Team:Bielefeld-Germany/Labjournal/week15

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

Monday August 6th

• Team Site Directed Mutagenesis: Plasmidisolation of E.coli_2307 V+VI
• Team Cellulose Binding Domain: Plated one colonie of Bba_I13522
  • Dephosphorilation of pSB1C3 Backbone
  • Ligation of CBDcex+pSB1C3 and CBDclos+pSB1C3
  • Transformed in KRX and plated on CM-selection-agar
• Team 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 E.coli P/S and E. coli HIS for plasmid isolation.

• Team Fungal Laccases: Plating positive colonies from cloning of Tv5 in pSb1C3 backbone.

Tuesday August 7th

• Team Bacterial Laccases: Plasmid isolation and control restriction of E.coli P/S and E. coli HIS in pSb1C3 showed correct fragment sizes in agarose gel. So we did a digest for prefix insertion of the new T7 promoter.

Wednesday August 8th

• Team Bacterial Laccases: We dephosphorylated the digested the plasmid 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 Laccases: Plasmid isolation of Tv5 laccase in pSB1C3 backbone.

Thursday August 9th

• Team Fungal Laccases: Again: Ligation of Tv35 in pSB1C3 backbone.
• Team Cultivation & Purification:
  • Got E.coli KRX with our own construct of B.pumilus, which has a chloramphenicol resistance 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 containing plasmids with [http://partsregistry.org/Part:BBa_K525710 BBa_K525710 (Ligase A)] and with our laccases from B.pumi, E.coli, B.halodurans, X.campestrisand ?T.thermophilus?.

Friday August 10th

• Team Bacterial Laccases:

• Again we did the digest of our new T7 promoter part and did the ligation in pSB1C3 backbone with ecol ORF 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 BBa_K863020 with the primers B.halo_FW and B.halo_RV for cloning the gene in pSB1C3 backbone without promoter and HIS tag.

• Team Cultivation & Purification:
• Flask cultivation of E.coli KRX without plasmid and containing [http://partsregistry.org/Part:BBa_K525710 BBa_K525710 (Ligase A)] or plasmids with laccases from E.coli, B.pumilus, B.halodurans, T.thermophilus or X.campestris to test various concentrations of chloramphenicol.

--> Settings: 100mL flasks without baffles, final volume: 10mL, autoinduction medium with 20/ 57,5/ 95/ 132,5/ 170µg/mL chloramphenicol, 37°C, 120rpm, double determination.

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 CueO, CotA (B. pumi), CotA (B. halo) and T.th 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.

Sunday August 12th

Sunday

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