Team:ETH Zurich/Notebook

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== Notebook ==  
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This is a template page. READ THESE INSTRUCTIONS.
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You are provided with this team page template with which to start the iGEM season.  You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki.  You can find some examples <a href="https://2008.igem.org/Help:Template/Examples">HERE</a>.
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===Week 1 (11.6-17.6)===
 +
* First meeting
 +
* Brainstorming
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{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"
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===Week 2 (18.6-24.6)===
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!align="center"|[[Team:ETH_Zurich|Home]]
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[[Image:20120925-iGem-5889.jpg|frameless|right|300px]]
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!align="center"|[[Team:ETH_Zurich/Team|Team]]
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Brainstorming
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!align="center"|[https://igem.org/Team.cgi?year=2012&team_name=ETH_Zurich Official Team Profile]
+
Possible candidate projects:  
-
!align="center"|[[Team:ETH_Zurich/Project|Project]]
+
* Bacteria sensing a small molecule (Vanillin) and navigates a robot towards the source  / Chemotaxis
-
!align="center"|[[Team:ETH_Zurich/Parts|Parts Submitted to the Registry]]
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!align="center"|[[Team:ETH_Zurich/Modeling|Modeling]]
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!align="center"|[[Team:ETH_Zurich/Notebook|Notebook]]
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!align="center"|[[Team:ETH_Zurich/Safety|Safety]]
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!align="center"|[[Team:ETH_Zurich/Attributions|Attributions]]
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|}
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 +
* Game Theory: Bacteria playing the Prisoners Dilemma Game
 +
* Sunburn warning system
-
You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summerIt is a very useful organizational tool as well.
+
* 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
 +
[[Image:IMG_1519.jpg|frameless|right|300px]]
 +
===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)
 +
[[Image:20120925-iGem-5876.jpg|frameless|right|300px]]
 +
===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.
 +
[[Image:20120924-iGem-5777.jpg|frameless|right|300px]]
 +
===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)===
 +
[[Image:IMG_1531.jpg|frameless|right|300px]]
 +
 
 +
* 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)===
 +
[[Image:IMG_1520.jpg|frameless|right|300px]]
 +
* 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)===
 +
 
 +
[[Image:20120924-iGem-5772.jpg|frameless|right|300px]]
 +
[[Image:20120925-iGem-5863.jpg|frameless|right|300px]]
 +
* 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
 +
[[Image:coomassie_spill.jpg|frameless|right|300px]]
 +
=== 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 [https://2012.igem.org/Team:Uppsala_University Team Uppsala iGEM 2012]
 +
* Transformation of Chromoproteins from [https://2012.igem.org/Team:Uppsala_University 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-
 +
* Cotransformation of UVR8-tetR-DBD in pSEVA with reporter in new strain (ROSETTA2)
 +
* 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 '''
 +
 
 +
{{:Team:ETH_Zurich/Templates/Footer}}

Latest revision as of 23:33, 26 October 2012

Eth ecolipseeth logo.png
Eth igem logo.png

Contents

Notebook

Week 1 (11.6-17.6)

  • First meeting
  • Brainstorming

Week 2 (18.6-24.6)

20120925-iGem-5889.jpg

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)

20120924-iGem-5772.jpg
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-
  • Cotransformation of UVR8-tetR-DBD in pSEVA with reporter in new strain (ROSETTA2)
  • 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

  • Brown, B. a, Headland, L. R., & Jenkins, G. I. (2009). UV-B action spectrum for UVR8-mediated HY5 transcript accumulation in Arabidopsis. Photochemistry and photobiology, 85(5), 1147–55.
  • Christie, J. M., Salomon, M., Nozue, K., Wada, M., & Briggs, W. R. (1999): LOV (light, oxygen, or voltage) domains of the blue-light photoreceptor phototropin (nph1): binding sites for the chromophore flavin mononucleotide. Proceedings of the National Academy of Sciences of the United States of America, 96(15), 8779–83.
  • 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.
  • Cloix, C., & Jenkins, G. I. (2008). Interaction of the Arabidopsis UV-B-specific signaling component UVR8 with chromatin. Molecular plant, 1(1), 118–28.
  • Cox, R. S., Surette, M. G., & Elowitz, M. B. (2007). Programming gene expression with combinatorial promoters. Molecular systems biology, 3(145), 145. doi:10.1038/msb4100187
  • Drepper, T., Eggert, T., Circolone, F., Heck, A., Krauss, U., Guterl, J.-K., Wendorff, M., et al. (2007). Reporter proteins for in vivo fluorescence without oxygen. Nature biotechnology, 25(4), 443–5
  • Drepper, T., Krauss, U., & Berstenhorst, S. M. zu. (2011). Lights on and action! Controlling microbial gene expression by light. Applied microbiology, 23–40.
  • EuropeanCommission (2006). SCIENTIFIC COMMITTEE ON CONSUMER PRODUCTS SCCP Opinion on Biological effects of ultraviolet radiation relevant to health with particular reference to sunbeds for cosmetic purposes.
  • Elvidge, C. D., Keith, D. M., Tuttle, B. T., & Baugh, K. E. (2010). Spectral identification of lighting type and character. Sensors (Basel, Switzerland), 10(4), 3961–88.
  • GarciaOjalvo, J., Elowitz, M. B., & Strogatz, S. H. (2004). Modeling a synthetic multicellular clock: repressilators coupled by quorum sensing. Proceedings of the National Academy of Sciences of the United States of America, 101(30), 10955–60.
  • Gao Q, Garcia-Pichel F. (2011). Microbial ultraviolet sunscreens. Nat Rev Microbiol. 9(11):791-802.
  • Goosen N, Moolenaar GF. (2008) Repair of UV damage in bacteria. DNA Repair (Amst).7(3):353-79.
  • Heijde, M., & Ulm, R. (2012). UV-B photoreceptor-mediated signalling in plants. Trends in plant science, 17(4), 230–7.
  • Hirose, Y., Narikawa, R., Katayama, M., & Ikeuchi, M. (2010). Cyanobacteriochrome CcaS regulates phycoerythrin accumulation in Nostoc punctiforme, a group II chromatic adapter. Proceedings of the National Academy of Sciences of the United States of America, 107(19), 8854–9.
  • Hirose, Y., Shimada, T., Narikawa, R., Katayama, M., & Ikeuchi, M. (2008). Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein. Proceedings of the National Academy of Sciences of the United States of America, 105(28), 9528–33.
  • Kast, Asif-Ullah & Hilvert (1996) Tetrahedron Lett. 37, 2691 - 2694., Kast, Asif-Ullah, Jiang & Hilvert (1996) Proc. Natl. Acad. Sci. USA 93, 5043 - 5048
  • Kiefer, J., Ebel, N., Schlücker, E., & Leipertz, A. (2010). Characterization of Escherichia coli suspensions using UV/Vis/NIR absorption spectroscopy. Analytical Methods, 9660. doi:10.1039/b9ay00185a
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