Team:ETH Zurich/Notebook

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Contents

Notebook

Week 1 (11.6-17.6)

  • First meeting
  • Brainstorming

Week 2 (18.6-24.6)

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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
IMG 1519.jpg

Week 3 (25.6-1.7)

  • Literature research on our different project ideas.

Week 4 (2.7-8.7)

  • Literature research on our different project ideas and final decision.

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)
20120925-iGem-5876.jpg

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)
  • Andreas Bosshart provided a pSEVA183 derived plasmid (pSEVA183-lacI), containing ampicillin resistance, constitutively expressed LacI from native promoter and Ptac promoter for cloned gene expression.
  • Ordered primers for full length tetR and truncated version (tetR-DBD) protein cloning
  • TetR controllable GFP expression system (BBa_I13522) was cloned from pSB1A2 to pSB1C3, tested size in agarose gel and sequenced.

Week 7 (23.7-29.7)

  • Cloning of YcgE & YcgF into psB1C3
  • Transformation of K.O. strains and inoculation for FACS
  • Cloning of tetR and tetR-DBD into pSEVA183-lacI and tested weather tetR-DBD is unable to repress GFP production from pSB1C3 plasmid.
  • Ordered primers for UVR8 fusions.
20120924-iGem-5777.jpg

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
  • Recloning of GFP reporter system (BBa_I13522) into pSB4K5 plasmid.
  • Cloning of UVR8 versions behind tetR-DBD and transforming fusion constructs (in pSEVA183-lacI) with GFP reporter system (in pSB4K5), later called as UVR8 system.

Week 9 (6.8-12.8)

IMG 1531.jpg
  • 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
  • First test of UVR8 constructs in platereader
  • Cloning ho1 (I15008) and pcyA (I15009) with RBS (B0034) into pSB1A3

Week 11 (20.8-26.8)

IMG 1520.jpg
  • Cloning LovTAP reporter (K322999) with a constitutive promoter (J23108) into vector pSB1C3
  • Testing of LovTap construct (Tecan plate reader)
  • Cloning Terminator (B0017) to RBS-ho1 (B0034-I15008) and RBS-pcyA (B0034-I15009)
  • New test of UVR8 constructs in platereader

Week 12 (27.8-2.9)

  • Testing of LovTap in different light conditions (6h incubation). Measuring RFP output with FACS.
  • Testing 312 nm UV-B response of UVR8 system on agar plates with different UV-B light regimes, distances from UV-B source and exposure times.
  • Isolation of cph8-sequence from pJT122 using PCR and cloning into pSB4A5

Week 13 (3.9-9.9)

  • Testing of LovTap in different light conditions (12h incubation). Measuring RFP output with FACS.
  • Testing UVR8 constructs repression dependency on induction (IPTG concentration) and UVR8 cell toxicity.

Week 14 (10.9-16.9)

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20120925-iGem-5863.jpg
  • 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 decoder. Ordering of Primers and inoculation of necessary parts.
  • Designing primers for Gibson ligation
  • Cloning pabA into vector containint pabB
  • Testing UVR8 systems in 25 and 50 mL LB medium in shaking flasks and characterization of UVR8 fusions in an SDS-acrylamide gels.
  • TetR-DBD-UVR8 and TetR-DBD-GGS-UVR8 were
  • Ordered primers for:
    • tetR-DBD-dUVR8 his tagged version
    • UVR8 mutagenesis
  • R146A and R286A mutations (single mutant has a destabilized dimer; double mutant cannot form dimmers)
  • Illegal PstI sites in UVR8 sequence.
  • Mutagenesis of R146A in tetR-DBD-UVR8 construct.
  • Cloning of RBS-ho1 with RBS-pcyA (BBa_K909000)
  • Site-directed-mutagenesis of cph8 to remove illegal PstI-site (K909002)

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
  • Testing of UVR8 system repression dependency on bacterial strain (Top10 and JM101)
  • Cloning of his-tagged versions of tetR-DBD-UVR8 and its R146A mutants.
  • Cloning of const. Promoter (BBa_J23108) to BBa_K909000 (BBA_K909001)
  • Cloning of terminator (B0017) to RBS-LacZ (BBa_I732017) (BBa_K909006)
  • Cloning of RBS (B0034) to cph8 (K909003)

Week 16 (24.09.-30.09.)

  • Interview with National Council Mr. Markus Ritter
  • finishing the wiki
Coomassie spill.jpg

Week 17 (01.10.-07.10.)

  • iGEM regional jamboree in Amsterdam
  • SDS-PAGE of pabA/B/C overexpressing strains
  • Western Blot of UVR8-TetR
  • Cloning hybrid promoters to eCFP (E0420), K9090005, mCherry (I01050)

Week 18 (08.10.-14.10.)

  • Analysis of dimer properties of UVR8 via Native gel
  • Detection of PABA - HPLC-
  • Analysis of possible inclusion body formation of UVR8-TetR fusion

Week 19 (15.10.-21.10.)

  • IPTG titration - Analysis of possible inclusion body formation of UVR8-TetR fusion
  • Detection of PABA -HPLC-
  • Transformation of low copy vectors from Team Uppsala iGEM 2012
  • Transformation of Chromoproteins from Team Uppsala iGEM 2012
  • Cloning of UVR-TetR fusions in a low copy vector
  • UVR8-TetR R146A R286A mutagenesis
  • Assembly of ptetci mCherry, placci mCherry (Decoder part 1)
  • Cotransformation of p SEVA and Decoder part1

Week 20 (22.10.-28.10.)

  • Transformation of the plasmid construct for PABA overproduction into a chorismate overproducing strain
  • Detection of PABA in new strain - HPLC-
  • FACS of Decoder part1
  • Testing of non-dimerizing UVR8-TetRDBD R146A R286A mutant
  • Purification and in vitro testing of UVR8-TetR his tagged protein
  • Assembly of whole decoder & cotransformation with pSEVA derived plasimd containing LacI and TetR genes
  • Parts preparation and submission
  • finishing the wiki again

Week 21 (29.10.-05.11.)

  • iGEM World Championship in Boston


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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