Difference between revisions of "Part:BBa K863020"
 
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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&nbsp;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&nbsp;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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We aimed to express this protein in E. coli to study the laccase activity on synthetic substrates at different pH levels. E. coli BL21Star cells were transformed with these parts, each of which contained a T7 promoter and termination sequence flanking the coding sequences. Expression was induced with IPTG and protein expression was performed, after which cells were harvested followed by sonication in protein extraction buffer. A pET160-GW/CAT construct expressing the chloramphenicol acetyltransferase (CAT) protein was used as a positive control for induction. The soluble fraction was analyzed on a 12% SDS-PAGE gel followed by Coomassie staining (Fig. 1). We observed expression of the positive control CAT protein (~32 kDa), but no evidence of tthl or bhal expression (Fig 2.)
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Fig 1. SDS PAGE of laccase proteins.
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==Cultivation, Purification and SDS-PAGE==
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Nevertheless, we assumed there would be some functional laccase present, and proceeded with decolorisation assays.
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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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We tested the laccases on the decolorization of bromophenol blue. We first experimented with a standard solution of laccase purified from Trametes versicolor by incubating bromophenol blue in a buffer containing copper sulphate with various quantities of Trametes laccase.
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===Purification===
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Next, we assayed the E. coli protein extracts for laccase activity at different pH levels. The positive control, from Trametes, worked very well as a positive control, but only at low pH. Aside from that, there was no significant difference between the absorbance of the laccases and the negative control (Fig 3.).
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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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Fig 3. Absorbance values of laccase proteins (Trametes = Positive control; CAT = Negatice control)
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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.&nbsp;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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==Added by LZU-HS-China-C==
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==Activity Analysis of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K863022 BHAL]==
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We think that this laccase may degrade antibiotics, but we are worried that the degradation antibiotic products of this laccase will have an adverse effect on the human body, so we studied the sulfadiazine degradation products of this laccase.
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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&nbsp;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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===Speculate the degradation pathway of sulfadiazine===
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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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In the experiment, liquid-mass spectrometry (HPLC-MS) was used in the resting state to explore the degradation pathway of SDZ. First, EcN-IL was inoculated into LB medium at 37°C overnight at 1% inoculum. After centrifugation to remove the supernatant, the bacteria were collected (6000 r/min, 10 min). The bacteria were washed twice with deionized water and then prepared into a bacterial suspension with a cell concentration of 1.0×109 CFU/mL, and 5 mL of the cell suspension was taken. Solution, add SDZ sodium salt with a final concentration of 5 mg/L to it, three replicates for each group. Incubate at 35°C for 3 hours and then centrifuge (10000 r/min, 10 min), collect the supernatant into a 2mL PE tube, and finally filter it into a special brown sample bottle for HPLC using a 0.22 μm organic filter membrane. The chromatographic analysis column is a C18 column (2.1 mm×100 mm; particle size: 3 μm) with a column temperature of 30°C; mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is methanol, flow rate is 0.3 mL/min, and sample volume 20 μL, electrospray ion source (ESI+).
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Elution program
  
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[[Image:T--LZU-HS-China-C--China-C1.jpg | thumb | center | 500px |Figure 1 ]]
  
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Laccase first breaks the S-N bond of sulfadiazine itself to divide it into two parts, and then undergoes a series of oxidation reactions to finally form an ion with m/z of 174.07 (C6H7NO3S) sulfanilic acid and a mass-to-charge ratio of 127.09.
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Latest revision as of 06:42, 11 October 2022

bhal laccase from Bacillus halodurans with T7 promoter, RBS and His-tag

bhal laccase from Bacillus halodurans with T7 and HIS

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 190
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


We aimed to express this protein in E. coli to study the laccase activity on synthetic substrates at different pH levels. E. coli BL21Star cells were transformed with these parts, each of which contained a T7 promoter and termination sequence flanking the coding sequences. Expression was induced with IPTG and protein expression was performed, after which cells were harvested followed by sonication in protein extraction buffer. A pET160-GW/CAT construct expressing the chloramphenicol acetyltransferase (CAT) protein was used as a positive control for induction. The soluble fraction was analyzed on a 12% SDS-PAGE gel followed by Coomassie staining (Fig. 1). We observed expression of the positive control CAT protein (~32 kDa), but no evidence of tthl or bhal expression (Fig 2.)


Fig 1. SDS PAGE of laccase proteins.

Nevertheless, we assumed there would be some functional laccase present, and proceeded with decolorisation assays.

We tested the laccases on the decolorization of bromophenol blue. We first experimented with a standard solution of laccase purified from Trametes versicolor by incubating bromophenol blue in a buffer containing copper sulphate with various quantities of Trametes laccase.

Next, we assayed the E. coli protein extracts for laccase activity at different pH levels. The positive control, from Trametes, worked very well as a positive control, but only at low pH. Aside from that, there was no significant difference between the absorbance of the laccases and the negative control (Fig 3.).




Fig 3. Absorbance values of laccase proteins (Trametes = Positive control; CAT = Negatice control)

Added by LZU-HS-China-C

We think that this laccase may degrade antibiotics, but we are worried that the degradation antibiotic products of this laccase will have an adverse effect on the human body, so we studied the sulfadiazine degradation products of this laccase.

Speculate the degradation pathway of sulfadiazine

In the experiment, liquid-mass spectrometry (HPLC-MS) was used in the resting state to explore the degradation pathway of SDZ. First, EcN-IL was inoculated into LB medium at 37°C overnight at 1% inoculum. After centrifugation to remove the supernatant, the bacteria were collected (6000 r/min, 10 min). The bacteria were washed twice with deionized water and then prepared into a bacterial suspension with a cell concentration of 1.0×109 CFU/mL, and 5 mL of the cell suspension was taken. Solution, add SDZ sodium salt with a final concentration of 5 mg/L to it, three replicates for each group. Incubate at 35°C for 3 hours and then centrifuge (10000 r/min, 10 min), collect the supernatant into a 2mL PE tube, and finally filter it into a special brown sample bottle for HPLC using a 0.22 μm organic filter membrane. The chromatographic analysis column is a C18 column (2.1 mm×100 mm; particle size: 3 μm) with a column temperature of 30°C; mobile phase A is 0.1% formic acid aqueous solution, mobile phase B is methanol, flow rate is 0.3 mL/min, and sample volume 20 μL, electrospray ion source (ESI+). Elution program

Figure 1

Laccase first breaks the S-N bond of sulfadiazine itself to divide it into two parts, and then undergoes a series of oxidation reactions to finally form an ion with m/z of 174.07 (C6H7NO3S) sulfanilic acid and a mass-to-charge ratio of 127.09.