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Team:Bielefeld-Germany/Labjournal/week18
From 2012.igem.org
Contents |
Week 18 (08/27 - 09/02/12)
Monday August 27th
- Team Cultivation & Purification:
- We made a buffer exchange of the promising E.coli laccase.
- 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
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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