Team:Bielefeld-Germany/Labjournal/week15

Week 15 (08/06 - 08/12/12)

Monday August 6th

• Team Site Directed Mutagenesis:
  • Plasmid-isolation of two more ecol-g2307a colonies.
• Team Cellulose Binding Domain:
  • Plated one colony of <partinfo>BBa_I13522</partinfo> on selection-agar.
  • Dephosphorylation of pSB1C3 Backbone.
  • Ligation of [http://partsregistry.org/Part:BBa_K863102 CBDcex(T7)] with the pSB1C3-Backbone and [http://partsregistry.org/Part:BBa_K863112 CBDclos(T7)] with the pSB1C3-Backbone.
  • Transformed both in KRX and plated on CM-selection-agar.
• Team Cloning of Bacterial Laccases:
  • No positive colonies after transformation of our assemblies from August 1st, but we realized that the primers we used for making the promoter parts can’t ligate with our insert an backbone because the primers are dephosphorylated and the plasmid backbone is dephosphorylated, too. Much effort in a mission which can't work but at least we know now why it doesn't work.

• Picking positive colonies from transformation of ecol and ecol_HIS in pSB1C3 for plasmid isolation.

• Team Fungal and Plant Laccases: Plating positive colonies from cloning of tvel5 in pSb1C3 backbone.

Tuesday August 7th

• Team Cloning of Bacterial Laccases:

• Plasmid isolation and control restriction of ecol and ecol_HIS in pSB1C3 showed correct fragment sizes in agarose gel. So we did a digest for prefix insertion of the new T7 promoter.

• Team Site Directed Mutagenesis:
  • Digestion of the two ecol-g2307a-mutants showed that one has lost the restriction-site. Prepared that one for sequencing.
• Team Cellulose Binding Domain:
  • Plasmid-isolation of <partinfo>Bba_I13522</partinfo>.
  • PCR with <partinfo>BBa_I13522</partinfo> as template and GFP_Freiburg and GFP_His6_compl-primers and clean-up through agarose-gel.
  • Restriktion of Linear Backbone, [http://partsregistry.org/Part:BBa_K863102 CBDcex(T7)] and [http://partsregistry.org/Part:BBa_K863121 GFP_His] with EcoRI and PstI.
  • Ligation of Linear Backbone with [http://partsregistry.org/Part:BBa_K863102 CBDcex(T7)] and with [http://partsregistry.org/Part:BBa_K863121 GFP_His], respectively.
  • Transformation of all that stuff and plating on selection-agar.

Wednesday August 8th

• Team Cloning of Bacterial Laccases:

• We dephosphorylated the digested the plasmids from day before and phosphorylated the promoter parts. After that we ligated the two parts and transformated the products into KRX electrocompetent cells.

• Team Fungal and Plant Laccases: Plasmid isolation of tvel5 laccase in pSB1C3 backbone.
• Team Site Directed Mutagenesis:
  • Made PCRs on tvel-t143g-mutants with Tv_lac10.P.FW and Tv_lac10.S.RV primers, with products of 1.6 kbp when there should be about 4.0 kbp.
• Team Cellulose Binding Domain:
  • Picked one not-red colony of [http://partsregistry.org/Part:BBa_K863103 CBDcex(T7)], [http://partsregistry.org/Part:BBa_K863112 CBDclos] and [http://partsregistry.org/Part:BBa_K863121 GFP_His] and plated it on seletion-agar
  • Restriktion of [http://partsregistry.org/Part:BBa_K863102 CBDcex(T7)]-PCR-product with AgeI and EcoRI with DpnI
  • Restriktion of [http://partsregistry.org/Part:BBa_K863121 GFP_His]-PCR-product with NgoMIV and PstI and DpnI
  • Freiburg-Assembly with [http://partsregistry.org/Part:BBa_K863102 CBDcex(T7)] and [http://partsregistry.org/Part:BBa_K863121 GFP_His] on the linearized pSB1C3 Backbone followed by plating on selection-agar.

Thursday August 9th

• Team Fungal and Plant Laccases: Again: Ligation of tvel35 in pSB1C3 backbone.
• Team Cultivation & Purification:
  • We got E.coli KRX with our own BioBrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000] in pSB1C3 like the rest.
  • We discussed if we did not produce anything because of the toxicity of our protein, which may reduce the plasmid stability. We searched for maximal used concentration of chloramphenicol and found out that concentrations up to 170µg/mL were used. Therefore we decided to start a new flask cultivation with concentrations of chloramphenicol varying in 5 steps from 20µg/mL to 170µg/mL. Today we made the 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] as well as [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863020 BBa_K863020], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863015 BBa_K863015] and ?[http://partsregistry.org/wiki/index.php?title=Part:BBa_K863010 BBa_K863010]?. We used E.coli KRX as negative and [http://partsregistry.org/Part:BBa_K525710 BBa_K525710] as positive control.
• Team Site Directed Mutagenesis:
  • Sequencing results arrived:
    • One bpul-plasmid is positive on both mutations
    • All Xccl-mutants are negative (there even was a part of 900 bp gone missing!)
  • plated six more colonies of the xccl-g3633c for plasmid-isolation
  • PCR with the original tvel10-plasmid and the Prefix/Suffix-primers, showed no product at all.
• Team Cellulose Binding Domain:
  • Plasmid-isolation of [http://partsregistry.org/Part:BBa_K863112 CBDclos(T7)], [http://partsregistry.org/Part:BBa_K863102 CBDcex(T7)] and [http://partsregistry.org/Part:BBa_K863121 GFP_His].
  • Transformated the assembly again with more of volume of the assembly-CBDcex-GFP_His-mix.

Friday August 10th

• Team Cloning of Bacterial Laccases:

• Again we did the digest of our new T7 promoter part and the ligation in pSB1C3 backbone with ecol ORF PCR products with and without HIS tag. After that we transformed the ligations in pSB1C3. Additionally we did the same with promoter J23110 instead of T7 promoter.
• We did PCR on [http://partsregistry.org/Part:BBa_K863020 BBa_K863020] with the primers B.halo_FW and B.halo_RV for cloning the gene in pSB1C3 backbone without promoter and HIS tag.
• We ligated the digested pSB1C3 plasmids with ecol and ecol_HIS with the new pT7 promoter and pSB1C3 backbone and transformed the approach in KRX.

• Team Cultivation & Purification:

• 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_K863020 BBa_K863020], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863015 BBa_K863015] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863010 BBa_K863010] to test various concentrations of chloramphenicol. We used E. coli KRX as negative and E. coli KRX with [http://partsregistry.org/Part:BBa_K525710 BBa_K525710] as positive control.

• • • Settings: 100 mL flasks without baffles, final volume: 10 mL, autoinduction medium with 20/ 57,5/ 95/ 132,5/ 170 µg/mL chloramphenicol, 37 °C, 120 rpm, double determination.
• Team Site Directed Mutagenesis:
  • Plasmid-isolation xccl-g3633c-colonies and test-digestion showed no positive mutated plasmids.
  • New pfu-PCR on the xccl-plasmid with xccl-g3633c primer-mix
• Team Cellulose Binding Domain:
  • Picked [http://partsregistry.org/Part:BBa_K863103 CBDcex(T7)+GFP_His] assembly colony and plated it on CM-selection-agar for plasmid-isolation.
  • Restricted [http://partsregistry.org/Part:BBa_K863112 CBDclos(T7)]with EcoRI and AgeI
    • Digested insert did not appear in the
  • Colony-PCR of [http://partsregistry.org/Part:BBa_K863112 CBDclos(T7)].
  • Picked and plated several colonies of the [http://partsregistry.org/Part:BBa_K863103 CBDcex(T7)+GFP_His]-assembly and plated the on CM-Agar.

Saturday August 11th

• Team Bacterial Laccases:

• Colony PCRs showed no bands. So we transformed the ligations from 10.08. again.
• We did the PCRs of the laccase genes ecol, bpul, bhal and lthl again. We used the …_FW / …_RV primers and the …_FW / …_RV_HIS primers of the different genes. Digestion of this PCR products and ligation with pT7 or promoter J23110 and the pSB1C3 plasmid backbone.

• Team Site Directed Mutagenesis:
  • Agarose-gel-electrophoresis of xccl-PCR (yesterday) showed bands at 3.2 kbp and over 12 kbp but not at 4 kbp as it should be.
• Team Cellulose Binding Domain:
  • A few of the plated pSB1C3+CDBcex+GFP-assembly colonies have grown
  • Colony-PCR [http://partsregistry.org/Part:BBa_K863112 CBDclos(T7)]-colonies showed no positive clones.

Sunday August 12th

• Team Bacterial Laccases: Cleanup from agarose gel of the PCR products from the day before. After that we did control restriction and got bands for ecol with J23110 promoter and with the new pT7. So we sent this plasmids for sequencing.
• Team Site Directed Mutagenesis:
  • pfu-PCR with xccl-plasmid and xccl-g2247c primers resulted in no product.
  • PCR with pre- and suffix primers for tvel10 with original Topo-plasmid as template with increasing temperature at each step (0.1°C) resulted in no product.
• Team Cellulose Binding Domain:
  • Colony-PCR for [http://partsregistry.org/Part:BBa_K863102 CBDcex(T7)], [http://partsregistry.org/Part:BBa_K863112 CBDclos(T7)] and [http://partsregistry.org/Part:BBa_K863103 CBDcex(T7)+GFP_His].
    • resulted in two positve [http://partsregistry.org/Part:BBa_K863112 CBDclos(T7)]-colonies.
  • Did another colony-pcr on [http://partsregistry.org/Part:BBa_K863102 CBDcex(T7)] and [http://partsregistry.org/Part:BBa_K863103 CBDcex(T7)+GFP_His] that brought up nine positve clones of [http://partsregistry.org/Part:BBa_K863102 CBDcex(T7)].

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