IGEM:MIT/2006/System brainstorming

Post your project ideas here.

See the brainstorming of last year's team.

Rough Ideas

1. Living Lamp (RS)
2. Bacterial scents (RS)
3. Diagnostic Bacteria (BC)
4. Analog Clock (AC)
5. Square Bacteria (JK)
6. Maze (JK)
7. Algae (organisms that eat photons) (JK)
8. Mitochondrial Liberation & Synthetic organelle(BC)
9. genome transfer (DE)
10. meso laundry list (DE)
11. rhodopsin (AC)
12. scrapped
13. wood eater (SS)
14. wood-o-genesis(SS)

Enabling Technologies

1. Characterization methods
   • FCS
   • MS2 bound to mRNA
2. Subset of composable promoters and RBSs
3. Plasmid-level parts
   • Single-copy
4. Synthetic Vesicles (Libchaber)
   • Proton pump
5. Synthetic genomes (a la T7)
6. Cool part domestication
   • Rhodopsin
   • Magnetotaxis
   • Competence
   • CC Signaling
   • Chemotaxis
   • Linear induction
7. Organism Domestication
   • meso, square bacteria, fast E. coli
8. Genome Boot-up
9. Assembly
   • in vitro, recombination
10. Phage

Organisms that eat photons

Cyanobacteria

Looks like cyanobacteria are significantly easier to genetically manipulate than algae (thanks to algae having cell walls, among other things). Cyanobacteria have Photosystem I/II and are thought to be the ancestor of chloroplasts (via endocytosis). Not sure why GreenFuel et al don't use them in their reactors, it is possibly because they don't have as high a lipid content as the algae (Dunaliella) that they use currently.

Synechocistis PCC 6806 is a naturally transformable cyanobacterium which will give you colonies on hard agar in 4 days. It is also heterotrophic, so you can grow it on glucose in the dark. (thanks to Peter Weigele for the info)

Another suggestion from Peter: "Something else you might want to consider is hydrogen from a photoheterotroph, such as Rhodopseudomonas palustris. You can get H2 from an acetate feedstock. Acetate is a waste product in many industrial fermentations and is an energy poor carbon source. Rhodo uses light to kick up the electrons to an energy level where they can be used to do work. Some protons get moved around too to make a gradient to drive ATP synthesis. The genome is sequenced and the strain is manipulable"

Rhodopsin

Might be cool to do something with the rhodopsin that is worked on by Ed Delong's group. Rhodopsin is a light-driven proton pump that has been shown to function in E.Coli.

Living lamp

According to this site, most homemade lava lamps are built using a mixture of mineral oil and 70-90% isopropyl alcohol (with possibly some supplemented chemicals to help the lamp work better.) Obviously such a method wouldn't work for us.

However, according the lava lamp patent descriptions, "The clear liquid is roughly 70/30% (by volume) water and a liquid which will raise the coefficient of cubic thermal expansion and encourage the movement. The patent recommends slip agents such as propylene glycol for this. However, glycerol, ethylene glycol, and polyethylene glycol (aka PEG) are also mentioned as being sufficient." This sentence implies that we ought to be able to use something other than alcohol.

The other relevant patent says, "A display device comprising a container having two substances therein, with one of the substances being of a heavier specific gravity and immiscible with the other substance, with the first substance being of such a nature that it is either substantially solid at room temperature or is so viscous at room temperature that neither will emulsify with the other liquid, and when heat is applied to the container, the first substance will become flowable and move about in the other substance.

...The liquid in which the globule is suspended is usually dyed water, but not necessarily so. The other liquid is chosen with very many considerations in mind, including the relative densities of the liquids at the desired operating temperature; the fact that the liquids must be immiscible; the fact that the surface tension must be such that the globule does not adhere to the walls of the container; the relative coefficients of thermal expansion of the liquids; and the shapes that are obtained during operation. A suitable liquid for the globule has been found to comprise mineral oil, paraffin, carbon tetrachloride and a dye or dyes. However, undue shaking or sharp impacts, especially during transport of the display device, can cause total or partial emulsification of the globule." My guess is that most homemade lava lamps are made from an alcohol mixture because it is cheaper and possibly also easier to achieve the lava effect.

Also note that they recommend putting a dimmer switch on the bulb below the lamp to be able to regulate the heat output.

Issues

1. We'd have to do some research to see if media or media supplemented with something would be
   1. nontoxic to cells
   2. have the necessary properties to achieve the lava effect at ~37°C
2. Luminescence requires oxygen so we'd have to oxygenate the contents of the lava lamp which might interfere with the lava effect. [from TK]

References

1. Lava Lamp how-to
2. US Patent #3,570,156
3. US Patent #3,387,396

Bacterial scents

Precursor	Enzyme	Compound	Scent	References
benzoic acid & S-adenosyl-L-methionine (SAM)	S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase (BAMT)	methyl benzoate	pleasant smell	[1]
trans-cinnamic acid & S-adenosyl-L-methionine (SAM)	?	methyl cinnamate	cinnamon?
jasmonic acid & S-adenosyl-L-methionine (SAM)	S-adenosyl-L-methionine:jasmonic acid carboxyl methyltransferase (JMT)	methyl jasmonate	jasmine
salicylic acid (SA) & S-adenosyl-L-methionine (SAM)	S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase (SAMT)	methyl salicylate	wintergreen	[2, 3, 4]

Expert advice

From Natalia Dudareva, Purdue University:

• thinks that you can smell wintergreen from E. coli cultures expressing SAMT with salicylic acid in the media

From Eran Pichersky, University of Michigan:

• E. coli cultures expressing SAMT with salicylic acid in the media will have a detectable wintergreen smell
• eliminate indole pathway (responsible for bad E. coli smell) to strengthen the scent.
• have shown production of several scent compounds in E. coli

SAMT

• C. breweri
  • DNA and protein sequence known
  • Expressed in E. coli
  • Methyl salicylate has been extracted from spent medium of E. coli cells when medium was supplemented with salicylic acid
  • Genbank AF133053
  • also can use benzoic acid as a substrate but with lower efficiency
  • crystal structure available
• A. majus (Snapdragon)
  • DNA and protein sequence known
  • Expressed in E. coli
  • Methyl salicylate has been extracted from spent medium of E. coli cells when medium was supplemented with salicylic acid
  • also can use benzoic acid as a substrate but with lower efficiency
  • Methyl benzoate has been extracted from spent medium of E. coli cells when medium was supplemented with benzoic acid
• S. floribunda
  • Genbank AJ308570
• A belladonna
  • Genbank AB049752

JMT

• A. thaliana AY008434

BAMT

• Snapdragon AF198492

References

1. Pott MB, Hippauf F, Saschenbrecker S, Chen F, Ross J, Kiefer I, Slusarenko A, Noel JP, Pichersky E, Effmert U, and Piechulla B. Biochemical and structural characterization of benzenoid carboxyl methyltransferases involved in floral scent production in Stephanotis floribunda and Nicotiana suaveolens. Plant Physiol. 2004 Aug;135(4):1946-55. DOI:10.1104/pp.104.041806 |
2. Ross JR, Nam KH, D'Auria JC, and Pichersky E. S-Adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, an enzyme involved in floral scent production and plant defense, represents a new class of plant methyltransferases. Arch Biochem Biophys. 1999 Jul 1;367(1):9-16. DOI:10.1006/abbi.1999.1255 |
3. Negre F, Kolosova N, Knoll J, Kish CM, and Dudareva N. Novel S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, an enzyme responsible for biosynthesis of methyl salicylate and methyl benzoate, is not involved in floral scent production in snapdragon flowers. Arch Biochem Biophys. 2002 Oct 15;406(2):261-70. DOI:10.1016/s0003-9861(02)00458-7 |
4. Zubieta C, Ross JR, Koscheski P, Yang Y, Pichersky E, and Noel JP. Structural basis for substrate recognition in the salicylic acid carboxyl methyltransferase family. Plant Cell. 2003 Aug;15(8):1704-16. DOI:10.1105/tpc.014548 |

Terpenes and terpenoids

• Terpenes are hydrocarbons: combinations of several isoprenes. (Sometimes encompasses terpenoids.)
• Terpernoids are modified terpenes with methyl groups added/removed or oxygens added
• From Wikipedia: "Terpenoids contribute to the scent of eucalyptus, the flavors of cinnamon, cloves and ginger and the color of yellow flowers. Well-known terpenoids include citral, menthol, camphor and the cannabinoids found in the Cannabis plant."

E. coli has the Δ³-isopentenyl-pyrophosphate pathway, and the enzymes to produce geranyl-PP. This pathway is less effective than the mevalonate pathway, but this has been cloned into an E. coli strain by Keastling's group. Many scented compounds can be made from isopentenyl-PP and geranyl-PP with one or two enzymes, including lemon, orange, pine, etc. See Ecocyc for the pathways (type IPP as a compound and look at the synthetic and reactant pathways that link to it).

Terpenes are also the precursor to rubber and many of the resins and gums.

Indole elimination

Indole is the precursor to and degradation product of tryptophan. We could knock out the relevant two enzymes and supply tryptophan exogenously. Also, we could supply tryptophan exogenously and see if that is sufficient to inhibit indole formation via feedback inhibition in a "normal" strain. [from TK]

Diagnostic bacteria

I'd like to be able to add a small number of diagnostic bacteria into a larger culture to detect the presence of cells containing engineered devices in the culture. Presumably there would be BB DNA floating around from lysed bacteria (does it get cut up?). The diagnostic bacteria would need to responsd to BB DNA by glowing green or smelling minty fresh:) The response might be mediated by uptake of DNA into the diagnostic bacteria and then use the mixed connective site as a riboregulator or maybe have a membrane protein that binds specific DNA sequences and triggers a two component system. Very sketchy proposal right now. Unless we could find easy ways of doing this it would be a protein engineering project.

Minimal Cellular Power Supply & Chassis

It would be really great to have a cell with the following properties:

• made from known components.
• works well with any system that's placed inside the cell.

The TK lab has done some foundational work on developing Mesoplasma florum as a standard cellular power supply and chassis (e.g., sequencing its genome).

Still, today, there is not a well-described simple cell that serves as a standard cellular power supply and chassis. Let's get on with it!

The goal of this project would be to take the development of Mesoplasma florum as a chassis to the next level. Specific parts of the project might include:

• making parts out of all the known Meso genes (e.g., ~600 new parts!)
• develop genome-scale engineering methods (e.g., cell and genome fusion techniques)
• measure the properties of a cell (e.g., transcription and translation load functions)
• design a new organism (e.g., minimal / modelable metabolism, DNA refactoring)

Strengths of this project would include:

• significant advisor interest and expertise
• some parts of the projects are, as near as possible, guaranteed to work (i.e., turn genes into new parts)
• the project would result in foundational contributions to the field (i.e., not another stupid bacteria trick)