Team:ETH Zurich/Biology/Journal

Week 1: 20.6-26.6

• First meeting

• Brainstorming

Week 2: 27.6-3.7

• Brainstorming

Week 3: 4.7-10.7

• Working on the design of the system

• Design of several Operons for AlcR and several PCR primers

Week 4: 11.7-17.7

• Ordering of the LacI_M1 and codon-optimized AlcR gene
• Making of competent DH5-α cells
• Cloning of the AlcR-testsystem

Week 5: 18.7-24.7

• First test of the AlcR-system in 96-well plates

Week 6: 25.7-31.7

• Transformation of the parts from the iGEM plates

Week 7: 1.8-7.8

• Growth test of DH5-α with AlcR-system in flasks

• Ligation and Transformation of
  • λ_P and luxI
  • λ_P and lacI
  • P_lac and GFP_LVA
  • P_lux and mCherry
  • P_tet and CI -had to be redone because of point mutation in the primer (week 9)
  • P_const and luxR

• First meeting with Dr. Oliver Frey (Bio engineering laboratory, Prof. Andreas Hierlemann, D-BSSE ETHZ) to exchange ideas about microfluidic design

Week 8: 8.8-14.8

• Synthesized LacI_M1 Gen arrived

• Ligation and Transformation of
  • P_lac-GFP_LVA and Terminator
  • P_lux-mCherry and Terminator
  • P_const-luxR and Terminator
  • P_lac-GFP_LVA-Terminator and P_lux-mCherry-Terminator on one plasmid
  • P_tet and LacI_M1
  • P_tet-LacI_M1 and Terminator

• Improving of the testsystem
  • Exchange of some rare codons in AlcR by PCR
  • His-tagging of AlcR for detection in Western blot
  • include sfGFP

• Transformation of λ_P

• Creation of competent JM101 cells
  • Check for transformation efficiency

Week 9: 15.8-21.8

• Growth and repression test of AlcR-system with M9-medium
• Ligation and Transformation of
  • λ_P and lacI
  • λ_P and luxI
  • P_tet and CI
  • λ_P-lacI and terminator
  • λ_P-luxI and terminator

Week 10: 22.8-28.8

• Transformation and Ligation
  • P_tet-CI and Terminator
• Preparation of chemically competent DH5-α and JM101 cells

Week 11: 29.8-4.9

• Transformation and Ligation
  • P_tet-CI and Terminator
• Diffusion test through tube system filled with agarose and cells
• Test of the improved AlcR system

Week 12: 5.9.-11.9

• Design of a system to characterize our new biobrick LacI_M1
  • Transformation and Ligation
    • P_Tet and LacI
    • P_Lac and GFP assam

• Design of an additional construct to test the band pass filter
  • Transformation and Ligation
    • P_const and TetR_LVA

• Growth-test of E. coli in M9 with reduced Ampicillin concentration

• AlcR test with reduced Ampicillin concentration

• Testdigest of λP-luxI-Terminator

• Alternative Sensor system: Xylene system
  • Transformation and Ligation
    • P_const and XylR
    • P_Xyl and CI
    • P_Xyl and LacI _M1

Week 13: 12.9.-18.9

• PCR of the pBR322 Ori
• PCR of the gen cluster for the upper TOL pathway
• first test of cell printing
• Transformation and Ligation of
  • P_const-TetR- P_Tet-LacI_M1 and λP-GFPassam
• Western Blot of AlcR
• Making of competent DH5-α cells
• Design and construction of Psb6A5

Week 14: 19.9.-25.9

• Characterization of LacI_M1
• Cloning of all biobricks into pSB1C3
• PCR of LacI and CI