Team:TU Dresden/Notebook/LabJournal



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Lab Journal

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Week 1: (20-26 July)

Investigation of P3 threshold for E. coli resistance

  • Plasmids were received (T18, ZHER2, LZT18, LZT25 and HER2 codon) and diluted. Also two pSB1C3 plasmids from iGEM containing RBS (BBa_E0020) and CFP (BBa_E0020) were diluted.
  • Preparation of E. coli GB05 and set cultures overnight.
  • Electroporation of the cells with the plasmids.
  • Transfected cells were streaked out on chloramphenicol (Cm) and kanamycin (kan) plates.
  • Colonies were picked and mixed with Cm and kan. These were stored in the fridge.
  • Streaking out colonies from DHM1, BTH101 and K12 plates.
Week 2 (27 July-2 August)

Investigation of P3 threshold for E. coli resistance

  • A miniprep was done with the seven cultures with the plasmids. A glycerol stock of each culture was saved.
  • The concentration of the plasmids was measured using the nanodrop.
  • A digestion of the plasmids was done and a gel was run.
Week 3 (3-9 August)

Correct folding study of target protein

  • pET28a vector preparation: digestion and dephosphorylation. Afterwards a gel was run and the DNA was extracted.
  • A PCR was set to create HER2 with NheI and NotI restriction sites. A digestion was done and the product was run on a gel and purified. The concentration was measured with the nanodrop. This process was repeated some days later since the concentration was too low.

    • Investigation of P3 threshold for E. coli resistance

  • 3 cultures for GB05 and K12 were set overnight.
  • Transformation of T25 and gene III plasmids and promoter (pLac) in E. coli: gene III in GB05 and K12; T25 in GB05; promoter in K12 and GB05.
  • Transformed colonies were plated on kan plates (gIII and T25) and Cm (pLac). A miniprep was done with the overnight cultures of pLac, T25 and gIII. The concentration of plasmid was measured with the nanodrop.
  • New back-up cultures were set overnight.
  • Cloning of P3 and RBS+CFP in pLac, followed by dephosphorylation and gel running and purification. The concentration was measured with the nanodrop.
      • Week 4 (10-16 August)
      • Ligation of pLac + P3 and RBS + CFP: first set up the cultures for transformation, and the transform the electrocompetent cells: pLac in K12, pLac+P3 in K12 and GB05, and RBS+CFP in GB05. Plate the transformed cells and incubate them overnight.
      • Restriction digestion for the biobrick constructs and BACTH buildup: ZHER2, HER2, LZT25, LZT18, T18, T25 and P3.
      • Restriction digestion of HER2 and pET28a. The products were run on a gel and afterwards the weight was measured.
      • Preparation of the plates needed for the setup: one colony of pLac in K12 and pLac+pIII in K12 were streaked out and incubated in Cm+LB.
      • Plasmid miniprep from RBS+CFP in GB05 and pLac+P3 in GB05. The concentration of the plasmids was measured with the nanodrop. The products were then digested, dephosphorylated and loaded on a gel.
      Week 5 (17-23 August)
      Week 6 (24-30 August)
      Week 7 (31 August-6 September)
      Week 8 (7-13 September)
      Week 9 (14-20th September)