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=Stick-to-everything proteins from mussels=
Revision as of 11:47, 1 June 2007
Ideas to chew on
Some ideas from 4/23/07 meeting
M1. Bacteria with squid reflecting protein (reflectin)
- Brian: 6 family members, all highly homologous
- Brian: biggest issue could be solubility problems (E. Coli)
- Brian: try expression in different systems where folding more likely to be correct (yeast, streptomyces, etc)
- Brian: only 1 major publication, so very little known about possible chaperones (see reference)
- Forrest: Paper on Methionine-Rich Repeat Proteins (MRRPs)
M2. Self mini-prepping bacteria
- comments: once triggered, will lyse, express RNases, and precipitate proteins and genomic DNA
M3. Bacteria with limited lifetime (telomeres)
- Brian: streptomyces bacteria have linear genome
- Brian: e. coli w/ linear genomes have been constructed (see reference)
- tk: The N15 plasmid (works in E. coli, commercially available from Lucigen) is linear.
M4. Bacteria with removed/non-functional DNA
- comments: "minicells" will grow for several weeks
M5. Incorporating biobrick parts into minicell
- comments: difficult to produce in large quatities
M6. Magnetic alignment of bacteria
- Brian: surface display of peptide which binds magnetic nanoparticles (iron oxide, cobalt oxide)
- Brian: can we control number of bound nanoparticles via concentration (i.e. one NP per bacteria)?
- Brian: feasibility: can we generate enough force and torque on NP to align bacteria (calculations)
- Forrest: Iron oxide nanoparticles tend to fall off the protein surface near neutral pH; cobalt oxide adheres better, but the synthesis conditions are quite toxic for cells
M7. Bacteria that illuminate when dark
M8. Bacteria which synthesize vitamins
- comments: Major vitamin deficiencies
- Brian: One of the most serious vitamin deficiencies in the current world is that of vitamin D (described as an epidemic in the USA). Although it can be produced by humans, the synthesis requires sunlight and many people do not receive sufficient UV radiation to produce the minimum daily requirement. Vitamin D is required for efficient calcium absorption in the gut, and deficiency leads to many bone disorders (rickets, osteoporosis, etc) as well as increasing the risk of autoimmune disease, diabetes, cancer, and cardiovascular disease. Current methods to synthesize vitamin D use extraction from sheep's wool. For more info on vitamin D, see wikipedia page epidemic cancer
- Brian: Another option is Vitamin B12, which is the main vitamin lacking in vegan diets (deficiency causes pernicious anemia). It is produced ONLY in prokaryotic organisms...
- Brian: Beriberi is caused by deficiency in thiamine (vitamin B1). It is very prevalent in Asian countries where many people rely entirely on white rice for their diet.
M9. Sensing pH
- Brian: idea -- use anthocyanins as pH sensor (expressed in plants such as red cabbage)
- Brian: E. Coli have been metabolically engineered to produce anthocyanin (see reference)
M10. Bacteria with kill switch
M11. Bacteria battle
- Forrest: Austin mentioned during the 4/23/07 meeting that this could be done in 2-D (on a dish)
- Forrest: Environmental conditions/stimuli can skew the outcome (e.g. shinning light or lowering pH causes on colony to have advantage over another)
- Brian: Could use F factor (bacterial conjugation) as the "weapon", where Strain A delivers a repressor gene lethal to Strain B and so on.
- Brian: Could have multiple fighting strains (e.g., A kills B, B kills C, C kills A)
- Brian: Possible to see population oscillations? Could easily model the system...
- tk: The idea of "phage wars" was an early incarnation of the IGEM competition. We rejected it because it seemed too yucky.
M12. Plastic binding bacteria
- comments: credit to Reshma
- Brian: bacteria bind to polymer plastic via surface display peptides
- Brian: one idea: couple to growth phase -- bacteria in stationary phase bind to side of plastic tube, which those still growing can be poured out (easy separation)
- Forrest: We have peptide sequences that bind to an electically conducting polymer (PPyCl) (NATURE MATERIALS 4 (6): 496-502 JUN 2005)
- tk: wouldn't a plastic making bacterium be more interesting?
- Forrest: Perhaps the bacteria can produce keratin, i.e. the plastic-like structural material in bird feathers; it would be very interesting to produce different keratin compositions (e.g. include pigments for color or strenghening) and microstructures (e.g. to diffract light like some feathers do)
- Forrest: Plastic Made by Bacteria Commercialized (Apr '07)
- Forrest: Some pionnering work is lead by MIT's Anthony Sinskey (Biopolymer Engineering)
M13. Luciferase Lava Lamp
- comments: credit to Reshma
M14. Organic Transister?
- using conductive M13 phage nanowires?
- Forrest: For electrical transister, M13 phage is not suitable because it's difficult to program both the head and tail to bind to electrodes. In the past, someone has been able to bind the tail end to an electrode and play with flow to get the head to make contact with another electrode. Not sure how much more we can improve on...
Random ideas from Superphage (Forrest)
F1. Engineering bacteria to operate in extreme environment (extremophiles)
- bacteria that die when not in artificially harsh environments (i.e. bacteria that 'escaped' from lab would not thrive)
F2. High protein bacteria/fungus
- Easy to grow, and highly-nutritious
- To be made into bread spread for poor or disaster-striken communities
F3. Blood clotting phage/bacteria
- function like Chitosan bandaids
F4. Bacteria that process animal waste to recover nutrients
- Recover proteins and other substances from pool of farm animal waste (e.g. the edible stuff floats to the top) and add back to animal feed
F5. Food spoilage detection
- Add non-harmful bacteria to milk, meat packaging, etc; these bacteria grow slightly more easily that the usual bacteria that make people sick, and are highly visible (e.g. bright purple) when they grow
- If consumer sees purple, if means that the food is possibly spoiled
F6. Fungus-based sensors
- Sensing: ammonia (NH3), CO2, light, stresses (osmotic shock, temperature, salt), UV, other fungi
- Fungi as sensors
- Fungus engineering
- Fungus eats radiation for breakfast
Random ideas from Cookb (Brian)
B1. RNA oligo synthesizing bacteria
- bacteria that produce and secrete RNA (mRNA, siRNA, RNAi, microRNA, etc)
- could be used to mass produce RNA-based therapies
- benefit from high-fidelity biological production (no error-prone commercial synthesis)
- commercial synthesis is limited to <20 bp (maybe 50 bp max)
- purification by HPLC later (and analyze by MS)
- protect RNA (chemicals protect 2'OH, could secrete as dsRNA)
- F factor secretion?
Olfactory sensing systems
- The idea is to capture the very wide diversity of the olfactory sensing systems in mammalian noses.
- Like antibodies, there is a very wide range of sequenced olfactory systems, especially from the new Dog genome.
- Here is a list of possible readings. Short version: proofs of concept in yeast have been made.
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- Brian -- I work on olfactory receptors (ORs), so I have a good deal of experience with them. But they are difficult to express (especially in bacteria and yeast) and this could be tricky to do in a summer, especially if trying to use multiple OR proteins.
Stick-to-everything proteins from mussels
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- Forrest -- ref.13 points:
- adhesive properties are from an "unusual amino acid 3,4-dihydroxy-l-phenylalanine (dopa)"
- "On inorganic surfaces the unoxidized dopa forms high-strength yet reversible coordination bonds, whereas on organic surfaces oxidized dopa is capable of adhering via covalent bond formation."
- Forrest --
- could perhaps express dopa on p3 (tail) or p8 (main body) protein of M13 phage
- after discussing with Rana, it seems that phage is probably not appropriate b/c 1) it is difficult/impossible to engineer phage to express tyrosine (perhaps too bulky) and 2) a phagemid approach would incorporate dopa arbitrarily and sparsely on phage; our best bet would be to engineer p3 of phage (p3 engineering is generally more forgiving than p8)
- Brian -- could express on E. Coli using fusion to surface proteins (e.g., OmpA, OmpX, FhuA, or LamB). see references below
- also possible to express on flagella via the Invitrogen FliTrx system
- a good consensus peptide appears to be AKPSYPPTYK, with tyrosines getting converted to L-DOPA. Also reported to have prolines converted to hydroxyproline, but not sure if required for stickiness.
- can convert tyrosine residues to L-DOPA by adding tyrosine hydroxylase (later could have bacteria secrete it)
- main question/concern is will bacteria stick to each other and clump up? separations obviously won't work if sticky (+) bacteria adhere to the non-sticky (-) bacteria we are trying to separate from.
- some papers on bacterial surface display:
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Adhesive bacteria with surface specificity
- Non-specific binding (mussel peptide) has certain disadvantages, such as exotic amino acids (DOPA, hydroxyproline) and possibility of cell clumping.
- Instead could investigate adhesive peptides which specifically bind common plastic/polymer surfaces.
- Reference (below): polystyrene binding peptides
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Getting it unstuck?
- Brian-- Once bacteria have adhered to surface, how do we get them off?
- Easy way would be to engineer a protease cleavage site into the displayed peptide. For instance, have a trypsin cleavage sequence and just add trypsin so cut the cells off the surface (just as is done in adherent mammalian cell culture).
- Hopefully the protease would not cause excessive damage to the bacteria