Difference between revisions of "Team:Stuttgart/Keratinases"

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           <tr><td>KerUS pet28</td><td>0,191</td><td>2991,667</td></tr>
 
           <tr><td>KerUS pet28</td><td>0,191</td><td>2991,667</td></tr>
 
           <tr><td>KerUS psD1C3</td><td>0,207</td><td>3391,667</td></tr>
 
           <tr><td>KerUS psD1C3</td><td>0,207</td><td>3391,667</td></tr>
 +
          <tr><td>KerUS BB</td><td>0,226</td><td>3866,667</td></tr>
 
           <tr><td>KerA pet28</td><td>0,194</td><td>3083,333</td></tr>
 
           <tr><td>KerA pet28</td><td>0,194</td><td>3083,333</td></tr>
 
           <tr><td>KerA psB1C3</td><td>0,213</td><td>3550,000</td></tr>
 
           <tr><td>KerA psB1C3</td><td>0,213</td><td>3550,000</td></tr>
 +
          <tr><td>KerA BB</td><td>0,201</td><td>3241,667</td></tr>
 +
          <tr><td>wild type <i>E.coli</i></td><td>0,248</td><td>4416,667</td></tr>
 
   </table>
 
   </table>
 
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             <tr><td>KerUS pet28</td><td>0,405</td><td>8358,333</td></tr>
 
             <tr><td>KerUS pet28</td><td>0,405</td><td>8358,333</td></tr>
 
           <tr><td>KerUS psD1C3</td><td>0,285</td><td>5350,000</td></tr>
 
           <tr><td>KerUS psD1C3</td><td>0,285</td><td>5350,000</td></tr>
 +
            <tr><td>KerUS BB</td><td>0,385</td><td>7841,667</td></tr>
 
             <tr><td>KerA pet28</td><td>0,482</td><td>10266,667</td></tr>
 
             <tr><td>KerA pet28</td><td>0,482</td><td>10266,667</td></tr>
 
             <tr><td>KerA psB1C3</td><td>0,585</td><td>12858,333</td></tr>
 
             <tr><td>KerA psB1C3</td><td>0,585</td><td>12858,333</td></tr>
 +
            <tr><td>KerA BB</td><td>0,410</td><td>8475,000</td></tr>
 +
            <tr><td>wild type <i>E.coli</i></td><td>0,615</td><td>13600,000</td></tr>
 
         </table>
 
         </table>
 
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<p>The equation of the trend line trough the measurement points of the BSA standard curve was used to determine the concentrations of the different keratinase samples.</p>
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<p>HIER STEHT EIN TEXT</p>
 
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   <b>Figure 1:</b> 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).
+
   <b>Figure 1:</b> : 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.
 
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   <b>Figure 1:</b> 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).  
+
   <b>Figure 1:</b> : 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.
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   <b>Figure 1:</b> : 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.
+
   <b>Figure 1:</b> Intracellular total protein concentration of three different keratinases (kerUS, kerA, kerP) each of them located in different vectors (pet28, pSB1C3 and pSB1K3).
 
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   <b>Figure 1:</b> : 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.
+
   <b>Figure 1:</b> Extracellular total protein concentration of three different keratinases (kerUS, kerA, kerP) each of them located in different vectors (pet28, pSB1C3 and pSB1K3).
 
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    <tr> <th></th> <th>Keratinases</th><th>Percentage extracellular protein</th> </tr>
 +
    <tr><td>KerUS pet 28</td><td>26%</td></tr>
 +
    <tr><td>KerUS psB1C3</td><td>39%</td></tr>
 +
    <tr><td>KerA pet 28</td><td>23%</td></tr>
 +
    <tr><td>KerA psB1C3</td><td>22%</td></tr>
 +
    <tr><th>KerP psB1C3</th> <th>22%</th></tr>
 +
    <tr><th>KerP psB1C3</th> <th>19%</th></tr>
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   <b>Figure 1:</b> Intracellular total protein concentration of three different keratinases (kerUS, kerA, kerP) each of them located in different vectors (pet28, pSB1C3 and pSB1K3).
+
   <b>Figure 1:</b> 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).
 
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   <b>Figure 1:</b> Extracellular total protein concentration of three different keratinases (kerUS, kerA, kerP) each of them located in different vectors (pet28, pSB1C3 and pSB1K3).
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   <b>Figure 1:</b> 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).  
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<p>SCHREIB WAS REIN :-)</p>
 
<p>SCHREIB WAS REIN :-)</p>
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Revision as of 02:18, 2 November 2017

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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Figure 1: : 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 1: : 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.

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Figure 1: Intracellular total protein concentration of three different keratinases (kerUS, kerA, kerP) each of them located in different vectors (pet28, pSB1C3 and pSB1K3).
Figure 1: 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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Figure 1: 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 1: 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).

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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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