Revision as of 10:00, 23 October 2017

To prove any enzyme activity a semi-quantitative hair degradation assay was performed.
First, cultures of E. coli containing kerA, kerUS, kerP plasmid and one wild-type E. coli were grown o/n at 37°C in sterile LB broth. Chloramphenicol (final concentration 35 µg/mL) was added to the cultures containing kerA and kerUS – Kanamycin (final concentration 50 µg/mL) to the cultures containing the kerP. After incubation OD600 was measured before inducing cultures containing kerA and kerUS with IPTG with a final concentration of 1mM.
Human hair was reduced to smaller pieces and then dried for 1 hour at 65°C. Afterwards the hair was distributed in 0.05 g aliquots and the full amount of each culture was added. The cultures + hair were incubated 120 hours at 37°C with slightly shaking.

Skim milk plate assay

This assay was performed to show qualitative enzyme activity.
The different keratinases (kerA, kerUS and kerP) should degrade the casein in the milk, seen as clear zones around the cultures itself or the supernatant of the cells.

Preparation of skim milk plates
First 100 mL LB/agar (see methods) and 5 g skim milk powder in 125 mL distilled water was prepared. Both solutions were autoclaved and mixed after cooling down. For the plates used for kerA and kerUS cultures chloramphenicol (35 µg/mL) was added – in case of kerP kanamycin (50 µg/mL) was used to prepare plates. The plates were poured and stored at 4°C.
Assay
kerA/kerUS: Before spreading both keratinases on the plates, cells were induced with 1mM IPTG o/n at 37°C. After incubation 10 µL of the induced cell cultures and supernatant, cell culture without IPTG induction and a wild-type E. coli control was spread on skim milk plates containing chloramphenicol. The plates were incubated at room temperature for four days.
kerP: 10 µL of cell culture, supernatant and a wild-type E. coli control was spread on skim milk plates containing kanamycin. The plates were incubated at room temperature for four days.

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 <ol><li>Preparation of skim milk plates
 <br>
-First 100 mL LB/agar (see methods) and 5 g skim milk powder in 125 mL distilled water was prepared. Both solutions were autoclaved and mixed after cooling down. For the plates used for kerA and kerUS cultures chloramphenicol (35 µg/mL) was added – in case of kerP kanamycin (50 µg/mL) was used to prepare plates. The plates were poured and stored at 4°C.</li>
+First 100 mL LB/agar (see <a href="https://2017.igem.org/Team:Stuttgart/Notebook">methods</a>) and 5 g skim milk powder in 125 mL distilled water was prepared. Both solutions were autoclaved and mixed after cooling down. For the plates used for kerA and kerUS cultures chloramphenicol (35 µg/mL) was added – in case of kerP kanamycin (50 µg/mL) was used to prepare plates. The plates were poured and stored at 4°C.</li>
 <li>Assay
 <br>

Difference between revisions of "Team:Stuttgart/Experiments"

Revision as of 10:00, 23 October 2017

Experiments

PART I - ESTERASES and LIPASES

PART II - KERATINASES

Semi-quantitative hair degradation assay

Skim milk plate assay

PART III - A LOVELY SCENT OF...

...ROSE FRAGRANCE

...LIMONENE FRAGRANCE