Team:Bielefeld-Germany/Labjournal/week18

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

Monday August 27th

• Team Cultivation & Purification:
  • We exchanged the buffer of the purificated E.coli laccase from 14th of august, which showed a promising band in the SDS-Page.
  • Made a flask 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 (without plasmid).

-->Settings: 1L flask without baffles, autoinduction medium, final volume: 250 mL, 60 µg/mL chloramphenicol, 37°C, 120 rpm for 12 hours, single determination

• • Cells from today's cultivation were disrupted via sonication and laccase was purified by using the HisTrap column.
  • Made precultures of E.coli KRX without plasmid and with plasmids containing laccases from B.halodurans, B.pumilus, E.coli, T.thermophilus or X.campestris. As postive control we used BBa_K525710 (Ligase A).

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 flask 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

• • Cells from today's cultivation were disrupted via sonication and laccase was purified by using the HisTrap column. This time the column was better cleaned by using the twofold volume of 500 mM imidazol.

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.

• Team Cultivation & Purification:
  • Made a SDS-Page of yesterday's cultivation, but it was too pale, so it had to be repeated
  • Installation of the two 3 L fermenters used for practical courses in our lab. Had to search for all of the components with the help of members of fermentation group and test electrodes.
  • Made precultures analogous to 08/27.

Thursday August 30th

• Team Cultivation & Purification:
  • Started another flask cultivation, the repetition of the cultivation on monday in a smaller scale but with a double determination. Cultivation of E.coli KRX with plasmids containing laccases from E.coli, B.pumilus, T.Thermophilus, 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, final volume: 60 mL, 37 °C, 120 rpm, 12 hours

• • Continuing Installation of the two 3 L fermenters.
  • Starting the first fermentation of E.coli KRX with plasmid containing laccase from E.coli

-->Settings: fermenter: Braun, autoinduction medium, final volume: 3 L, 37 °C, stirrer on cascade to hold a pO2 of 50 %, airflow: 2 NF/m, 12 hours. We had some problems with the controllation of settings.

• • ** Made precultures analogous to 08/27.

Friday August 31th

• Team Cultivation & Purification:
  • Cells of the yesterday's flask cultivation and a sample of equal size of E.coli fermentation were disrupted via sonication and laccase was purified by using the HisTrap column. The purificated sample of the fermentation did not show any activity, so we decided not to purify the whole sample.
  • First fermentation of E.coli KRX with laccase from B.pumilus

-->Settings: fermenter: Infors, autoinduction medium, final volume: 3 L, 37 °C, stirrer on cascade to hold a pO2 of 50 %, airflow: 2 NF/m, 12 hours.

Saturday September 1st

• Team Cultivation & Purification:
  • Harvesting and centrifugation of fermentation of B.pumilus laccase. Store pellet at 4°C

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.
• Team Cultivation & Purification:
  • • • Made precultures analogous to 08/27.

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