IGEM:MIT/2007/Ideas

Ideas to chew on

Chitin/Chitosan Links

• Chitin synthesis and localization in cell division cycle mutants of Saccharomyces cerevisiae
• Chs1p and Chs3p, two proteins involved in chitin synthesis, populate a compartment of the Saccharomyces cerevisiae endocytic pathway
• Chitin synthetase zymogen is attached to the yeast plasma membrane
• Activating System of Chitin Synthetase from Saccharomyces cerevisiae
• [1]

More Random Ideas

(based on some articles but I'm not sure if any are feasible)

"Environment"

• Bacteria that will help turn rock into fertile soil and can live in extreme environments to colonize Mars
  • [2]
    • or maybe create a self-sustaining biosystem of extremophiles
• bacteria to degrade trash
  • styrofoam: [3]
• Other issues: Waterlogging, Oil spill cleanup, water filtration, non-chemical alternative to pesticides

"General"

• Harvesting Light Energy (this is nothing new, but it is now possible to convert e.coli in a single step) [4]
  • perhaps we could try to take this further by turning the mechanical energy of the bacteria into another form (electrical,biofuel...)that can be used by humans?
• oscillatory system (biological clock)

"Medical"

• bacteria to produce chitosan(currently used in bandages)layer to protect open wounds (burns) from infection and encourage healing/clotting
  • [5]

---

(Bernice)

Possible Project Ideas from the 5.23.07 Meeting (Apologies if some of these are repeats)

GENERAL IDEAS

• Bioluminescence
• Building a biofilm: a mechanism to adhere yeast to surfaces when stimulated (through amino acids)
  • Similar to mollusk muscles
• A “lab kit” – for quick separations (make wanted bacteria sticky and then save the rest)
  • Separate different types/kinds of bacteria?
• Squid reflective (crystal) plates [a reflective protein]
  • Tyr, Trp, Met – pumps protein out through secretory valves (six types?)
  • Possible problem could arise because proteins are not soluble?

ENVIRONMENTAL ISSUES

• De-salinization (?)
• Biofilm with pores – selectively push ions to desired positions [like spider silk?]
• Making efficient/earth-friendly fuel
• Healthy/nutritious bacteria for consumption (vegemite?)

MEDICAL/HEALTH ISSUES

• Bacteria in the gut that could digest lactase OR insulin, etc.
  • Possible barrier could occur when testing bacteria unless we used model system
• Bacteria that synthesize vitamins (such as D, A, B12)
  • Possible problem could be overdosing on certain vitamin

_ _ _ _ _

I just finished a class on microfluidic devices and engineering with cells, etc. if we possibly want to incorporate that sort of technology into our final project? (I don’t think it’d be feasible to use microfluidics as the actual basis of the project…but just in case…)

[from Semmie]

Request for Scope

The ideas are great, guys, and thanks for the links too. But can someone go through the ideas and give them a sense of complexity? A lot of these sound really cool but I'm scared they are beyond the scope of a summer project.--Toan 01:16, 2 June 2007 (EDT)

Some ideas from 4/23/07 meeting

M1. Bacteria with squid reflecting protein (reflectin) NO

• comments:
  • 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)
  • Robbie: Nature materials paper from the air force research laboratory describing the production of reflectin in E. coli. They purified the reflectin and made a biofilm of it. [6]

M2. Self mini-prepping bacteria

• comments: once triggered, will lyse, express RNases, and precipitate proteins and genomic DNA

M3. Bacteria with limited lifetime (telomeres)

• comments:
  • 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

• comments:
  • 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

• comments:

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

• comments:
  • 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

• comments:

M11. Bacteria battle

• comments:
  • 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?

• comments:
  • 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)

http://environment.newscientist.com/article/dn11614-extremeliving-bacteria-has-genome-sequenced.html

• Genes from Syntrophus bacterium
• comments:

F2. High protein bacteria/fungus

• Easy to grow, and highly-nutritious
• To be made into bread spread for poor or disaster-striken communities
• comments:
  • Jess: Whey protein (whey is milk plasma, the liquid that remains after milk is curdled) is used by athletes and body builders to increase muscle mass. It's mostly beta-lactoglobulin (~65%), alpha-lactalbumin (~25%), and serum albumin (~8%), so maybe we could engineer bacteria to create these proteins. (Perhaps make them a byproduct of storage medium digestion for easy transportation to the communities.)
  • Whey Protein

F3. Blood clotting phage/bacteria

• function like Chitosan bandaids

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

• comments:

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
• comments:

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
• comments:

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
• comments:

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.

1. Katada S, Nakagawa T, Kataoka H, and Touhara K. Odorant response assays for a heterologously expressed olfactory receptor. Biochem Biophys Res Commun. 2003 Jun 13;305(4):964-9. DOI:10.1016/s0006-291x(03)00863-5 |
2. Pajot-Augy E, Crowe M, Levasseur G, Salesse R, and Connerton I. Engineered yeasts as reporter systems for odorant detection. J Recept Signal Transduct Res. 2003;23(2-3):155-71. DOI:10.1081/rrs-120025196 |
3. Levasseur G, Persuy MA, Grebert D, Remy JJ, Salesse R, and Pajot-Augy E. Ligand-specific dose-response of heterologously expressed olfactory receptors. Eur J Biochem. 2003 Jul;270(13):2905-12. DOI:10.1046/j.1432-1033.2003.03672.x |
4. Minic J, Sautel M, Salesse R, and Pajot-Augy E. Yeast system as a screening tool for pharmacological assessment of g protein coupled receptors. Curr Med Chem. 2005;12(8):961-9. DOI:10.2174/0929867053507261 |
5. Minic J, Persuy MA, Godel E, Aioun J, Connerton I, Salesse R, and Pajot-Augy E. Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening. FEBS J. 2005 Jan;272(2):524-37. DOI:10.1111/j.1742-4658.2004.04494.x |
6. Touhara K. Odor discrimination by G protein-coupled olfactory receptors. Microsc Res Tech. 2002 Aug 1;58(3):135-41. DOI:10.1002/jemt.10131 |
7. Shirokova E, Schmiedeberg K, Bedner P, Niessen H, Willecke K, Raguse JD, Meyerhof W, and Krautwurst D. Identification of specific ligands for orphan olfactory receptors. G protein-dependent agonism and antagonism of odorants. J Biol Chem. 2005 Mar 25;280(12):11807-15. DOI:10.1074/jbc.M411508200 |
8. Radhika V, Proikas-Cezanne T, Jayaraman M, Onesime D, Ha JH, and Dhanasekaran DN. Chemical sensing of DNT by engineered olfactory yeast strain. Nat Chem Biol. 2007 Jun;3(6):325-30. DOI:10.1038/nchembio882 |

Comments:

• 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.

Sticky bacteria/viruses

Stick-to-everything proteins from mussels

9. Hwang DS, Yoo HJ, Jun JH, Moon WK, and Cha HJ. Expression of functional recombinant mussel adhesive protein Mgfp-5 in Escherichia coli. Appl Environ Microbiol. 2004 Jun;70(6):3352-9. DOI:10.1128/AEM.70.6.3352-3359.2004 |
10. Hwang DS, Gim Y, and Cha HJ. Expression of functional recombinant mussel adhesive protein type 3A in Escherichia coli. Biotechnol Prog. 2005 May-Jun;21(3):965-70. DOI:10.1021/bp050014e |
11. Hwang DS, Gim Y, Yoo HJ, and Cha HJ. Practical recombinant hybrid mussel bioadhesive fp-151. Biomaterials. 2007 Aug;28(24):3560-8. DOI:10.1016/j.biomaterials.2007.04.039 |
12. Hwang DS, Gim Y, Kang DG, Kim YK, and Cha HJ. Recombinant mussel adhesive protein Mgfp-5 as cell adhesion biomaterial. J Biotechnol. 2007 Jan 20;127(4):727-35. DOI:10.1016/j.jbiotec.2006.08.005 |
13. Lee H, Scherer NF, and Messersmith PB. Single-molecule mechanics of mussel adhesion. Proc Natl Acad Sci U S A. 2006 Aug 29;103(35):12999-3003. DOI:10.1073/pnas.0605552103 |
14. Lin Q, Gourdon D, Sun C, Holten-Andersen N, Anderson TH, Waite JH, and Israelachvili JN. Adhesion mechanisms of the mussel foot proteins mfp-1 and mfp-3. Proc Natl Acad Sci U S A. 2007 Mar 6;104(10):3782-6. DOI:10.1073/pnas.0607852104 |
15. Wang J, Liu C, Lu X, and Yin M. Co-polypeptides of 3,4-dihydroxyphenylalanine and L-lysine to mimic marine adhesive protein. Biomaterials. 2007 Aug;28(23):3456-68. DOI:10.1016/j.biomaterials.2007.04.009 |

Comments:

• 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)
• Forrest --
  • we can create a bacteria smart glue: a substance that becomes sticky when a certain temperature is reached, or if a certain chemical is present, etc (a "programmable glue", so to speak)

• 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:

16. Rice JJ, Schohn A, Bessette PH, Boulware KT, and Daugherty PS. Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands. Protein Sci. 2006 Apr;15(4):825-36. DOI:10.1110/ps.051897806 |
17. Bessette PH, Rice JJ, and Daugherty PS. Rapid isolation of high-affinity protein binding peptides using bacterial display. Protein Eng Des Sel. 2004 Oct;17(10):731-9. DOI:10.1093/protein/gzh084 |
18. Etz H, Minh DB, Schellack C, Nagy E, and Meinke A. Bacterial phage receptors, versatile tools for display of polypeptides on the cell surface. J Bacteriol. 2001 Dec;183(23):6924-35. DOI:10.1128/JB.183.23.6924-6935.2001 |
19. Hall SS, Mitragotri S, and Daugherty PS. Identification of peptide ligands facilitating nanoparticle attachment to erythrocytes. Biotechnol Prog. 2007 May-Jun;23(3):749-54. DOI:10.1021/bp060333l |
20. Samuelson P, Gunneriusson E, Nygren PA, and Ståhl S. Display of proteins on bacteria. J Biotechnol. 2002 Jun 26;96(2):129-54. DOI:10.1016/s0168-1656(02)00043-3 |
21. Wernérus H and Ståhl S. Biotechnological applications for surface-engineered bacteria. Biotechnol Appl Biochem. 2004 Dec;40(Pt 3):209-28. DOI:10.1042/BA20040014 |

Adhesive bacteria with surface specificity

• Brian--
  • 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

22. Gebhardt K, Lauvrak V, Babaie E, Eijsink V, and Lindqvist BH. Adhesive peptides selected by phage display: characterization, applications and similarities with fibrinogen. Pept Res. 1996 Nov-Dec;9(6):269-78.
23. Kenan DJ, Walsh EB, Meyers SR, O'Toole GA, Carruthers EG, Lee WK, Zauscher S, Prata CA, and Grinstaff MW. Peptide-PEG amphiphiles as cytophobic coatings for mammalian and bacterial cells. Chem Biol. 2006 Jul;13(7):695-700. DOI:10.1016/j.chembiol.2006.06.013 |
24. Adey NB, Mataragnon AH, Rider JE, Carter JM, and Kay BK. Characterization of phage that bind plastic from phage-displayed random peptide libraries. Gene. 1995 Apr 14;156(1):27-31. DOI:10.1016/0378-1119(95)00058-e |
25. Menendez A and Scott JK. The nature of target-unrelated peptides recovered in the screening of phage-displayed random peptide libraries with antibodies. Anal Biochem. 2005 Jan 15;336(2):145-57. DOI:10.1016/j.ab.2004.09.048 |

Comment from Brian (email): As for the sticky bacteria, we had a bunch of ideas concerning the control of crosslinking (via oxidation/reduction). Perhaps even to use crosslinking (bacteria sticking/clumping) as a sensor readout. Possibility also of using leucine zippers (protein/protein interaction) for cells clumping.

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

Bacterial photoresist

• Forrest-- have light as stimulus (e.g. rhodopsin approach), and stickiness as response (e.g. dopa or other peptide sequence); expose biofilm to light to cause stickiness
• Brian-- can do 3D printing; concerned about exposure time (might take too long)
• Forrest-- time for bacterial photography is 4-12hrs; does seem like a long time, but might be acceptable for project if we can make it work
• Forrest-- can also do lithography; portions of film exposed to light stick together to the substrate, and unexposed regions can be washed away or killed

Some applications of bacterial glue (from news)

Fiber-Hungry Bacteria Could Form Natural "Bond" With Wood Industry (Jul 2004)

Bacterial Glue Could Become Medical Adhesive (Apr 2006)

Water Decontamination

• Water is collected from a river (or other source) into a filtration setup
• Bacteria is added to the water
• The bacteria bind to or take in metals or other pollutants (input 1: detection/uptake of pollutant)
• Input causes bacteria to be able to bind to the filter material (output 1: stickiness to filter material)
• The water is now pollutant-free
• The filter can be cleaned by rinsing/soaking it with water while shining light on it (input 2: light)
• This second input causes the bacteria to unbind from the filter material (output 2: loss of stickiness to filter material)