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=
 * All about Luciferin
 * Imaging of light emission from the expression of luciferases in living cells and organisms: a review
 * http://www3.interscience.wiley.com/cgi-bin/fulltext/89014035/PDFSTART
 * General info to put on background info slides


 * 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
 * http://www.ncbi.nlm.nih.gov/sites/entrez
 * 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
 * link expression of luciferin 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