IGEM:Harvard/2010/Brainstorming/food

Strains, Plasmids, and Trasformation

references

1. Broothearts et. al. Gene transfer to plants by diverse species of bacteria. Nature, 2004.

2. Galvin [1] Agrobacterium-Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool

Agrobacterium infection diagram

3. Antunes, et. al. [2] A synthetic de-greening gene circuit provides a reporting system that is remotely detectable and has a re-set capacity

protocols

• Book: Agrobacterium Protocols

Nathan Lord (Harvard) and Mike Thomashow (Michigan State) will be giving us detailed protocols for transformation of Arabidopsis plants with agrobacterium. Some things we've learned so far from these experts:

• directed gene integration in plants is very tricky --- best to do random
  
• flower-dipping is the best way to transform arabidopsis
  *best to put all genes on one plasmid, though selecting for two different plasmids by two agro strains might be possible, too
  
• from flower-dipping, can take 2 months to get primary transformants
  
• kanamycin and basta are common markers for positive transformants
  
• we will need to buy "surfactin" and "gambord" for the flower dipping procedure
  

RNAi

1. Chuang CF and Meyerowitz EM. Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc Natl Acad Sci U S A. 2000 Apr 25;97(9):4985-90. DOI:10.1073/pnas.060034297 |

Markers and Genetic Fences

Dual selection system ideas

• use selectable markers such as URA3 to control whether an integration plasmid is replicated or not
  
• cells with the URA3 gene can grow on media without uracil, but get killed by 5-Fluoroorotic acid (5-FOA)
  
• cells without the URA3 gene die on media without uracil, but can survive exposure to 5-FOA
  

De-Greening

• this paper has some parts that could be useful as kill-switches
• A synthetic de-greening gene circuit provides a reporting system that is remotely detectable and has a re-set capacity.
• steroid-inducible chlorophyll knockdown

Downstream effectors

allergens

Peanut and Tomatoes review

1. Singh MB and Bhalla PL. Genetic engineering for removing food allergens from plants. Trends Plant Sci. 2008 Jun;13(6):257-60. DOI:10.1016/j.tplants.2008.04.004 |

Peanut profilins: Ara h knock-down

1. Dodo H, Konan K, and Viquez O. A genetic engineering strategy to eliminate peanut allergy. Curr Allergy Asthma Rep. 2005 Jan;5(1):67-73. DOI:10.1007/s11882-005-0058-0 |

Tomato profilins: Lyc e knock-down

1. Design of tomato fruits with reduced allergenicity by dsRNAi-mediated inhibition of ns-LTP (Lyc e 3) expression. Le LQ, Lorenz Y, Scheurer S, Fötisch K, Enrique E, Bartra J, Biemelt S, Vieths S, Sonnewald U. Plant Biotechnol J. 2006 Mar;4(2):231-42. pmid:17177799
2. Reduced allergenicity of tomato fruits harvested from Lyc e 1-silenced transgenic tomato plants. Le LQ, Mahler V, Lorenz Y, Scheurer S, Biemelt S, Vieths S, Sonnewald U. J Allergy Clin Immunol. 2006 Nov;118(5):1176-83. Epub 2006 Sep 8. pmid:17088146

Orange profilin: Cit s 2 (characterized, but not knocked down yet)

1. Isolation, cloning and allergenic reactivity of natural profilin Cit s 2, a major orange allergen. López-Torrejón G, Ibáñez MD, Ahrazem O, Sánchez-Monge R, Sastre J, Lombardero M, Barber D, Salcedo G. Allergy. 2005 Nov;60(11):1424-9. pmid:16197476

flavors

• Growth dependent banana odor generator, MIT iGEM 2006

• [3] Tieman et al. Tomato aromatic amino acid decarboxylases participate in synthesis of the flavor volatiles 2-phenylethanol and 2-phenylacetaldehyde

• (Phenylalanine Ammonia-lyase) catalyzes the conversion of phenylalanine to cinnamic acid, MIT iGEM 2006

• phase dependent wintergreen odor generator, MIT iGEM 2006

VANILLA: http://www.3dchem.com/moremolecules.asp?ID=307&othername=Vanilla#

1. Sinha AK, Sharma UK, and Sharma N. A comprehensive review on vanilla flavor: extraction, isolation and quantification of vanillin and others constituents. Int J Food Sci Nutr. 2008 Jun;59(4):299-326. DOI:10.1080/09687630701539350 |

De novo biosynthesis of vanillin in fission yeast (Schizosaccharomyces pombe) and baker's yeast (Saccharomyces cerevisiae). http://www.ncbi.nlm.nih.gov.ezp-prod1.hul.harvard.edu/pubmed/19286778

miraculin

• Functional expression of the taste-modifying protein, miraculin, in transgenic lettuce

• Functional Expression of Miraculin, a Taste-Modifying Protein in Escherichia Coli

1. Theerasilp S and Kurihara Y. Complete purification and characterization of the taste-modifying protein, miraculin, from miracle fruit. J Biol Chem. 1988 Aug 15;263(23):11536-9.

spices

Capsaicin: http://en.wikipedia.org/wiki/Capsaicin

1. Stewart C Jr, Kang BC, Liu K, Mazourek M, Moore SL, Yoo EY, Kim BD, Paran I, and Jahn MM. The Pun1 gene for pungency in pepper encodes a putative acyltransferase. Plant J. 2005 Jun;42(5):675-88. DOI:10.1111/j.1365-313X.2005.02410.x |

2. Prasad BC, Kumar V, Gururaj HB, Parimalan R, Giridhar P, and Ravishankar GA. Characterization of capsaicin synthase and identification of its gene (csy1) for pungency factor capsaicin in pepper (Capsicum sp.). Proc Natl Acad Sci U S A. 2006 Sep 5;103(36):13315-20. DOI:10.1073/pnas.0605805103 |

From wikipedia:

• TRPV1, the only known receptor (a transient receptor potential channel) for capsaicin.
• Piperine, the active piquant chemical in black pepper
• Allyl isothiocyanate, the active piquant chemical in mustard, radishes, horseradish, and wasabi
• Allicin, the active piquant flavor chemical in uncooked garlic and onions (see those articles for discussion of other chemicals in them relating to pungency, and eye irritation)
• Naga Jolokia pepper, the world's most capsaicin-rich fruit
• Discovery and development of TRPV1 antagonists
• Capsinoids are similar in structure to capsaicin, but lack the extreme pungency.

others

1. Raksakulthai R and Haard NF. Exopeptidases and their application to reduce bitterness in food: a review. Crit Rev Food Sci Nutr. 2003;43(4):401-45. DOI:10.1080/10408690390826572 |

http://en.wikipedia.org/wiki/Limonene

caffeine etc.

1. Ashihara H, Sano H, and Crozier A. Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. Phytochemistry. 2008 Feb;69(4):841-56. DOI:10.1016/j.phytochem.2007.10.029 |

2. http://www.plantcell.org/cgi/content/abstract/6/5/723

Questions

can we eat it for the iGEM project (human testing)? Can we hand it out to be eaten (FDA)? Patent/copyright issues? Can we have actual tranformed plants ready by the jamboree