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| − | First some trials of shaking flask cultivations were made with various parameters to identify the best conditions for production of the His tagged laccase Lbh1 from [http://www.dsmz.de/catalogues/details/culture/DSM-18197.html?tx_dsmzresources_pi5 ''Bacillus halodurans'' C-125 ] named BHAL. Due to inactivity of the enzyme in the cell lysate a purification method was established (using Ni-NTA-Histag resin). BHAL could not be detected by SDS-PAGE (theoretical molecular weight of 56 kDa) or activity test by using the BioBrick <partinfo>BBa_K863020</partinfo> and ''E. coli'' KRX as expression system. Due to this results the new BioBrick <partinfo>BBa_K863022</partinfo> was constructed and expressed ''E. coli'' Rossetta-Gami 2. With this expression system the laccase could be produced and analysed via SDS-PAGE. A small scale Ni-NTA-column was used to purify the laccase. The fractionated samples were tested regarding their activity with ABTS and showed ability in oxidizing ABTS. A scale up was not yet performed.
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| − | ==Cultivation, Purification and SDS-PAGE==
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| − | ===Cultivation===
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| − | The first trials to produce the Lbh1 - laccase from ''Bacillus halodurans'' (named BHAL) were performed in shaking flasks with various flask designs (from 100 mL-1 to 1 L flasks, with and without baffles) and under several conditions. The varied parameters in our screening experiments were temperature (27 °C,30 °C and 37 °C), concentration of chloramphenicol (20-170 µg mL<html><sup>-1</sup></html>), induction strategy (autoinduction and manual induction with 0,1 % rhamnose) and cultivation time (6 to 24 h). Furthermore we cultivated with and without 0.25 mM CuCl<html><sub>2</sub></html> to provide a sufficient amount of copper, which is needed for the active center of the laccase. ''E.coli'' KRX was not able to produce active BHAL under the tested conditions, therefore another chassis was chosen. For further cultivations ''E. coli'' Rosetta-Gami 2 was transformed with BBa_K863012, because of its ability to translate rare codons. BHAL was produced under the following conditions:
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| − | * flask design: shaking flask without baffles
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| − | * medium: [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#LB_medium LB]-Medium
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| − | * antibiotics: 60 µg mL<html><sup>-1</sup></html> chloramphenicol and 300 µg mL<html><sup>-1</sup></html> ampicillin
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| − | * temperature: 37 °C
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| − | * cultivation time: 24 h
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| − | ===Purification===
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| − | The cells were harvested and resuspended in Ni-NTA-equilibration buffer, mechanically lysed by sonification and centrifuged. After preparing the cell paste the BHALlaccase could not be purified with the 15 mL column, because of the column was not available. For this reason a small scale purification (6 mL) of the supernatant of the lysate was performed with a [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Production#Syringe_method 1 mL Ni-NTA-column]. The elution was collected in 1 mL fractions.
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| − | ===SDS-PAGE===
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| − | [[Image:Bielefeld2012_0913.jpg|450px|thumb|left|'''Figure 1:'''SDS-PAGE of purified lysate derived from a flask cultivation of ''E. coli'' Rosetta-Gami 2 carrying <partinfo>BBa_K863022</partinfo>. Lanes 2 to 7 show the flow-through, the wash and the elution fractions 1 to 4. BHAL has a molecular weight of 56 kDa and is marked with an arrow.]]
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| − | In figure 1 the different fractions of the purified cell lysate of ''E. coli'' Rosetta-Gami 2 with <partinfo>BBa_K863022</partinfo> are shown in a SDS-PAGE. BHAL has a molecular weight of 56 kDa. In lane 5, which corresponds to the elution fraction 2, a faint band of 56 kDa is visible. Therefore the fractions were further analysed by activity test and MALDI-TOF.
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| − | ==Activity Analysis of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K863022 BHAL]==
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| − | The resulting fractions of the cultivation and purification of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K863022 BHAL] (fraction 1 to 5) were analysed with activity tests. After rebuffering into deionized H<sub>2</sub>O and incubation with 0.4 mM CuCl<sub>2</sub> for 2 hours, the samples were measured with 140 µL sample, 0.1 mM ABTS, 100 mM sodium acetate buffer to a final volume of 200 µL. The change in optical density was measured at 420 nm, reporting the oxidation of ABTS for 5 hours at 25°C. An increase in ABTS<sub>ox</sub> can be seen (Figure 2), indicating produced [https://parts.igem.org/wiki/index.php?title=Part:BBa_K863022 BHAL] laccase in each fraction. Fraction 2 shows the highest amount of ABTS<sub>ox</sub> (55%) reaching saturation after 3 hours. Similar to [https://parts.igem.org/wiki/index.php?title=Part:BBa_K863000 BPUL] laccase, [https://parts.igem.org/wiki/index.php?title=Part:BBa_K863022 BHAL] is capable to reach saturation after 3 hours with approximately oxidizing 55% of the supplied ABTS. Therefore [https://parts.igem.org/wiki/index.php?title=Part:BBa_K863022 BHAL] is going to be characterized further.
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| − | [[Image:Bielefeld2012_17_09_BHAL1.jpg|thumbnail|center|450px|'''Figure 2''': Activity test of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K863022 BHAL] fractions after purification. Reaction setup includes 140 µL fraction sample (CuCl2 incubated), 0.1 mM ABTS and 100 mM sodium actetate buffer (pH 5) to a final volume of 200 µL. Measurements were done at 25°C and over a time period of 5 hours. Each fraction shows activity, especially fraction 2, which therefore contains most [https://parts.igem.org/wiki/index.php?title=Part:BBa_K863022 BHAL] laccase. (n=4)]]
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