Team:Stuttgart/Keratinases

Results

Keratinases

BCA Assay

Protein concentration of supernatant and cell disruption of E.coli carrying different keratinase plasmids (KerP, KerUS and KerA in different vectors) was measured with BCA Assay method (see chapter methods). The figure 1 below shows the BSA standard curve (blank corrected, arithmetic average of triplicates). Table 7 and 8 show the results of sample measurements (OD values: blank corrected, arithmetic average of triplicates). The concentrations are calculated with the calibration curve (BSA standard curve). Expectetly protein concentration is much more higher in cell disruption than in supernatants. It is also noticeable that protein concentraion of E.coli wild type is very high compared to engineered cultures. Unfortunately we do not have OD values, but we explain it by a higher amount of wild type cells after cultivation.

Table: supernatant (dilution 1:25):

sample OD562concentration [µg/mL]
KerP psD1K30,1943066,667
KerP psD1C30,1953091,667
KerUS pet280,1912991,667
KerUS psD1C30,2073391,667
KerUS BB0,2263866,667
KerA pet280,1943083,333
KerA psB1C30,2133550,000
KerA BB0,2013241,667
wild type E.coli0,2484416,667

Table: cell disruption (dilution 1:25):

sample OD562concentration [µg/mL]
KerP psD1K30,51411075,000
KerP psD1C30,61113491,667
KerUS pet280,4058358,333
KerUS psD1C30,2855350,000
KerUS BB0,3857841,667
KerA pet280,48210266,667
KerA psB1C30,58512858,333
KerA BB0,4108475,000
wild type E.coli0,61513600,000
Figure 1: BSA standard calibration curve.

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Enzyme activity

To evaluate which keratinase shows the most efficient enzyme activity we focused on three different keratinases that originated from distinct organisms and were known to be successfully expressed in E. coli (Gupta and Gupta 2010, Hu et al. 2013, Jaouadi et al. 2013). KerUS that is originally expressed by Bacillus brevis and KerA originating from Bacillus Licheniformis are both Keratinases that the iGEM Team Canmore has worked with in previous projects. They were so kind to send us these enzymes on different backbones (pet28, pSB1C3). KerP from Pseudomonas aeruginosa was synthesized and cloned in a pSB1K3 and a pSB1C3 backbone.

To avoid confusions, we have to state that clonations from KerUS and KerA that were located on a pSB1C3 backbone and that we obtained from iGEM Team Canmore 2016 were not successful. During transformation process of these plasmids into our competent Dh5α cells, a transposon interrupted our keratine coding sequence and rendered these cells dysfunctional. This was proved by sequencing our transformed cells. Therefore, cell modifications by exchanging various promoters (BBa_J23115, BBa_J23119, BBa_K206000) and signal sequences (pelB and OmpA) were not successful (see gels in notebook) and no enzyme activity assay could be performed using these strains.

Figure 2: : Extinction of Azo-keratine conversion over time of three different keratinases (kerUS, kerA, kerP) each of them located in different vectors (pet28, pSB1C3 and pSB1K3). Measurements of enzyme activity was performed at three timepoints (0h, 3h, 24h). Intracellular enzyme activity was measured.
Figure 3: : Extinction of Azo-keratine conversion over time of three different keratinases (kerUS, kerA, kerP) each of them located in different vectors (pet28, pSB1C3 and pSB1K3). Measurements of enzyme activity was performed at three timepoints (0h, 3h, 24h). Extracellular enzyme activity was measured.

To overcome this problem, we performed clonation using KerUS and KerA from Team Canmore 2016 again. KerUS and KerA were located once on a pet28b+ backbone and on a pSB1C3 backbone. Clonation was performed using NEB 5-alpha Competent E. coli this time. Another sequencing was performed to assure correct protein expression and to verify that the transposon incidence occurred during our transformation.

Transformation of the keratinases was successful this time and enzyme activity assays using these strains with additionally including KerP on a PSB1K3 and on a pSB1C3 backbone were performed.

Enzyme activity was observed by performing an enzyme activity using keratin azure substrate (Sigma-Aldrich). During active keratinolytic activity azo-keratine is converted, so that azo dye is released. This extinction can be measured in the filtrate at 440 nm. As a control we used water + substrate. We refered higher enzyme activity to an increasing extinction rate. This extinction rate was measured over time at three time points (0h, 3h and 24h) and was compared between our KerA, KerUS and KerP located on the different backbones.

To verify if our signal peptides on the keratinase constructs (pelB for KerUS and KerA, and natural signal sequence of Pseudomonas aeruginosa for KerP) were definitely functional, we compared the extinction time of keratine azure intracellular (fig.2) and extracellular (fig.3) over time. Cells were lysated and the intracellular enzyme activity was evaluated by an increasing extinction rate. Same was done using the supernatant of the keratinases, to see how many active enzymes were extracellularly secreted.

In both different assays KerP located in the pSB1C3 backbone showed highest activity if we can refer it to the amount of converted keratine azure. Interestingly KerP in pSB1K3 showed the second highest enzyme activity intracellularly, while it showed barely enzyme activity extracellularly. As they both have the same natural signal sequence, we wonder if this might due to the different antibiotic resistances on the plasmid.

KerUS on the pet28b+ backbone seemed showed intracellular lower activity, but was clearly high on the extracellular level. This may lead to the assumption that KerUS is quite efficiently secreted out of the cell

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Nevertheless, to make valid postulations we have to put these substrate extinctions in relation to the protein concentration or their OD600 of the used enzymes respectively. For this we measured total protein concentrations via BSA Assay as already described above intracellular (fig. 4) and extracellular (fig. 5).

It is important to mention that enzyme activity assays from fig 2 and fig 3 were unfortunately independently performed. The total protein concentration extracellular in fig 5, just gives an idea of how much the distinct keratinase strains express proteins in total, with KerA pet28b+ and KerUS pSB1C3 secreting the highest total protein amount, but cannot be put in relation to the extinction rates. Nevertheless, to get an idea and as we had the OD600 measured for the extracellular assay measured, we put it in relation to the OD600 (fig. 7).

Figure 4: Intracellular total protein concentration of three different keratinases (kerUS, kerA, kerP) each of them located in different vectors (pet28, pSB1C3 and pSB1K3).
Figure 5: Extracellular total protein concentration of three different keratinases (kerUS, kerA, kerP) each of them located in different vectors (pet28, pSB1C3 and pSB1K3).
KeratinasesPercentage extracellular protein
KerUS pet 2826%
KerUS psB1C339%
KerA pet 2823%
KerA psB1C322%
KerP psB1C3 22%
KerP psB1C3 19%

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Intracellular extinction rate was performed simultaneously to protein concentration determination and could be set in relation to each other (fig. ). KerP in pSB1C3 backbone and KerA in pet28b+ backbone showed highest protein amount intracellular, with KerUS in psB1C3 vector showing the lowest expression. This goes in line with our extinction rates observing these keratinases.

Regarding the time courses of enzyme activity intracellular (in relation to the protein concentration) fig.6 and extracellular (in relation the their OD600) (fig. 7), we see that KerUS pSB1C3 shows the significantly highest enzyme activity, while extracellular KerUS in pet28b+ shows highest enzyme activity extracellularly. This might lead to the assumption that KerUS is highly expressed in the cell on a pet28b+ backbone but can be also successfully secreted when being located on the pSB1C3 backbone.

Figure 6: Ratio extinction azokeratine/protein concentraion of intracellular keratinases over time. To see is extinction (440nm) of converted azo-keratine in relation to total protein concentration (c) of three different keratinases (kerUS, kerA, kerP) each of them located in different vectors (pet28, pSB1C3 and pSB1K3).
Figure 7: Ratio extinction azokeratine/OD600 of extracellular keratinases over time. To see is extinction (440nm) of converted azo-keratine in relation to total protein concentration (c) of three different keratinases (kerUS, kerA, kerP) each of them located in different vectors (pet28, pSB1C3 and pSB1K3).
Figure 8: Alignment with sequencing data of kerA and sequence of kerA (with standard iGEM primers).
Alignment with sequencing data of kerA and sequence of kerA (with standard iGEM primers). Forward primer binds between insert point 3467 and insert point 300. R evers primer binds between insert point 687 and insert point 1587. There is a gap between 300 and 687 (387 bp long) at the begining of kerA gene.
Figure 9: Alignment with sequencing data of kerUS and sequence of kerUS (with standard iGEM primers).
Alignment with sequencing data of kerUS and sequence of kerUS (with standard iGEM primers). Forward primer binds between insert point 3489 and insert point 277. Revers primer binds between insert point 783 and insert point 1605. There is a gap between 277 and 783 (506 bp long) at the beginning of kerUS gene ( fig. 9)

Sequencing kerUS forward

Figure 10:

Blue: Alignment with backbone and lac promotor, RBS

Red : transposon

Figure 11: BLAST Hits with 100% ident. in different organisms.

With BLAST we detected a transposon sequence (fig. 11).

Sequencing kerA forward

Figure 12:

Green: alignment with backbone, lac, RBS

Blue: Alignment with pelB seq

Orange: Alignment with kerA

Red: transposon

Figure 13:

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Figure 14:

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Figure 15: Digest of psB1C3 KerP with the rstriction enzymes EcoRI and PstI

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Figure 1: Digest of kerP plasmids

FÜLL MICH MIT WORTEN

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Figure 1:

SCHREIB WAS REIN :-)

Figure 1: Agaraose-gel electrophoresis of purified rose-PCR products.

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REFERENCES

  1. Hu, Hong; He, Jun; Yu, Bing; Zheng, Ping; Huang, Zhiqing; Mao, Xiangbing et al. (2013): Expression of a keratinase (kerA) gene from Bacillus licheniformis in Escherichia coli and characterization of the recombinant enzymes. In: Biotechnology letters 35 (2), S. 239–244.
  2. Jaouadi, Nadia Zaraî; Rekik, Hatem; Badis, Abdelmalek; Trabelsi, Sahar; Belhoul, Mouna; Yahiaoui, Amina Benkiar et al. (2013): Biochemical and molecular characterization of a serine keratinase from Brevibacillus brevis US575 with promising keratin-biodegradation and hide-dehairing activities. In: PloS one 8 (10), e76722.
  3. Sharma, Richa; Gupta, Rani (2010): Extracellular expression of keratinase Ker P from Pseudomonas aeruginosa in E. coli. In: Biotechnology letters 32 (12), S. 1863–1868.

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