Team:Bielefeld-Germany/Results/Summary

From 2012.igem.org

Summary

BPUL - Laccase from Bacillus pumilus DSM 27 (ATCC7061)

The first trails to produce the BPUL were produced in shaking flask with various designs (from 100 mL^-1 to 1 L flasks, with and without baffles) and under different conditions. The parameters we have changed during our screening experiments were the temperature (27 °C,30 °C and 37 °C), different concentrations of chloramphenicol (20-170 µg mL^-1), various induction strategies (autoinduction and manual induction), several cultivation times (6 - 24 h) and in absence or presence of 0,25 mM CuCl₂. Due to the screening experiments we identified the best conditions under which BPUL was expressed:

• flask design: shaking flask without baffles
• medium: autoinduction medium
• antibiotics: 60 µg mL^-1 chloramphenicol
• temperature: 37 °C
• cultivation time: 12 h

The reproducibility and repeatability of the measured data and results were investigated for the shaking flask and bioreactor cultivation with n≤3.

The Results of the SDS-PAGE analysis are shown in the following images:

SDS PAGES!!!!!

ACTIVITÄTSMESSUNGEN VOM ERSTEN AKTIVEN B PUMI!!!!!!

After the measurement of activity of BPUL we made a scale-up and fermented E. coli KRX with <partinfo>BBa_K863000</partinfo> in Braun Biostat B with a total volume of 3 L. Agitation speed, pO₂ and OD₆₀₀ were determined and illustrated in Figure 1. We got a long lag phase of 2 hours due to a relativly old preculture. The cell growth caused a decrease in pO₂ and after 3 hours the value fell below 50 %, so that the agitation speed increased automatically. After 8,5 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 OD₆₀₀ of 3,53 was reached after 10 hours, which means a decrease of 28 % in comparison to the fermentation of E. coli KRX under the same conditions(OD_600,max =4,86 after 8,5 hours, time shift due to long lag phase). The cells were harvested after 11 hours.

Figure 1: Fermentation of E. coli KRX with <partinfo>BBa_K863000</partinfo> (BPUL) in Braun Biostat B, scale: 3 L, autoinduction medium + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation on cascade to hold a pO₂ of 50 %, OD₆₀₀ taken every 30 minutes.

The harvested cells were resuspended in Ni-NTA-equilibrationbuffer, mechanically lysed by homogenization and centrifuged. The supernatant of the lysed cell paste was loaded on the Ni-NTA-column (15 mL Ni-NTA resin) with a flowrate of 1 mL min^-1 cm^-2. Then the column was washed by 10 column volumes (CV) with Ni-NTA-equilibrationbuffer. The bound proteins were eluted by an increasing Ni-NTA-elutionbuffer gradient from 0 % to 100 % with a total volume of 100 mL and the elution was collected in 10 mL fractions. The chromatogramm of the BPUL-elution is shown in figure 2:

Figure 2: Chromatogramm of wash and elution from FLPC Ni-NTA-Histag Purification of BPUL produced by 3 L fermentation of E. coli KRX with <partinfo>BBa_K863000</partinfo>. BPUL was eluted between a process volume of 460 mL to 480 mL with a maximal UV-detection signal of 69 mAU

The chromatogramm shows a remarkable widespread peak between the process volume of 460 mL to 480 mL with the highest UV-detection signal of 69 mAU , which can be explained by the elution of bound proteins. The corresponding fractions were analysed 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 mL there are several strong peaks (up to a UV-detection-signal of 980 mAU) detectable. These results are caused by an accidental detachement in front of the UV-detector. 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.

Another scale-up of the fermentation of E. coli KRX with <partinfo>BBa_K863000</partinfo> was made up to a final working volume of 6 L in Bioengineering NFL22. Agitation speed, pO₂ and OD₆₀₀ 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 rpm after one hour due to control problems. Then the pO₂ sank until a cultivation time of 4,75 hours, when the deceleration phase started. Then it increased again. There is no visible break through induction of protein expression. A maximal OD₆₀₀ of 3,68 was reached after 7/8 hours of cultivation, which is similar to the 3 L fermentation (OD₆₀₀ = 3,58 after 10 hours, time shift due to long lag phase). The cells were harvested after 12 hours.

Figure 3: Fermentation of E. coli KRX with <partinfo>BBa_K863000</partinfo> (BPUL) in Bioengineering NFL22, scale: 6 L, autoinduction medium + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation increased when pO₂ was below 30 %, OD₆₀₀ taken every hour.

The harvested cells were prepared in Ni-NTA-equilibrationbuffer, mechanically lysed by homogenization and centrifuged. The supernatant of the lysed cell paste was loaded on the Ni-NTA-column (15 mL Ni-NTA resin) with a flowrate of 1 mL min^-1 cm^-2. Then the column was washed by 5 column volumes (CV) with Ni-NTA-equilibrationbuffer.The bound proteins were eluted by an increasing elutionbuffer gradient from 0 % (equates to 20 mM Imidazol) to 100 % (equates to 500 mM imidazol) with a total volume of 200 mL. This strategy was chosen to improve of purification by a slower increase of Ni-NTA-elutionbuffer concentration. The elution was collected in 10 mL fractions. The chromatogramm of the BPUL-elution is shown in figure 2:

Figure 4: Chromatogramm of wash and elution from FLPC Ni-NTA-Histag Purification of BPUL produced by 6 L fermentation of E. coli KRX with <partinfo>BBa_K863000</partinfo>. BPUL was eluted between a process volume of 832 mL and 900 mL with a maximal UV-detection signal of 115 mAU.

The chromatogramm shows a strong 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 mL and 900 mL there is remarkable widespread peak with a UV-detection signal of 115 mAU. This peak corresponds to an elution of bound proteins at a Ni-NTA-elutionbuffer concentration between 10 % and 20 % (equates to 50-100 mM imidazole) . 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 bound proteins was already eluted. Just to be on the safe side, all fractions were analysed by SDS-PAGE analysis to detect BPUL. The results of the SDS-PAGE are shown in the following pictures.

ECOL - Laccase from Escherichia coli BL21 (DE3)

The first trails to produce ECOL were produced in shaking flask with various designs (from 100 mL^-1 to 1 L flasks, with and without baffles) and under different conditions. The parameters we have changed during our screening experiments were the temperature (27 °C,30 °C and 37 °C), different concentrations of chloramphenicol (20-170 µg mL^-1), various induction strategies (autoinduction and manual induction), several cultivation times (6 - 24 h) and in absence or presence of 0,25 mM CuCl₂. Due to the screening experiments we identified the best conditions under which ECOL was expressed:

• flask design: shaking flask without baffles
• medium: autoinduction medium
• antibiotics: 60 µg mL^-1 chloramphenicol
• temperature: 37 °C
• cultivation time: 12 h

The Results of the SDS-PAGE analysis are shown in the following images:

SDS PAGES!!!!!

ACTIVITÄTSMESSUNGEN VOM ERSTEN AKTIVEN ECOL!!!!!!

After the measurement of activity of ECOL we made a scale-up and fermented E. coli KRX with <partinfo>BBa_K863005</partinfo> in Infors Labfors with a total volume of 3 L. Agitation speed, pO₂ and OD₆₀₀ were determined and illustrated in Figure 1. The exponential phase started after 1,5 hours of cultivation. The cell growth caused a decrease in pO₂. After 2 hours of cultivation the agitation speed increased up to 629 rmp (5,9 hours) to hold the minimal pO₂ level of 50 %. Then, after 4 hours there was a break in cell growth due to induction of protein expression. The maximal OD₆₀₀ of 2,78 was reached after 5 hours. In comparison to E. coli KRX (OD_600,max =4,86 after 8,5 hours) and to E. coli KRX with <partinfo>BBa_K863000</partinfo> (OD_600,max =3,53 after 10 hours, time shift due to long lag phase) the OD_600,max is 22 % or rather 43 % 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 hours.

Figure 1: Fermentation of E. coli KRX with <partinfo>BBa_K863005</partinfo> (ECOL) in Infors Labfors Bioreactor, scale: 3 L, autoinduction medium + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation on cascade to hold a pO₂ of 50 %, OD₆₀₀ taken every 30 minutes.

The harvested cells were resuspended in Ni-NTA-equilibrationbuffer, mechanically lysed by homogenization and centrifuged. The supernatant of the lysed cell paste was loaded on the Ni-NTA-column (15 mL Ni-NTA resin) with a flowrate of 1 mL min^-1 cm^-2. Then the column was washed by 10 column volumes (CV) with Ni-NTA-equilibrationbuffer. The bound proteins were eluted by an increasing Ni-NTA-elutionbuffer step elution from 5 % (equates to 25 mM imidazol) with a total volume of 60 mL, to 50 % (equates to 250 mM Imidazol) with a total volume of 60 mL, to 80 % (equates to 400 mM imidazol) with a total volume of 40 mL and finaly to 100 % (equates to 500 mM imidazol) with a total volume of 80 mL. This strategies was chosen to improve the purification caused by a step by step increasing Ni-NTA-elutionbuffer concentration. The elution was collected in 10 mL fractions. The chromatogramm of the BPUL-elution is shown in figure 2:

Figure 2: Chromatogramm of Wash and Elution from FLPC Ni-NTA-Histag Purification of ECOL produced by 3 L fermentation of E. coli KRX with <partinfo>BBa_K863005</partinfo>. ECOL was eluted by a concentration of 50 % (equates to 250 mM imidazol) with a maximal UV-detection signal of 292 mAU.

The chromatogramm shows two remarkable peaks. The first peak was detected by a Ni-NTA-equilibrationbuffer concentration of 5 % (equates 25mM Imidazol) and resulted from the elution of weakly bound proteins. After increasing the Ni-NTA-elutionbuffer concentration to 50 % (equates to 250 mM imidazol) a large peak up to a UV-detection signal of 292 mAU was measured. The strength 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 Ni-NTA-elutionbuffer. The corresponding SDS-PAGES are shown in the following images.

Figure 3: Fermentation from 09/07 of E. coli KRX containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005], 6 L in NFL22, autoinduction medium, 37 °C, pO₂ 30 % for 12 hours

Another scale-up of the fermentation of E. coli KRX with <partinfo>BBa_K863005</partinfo> was made up to a final working volume of 6 L in Bioengineering NFL22. Agitation speed, pO₂ and OD₆₀₀ were determined and illustrated in Figure 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 hours of cultivation, which results in an decrease of pO₂ value and therefore in an increase of agitation speed. After 4 hours of cultivation the maximal OD₆₀₀ of 2,76 was reached, which is comparable to the 3 L fermentation of E. 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 reported by the [http://www.dbu.de/OPAC/ab/DBU-Abschlussbericht-AZ-13191.pdf DBU]. In comparison to the fermentation of E. coli KRX with <partinfo>BBa_K863000</partinfo> under the same conditions (OD_600,max= 3,53), the OD_600,max was 22 %. Cells were harvested after 12 hours.

Figure 3: Fermentation of E. coli KRX with <partinfo>BBa_K863005</partinfo> (ECOL) in Bioengineering NFL22, scale: 6 L, autoinduction medium + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation increased when pO₂ was below 30 %, OD₆₀₀ taken every hour.

The harvested cells were resuspended in Ni-NTA-equilibrationbuffer, mechanically lysed by homogenization and centrifuged. The supernatant of the lysed cell paste was loaded on the Ni-NTA-column (15 mL Ni-NTA resin) with a flowrate of 1 mL min^-1 cm^-2. Then the column was washed by 10 column volumes (CV) with Ni-NTA-equilibrationbuffer. The bound proteins were eluted by an increasing Ni-NTA-equilibrationbuffer gradient from 0 % to 100 % with a total volume of 200 mL and the elution was collected in 10 mL fractions. The chromatogramm of the BPUL-elution is shown in figure 4:

Figure 4: Chromatogramm of Wash and Elution from FLPC Ni-NTA-Histag Purification of ECOL produced by 3 L fermentation of E. coli KRX with <partinfo>BBa_K863005</partinfo>. ECOL was eluted between a process volume 670 mL to 750 mL with a maximal UV-detection signal of 189 mAU.

After washing the column with 10 CV Ni-NTA-elutionbuffer the elution process was started. At a process volume of 670 mL to 750 mL the chromatogramm shows a strong widespread peak (UV-detection signal 189 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 (see purification of the 3 L Fermentation above). To detect ECOL the corresponding fractions were analysed by SDS-PAGE analysis.

!!SDS-Page!!

XCCL - Laccase from Xanthomonas campestris pv. campestris B100

BHAL - Laccase from Bacillus halodurans C-125

TTHL - Laccase from Thermus thermophilus HB27

The first trails to produce the Thermo thermophilus-laccase (TTHL) were produced in shaking flask with various designs (from 100 mL^-1 to 1 L flasks, with and without baffles) and under different conditions. The parameters we have changed during our screening experiments were the temperature (27 °C,30 °C and 37 °C), different concentrations of chloramphenicol (20-170 µg mL^-1), various induction strategies (autoinduction and manual induction), several cultivation times (6 - 24 h) and in absence or presence of 0,25 mM CuCl₂. 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
• medium: LB-Medium
• antibiotics: 60 µg mL^-1 chloramphenicol and 300 µg mL^-1 ampicillin
• temperature: 37 °C
• cultivation time: 24 h

The reproducibility and repeatability of the measured data and results were investigated for the shaking flask and bioreactor cultivation with n≤3.

The Results of the SDS-PAGE analysis are shown in the following images:

SDS PAGES!!!!!

ACTIVITÄTSMESSUNGEN VOM ERSTEN AKTIVEN B PUMI!!!!!!

Fermentation of E. coli KRX with <partinfo>BBa_K863012</partinfo>

</center> After measuring activity of TTHL we made a scale-up and fermented E. coli Rosetta-Gami 2 with <partinfo>BBa_K863000</partinfo> in Bioengineering NFL22 with a total volume of 6 L. Agitation speed, pO₂ and OD₆₀₀ were determined and illustrated in Figure 1. The cells immediatly began to grow and therefore the pO₂ decreased up to a value of 0 %, because the breakdown of the control unit. After a cultivation time of 9 hours the agitation speed was increased up to a 500 rpm, which resulted in a pO₂ value of more than 100 % for the rest of the cultivation. During the whole process the OD₆₀₀ increased slowly in comparison to the fermentation of E. coli KRX with <partinfo>BBa_K863000</partinfo> or <partinfo>BBa_K863005</partinfo>. The maximal OD₆₀₀ was reached after 19 hours of cultivation, when the cells were harvested.

Figure 1: Fermentation of E. coli Rosetta-Gami 2 with <partinfo>BBa_K863012</partinfo> (TTHL) in Bioengineering NFL22, scale: 6 L, autoinduction medium + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation increased when pO₂ was below 30 %, OD₆₀₀ taken every hour.

The cells were harvested and resuspended in Ni-NTA-equilibrationbuffer, mechanically lysed by homogenization and centrifuged. After preparing the cell paste we did not have the possibility to purificate the TTHL with the 15 mL. To detect and to analyse our produced TTHL we implement a small scale purification of 6 mL of the supernatant with a 1 mL Ni-NTA-column. The results of the SDS-PAGE analysis are shown the following images:

!!SDS-Page!!

PCIL - Laccase from Pycnoporus cinnabarinus

TVEL5 - Laccase from Trametes versicolor

TVEL10 - Laccase from Trametes versicolor

TVEL13 - Laccase from Trametes versicolor

TVEL20 - Laccase from Trametes versicolor

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