Team:Stuttgart/Notebook

Notebook

Notebook - important experiments

26.07.2017

  • transformation of kerUS (BBa_K1498000)
  • preparation of antibiotic stock solutions: chloramphenicol (35 mg/mL) and ampicillin (100 mg/mL)

27.07.2017

  • growing of a single colony (kerUS (BBa_K1498000)) in 5 mL LB media chloramphenicol (35 µg/mL] at 37 °C
  • single colonies are plated on agar plates and incubated at 37 °C over night

28.07.2017

  • Preparation of: miniprep (Jena Biosciences Kit)and glycerol stock (kerUS (BBa_K1498000))

03.08.2017

  • transformation of BBa_K1149002 and BBa_K1149003

04.08.17

  • single colonies (BBa_K1149002 and BBa_K1149003) are plated on agar plates and incubated at 37 °C over night

09.08.17

  • growing of a single colony (BBa_K1149002 and BBa_K1149003) in 5 mL LB media + chloramphenicol (35 µg/mL] at 37 °C

10.08.17

  • Preparation of miniprep (Jena Biosciences Kit) and glycerol stock (storage: -80 °C) (BBa_K1149002 and BBa_K1149003)
  • SOC media preparation
  • transformation pet19-LipB

21.08.2017

  • preparation: preliminary test of esterase assay with EstCS2: Bba_K1149002 - preparation of o/n cultures (37°C)

22.08.2017

  • preparation: preliminary test of esterase assay with EstCS2: Bba_K1149002 - glycerol stocks and induction with arabinose

23.08.2017

  • preliminary test of esterase assay with EstCS2: Bba_K1149002 (link results)

28.08.2017

  • preparation of electro-competent cells

31.08.2017

  • transformation/electroporation with iGEM competent cells test kit DNA (o/n incubation, 37°C - transformation failed)

01.09.2017

  • transformation/electroporation with pUC19 plasmid (o/n incubation, 37°C - transformation failed)

06.09.2017-08.09.17

  • enzyme activity assay (cell pellet and supernatant) with BBa_K1149002 and pet19-LipB

12.09.2017

  • preparation of chemo-competent cells

13.09.2017

  • transformation with iGEM competent cells test kit DNA and pUC19 plasmid (o/n incubation, 37°C) - transformation successful


14.09.2017

  • calculation - efficiency of the chemo-competent cells/pUC19:
    efficiency E =(56cfu/0.001ng)*1000ng/(µL)=5.6*((10)^7 )cfu/µL

18.09.2017

  • preparation InterLab study - transformation of 8 parts into DH5alph E. coli cells

19.09.17 – 21.09.17

  • InterLab study LUDOX measurement
  • enzyme activity assay with BBa_K1149002 of the supernatant - measurement in biological triplicates
  • InterLab study Fluorescein measurement

22.09.2017

  • InterLab study sample measurement (link results)

27.09.17 - 29.09.17

  • enzyme activity assay with pet19-LipB of the supernatant - measurement in biological triplicates

06.10.2017

  • Collaboration iGEM team Heidelberg: preparation mutagenesis plasmid activity assay - preparation of media, antibiotic-stocks and sugar-stocks + agar plates with different antibiotics

10.10.2017

  • Collaboration iGEM team Heidelberg: preparation of different cultures + induction of the cells with arabinose (o/n incubation, 37°C)

11.10.2017

  • Collaboration iGEM team Heidelberg: spread the o/n cultures on prepared agar plates (o/n incubation, 37°C)

16.10.2017

  • preparation semi-quantitative keratinase-assay: spread kerUS, KerA and KerP on chloramphenicol/kanamycin agar plates (o/n incubation, 37°C)
  • Preparation skim-milk-plate assay: preparation of skim-milk-agar-plates with chloramphenicol/kanamycin

18.10.2017

  • M9 media preparation
  • preparation semi-quantitative keratinase-assay: preparation of o/n cultures (37°C)
  • Preparation skim-milk-plate assay: preparation of o/n cultures (37°C)

19.10.2017

  • PCR - amplification of LipB - cloning of LipB in the iGEM standard plasmid (BBa_K1149002 is used)
  • PCR - amplification of BBa_K1149002 (without EstCS2)
  • semi-quantitative keratinase-assay: add 0.005 g of dried hair (65 °C, 1h) to o/n cultures (KerUS and KerA are induced with 1mM IPTG before) - 5 days incubation, 37°C (link results)
  • skim-milk-plate assay kerP - spread on prepared skim-milk agar plates (supernatant and cells) - 4 days incubation, room temperature (link results)

20.10.2017

  • SDS page, Gibson Assembly, Transformation (Zymos) - cloning of LipB in the iGEM standard plasmid (BBa_K1149002 is used)
  • skim-milk-plate assay kerUS and KerA - spread on prepared skim-milk agar plates (supernatant and cells) - 4 days incubation, room temperature (link results)