Team:Bielefeld-Germany/Results/thermo

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Summary

First some trials of shaking flask cultivations were made with different parameters to define the best conditions for the production of the His-tagged laccase Ltth from Thermus thermophilus HB27 named TTHL. Because of no measured activity in the cell lysate a purification method was established (using Ni-NTA-Histag resin). Using E. coli KRX containing BioBrick BBa_K863010 TTHL could not be detected by SDS-PAGE (molecular weight of 53 kDa) or by activity test. Therefore a new BioBrick BBa_K863012 was constructed and expressed in E. coli Rosetta-Gami 2. With this expression system the TTHL could be detected by SDS-PAGE (molecular weight of 53 kDa). To prove the activity of the produced laccase we used a small scale Ni-NTA-column to purify it. The fractionated samples were tested concerning their activity. Activity in oxidizing ABTS has been detected. After measuring activity of TTHL a scale up was made up to 6 L.


Contents


Shaking Flask Cultivation

The first trials to produce the Ltth-laccase from Thermo thermophilus HB27 (named TTHL) were performed in shaking flasks with various volumes (from 100 mL to 1 L flasks, with and without baffles) and under different conditions. The used BioBrick was BBa_K863010 and expressed in E. coli KRX. The changed parameters during the screening experiments were temperature (27 °C, 30 °C and 37 °C), concentration of chloramphenicol (20-170 µg mL-1), induction strategy (autoinduction and manual induction with 0,1 % rhamnose) and cultivation time (6 to 24 h). Furthermore, E. coliwas cultivated with and without 0,25 mM CuCl2 to provide a sufficient amount of copper, which is needed for the active center of the laccase. Under the screened conditions it was not observable that E. coli KRX produces active TTHL. Therefore another BioBrick was constructed and another chassi was chosen. For further cultivations the BioBrick BBa_K863012 was used, which has a constitutive promoter insteat of the T7 promoter system. Additionally, the strain E. coli Rosetta-Gami 2 was chosen, because of its ability to translate rare codons. TTHL was then produced under the following conditions:

  • flask design: shaking flask without baffles
  • medium: LB-Medium
  • antibiotics: 60 µg mL-1 chloramphenicol and 300 µg mL-1 ampicillin
  • temperature: 37 °C
  • cultivation time: 24 h

The reproducibility of the measured data and results were investigated for the shaking flask cultivation, but not yet for the bioreactor cultivation.

Fermentation of E. coli KRX with BBa_K863012

Figure 1: Fermentation of E. coli Rosetta-Gami 2 with BBa_K863012 (TTHL) in a Bioengineering NFL22. Conditions: 6 L of autoinduction medium + 60 µg/mL chloramphenicol at 37 °C, pH 7. Agitation increased when pO2 was below 30 % and OD600 was measured each hour.

After measuring activity of TTHL we made a scale-up and fermented E. coli Rosetta-Gami 2 with BBa_K863000 in a Bioengineering NFL22 with a total volume of 6 L. Agitation speed, pO2 and OD600 were monitored and are illustrated in Figure 1. There is no noticeable lag phase. Due to the cell growth the pO2 decreased to a value of 0 %, which was additionally caused by the breakdown of the control unit. After a cultivation time of 9 hours the agitation speed was increased manually up to 500 rpm, which resulted in a higher pO2 value of more than 100 % for the rest of the cultivation. During the whole process the OD600 increased slower when compared to the fermentation of E. coli KRX with BBa_K863000 or BBa_K863005. The maximal OD600 was reached after 19 hours of cultivation time so that the cells were harvested.


Purification of TTHL

The cells were harvested and resuspended in Ni-NTA-equilibrationbuffer, mechanically lysed by homogenization and centrifuged. After preparing the cell paste the TTHL could not be purificate with the 15 mL column, because of its damage. For this reason a small scale purification (6 mL) of the supernatant of the lysate was made with a 1 mL Ni-NTA-column.

SDS-PAGE of purification TTHL

Figure 2: SDS-PAGE of purified E. coli Rosetta-Gami 2 containing BBa_K863012 lysate (fermented in 6 L Bioengineering NFL22). The flow-through, wash and elution fraction 1 to 5 are shown. The arrow marks the TTHL band with a molecular weight of 53 kDa.

Figure 2 shows the SDS-PAGE of the purified E. coli Rosetta-Gami 2 lysates fermented in 6 L Bioengineering NFL22 including the flow-through, wash and all elution fractions (1 to 5). TTHL has a molecular weight of 53 kDa and the corresponding band was marked with a red arrow. The TTHL band appears in fractions 1 to 3, but not in the other two elution fractions. Furthermore there are some other non-specific bands, which could not be identified. To improve the purification the 15 mL column and ÄKTA method could be used.


Activity Analysis of TTHL

Although there was no activity measurable after cultivation and purification of TTHL under a T7 promoter, activity tests of TTHL under a constitutive promoter did reveal TTHL laccases capable of oxidzing ABTS. Fractions 1 to 5 of the purification above were rebuffered into deionized H2O and icubated with 0.4 mM CuCl2 for 2 hours. Activity measurements took place using 100 mM sodiume acetate buffer, 140 µL sample, 0.1 mM ABTS, ad 200 µL deionized H2O. The change in optical density at 420 nm was detected, reporting the oxidization of ABTS through laccases. Fractions 2 to 5 show activity (figure 3). Fraction 2 seems to contain most of TTHL showing the highest activity compared to the other fractions: 40% of the used ABTS have been oxidized after 2 hours. With this in mind protein concentrations have to be determined and the activity of the TTHL laccase can be characterized in further experiments including pH optimum and activity in regard of temperature shifts.

Figure 3: Activity test of TTHL fractions resulting from the purification. Reaction setup includes 100 mM sodium actetate buffer (pH 5), 140 µL fraction sample (CuCl2 incubated), 0.1 mM ABTS, ad 200 µL deionized H2O. Measurements were done at 25°C and over a time period of 5 hours. TTHL shows activity in oxidizing ABTS except fractions 1 seems to have no active TTHL. (n=4)



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