Difference between revisions of "Team:Stuttgart/Keratinases"

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<p> Digest of keratinase plasmids pSB1K3-kerP and pSB1C3-kerP with EcoR1 and Pst1. Line 1: Marker, DNA ladder (Thermofisher +1 kb). Line 2-9: Plasmids from transformed colonies 1-4 cloned in pSB1K3. Line 10: pSB1K3-kerP new transformation. Line 11-12: kerP cloned in pSB1C3 FÜLL MICH MIT WORTEN</p>
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<p> Digest of keratinase plasmids pSB1K3-kerP and pSB1C3-kerP with EcoR1 and Pst1. Line 1: Marker, DNA ladder (Thermofisher +1 kb). Line 2-9: Plasmids from transformed colonies 1-4 cloned in pSB1K3. Line 10: pSB1K3-kerP new transformation. Line 11-12: kerP cloned in pSB1C3</p>
  
  

Revision as of 20:54, 1 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.

HIER STEHT EIN TEXT

AB HIER DÜRFT IHR EINFÜGEN :-) AB HIER DÜRFT IHR UNTEN EINFÜGEN :-) AB HIER DÜRFT IHR UNTEN EINFÜGEN :-) AB HIER DÜRFT IHR UNTEN EINFÜGEN
Figure 1: Digest of kerP plasmids

Digest of keratinase plasmids pSB1K3-kerP and pSB1C3-kerP with EcoR1 and Pst1. Line 1: Marker, DNA ladder (Thermofisher +1 kb). Line 2-9: Plasmids from transformed colonies 1-4 cloned in pSB1K3. Line 10: pSB1K3-kerP new transformation. Line 11-12: kerP cloned in pSB1C3

Text hier einfügen

Figure 1: Figure 12: Digest of promotors

SCHREIB WAS REIN :-)

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

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Figure 1: Sucessful transformation of assembled rose plasmid in DH5alpha E.coli cells.

text hier einfügen

Figure 1: Restriction digest of purified rose plasmids from different transformations and E.coli colonys. Cut with EcoRI.
Figure 1: Restriction digest of purified rose plasmids from different transformations and E.coli colonys. Cut with SpeI.
Figure 1: Colony PCR with 12 E.coli colonys after transformation of assembeld rose plasmid.