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After Washing the Column with 10 CV equilibrationbuffer the elution process was started. The chromatogramm shows a great peak caused by the  elution of a high amount of proteins. The run of the curve show a little 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-by-step increasing of the elutionbuffer concentration ([https://2012.igem.org/Team:Bielefeld-Germany/Results/Summary#Purification_of_ECOL see purification of the 3 L Fermentation above]).To detect our protein the corresponding fraction were analysed by SDS-PAGE analysis.
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== XCCL - Laccase from ''Xanthomonas campestris pv. campestris B100'' ==
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== BHAL - Laccase from ''Bacillus halodurans C-125'' ==
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== TTHL - Laccase from ''Thermus thermophilus HB27'' ==
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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.
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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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===Purification===
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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. During the preparation of the Ni-NTA-column the pressure increased immediality and led to a damage of the column. Due to the damaged column a Ni-NTA-Purification with the 15&nbsp;mL Ni-NTA-resin was not possible. To detect and to analyse our produced ''T. thermophilus""-laccase we implement a small scale purificatoin of  6&nbsp;mL ot the supernatant with a 1&nbsp;ml Ni-NTA-column. The results of the SDS-PAGE analysis is shown the following images:
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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

  • [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)
  • [http://www.dsmz.de/catalogues/details/culture/DSM-18197.html?tx_dsmzresources_pi5 Bacillus halodurans C-125 ] (named BHAL) and from
  • [http://www.dsmz.de/catalogues/details/culture/DSM-7039.html?tx_dsmzreso Thermus thermophilus HB27] (named TTHL)

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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K729006 BBa_K729006] from the University College 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. For a comparison E. 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. 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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