20.109(F09):HC^2

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Goal

To engineer a plant to emit luciferase in applications to light up street paths in the developing world.

-2CS shown to work in Arabidopsis thaliana, ecotype Columbia (Col-0) -Cross check genetic similarity between tobacco plants and A. thaliana -

Notes

[Word outline] [PPT outline]

Engineering A. thaliana to express 2CS

Procedure:

  • Plants were transformed with Agrobacterium GV3011

Notes from December 3, 2009

From personal communication with Angie and Natalie how bright luciferin grind, measure leaf brightness photodetector develop on film CDD count measure emission of light per lefa basis, scale with surface area digitally capture - optical output of LED - ultimately matters how we see it measure output of light parameters important to human eye throw off energy balance of plant?

Tasks list

Hattie:

  • light emission measurements, power. optical output.
  • luciferin production

Helen:

  • understand his paper better
  • energy balance of plants - luciferin

Both:

  • Levskaya paper- understand methods to make chimeric photoreceptor

phytochrome B - detector for photosynthesis: detects peak wavelength of sunlight


Genetics:

  • 2CS shown to work in plants, at least specific to (check what kind of plants)
  • Cross check genetic similarity between tobacco plants and plant used

[Synthetic Biology] Can conserved-signaling components from bacteria and plants be adapted to provide key components of synthetic signal transduction pathways in mammalian cells?

  • Antunes, Morey, Tewari-Singh, Bowen, Smith, Webb, Hellinga, and Medford. "Engineering key components in a synthetic eukaryotic signal transduction pathway." Molecular Systems Biology 2009; 5:270.
This experiment tested whether conserved modular domains from highly evolved bacterial systems could retain functionality in a eukaryotic system. The requirement for nuclear translocation of a phosphorylated carrier protein is a key difference between bacteria and plant histidine kinase (HK) signal transduction systems. The researchers discovered that PhoB-GFP and OmpR-GFP can translocate to the plant cell nucleus in response to a cytokinininduced HK signal.
  • Weber W, Schoenmakers R, Keller B, Gitzinger M, Grau T, Daoud-El Baba M, Sander P, Fussenegger M: A synthetic mammalian gene circuit reveals antituberculosis compounds. Proc Natl Acad Sci U S A 2008, 105:9994-9998.
A synthetic Mycobacterium tuberculosis circuit implanted into mammalian cells was successfully used to discover novel antituberculosis compounds that are able to switch off the intrinsic etionamide resistance of this pathogen.
  • Weber W, Luzi S, Karlsson M, Sanchez-Bustamante CD, Frey U, Hierlemann A, Fussenegger M: A synthetic mammalian electro-genetic transcription circuit. Nucleic Acids Res 2009, 37:e33.
Electricity-inducible gene circuit enabling voltage-dependent transcription fine-tuning in mammalian cells.
  • Kramer BP, Fischer M, Fussenegger M: Semi-synthetic mammalian gene regulatory networks. Metab Eng 2005, 7:241-250.
Host-interfaced ‘semi-synthetic’ regulatory cascade that plugs into the mammalian oxygen response system and accepts native hypoxiainduced factor 1a signal input as well as synthetic signal input from administered small-molecule drugs.
  • Stricker J, Cookson S, Bennett MR, Mather WH, Tsimring LS, Hasty J: A fast, robust and tunable synthetic gene oscillator. Nature 2008, 456:516-519.
Latest generation prokaryotic oscillator with small molecule responsive tunability of the transgene expression period.
  • Weber W, Daoud-El Baba M, Fussenegger M: Synthetic ecosystems based on airborne inter- and intrakingdom communication. Proc Natl Acad Sci U S A 2007, 104:10435-10440.
Design and characterization of gene networks that control transcription signals within and across different populations of the same or different species. Highlights: synthetic hormone systems operating in mice and predator–prey ecosystems with oscillating population dynamics.
Why we had to use Cph1 instead of natural plant phytochrome
"The cyanobacterial phytochrome Cph1 is a light-regulated His protein kinase and mediates phosphotransfer to the response regulator Rcp1 (Yeh et al., 1997). This discovery and sequence similarity of plant phytochromes to bacterial His protein kinases suggested that higher plant phytochromes might be His protein kinases and that light signaling in higher plants could use a light–regulated phosphotransfer mechanism. In Arabidopsis, there are five photoreceptor phytochromes: PHYA, PHYB, PHYC, PHYD, and PHYE (Sharrock and Quail, 1989; Clack et al., 1994). These phytochromes have two major structural domains (Fig. 2). The aminoterminal domain has a covalently attached linear tetrapyrrole chromophore for light absorption and photoreversibility. The carboxy terminus consists of two PAS domains and a domain related to the His protein kinase for signal transduction. Plant phytochromes are soluble proteins with structural features similar to those of sensor His protein kinases, with an N-terminal sensor and a C-terminal His protein kinase domain. However, none of the phytochromes contain the five conserved motifs essential for His protein kinase activity (Fig. 4)."
Luciferase is degraded rapidly
In the absence of osmolytes, the remaining activity was about zero after 1000 min at 23 °C, whereas sucrose, glycine and DMSO kept about 70%, 65% and 20% of luciferase original activity after 1000 min, respectively (Fig. 4A). At 37 °C, the results showed that the native luciferase activity rapidly decreased in the absence of osmolytes and was almost zero after 40 min. With adding sucrose or glycine, the luciferase inactivation rate gradually slowed, but the effect of DMSO was negligible. As indicated in Fig. 4B, sucrose and glycine kept 35% and 25% of original activity after 1000 min, respectively, whereas in the presence of DMSO about 95% of the activity was lost within 40 min

Possibilities

  • Design plants to make something: as a sensor, bioreactor....
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