Team:Bielefeld-Germany/Labjournal/week18

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

Monday August 27th

• Team Cultivation & Purification:
  • We exchanged the buffer of the purified ECOL from 08/14, which showed a promising band in the SDS-Page.
  • Made a flask cultivation of E.coli KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863010 BBa_K863010], B.halodurans and X.campestris with positive ([http://partsregistry.org/Part:BBa_K525710 BBa_K525710]) and negative control (without plasmid).

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

• • Cells from today's cultivation were disrupted via sonification and laccase was purified by using the HisTrap column.
  • Made precultures of E.coli KRX without plasmid and with laccases from B.halodurans, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863010 BBa_K863010] or X.campestris. As negative control we used E.coli KRX and as positive control we used E.coli KRX with [http://partsregistry.org/Part:BBa_K525710 BBa_K525710].

• Team Bacterial Laccases: Digest of our new plasmid pSB1C3 with pT7 promoter (hopefully, cause we have to wait for sequencing results). Dephosphorylation of digested vector and purification over agarose gel. After that, the backbone was ligated with ecol_HIS, tthl_HIS, bpul_HIS and bhal_HIS inserts.

Again ligation of J231110 in pSB1C3 backbone.

Tuesday August 28th

• Team Cultivation & Purification:
  • The band appearing in the SDS-Page of the cultivation of the 08/14 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 cultivation of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005], so we reproduced our result:) It seems, that the purification and the higher temperature had the essential influence on the production.
  • Started a new flask cultivation of E.coli KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863010 BBa_K863010], B.halodurans and X.campestris with positive ([http://partsregistry.org/Part:BBa_K525710 BBa_K525710]) and negative control (E.coli KRX without plasmid).

-->Settings: 300 mL flasks without baffles, final volume: 60 mL, autoinduction medium, 0,35 mM CuCl2, 37 °C, 120 rpm, durance: 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.

• Team Bacterial Laccases: Ligation of pSB1C3 with promoters J23117, J23110 and J23103. The promoters (which are again primer pairs, which have to be annealed) were diluted 1:1000 (0,1pmol) before and boiled with 98°C and slowly cooled down for ligation of the forward and the reverse primers.

• Colony PCR of ligated products with T7 promoter and insert. The PCR showed only for the tthl laccase a band in the right height. So we picked this colony and plated it to isolate the plasmids.

• Team Fungal Laccases: Digest of tvel35 PCR product for cloning in pSB1C3.

Wednesday August 29th

• Team Cellulose Binding Domain:
• Isolation of CBDcex+GFP VI and CBDcex X+XII
• Restriction 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 xccl 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 of E.coli KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863010 BBa_K863010], B.halodurans and X.campestris as well as of positive ([http://partsregistry.org/Part:BBa_K525710 BBa_K525710]) and negative control (E.coli KRX without plasmid).
• Team Bacterial Laccases: Restriction of ecol_HIS, bpul_HIS, tthl_HIS and bhal_HIS estimated for the hundredth time.

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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863010 BBa_K863010], B.halodurans and X.campestris with positive ([http://partsregistry.org/Part:BBa_K525710 BBa_K525710]) 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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005]

-->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 of E.coli KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863010 BBa_K863010], B.halodurans and X.campestris as well as of positive ([http://partsregistry.org/Part:BBa_K525710 BBa_K525710]) and negative control (E.coli KRX without plasmid).

• Team Bacterial Laccases:

• Ligation of the restriction from the day before in J61101 J23100 plasmid backbone.

Picking 20C , 20O and 18 I from this years iGEM plates and transformation in KRX.

• Team Fungal Laccases:

• Digest of tvel35 again.
• PCR of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K50002 BBa_K500002] with K500002_FW and K500002_RV primers.

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 containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000]

-->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 culture of fermentation on 08/31 (E.coli KRX containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000]). 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 of E.coli KRX containing either [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000] or a plasmid with His-tagged GFP.

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