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In their search for ''inter''cellular protein trafficking mechanisms, researchers have discovered different secretion mechanisms that they have creatively termed “type I secretion,” “type II secretion,” etc. It is mostly pathogenic Gram-negative bacteria that employ these pathways. These mechanisms transport proteins from the cytoplasm, ''across'' the periplasmic space, to the exterior of the organism, generally destined for a eukaryotic host. Interestingly, the bacterial flagellum employs type III secretion (TTS), shuttling the flagellar precursors, in addition to non-flagellar proteins, through the growing flagellar shaft <cite>Journet</cite>. If the "cap" of the flagellum is deleted, flagellar polymerization is eliminated and "secretion-selected" proteins leak into the growth media <cite>Majander</cite>. Furthermore, it has been reported that systems have been created to use these secretion mechanisms for heterologous protein and DNA delivery <cite>
In their search for ''inter''cellular protein trafficking mechanisms, researchers have discovered different secretion mechanisms that they have creatively termed “type I secretion,” “type II secretion,” etc. It is mostly pathogenic Gram-negative bacteria that employ these pathways. These mechanisms transport proteins from the cytoplasm, ''across'' the periplasmic space, to the exterior of the organism, generally destined for a eukaryotic host. Interestingly, the bacterial flagellum employs type III secretion (TTS), shuttling the flagellar precursors, in addition to non-flagellar proteins, through the growing flagellar shaft <cite>Journet</cite>. If the "cap" of the flagellum is deleted, flagellar polymerization is eliminated and "secretion-selected" proteins leak into the growth media <cite>Majander</cite>. Furthermore, it has been reported that systems have been created to use these secretion mechanisms for heterologous protein and DNA delivery <cite>Spreng</cite>.
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Revision as of 07:34, 28 May 2006
Post your project ideas here.
See the brainstorming of last year's team.
- Bacterial scents (RS)
- Living Lamp (RS)
- Fast time scale switching
- Analog Clock (AC)
- Cool part domestication
- CC Signaling
- Linear induction
- Organism Domestication
- meso, square bacteria, fast E. coli, cyanobacteria
- Do some work with stationary phase, auto-inducing media (a la Studier)
- Multiple genetic populations interacting
- Organisms that eat photons (JK)
- rhodopsin (AC)
- Synthetic Vesicles (Libchaber)
- meso (DE)
- laundry list of parts
- put genome in a YAC
- knock out restriction system
- genome boot-up
- genome transfer (DE)
- autolyse cells
- Cell/Electrical connections
Some may be resurrected
- Mitochondrial Liberation & Synthetic organelle(BC)
- Square Bacteria (JK)
- Maze (JK)
- wood eater (SS)
- Diagnostic Bacteria (BC)
- Characterization methods
- MS2 bound to mRNA
- Subset of composable promoters and RBSs
- Plasmid-level parts
- Synthetic genomes (a la T7)
- in vitro, recombination
Connect cells to some kind of electrical system. Would allow for easy monitoring, interesting feedback systems, and allow creation of hybrid systems.
- rhodopsin (proton pump changes pH)
- Magnetic bacteria generating electricity
- Genetic controlled expression of cell surface enzyme that interacts with substrate on surface that changes electrical properties: 
- Collier JH and Mrksich M. . pmid:16461913.
Under induction control, express
- Lysis gene
- either RNase or DNase (depending on whether prepping DNA/RNA)
Spin down cells, perhaps do a ethanol precipitation. Can easily select for proper performing cells.
Things to think about
- Is there any way to enzymatically separate chromosomal from plasmid DNA?
- If have different control pathways that also expresses BioBrick enzymes, then may be able to prep and cut at the same time. A bit further out in fantasy land: using 3-antibiotic selection or other method, we could have cells auto-assemble parts. Grow up one cell line, induce with X which preps and cuts with ES, grow up another cell with second part, induce with Y which preps and cuts with XP, mix two lysates together, add destination plasmid, ligase, and transform new cells.
- Would be pretty modular in being able to be built incrementally by externally supplying other enzymes.
- Ideally, would require no chemicals (i.e. ethanol). Probably no way to get away from one centrifuge step to get rid of cell debris (what else other than nucleic acids would remain soluble?)
DNA prep only
An alternative is DNA production only. Find a DNA secreting system (Agrobacterium for instance), put the secreting signal on the plasmid. The cells continually make and secrete the desired DNA (DNA bioreactor). By not lysing the cells, it makes it easier to separate the cells from the DNA.
- Garrett J, Fusselman R, Hise J, Chiou L, Smith-Grillo D, Schulz J, and Young R. . pmid:6457237.
- Jain V and Mekalanos JJ. . pmid:10639478.
Organisms that eat photons
Looks like cyanobacteria are significantly easier to genetically manipulate than algae. 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. Two popular cyanobacteria strains are PCC7942 and PCC6803. Peter has both of these strains available, as well as some of the plasmids for genetic manipulation, he has also made & tested the growth media BG11.
- Synechocistis PCC6803 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)
- Knock in/out system available for 6803 w/ a sucrose selection/counter selection. Keith Tyo in the Stephanopolous lab has the plasmids, etc, but Peter is in the process of getting them from him.
- S.elongus (7942) is the model system for circadian clocks in prokaryotes (its used in the vanO lab), so that might be a cool hook to play with as well.
- Genetic manipulation was worked out for 7942 largely by Sue Golden.
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"
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.
The H2 sensing circuit has been studied in detail in Ralstonia eutropha. The network consists of a hydrogenase-like protein to control gene expression and also a two-component regulatory system. This bacterium can metabolize Hydrogen gas as an energy source and the hydrogen sensor is used to regulate the synthesis of the metabolic enzymes.
Ralstonia eutropha is a gram negative bacteria. Peter Weigele has the strain and the Sinskey lab has plasmids to transform it. It expresses two hydrogenases, one membrane bound (MBH), the other cytoplasmic (SH). The operons for both of the multi-protein complexes are co-regulated. HoxA is the key regulator. HoxB,C and J form the signal transduction network. HoxBC appears to be the hydrogen receptor and HoxJ inactivates HoxA by phosphorylation. When HoxBC is bound to Hydrogen, it inactivates HoxJ .
Kamachi and coworkers have produced a Light-driven hydrogen production system in Synechocystis. Not directly related to the H2 sensor but maybe cool for the photon eating stuff above.
- Kleihues L, Lenz O, Bernhard M, Buhrke T, and Friedrich B. . pmid:10781538.
- Lenz O, Bernhard M, Buhrke T, Schwartz E, and Friedrich B. . pmid:11931556.
- Ihara M, Nishihara H, Yoon KS, Lenz O, Friedrich B, Nakamoto H, Kojima K, Honma D, Kamachi T, and Okura I. . pmid:16542111.
Samantha looking into this.
Very recent paper showing that Saccharophagus degradans can break down cellulose and all other major polysaccharides . There could be uses in converting plant biomass to energy.
E. coli has receptors for blue light. Could perhaps get another light control channel.
- Wright S, Walia B, Parkinson JS, and Khan S. . pmid:16707688.
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.
- We'd have to do some research to see if media or media supplemented with something would be
- nontoxic to cells
- have the necessary properties to achieve the lava effect at ~37°C
- Luminescence requires oxygen so we'd have to oxygenate the contents of the lava lamp which might interfere with the lava effect. [from TK]
|benzoic acid & S-adenosyl-L-methionine (SAM)||S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase (BAMT)||methyl benzoate||pleasant smell|||
|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||[11, 12, 13]|
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
- 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
- A. thaliana AY008434
- Snapdragon AF198492
- Pott MB, Hippauf F, Saschenbrecker S, Chen F, Ross J, Kiefer I, Slusarenko A, Noel JP, Pichersky E, Effmert U, and Piechulla B. . pmid:15310828.
- Ross JR, Nam KH, D'Auria JC, and Pichersky E. . pmid:10375393.
- Negre F, Kolosova N, Knoll J, Kish CM, and Dudareva N. . pmid:12361714.
- Zubieta C, Ross JR, Koscheski P, Yang Y, Pichersky E, and Noel JP. . pmid:12897246.
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 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]
Indole can act as an extracellular signal so indole can probably get in and out of the cell.
In Pathway Reactions as a Reactant:
tryptophan biosynthesis : indole + L-serine = L-tryptophan + H2O
In Pathway Reactions as a Product:
tryptophan biosynthesis : indole-3-glycerol-phosphate = indole + D-glyceraldehyde-3-phosphate
tryptophan degradation II (via pyruvate) : L-tryptophan + H2O = indole + pyruvate + ammonia
trpB (biosynthesis) and tnaA (degradation)
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)
Random, Environmentally-Sensitive Design Generator
In 2000, Elowitz and Leibler created a ring oscillator system in bacteria which allowed for the oscillation of green fluorescent protein expression. I would like to develop many plasmids of this kind with a few modifications. First, each plasmid’s expression of the ocscillating system would be dependent on the sensing of a particular type of light (specifically, red, yellow, or blue). In addition, following each of the three repressors constituting the oscillator would be a unique fluorescent color protein coding region (again, red, yellow, or blue). These plasmids would be randomly distributed onto a field of cells. Then, once a light is shined on a particular region of the field, environmentally-sensitive, multi-color light patterns will be developed on the field of cells. Reference: Elowitz, M. B. & Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335–338 (2000).
Strength: Cool Demo
Weakness: Not any real significant scientific value
In 2004, a team of students from the University of Texas at Austin created a system engineered inside bacteria which produced a black precipitate in the absence of red light. This system was then used to take pictures of a projected slide with the aid of modern imaging technology. I would like to explore the possibility of connecting the light sensor component of their system to another genetic circuit. This genetic circuit would consist of a promoter, which would trigger subsequent red light production when enough polymerases per second (PoPS) are received from the output of the light-sensing device. Thus, by shining red light on one cell, all of the cells in a bacterial lawn will be lit up red. One could then place yellow, green, and blue light-sensing and light-producing systems similar to these systems in the same cells, inducing the whole lawn to amplify a light signal directed towards a single cell. Reference: Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, Davidson EA, Scouras A, Ellington AD, Marcotte EM, Voigt CA. Synthetic biology: engineering Escherichia coli to see light. Nature. 2005 Nov 24;438(7067):441-442.
Strength: Could be used as an indirect measurement of PoPS which is integral to the development of synthetic biology Physics professors may be interested Could be used in the future as a cheaper light source
Weakness: May seem too simple or unuseful at first glance
Vibrio furnissii hydrocarbon production
Park has discovered a strain of Vibrio furnissii which produces significant quantities of long chain hydrocarbons from fatty acids [16, 17, 18]. The key pathway is from the aldehyde hexadecanal to the alcohol hexadecanol to the hydrocarbon hexadecane. Remarkably, this reduction appears to take place in an oxygen environment, making the enzyme especially interesting.
A biological sensor for hexadecane could be made from the system of Holden  using the hydrocarbon sensor of Pseudomonas aeruginosa.
The project would be to enrich for V. furnissii strains on alkaline peptone water (with 7% NaCl see here), plate out on TCBS agar, isolate Vibrio colonies, and select hydrocarbon producers. We would then shotgun clone genomic DNA into E. coli, add exogenous hexadecanol, and attempt to isolate clones producing hexadecane. Sequencing selected strains might reveal the important enzyme.
Several diffficulties arise: potential pathogenicity of V. furnissii, likelihood of obtaining a hydrocarbon producing strain, cross reaction of the Holden sensor between hexadecanol and hexadecane. The strain NCTC 11218 is alleged to be non-pathogenic, although the evidence for this seems thin. It is used as a validation test for TCBS agar in microbiology labs. See Taylor .
Wackett at the University of Minnesota has started looking at this with this short proposal:
Petroleum Fuels in Real-Time from Renewable Resources Lead: Lawrence P. Wackett (BMBB) Funding Amount: $270,000 Project Abstract: Petroleum hydrocarbons took hundreds of millions of years to form naturally, but are being harvested and combusted in centuries. Either our vehicles and industry will change dramatically, or a renewable source of petroleum-like alkanes will need to be developed. Recently, bacteria have been shown to biosynthesize middle chain-length alkanes in the time frame of enzyme-catalyzed reactions (a typical enzyme second order rate constant is 106 M-1s-1). Middle chain-length alkanes are the “sweet” part of petroleum that is used as liquid fuels. Longer chain-length alkanes are less preferred as fuels and short-chain alkanes are gaseous and thus not amenable for use as transportation fuels. In traditional petroleum fuels, sulfur-containing ring compounds cause polluting sulfurous oxides during combustion. Bacterially-produced middle chain-length alkanes can provide a fuels source for society that derives from renewable resources and combusts efficiently and with much less pollution. Moreover, alkane-producing metabolism could be tuned to generate different chain-length alkanes to be used for different purposes; for example, C8– C14 alkanes can be used in automobile engines and C14 – C22 alkanes used in diesel engines. Research is proposed here to better understand and engineer bacterial fermentations that transform common renewable resources into fuel hydrocarbons. A bacterium has been described that is capable of producing C14 – C22 alkanes (diesel fuel) from glucose, xylose, acetate, sugar cane molasses, xylan or chitin. The pathway and enzymes involved in lipid reduction to alkanes is proposed but still requires experimental investigation. In this project, the researchers will begin to: (1) identify the key enzyme(s) reducing alcohols to alkanes, (2) elucidate the pathway for alkane production, (3) metabolically engineer superior alkane producing strains, (4) isolate superior alkane producing bacteria from nature, and (5) analyze the optimal economic outcomes for biobased production of alkanes from various renewable resources. This project has a very high probability of generating new intellectual property and providing the basis for external funding from the Department of Energy, the National Science Foundation and other agencies.
See also Wackett's presentation: 
- Park MO, Tanabe M, Hirata K, and Miyamoto K. . pmid:11549018.
- Park MO, Heguri K, Hirata K, and Miyamoto K. . pmid:15659187.
- Park MO. . pmid:15687207.
- Holden PA, LaMontagne MG, Bruce AK, Miller WG, and Lindow SE. . pmid:11976128.
- Taylor JA and Barrow GI. . pmid:7229102.
- Brenner DJ, Hickman-Brenner FW, Lee JV, Steigerwalt AG, Fanning GR, Hollis DG, Farmer JJ 3rd, Weaver RE, Joseph SW, and Seidler RJ. . pmid:6630464.
- Oró J, Tornabene TG, Nooner DW, and Gelpi E. . pmid:6025301.
- Birkeland N-K, The microbial diversity of deep subsurface oil reservoirs, Chapter 14, Studies in Surface Science and Catalysis 151 Vazuez-Duhalt R and Quintero-Ramirez R (eds.), (2004) Elesevier B. V.
- Valderrama B, Bacterial hydrocarbon biosynthesis revisited, Chapter 13, Studies in Surface Science and Catalysis 151 Vazuez-Duhalt R and Quintero-Ramirez R (eds.), (2004) Elesevier B. V.
- Haight RD and Morita RY. . pmid:5924270.
- Hickman-Brenner FW, Brenner DJ, Steigerwalt AG, Schreiber M, Holmberg SD, Baldy LM, Lewis CS, Pickens NM, and Farmer JJ 3rd. . pmid:6746884.
- O'Hara CM, Sowers EG, Bopp CA, Duda SB, and Strockbine NA. . pmid:14662957.
- Stone RW and Zobel CE, Bacterial aspects of the origin of petroleum, Industrial and Engineering Chemistry, Vol 44 No 11 pp 2564-7 (1952).
Protein/Nucleic Acid/BioBrick(??) Intercellular Translocation
Hey guys. These ideas are definitely in their beginning stages. I will be editing and putting up more references throughout the next few days. I just wanted to put this up if people wanted to start thinking about it some more.
In their search for intercellular protein trafficking mechanisms, researchers have discovered different secretion mechanisms that they have creatively termed “type I secretion,” “type II secretion,” etc. It is mostly pathogenic Gram-negative bacteria that employ these pathways. These mechanisms transport proteins from the cytoplasm, across the periplasmic space, to the exterior of the organism, generally destined for a eukaryotic host. Interestingly, the bacterial flagellum employs type III secretion (TTS), shuttling the flagellar precursors, in addition to non-flagellar proteins, through the growing flagellar shaft . If the "cap" of the flagellum is deleted, flagellar polymerization is eliminated and "secretion-selected" proteins leak into the growth media . Furthermore, it has been reported that systems have been created to use these secretion mechanisms for heterologous protein and DNA delivery .
So…it would be kinda cool to make the following:
- Customizable protein translocators/channels
- “Easily” and “reliably” transfer BioBricks from one cell to another by creating some type of “secretion tag”
- I would imagine if you are getting bacteria to mass produce some type of protein for you, it would be useful if the bacteria would just spit the proteins out into the media.
- Insert ideas here
There is much to be worked out here. Bacteria pathogenicity. Lack of a basic understanding of some aspects of the secretion mechanisms. ... We’ll see what happens, though.