Team:Bielefeld-Germany/Labjournal/week18

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

Monday August 27th

• Team Cloning of 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.
• Team Cellulose Binding Domain:
  • Colony-PCR [http://partsregistry.org/Part:BBa_K863102 CBDcex(T7)], [http://partsregistry.org/Part:BBa_K863121 GFP_His] and [http://partsregistry.org/Part:BBa_K863103 CBDcex(T7)+GFP_His] with no positive result at all.
  • Restriction of pSB1C3+[http://partsregistry.org/Part:BBa_K863103 CBDcex(T7)+GFP_His] with PstI and AgeI followed by dephosphorylation.
  • Restriction of pSB1C3+RFP with XbaI and PstI, followed by dephosphorylation.
  • Restriction of [http://partsregistry.org/Part:BBa_K863102 CBDcex(T7)]-PCR-product with XbaI and PstI
  • Restriction of [http://partsregistry.org/Part:BBa_K863112 CBDclos(T7)] and [http://partsregistry.org/Part:BBa_K863102 CBDcex(T7)]-PCR-products with XbaI and AgeI
  • Transformed the [http://partsregistry.org/Part:BBa_K863113 CBDclos(T7)+GFP_His]-ligation into KRX and plated cell on selection-agar.
• Team Site Directed Mutagenesis:
  • Digestion of two tvel10-plasmids with BamHI and PstI to define direction of the insert. One is positive, one negative.
  • Made the positive tvel10-plasmid ready for sequencing.
• 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], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863015 BBa_K863015] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863020 BBa_K863020] 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 with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863015 BBa_K863015], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863020 BBa_K863020], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863010 BBa_K863010]. 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].

Tuesday August 28th

• Team Cloning of 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 Cellulose Binding Domain:
  • Gel-clean up of the digested pSB1C3-backbone.
  • Colony-PCR of [http://partsregistry.org/Part:BBa_K863102 CBDcex(T7)] and [http://partsregistry.org/Part:BBa_K863103 CBDcex(T7)+GFP_His] with four positive colonies of [http://partsregistry.org/Part:BBa_K863102 CBDcex(T7)].
  • Plasmid-isolation of one [http://partsregistry.org/Part:BBa_K863103 CBDcex(T7)+GFP_His] followed by restriction with no positive result.
  • Plated another [http://partsregistry.org/Part:BBa_K863103 CBDcex(T7)+GFP_His]-colony for plasmid-isolation.
  • Plated small dots of 25 Colonies of the [http://partsregistry.org/Part:BBa_K863113 CBDclos(T7)+GFP_His]-transformation to look if they are red.
  • Plasmidisolation of <partinfo>J61101</partinfo> and digestion with SpeI and PstI
• Team Fungal and Plant Laccases:
  • Digest of tvel35 PCR product for cloning in pSB1C3.
• 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], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863015 BBa_K863015] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863020 BBa_K863020] 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.

Wednesday August 29th

• Team Bacterial Laccases:
  • Restriction of ecol_HIS, bpul_HIS, tthl_HIS and bhal_HIS estimated for the hundredth time.
• Team Cellulose Binding Domain:
  • Plasmid-isolation of two plated [http://partsregistry.org/Part:BBa_K863102 CBDcex(T7)]- and the [http://partsregistry.org/Part:BBa_K863103 CBDcex(T7)+GFP_His]-coloniy and test-digestion of the plasmid with NotI showed fragments of the correct size.
  • Colony-PCR of [http://partsregistry.org/Part:BBa_K863113 CBDclos(T7)+GFP_His]-colonies brought up no positive colonies.
  • To have a comparison to our His-tagged-GFP-fusion-protein we decided to use an exact copy of the His-tagged-GFP. Therefore we made primers to add the His-tag the <partinfo>I13522</partinfo>.
    • Made gradient-pcr with <partinfo>I13522</partinfo> as template and the GFP_FW and GFP_His_compl primers. the most correct product was at 62°C.
• 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], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863015 BBa_K863015] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863020 BBa_K863020] as well as of positive ([http://partsregistry.org/Part:BBa_K525710 BBa_K525710]) and negative control (E.coli KRX without plasmid).
• Team Substrate Analytic: Marcus Persicke has detected the Estrone and Estradiol limit.

Thursday August 30th

• Team Bacterial Laccases:
  • Ligation of the digested PCR products from the day before in BBa_J61101_pSB1A2 plasmid backbone.
  • Ligation of J23110 and J23103 (annealed primers) in pSB1C3 backbone.

• Team Cellulose Binding Domain:
  • Pooled the six good and the six other PCR-fractions with producd of [http://partsregistry.org/Part:BBa_K863122 const.GFP_His]-gradient-PCR.
    • Clean-Up of the PCR-products
    • Start of Restriktion GFP-His6tag with EcoRI, PstI and DpnI in O-Buffer .
  • Colony-PCRs of [http://partsregistry.org/Part:BBa_K863121 GFP_His] - All negativ.

• Team Fungal Laccases:
  • Digest of tvel35 again and ligation in pSB1C3.
  • PCR of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K500002 BBa_K500002] with K500002_FW and K500002_RV primers.

• 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], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863015 BBa_K863015] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863020 BBa_K863020] 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], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863015 BBa_K863015] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863020 BBa_K863020] as well as of positive ([http://partsregistry.org/Part:BBa_K525710 BBa_K525710]) and negative control (E.coli KRX without plasmid).

Friday August 31th

• Team Bacterial Laccases:
  • Colony-PCRs of the ligations with J23100 + J61101 + ecol_HIS, bpul_HIS, tthl_HIS and bhal_HIS. Positive colonies for for ecol_HIS and tthl_HIS and bpul_HIS were plated on LB + AMP.

• Team Fungal Laccases:
  • Purification of PCR product [http://partsregistry.org/wiki/index.php?title=Part:BBa_K500002 BBa_K500002].

• Team Cellulose Binding Domain:
  • Ligation of [http://partsregistry.org/Part:BBa_K863122 const.GFP_His] with a pSB1C3 and transformation of KRX with the product of the ligation.

• Team Site Directed Mutagenesis:
  • sequencing of tvel10-plasmid showed a mutation at base 1573, altering the second last amino acid from threonine to asparagine

• 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 Bacterial Laccases:

• Plasmid isolations on the plated colonies and control restriction with NotI. Control digest showed for J23100 + J61101+ tthl_HIS, J23100 + J61101 + ecol_HIS positive bands.

• Team Fungal Laccases: Plating two colonies from tvel35+pSB1C3 transformation.

• Team Cellulose Binding Domain:
  • Got green colonies on [http://partsregistry.org/Part:BBa_K863122 const.GFP_His]-dish. Picked three and plated them on CM-select-agar.

• 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 Bacterial Laccases:
  • Control digest of tvel35 in pSB1C3 was positive.
  • Plasmid isolation from pSB1C3 + J23110, pSB1C3 + J23110 + J23103 and the plasmids from the distribution plates (18I, 20C and 20 O), for J23100 + J61101+ bpul_HIS. Control digest showed correct bands for J23100 + J61101+ bpul_HIS, pSB1C3 + J23103 and pSB1C3 + J23110.

• Team Cellulose Binding Domain:
  • unsuccessful Colony-PCR of [http://partsregistry.org/Part:BBa_K863113 CBDclos(T7)+GFP_His].

• Team Site Directed Mutagenesis:
  • Gradient-PCR of tvel10-plasmid with tvel10-t243g primer-mix.

• 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.

• 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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