Team:Bielefeld-Germany/Labjournal/week18

Week 18 (08/27 - 09/02/12)

Monday August 27th

• Team Cultivation & Purification:
  • We made a buffer exchange of the promising E.coli laccase from 14th of august.
  • We cultivated E.coli KRX with plasmids containing laccases from E.coli, B.pumilus, T.thermophilus, B.halodurans and X.campestris in 1 L flasks without baffles. As references E.coli KRX without plasmid and with plasmid containing Ligase A were used -->Settings: Use of Autoinduction medium, final volume: 250 mL, 60 µg/mL chloramphenicol, 37°C, 120 rpm for 12 h
  • Cells were harvested and then disrupted in equilibration buffer via sonication. After centrifugation the supernatant was purified by using the HisTrap column
  • A new preculture was made for the next cultivation

Tuesday August 28th

• • Team Cultivation & Purification:
    • The band appearing in the SDS-Page of the cultivation of the 14th of august was analysed via Maldi-Tof and we a positive result: we got our laccase!
    • We made a SDS-Page from yesterday's cultivation and got the same band for E.coli cultivation, so we reproduced our result:) It seems, that the higher temperature had the essential influence on the production.
    • Started a new cultivation of E.coli KRX with plasmids containing laccases from E.coli, B.pumilus, '#T.thermophilus, B.halodurans and X.campestris with positive (Ligase A) and negative control (E.coli KRX without plasmid). -->Settings: 300 mL flasks without baffles, autoinduction medium with 0,35 mM CuCl2 added, final volume: 60 mL, 37 °C, 120 rpm, 12 hours
    • Made cell disruption via sonification and purificated the protein via HisTrap column. This time the column was better cleaned by using the twofold volume of 500 mM imidazol.
    • New precultes were prepared for the next cultivation

• Vorkultur ansetzen E.coli 15 mL, K529710 mit Ligase A, LB,37°C, Schikanen

Wednesday August 29th

• Team Cellulose Binding Domain:

Isolation of CBDcex+GFP VI and CBDcex X+XII Restriktion of them with NotI showed right bands (about 400 bp for CBDcex and 1200 bp for CBDcex+GFP)

• Team Site Directed Mutagenesis:

New Primers for X.campestris arrived.

Thursday August 30th

Friday August 31th

Saturday September 1st

Sunday September 2nd

• Team Activity Tests: This week we had Team Modeling over and they told us about their concerns. To continue modeling they wanted to have a look at the activity of our laccase from T. versicolor but with different ABTS concentrations. Especially the were interested in the first time points after adding ABTS. This should give them enough information to calculate the enzyme activity. We didn't want to wait, so we started immediately with our standard activity test. Our tested ABTS concentrations were: 0.5µl, 1µl, 2µl, 4µl and 8µl. We got nice activity curves but also noticed, that the activity saturated quickly and therefore the initial activity of our laccase can not be measured accurately. Of course Team Modeling got our data just in time, but we also want to start new activity tests with half of the amount of laccase. So we are still trying to keep our lovely Team Modeling satisfied.

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