Team:ETH Zurich/Notebook

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(Difference between revisions)
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* Ordering of additional parts from the iGEM headquater  
* Ordering of additional parts from the iGEM headquater  
* Ordering primers for YcgF & YcgE
* Ordering primers for YcgF & YcgE
 +
* Ordered cDNA of UVR8 from prof. dr. Ronald Urm (Geneva)
 +
* Brainstorming on tetR-DBD and UVR8 fusion strategies:
 +
** Native UVR8 fusion with tetR-DBD (tetR-DBD-UVR8)
 +
** Truncated version of UVR8 fusion with tetR-DBD (tetR-DBD-dUVR8)
 +
** tetR-DBD-UVR8 fusion extended with [GGS]2 linker (tetR-DBD-GGS-UVR8)
 +
===Week 6 (16.7-22.7)===
===Week 6 (16.7-22.7)===

Revision as of 11:50, 25 September 2012

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Eth igem logo.png

Contents

Notebook

Week 1 (11.6-17.6)

  • First meeting
  • Brainstorming

Week 2 (18.6-24.6)

Brainstorming: possible candidate projects:
  • Bacteria sensing a small molecule (Vanillin) and navigates a robot towards the source / Chemotaxis
  • Game Theory: Bacteria playing the Prisoners Dilemma Game
  • Sunburn warning system
  • Early-warning-system for water lack in plants using Abscisic Acid (ABA) detection
  • frequency dependent music tuning device / Mechanical receptor sensing
  • tightly regulated expression system without leakiness
  • C-PS (Cell Positioning System): GPS for a cell
  • Temperature sensing yeast used in beer brewing

Week 3 (25.6-1.7)

Week 4 (2.7-8.7)

Week 5 (9.7-15.7)

  • Ordering of additional parts from the iGEM headquater
  • Ordering primers for YcgF & YcgE
  • Ordered cDNA of UVR8 from prof. dr. Ronald Urm (Geneva)
  • Brainstorming on tetR-DBD and UVR8 fusion strategies:
    • Native UVR8 fusion with tetR-DBD (tetR-DBD-UVR8)
    • Truncated version of UVR8 fusion with tetR-DBD (tetR-DBD-dUVR8)
    • tetR-DBD-UVR8 fusion extended with [GGS]2 linker (tetR-DBD-GGS-UVR8)


Week 6 (16.7-22.7)

  • Cloning of YcgZ promoter (K238013) and GFP (E0840) into pSB1AK3
  • Cloning of YcgE & YcgF from bacterial genome (PCR)
  • Preparation of competent K.O. strains (Δrpos, ΔYcgE, ΔYcgF, parent)

Week 7 (23.7-29.7)

  • Cloning of YcgE & YcgF into psB1C3
  • Transformation of K.O. strains and inoculation for FACS

Week 8 (30.7-5.8)

  • Cloning of LacZ downstream to the YcgZ promoter into pSB1C3, tranformation, colony PCR, sequencing
  • Single cell analysis of K23013-E0840 using FACS
  • Transformation of K.O. strains with construct K23013-LacZ and inoculation for Miller Assay

Week 9 (6.8-12.8)

  • Cloning of RBS B0034 upstream to YcgE & YcgF, transformation, colony PCR, sequencing
  • Designing YcgZ promoter with multiple operator sites
  • Test construct K23013-LacZ with the Miller assay

Week 10 (13.8-19.8)

  • Cloning pabB (S04039) with pabA (K137055) into vector pSB1C3; LovTAP reporter (K322999) with a constitutive promoter (J23108) into vector pSB1C3
  • Fusing designed YcgZ promoters to LacZ

Week 11 (20.8-26.8)

  • Cloning LovTAP reporter (K322999) with a constitutive promoter (J23108) into vector pSB1C3
  • Testing of LovTap construct (Tecan plate reader)

Week 12 (27.8-2.9)

  • Testing of LovTap in different light conditions (6h incubation). Measuring RFP output with FACS.

Week 13 (3.9-9.9)

  • Testing of LovTap in different light conditions (12h incubation). Measuring RFP output with FACS.

Week 14 (10.9-16.9)

  • UVR8 System : Testing of different exposure invervals and UV intensities.
  • Changing the read-out of the UVR8 system from GFP to Galactosidase
  • Cloning of new read-out system for LovTap from RFP to Galactosidase due to observed bleaching upon light exposure.
  • Cloning of PabA and PabB in one verctor
  • Exact planning of the multiplexer. Ordering of Primers and inoculation of necessary parts.
  • Designing primers for Gibson ligation
  • Cloning pabA into vector containint pabB

Week 15 (17.9-23.9)

  • Cloning of new read-out system for LovTap with LacZ
  • Cloning protein coding region of LacZ and TetR with a constitutive promoter (Decoder)
  • Cloning all parts in the pSB1C3 backbone



References

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  • Christie, J. M., Arvai, A. S., Baxter, K. J., Heilmann, M., Pratt, A. J., O’Hara, A., Kelly, S. M., et al. (2012). Plant UVR8 photoreceptor senses UV-B by tryptophan-mediated disruption of cross-dimer salt bridges. Science (New York, N.Y.), 335(6075), 1492–6.
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