iGEM 2012 — Team ETH_Zurich

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# Team:ETH Zurich/Notebook

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* [Home](/Team%3AETH_Zurich "Team:ETH Zurich")
* [Project](/Team%3AETH_Zurich/Project_overview "Team:ETH Zurich/Project overview")
  + [Overview](/Team%3AETH_Zurich/Project_overview "Team:ETH Zurich/Project overview")
  + [Motivation](/Team%3AETH_Zurich/Motivation "Team:ETH Zurich/Motivation")
  + [Applications](/Team%3AETH_Zurich/Applications "Team:ETH Zurich/Applications")
  + [Lab & Modelling](/Team%3AETH_Zurich/Interplay "Team:ETH Zurich/Interplay")
* [Parts](/Team%3AETH_Zurich/Project "Team:ETH Zurich/Project")
  + [Overview](/Team%3AETH_Zurich/Project "Team:ETH Zurich/Project")
  + [Direct: UVR8-TetRDBD](/Team%3AETH_Zurich/UVR8 "Team:ETH Zurich/UVR8")
  + [Indirect: Decoder](/Team%3AETH_Zurich/Decoder "Team:ETH Zurich/Decoder")
  + [Output: PABA](/Team%3AETH_Zurich/PABA "Team:ETH Zurich/PABA")
  + [Parts](/Team%3AETH_Zurich/Parts "Team:ETH Zurich/Parts")
* [Modeling](/Team%3AETH_Zurich/Modeling "Team:ETH Zurich/Modeling")
  + [Overview](/Team%3AETH_Zurich/Modeling "Team:ETH Zurich/Modeling")
  + [Photoinduction](/Team%3AETH_Zurich/Modeling/Photoinduction "Team:ETH Zurich/Modeling/Photoinduction")
  + [UVR8-TetRDBD Circuit](/Team%3AETH_Zurich/Modeling/UVR8 "Team:ETH Zurich/Modeling/UVR8")
  + [Sun Protection Factor](/Team%3AETH_Zurich/Modeling/SPF_model "Team:ETH Zurich/Modeling/SPF model")
  + [Decoder Circuit: LovTAP/Cph8](/Team%3AETH_Zurich/Modeling/Construct2 "Team:ETH Zurich/Modeling/Construct2")
  + [Parameters](/Team%3AETH_Zurich/Modeling/Parameters "Team:ETH Zurich/Modeling/Parameters")
* **Notebook**
  + **Overview**
  + [Material & Methods](/Team%3AETH_Zurich/MaterialMethods "Team:ETH Zurich/MaterialMethods")
* [Achievements](/Team%3AETH_Zurich/Achievements "Team:ETH Zurich/Achievements")
  + [Medal Achievements](/Team%3AETH_Zurich/Achievements "Team:ETH Zurich/Achievements")
  + [Project Achievements](/Team%3AETH_Zurich/Achievements_overall "Team:ETH Zurich/Achievements overall")
* [Safety](/Team%3AETH_Zurich/Safety "Team:ETH Zurich/Safety")
* [Human practice](/Team%3AETH_Zurich/HumanPractice "Team:ETH Zurich/HumanPractice")
* [Team](/Team%3AETH_Zurich/Team "Team:ETH Zurich/Team")
  + [Team description](/Team%3AETH_Zurich/Team "Team:ETH Zurich/Team")
  + [Attributions & Acknowledgements](/Team%3AETH_Zurich/Attributions "Team:ETH Zurich/Attributions")
  + [Official Team Profile](https://igem.org/Team.cgi?year=2012&team_name=ETH_Zurich)
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|  |
| --- |
| Contents   * [1 Notebook](#Notebook)   + [1.1 Week 1 (11.6-17.6)](#Week_1_.2811.6-17.6.29)   + [1.2 Week 2 (18.6-24.6)](#Week_2_.2818.6-24.6.29)   + [1.3 Week 3 (25.6-1.7)](#Week_3_.2825.6-1.7.29)   + [1.4 Week 4 (2.7-8.7)](#Week_4_.282.7-8.7.29)   + [1.5 Week 5 (9.7-15.7)](#Week_5_.289.7-15.7.29)   + [1.6 Week 6 (16.7-22.7)](#Week_6_.2816.7-22.7.29)   + [1.7 Week 7 (23.7-29.7)](#Week_7_.2823.7-29.7.29)   + [1.8 Week 8 (30.7-5.8)](#Week_8_.2830.7-5.8.29)   + [1.9 Week 9 (6.8-12.8)](#Week_9_.286.8-12.8.29)   + [1.10 Week 10 (13.8-19.8)](#Week_10_.2813.8-19.8.29)   + [1.11 Week 11 (20.8-26.8)](#Week_11_.2820.8-26.8.29)   + [1.12 Week 12 (27.8-2.9)](#Week_12_.2827.8-2.9.29)   + [1.13 Week 13 (3.9-9.9)](#Week_13_.283.9-9.9.29)   + [1.14 Week 14 (10.9-16.9)](#Week_14_.2810.9-16.9.29)   + [1.15 Week 15 (17.9-23.9)](#Week_15_.2817.9-23.9.29)   + [1.16 Week 16 (24.09.-30.09.)](#Week_16_.2824.09.-30.09..29)   + [1.17 Week 17 (01.10.-07.10.)](#Week_17_.2801.10.-07.10..29)   + [1.18 Week 18 (08.10.-14.10.)](#Week_18_.2808.10.-14.10..29)   + [1.19 Week 19 (15.10.-21.10.)](#Week_19_.2815.10.-21.10..29)   + [1.20 Week 20 (22.10.-28.10.)](#Week_20_.2822.10.-28.10..29)   + [1.21 Week 21 (29.10.-05.11.)](#Week_21_.2829.10.-05.11..29)   + [1.22 References](#References) |

## Notebook

### Week 1 (11.6-17.6)

* First meeting
* Brainstorming

### Week 2 (18.6-24.6)

[![20120925-iGem-5889.jpg](/wiki/images/b/bf/20120925-iGem-5889.jpg)](/File%3A20120925-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](/wiki/images/f/fc/IMG_1519.jpg)](/File%3AIMG_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](/wiki/images/8/88/20120925-iGem-5876.jpg)](/File%3A20120925-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](/wiki/images/c/c7/20120924-iGem-5777.jpg)](/File%3A20120924-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](/wiki/images/6/6d/IMG_1531.jpg)](/File%3AIMG_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](/wiki/images/5/5b/IMG_1520.jpg)](/File%3AIMG_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](/wiki/images/7/7a/20120924-iGem-5772.jpg)](/File%3A20120924-iGem-5772.jpg)

[![20120925-iGem-5863.jpg](/wiki/images/2/2a/20120925-iGem-5863.jpg)](/File%3A20120925-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](/wiki/images/5/56/Coomassie_spill.jpg)](/File%3ACoomassie_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](https://2012.igem.org/Team%3AUppsala_University)
* Transformation of Chromoproteins from [Team Uppsala iGEM 2012](https://2012.igem.org/Team%3AUppsala_University)
* 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
* Kinkhabwala, A., & Guet, C. C. (2008). Uncovering cis regulatory codes using synthetic promoter shuffling. PloS one, 3(4), e2030.
* Krebs in Deutschland 2005/2006. Häufigkeiten und Trends. 7. Auflage, 2010, Robert Koch-Institut (Hrsg) und die Gesellschaft der epidemiologischen Krebsregister in Deutschland e. V. (Hrsg). Berlin.
* 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.
* Levskaya, A. et al (2005). Engineering Escherichia coli to see light. Nature, 438(7067), 442.
* Mancinelli, A. (1986). Comparison of spectral properties of phytochromes from different preparations. Plant physiology, 82(4), 956–61.
* Nakasone, Y., Ono, T., Ishii, A., Masuda, S., & Terazima, M. (2007). Transient dimerization and conformational change of a BLUF protein: YcgF. Journal of the American Chemical Society, 129(22), 7028–35.
* Orth, P., & Schnappinger, D. (2000). Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system. Nature structural biology, 215–219.
* Parkin, D.M., et al., Global cancer statistics, 2002. CA: a cancer journal for clinicians, 2005. 55(2): p. 74-108.
* Rajagopal, S., Key, J. M., Purcell, E. B., Boerema, D. J., & Moffat, K. (2004). Purification and initial characterization of a putative blue light-regulated phosphodiesterase from Escherichia coli. Photochemistry and photobiology, 80(3), 542–7.
* 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.
* Roux, B., & Walsh, C. T. (1992). p-aminobenzoate synthesis in Escherichia coli: kinetic and mechanistic characterization of the amidotransferase PabA. Biochemistry, 31(30), 6904–10.
* Strickland, D. (2008). Light-activated DNA binding in a designed allosteric protein. Proceedings of the National Academy of Sciences of the United States of America, 105(31), 10709–10714.
* 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.
* Tabor, J. J., Levskaya, A., & Voigt, C. A. (2011). Multichromatic Control of Gene Expression in Escherichia coli. Journal of Molecular Biology, 405(2), 315–324.
* Thibodeaux, G., & Cowmeadow, R. (2009). A tetracycline repressor-based mammalian two-hybrid system to detect protein–protein interactions in vivo. Analytical biochemistry, 386(1), 129–131.
* Tschowri, N., & Busse, S. (2009). The BLUF-EAL protein YcgF acts as a direct anti-repressor in a blue-light response of Escherichia coli. Genes & development, 522–534.
* Tschowri, N., Lindenberg, S., & Hengge, R. (2012). Molecular function and potential evolution of the biofilm-modulating blue light-signalling pathway of Escherichia coli. Molecular microbiology.
* Tyagi, A. (2009). Photodynamics of a flavin based blue-light regulated phosphodiesterase protein and its photoreceptor BLUF domain.
* Vainio, H. & Bianchini, F. (2001). IARC Handbooks of Cancer Prevention: Volume 5: Sunscreens. Oxford University Press, USA
* Quinlivan, Eoin P & Roje, Sanja & Basset, Gilles & Shachar-Hill, Yair & Gregory, Jesse F & Hanson, Andrew D. (2003). The folate precursor p-aminobenzoate is reversibly converted to its glucose ester in the plant cytosol. The Journal of biological chemistry, 278.
* van Thor, J. J., Borucki, B., Crielaard, W., Otto, H., Lamparter, T., Hughes, J., Hellingwerf, K. J., et al. (2001). Light-induced proton release and proton uptake reactions in the cyanobacterial phytochrome Cph1. Biochemistry, 40(38), 11460–71.
* Wegkamp A, van Oorschot W, de Vos WM, Smid EJ. (2007 )Characterization of the role of para-aminobenzoic acid biosynthesis in folate production by Lactococcus lactis. Appl Environ Microbiol. Apr;73(8):2673-81.

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