20.109(F08): T/R Team Purple's Research Proposal

Proposal

We propose to create a bonsai nightlight by genetically altering a bonsai plant to introduce three functions:

• luminescence of floral parts (probably petals if not the whole flower as petals are the prominent part of the flower)
• regulation of luminescence by day/night cycles
• switch (possibility to "turn off" the luminescence within a half-an-hour time period)

Stuff to Consider

• Species of bonsai
• Gene expression in flowers
  • emphasize proteins expressed ONLY in flowers (probably use petals because theyre the prominent part?...)
• Gene/protein used for glowing
  • Luciferase
  • Luciferin substrate
  • fusion effects?
• Day/night cycles - luminescence only at night
  • Looking at time-of-the-day-sensitive plants and genes responsible for sensitivity to sunlight
  • How to link luciferase expression with genes associated with day/night cycles
  • Perhaps use genes associated with time-sensitive proteins (is there such thing?) instead of light sensitivity to avoid the weird "feedback loop problem"
• Possibility to turn luminescence "off" (by spraying or watering, for example)
  • Compound that catalyzes degradation of luciferase
  • Receptors that would allow the compound into the cell
    • modify receptors already present
    • introduce receptors from another species/specifically engineered receptors
    • append ligands to luciferase degraders
• Side effects
  • Will light from glowing flowers mess up natural day/night cycle of bonsai?
  • Will it mess up year-long cycles?
  • Will life expectancy of bonsai decrease?
  • Harmful to people? (It shouldn't be...)
  • Actual trials with the genetically engineered plant will answer the above questions (except for harmful to people - probably do animal trials?)
  • Additional requirements for plant upkeep? (because it might spend a lot of energy producing the glow) - trials will show if more ATP is needed, in which case, more genetic alteration is needed

Research

• Luciferase Expression
  • Firefly Luciferase Gene: Structure and Expression in Mammalian Cells
    • http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=365129
    • Luciferase cDNA
  • Transient and Stable Expression of the Firefly Luciferase Gene in Plant Cells and Transgenic Plants
    • http://www.jstor.org/stable/pdfplus/1698089.pdf
    • pretty much says luciferase works in plants - has that glowing picture of a plant that we can use in presentation
  • What has been done with luciferin and GFP (along with controversies)
    • http://www.bio.davidson.edu/people/kabernd/seminar/2002/edible/pt.htm
    • First luminescent plant - orchid (needs addition of luciferin substrate)

• Luciferin
  • Tracer studies on dinoflagellate luciferin with [15N]-glycine and [15N]-l-glutamic acid in the dinoflagellate Pyrocystis lunula
    • http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6THS-47T7SX0-3M&_user=501045&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000022659&_version=1&_urlVersion=0&_userid=501045&md5=b0bbe109e31508e26827a9e054e655c6
    • Luciferin biosynthesis pathway (or rather first draft of it)

• Finding Genes for Petal Pigmentation
  • Control of Pigment Biosynthesis Genes during Petal Development
    • http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=160358&blobtype=pdf
    • an explanation on the process of pigment synthesis in petals of plants, can look through references of this paper to find more potentially useful articles?
  • Pigment Composition and Characterazation of Anthocyanin Synthese Gene in Dianthus
    • http://sciencelinks.jp/j-east/article/200317/000020031703A0537067.php
    • description of specific genes relating to pigmentation of Dianthus flowers (major pigment cyanidin)

• Switch: Luciferase inhibitor
  • Kinetics of inhibition of firefly luciferase by oxyluciferin and dehydroluciferyl-adenylate
    • http://www.rsc.org/publishing/journals/PP/article.asp?doi=b809935a
    • Byproducts of luciferase-luciferin luminescence inhibit luciferase activity (in vitro only)
  • False Positives in a Reporter Gene Assay: Identification and Synthesis of Substituted N-Pyridin-2-ylbenzamides as Competitive Inhibitors of Firefly Luciferase
    • http://pubs.acs.org/cgi-bin/doilookup/?10.1021/jm8004509
    • luciferase inhibitors (maybe better internalization possible?..)

• Cryptochrome (a possible gene we could use associated with light sensitivity / day-night cycles
  • CIRCADIAN RHYTHMS: Clock Photoreceptor Shared By Plants and Animals
    • http://www.sciencemag.org/cgi/content/full/282/5394/1628?ck=nck
    • look for the gene for cryptochrome or a homolog of it in bonsai, and try linking it with the gene for luciferase
    • may not work due to sensitivity to light - trials will show

Proposed (General) Plan of Action

• insert genes involved in luciferin biosynthetic pathway in bonsai DNA
• fuse one gene involved in lucifering biosynthetic pathway to a gene expressed only in floral parts in order to constitutively synthesize luciferin in flower region
• insert luciferase gene into bonsai DNA and link it to a gene expressed at night (find a gene associated with day/night cycles)
  • that way, light will only be produced at night, when luciferin and luciferase are both produced in plant flower cells
• clone gene coding for a receptor responsible for transport of the luciferase inhibitor inside the cell
• the spray will place luciferase inhibitor on the cell surface allowing for internalization through the receptor and inactivation of luciferase

Future Improvements to the Project

• different colors
• reducing costs to make plant cheaper for public
  • cheaper methods of producing both plant and spray
  • perhaps more research to make this trait heritable through seeds