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Team:ETH Zurich/Notebook
From 2012.igem.org
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===Week 2 (18.6-24.6)=== | ===Week 2 (18.6-24.6)=== | ||
| + | [[Image:20120925-iGem-5889.jpg|frameless|right|300px]] | ||
Brainstorming | Brainstorming | ||
| - | |||
Possible candidate projects: | Possible candidate projects: | ||
* Bacteria sensing a small molecule (Vanillin) and navigates a robot towards the source / Chemotaxis | * Bacteria sensing a small molecule (Vanillin) and navigates a robot towards the source / Chemotaxis | ||
| Line 26: | Line 26: | ||
* Temperature sensing yeast used in beer brewing | * Temperature sensing yeast used in beer brewing | ||
| - | + | [[Image:IMG_1519.jpg|frameless|right|300px]] | |
===Week 3 (25.6-1.7)=== | ===Week 3 (25.6-1.7)=== | ||
* Literature research on our different project ideas. | * Literature research on our different project ideas. | ||
| Line 39: | Line 39: | ||
** Truncated version of UVR8 fusion with tetR-DBD (tetR-DBD-dUVR8) | ** Truncated version of UVR8 fusion with tetR-DBD (tetR-DBD-dUVR8) | ||
** tetR-DBD-UVR8 fusion extended with [GGS]2 linker (tetR-DBD-GGS-UVR8) | ** 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)=== | ===Week 6 (16.7-22.7)=== | ||
* Cloning of YcgZ promoter (K238013) and GFP (E0840) into pSB1AK3 | * Cloning of YcgZ promoter (K238013) and GFP (E0840) into pSB1AK3 | ||
| Line 48: | Line 47: | ||
* Ordered primers for full length tetR and truncated version (tetR-DBD) protein cloning | * 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. | * 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)=== | ===Week 7 (23.7-29.7)=== | ||
| Line 55: | Line 53: | ||
* Cloning of tetR and tetR-DBD into pSEVA183-lacI and tested weather tetR-DBD is unable to repress GFP production from pSB1C3 plasmid. | * 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. | * Ordered primers for UVR8 fusions. | ||
| - | + | [[Image:20120924-iGem-5777.jpg|frameless|right|300px]] | |
| - | + | ||
===Week 8 (30.7-5.8)=== | ===Week 8 (30.7-5.8)=== | ||
* Cloning of LacZ downstream to the YcgZ promoter into pSB1C3, tranformation, colony PCR, sequencing | * Cloning of LacZ downstream to the YcgZ promoter into pSB1C3, tranformation, colony PCR, sequencing | ||
| Line 63: | Line 60: | ||
* Recloning of GFP reporter system (BBa_I13522) into pSB4K5 plasmid. | * 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. | * 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)=== | ===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 | * Cloning of RBS B0034 upstream to YcgE & YcgF, transformation, colony PCR, sequencing | ||
* Designing YcgZ promoter with multiple operator sites | * Designing YcgZ promoter with multiple operator sites | ||
* Test construct K23013-LacZ with the Miller assay | * 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 | * 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 | * 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)=== | ===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 | * Cloning LovTAP reporter (K322999) with a constitutive promoter (J23108) into vector pSB1C3 | ||
* Testing of LovTap construct (Tecan plate reader) | * 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)=== | ===Week 12 (27.8-2.9)=== | ||
* Testing of LovTap in different light conditions (6h incubation). Measuring RFP output with FACS. | * 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. | * 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)=== | ===Week 13 (3.9-9.9)=== | ||
* Testing of LovTap in different light conditions (12h incubation). Measuring RFP output with FACS. | * 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. | * Testing UVR8 constructs repression dependency on induction (IPTG concentration) and UVR8 cell toxicity. | ||
| - | |||
===Week 14 (10.9-16.9)=== | ===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. | * UVR8 System : Testing of different exposure invervals and UV intensities. | ||
* Changing the read-out of the UVR8 system from GFP to Galactosidase | * 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 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 | * Cloning of PabA and PabB in one verctor | ||
| - | * Exact planning of the | + | * Exact planning of the decoder. Ordering of Primers and inoculation of necessary parts. |
* Designing primers for Gibson ligation | * Designing primers for Gibson ligation | ||
* Cloning pabA into vector containint pabB | * Cloning pabA into vector containint pabB | ||
| Line 102: | Line 107: | ||
* Illegal PstI sites in UVR8 sequence. | * Illegal PstI sites in UVR8 sequence. | ||
* Mutagenesis of R146A in tetR-DBD-UVR8 construct. | * 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)=== | ===Week 15 (17.9-23.9)=== | ||
| Line 109: | Line 116: | ||
* Testing of UVR8 system repression dependency on bacterial strain (Top10 and JM101) | * 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 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}} | {{:Team:ETH_Zurich/Templates/Footer}} | ||
Latest revision as of 23:33, 26 October 2012
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)
- 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)
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.
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)
- 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)
- 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)
- 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
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
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- 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.
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- Lamparter, T., Michael, N., Mittmann, F., & Esteban, B. (2002). Phytochrome from Agrobacterium tumefaciens has unusual spectral properties and reveals an N-terminal chromophore attachment site. Proceedings of the National Academy of Sciences of the United States of America, 99(18), 11628–33.
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- Rizzini, L., Favory, J.-J., Cloix, C., Faggionato, D., O’Hara, A., Kaiserli, E., Baumeister, R., et al. (2011). Perception of UV-B by the Arabidopsis UVR8 protein. Science (New York, N.Y.), 332(6025), 103–6.
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- Sinha RP, Häder DP. UV-induced DNA damage and repair: a review. Photochem Photobiol Sci. (2002). 1(4):225-36
- Sambandan DR, Ratner D. (2011). Sunscreens: an overview and update. J Am Acad Dermatol. 2011 Apr;64(4):748-58.
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