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From 2012.igem.org

Contents 1 Week 24 (10/08 - 10/14/12) 1.1 Monday October 08th 1.2 Tuesday October 09th 1.3 Wednesday October 10th 1.4 Thursday October 11th 1.5 Friday October 12th 1.6 Saturday October 13th 1.7 Sunday October 14th

Week 24 (10/08 - 10/14/12)

Monday October 08th

All: Day off in Amsterdam!!!

Tuesday October 09th

Wednesday October 10th

• Team Shuttle Vector:
  • Ligation of pSB1C3::BBa_K863202 construct in KRX cells was done.
• Team Cellulose Binding Domain:
  • Gradient-PCRs of CBDcex_Freiburg; CBDclos_Freiburg; GFP_Freiburg and ecol_Freiburg (47 °C to 67°C)
    • Product:
      • CBDcex_Freiburg at all temperatures
      • CBDclos_Freiburg at all temperatures
      • GFP_Freiburg at all temperatures
      • ecol_Freiburg at 53 °C to 61°C (best at 57 to 59 °C)
    • pooled fractions for gel clean up
• Team Site Directed Mutagenesis:
  • Ordered Primers for the SDM of the illegal XbaI restriction site in the shuttle vector
• Team Cultivation and Purification:
  • Made precultures of E. coli KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005]for 19L fermentation (500mL preculture)

Thursday October 11th

• Team Cellulose Binding Domain:
  • Clean-Up of the Gel-pieces (from PCR-products)
  • Restriction of PCR-products (3h):
    • GFP_Freiburg:
      • XbaI+PstI Tango-Buffer for Ligation
      • XbaI+AgeI Tango 4xAgeI for Assembly
    • CBDclos/cex:
      • NgoMIV+PstI P.4+BSA 2xPstI for Assembly
  • Assembly and Ligation (1h with T4):
    • GFP_Freiburg + CBDcex_Freiburg + J61101
    • GFP_Freiburg + CBDclos_Freiburg + J61101
    • GFP_Freiburg + J61101
  • Transformation in E. coli KRX (3 µL, chemical):
    • GFP_Freiburg + CBDcex_Freiburg + J61101
    • GFP_Freiburg + CBDclos_Freiburg + J61101
    • GFP_Freiburg + J61101
  • All Transformations were plated on AMP-select-agar

• Team Cultivation and Purification
  • 12 L Cultivation of E. coli KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005]
    • Settings:

Bioengineering NFL 19L fermenter, autoinduction HSG medium, 60 µg/mL chloramphenicol, 19 L, 37 °C, stirrer on cascade to hold a pO2 of 50 %, airflow: 12 NL/m, 16 hours. HSG medium was choosen to get a high biomass concentration with hope for a higher amount of laccases.

• • Made precultures of E. coli Rosetta Gami 2 containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 BBa_K863012](09/09) and <partinfo>BBa_K863022</partinfo> (09/09) behind a constitutive promoter. (500mL preculture)

Friday October 12th

• Team Cellulose Binding Domain:
  • A lot of colonies on all three dishes (J61101+GFP_Freibug;J61101+GFP_Freibug+CBDcex;J61101+GFP_Freibug+CBDclos)
    • no obvious green fluorescence, not even at J61101+GFP
    • Maybe the combination of J23100 and J61101 isn't strong enough to express a visible amount of GFP
  • Colony-PCR of 16 colonies of every dish
    • J61101 + GFP_Freiburg:
      • 16/16 positive
      • plated two on select-agar
    • J61101 + GFP_Freiburg + CBDcex:
      • 8/16 positve
      • plated three on select-agar
    • J61101 + GFP_Freiburg + CBDclos:
      • 7/16 positive
      • plated three on select-agar
• Team Shuttle Vector:
  • The induction with 0.5% (v/v) methanol of P. pastoris GS115 cells with integrated BBa_K863204::GFP was started.
• Team Substrate Analysis:
  • Since we have seen some new peaks after Estradiol treated with Laccases in the LC-MS, we wanted to have more degradation products to do an MS so we used a higher concentradion of Estradiol and Laccase and let them incubate for 66 hours.

• Team Cultivation and Purification
  • 12L Cutlivations of E. coli KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005] were harvested and stored at 4°C until purification.
  • 12 L Cultivation of E. coli Rosetta Gami 2 containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 BBa_K863012](09/09) and <partinfo>BBa_K863022</partinfo> (09/09)
    • Settings:

Bioengineering NFL 19L fermenter, HSG medium, 60 µg/mL chloramphenicol, 300 µg/mL ampicillin, 12 L, 37 °C, stirrer on cascade to hold a pO2 of 50 %, airflow: 12 NL/m, 22-24 hours. HSG medium was chosen to get a high biomass concentration with hope for a higher amount of laccases.

Saturday October 13th

• Team Shuttle Vector:
  • The P. pastoris GS115 cells integrated with BBa_K863204::GFP were also inducted with 0.5% (v/v) methanol (100%).

• Team Cultivation and Purification
  • 12L Cutlivations of E. coli Rosetta Gami 2 containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 BBa_K863012](09/09) and <partinfo>BBa_K863022</partinfo> (09/09) were harvested and stored at 4°C until purification.

Sunday October 14th

• Team Shuttle Vector:
  • The P. pastoris GS115 cells integrated with BBa_K863204::GFP were also inducted with 0.5% (v/v) methanol (100%).

• Team Fungal and Plant Laccases:
  • Plasmid isolation of pSB1C3::BBa_K863204::TV5 for transformation into P. pastoris GS115 was done. After isolation the plasmid was digested with NotI, purified with the PCR clean up kit and transformed via protocol in P. pastoris GS115.

• Team Cellulose Binding Domain:
  • Tried to isolate all plates, but only got low concentrations (5 ng/µL - 20 ng/µL)
  • plated eight different colonies for isolation

• Team Substrate Analysis:
  • We took sample from the reaction of the 12th October.

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