|Line 84:||Line 84:|
Revision as of 02:02, 3 May 2007
Hi all! Great meeting yesterday, with a lot of terrific ideas proposed. Now comes the hard part ... deciding which project to pursue this summer. I put an overview slide of some of the projects proposed over the last three brainstorming sessions here ( Brainstorming Overview) I hope it's of use. You can use the section below (between the ---) to hash out final ideas, proposals, and to lobby for your favorite projects. If you have any questions on updating the wiki you can contact me or Nick via email. Thanks, Mike (4/24/07)
Final Project Ideas, Discussions, and Lobbying (post 4-23-07)
I really like the idea of pathway engineering. I think we should stick to E. coli for several reasons: 1) the knowledge of E. coli, both within our group members and from outside papers, seems to have a comparative advantage over other organisms; 2) many of the pathways we're considering have been explored using E. coli already; 3) E. coli seems relatively easy to work with (though I can't argue this from experience). I found a really nice paper that summarizes some of the pathways we've considered, and what we might be able to do. Commercial Production of Chemicals Using Pathway Engineering. I particularly like the following excerpt:
We have constructed a strain of E. coli containing the genes from S. cerevisiae for glycerol production and the genes from K. pneumoniae for 1,3-propanediol production. E. coli provides several advantages of other systems. E. coli is the most completely studied organism. E. coli provides a rich set of genetic tools: sequenced genome, vectors, promoters, etc. E. coli's metabolism and physiology are well characterized and a large number of metabolic mutants have been constructed and analyzed. E. coli has been used in large scale fermentations and production on an industrial level. In addition, E. coli is closely related to the natural 1,3-propanediol producers: K. pneumoniae and C. freundii. Since E. coli does not naturally produce glycerol or 1,3-propanediol, there is no natural regulation to overcome. Through the construction of arti¢cial operons for the optimized expression of the genes for the 1,3-propanediol pathway, we have built an E. coli strain, which can produce 1,3-propanediol from glucose. Currently, the 1,3-propanediol production performance of our glucose to 1,3-propanediol organism equals or surpasses that of any glycerol to 1,3-propanediol natural organism.
[As a side note, 1,3-propanediol is a monomer with potential utility for production of polyester fibers and manufacture of polyurethanes. In itself, it's not a project I'm suggesting, but I think the excerpt captures the relative ease of working with E.coli for engineering pathways, and it shows the efficacy of E.coli in other engineering studies.]
... So I think I've done enough encouragement for using E. coli in general, and from personal discussions I've had with some of you, it seems like we're in agreement. I think the engineering for EtOH synthesis was a popular topic, but I'm not sure exactly where we want to go with it- it seems that a lot of groups have already engineered relatively efficient strains, and with our time restraint, I doubt that we'll get results that even come close to meeting the standard that has already been set. I like the idea of vitamin synthesis - it seems that plant engineering has been extremely popular in that regard, but not as much attention has been given to organisms such as E. coli - so I think it might be an interesting path to try. Thoughts?
Hi Stephanie, thanks for posting your ideas on the web. In response to your post, yes, I agree that E. coli would be the most tractable organism to use this summer, and I do like the idea of metabolic engineering as well as surface engineering. I’ll post some examples from the literature on engineering pathways in microorganisms in the next few days. Two papers you may be interested in:
1. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Martin VJ, Pitera DJ, Withers ST, Newman JD, Keasling JD. ( Nat Biotechnol. 2003 Jul;21(7):796-802.)
2. Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Pfleger BF, Pitera DJ, Smolke CD, Keasling JD. ( Nat Biotechnol. 2006 Aug;24(8):1027-32. )
You may also want to examine how many steps (reactions or proteins) are involved in the pathways you are interested in, since this will give you an idea of the complexity of the potential project. Thanks again for posting your idea. I'll add some more feedback in the next day or so.
Wow, thanks for the papers. I think it would be a good idea for everyone to read them - but if anyone on the team is willing to read/bullet/summarize the papers so it is easy for us the extract what we think is important, that would be wonderful. I will probably be reading them within the next few days, and I'll post my thoughts afterward.
Bacterial Surface Expression and Cellular Targeting (compiled 5/2/07 by Mike)
Hi all, I put together a project outline here related to Perry’s and Stephanie’s presentation on surface targeting of bacteria, focusing on an area that many of you expressed interest in: Medical Applications of Synthetic Biology. Importantly, it is a project that is feasible to finish during the summer time limit (a big consideration), a project that would be a great resource for future researchers, has the potential for quality research with wide ranging applications, and a project that each of you could really contribute to. Also it embraces the iGEM idea of creating modular genetic systems.
The project outline involves the creation of a modular system used to express a peptide library on the surface of E. coli. This would utilize the power of combinatorial constructions, selections, and surface expression (Three underutilized strengths George Church talked about during our first meeting).
Essentially, we could use the BioBrick strategy to create two "parts": 1 ) a membrane protein that will anchor a protein or peptide library of interest to the outer membrane of E. coli, and 2) a random peptide library that we can fuse to the outer membrane portion. We could use this to select peptides that bind various tissue types or cells, enabling us to target bacteria to specific cell types.
An outline of the strategy is as follows:
1) PCR or have synthesized the genes for two or three membrane proteins that will serve as the membrane anchors. PCR primers will contain the BioBrick sequence ends to create fusion proteins. Two candidate membrane proteins are OmpA and AIDA1 (see references). We can also use some constructs already made by myself, Perry, and the MCB100 students last quarter to facilitate the project.
References: Display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines. Georgiou G, Stathopoulos C, Daugherty PS, Nayak AR, Iverson BL, Curtiss R 3rd. Nat Biotechnol. 1997 Jan;15(1):29-34. Review. Media:Georgiou.pdf
Autodisplay: efficient bacterial surface display of recombinant proteins. Jose J. Appl Microbiol Biotechnol. 2006 Feb;69(6):607-14. Epub 2005 Dec 20. Review. Media:Jose.pdf
2. Next we can synthesize a double stranded nucleic acid sequence that encodes a random peptide library. This is straightforward to have synthesized since we can synthesize a random oligonucleotide (equal mix of A,T,C,G at each position) flanked by the BioBrick sequences.
3. Next we can ligate the Membrane Portion and Random Peptide Library into an expression plasmid and overexpress the membrane protein-peptide library in E. coli.
4. We can then characterize the overexpression by running protein denaturing gels, assay transformation effeciency and coverage of the library, and assay for surface expression by using a variety of control surface proteins like histidine tags (bind nickel) and streptavidin binding tags (bind streptavidin). I have some control vectors made that we can use during our testing.
5. After we construct the fusion library we can use the power of selection to identify surface peptide sequences that target our bacteria to various tissue types or cell types we are interested in. This has important medical implications since we may be able to target "microbial factories" to various areas of the body using this method (related to many of your brainstorming ideas realting to synthetic symbiosis, ie for vitamin production etc). We can also select for peptides that bind to other cell types. The list goes on.
This system is analogous to what has been done using phage (see reference), but in our case we can use bacteria, since bacteria have the added advantage that once bound they can produce desired products or perform desired functions (see references), such as targeting cancer cells (see references).
- Organ targeting in vivo using phage display peptide libraries. Pasqualini R, Ruoslahti E.
Nature. 1996 Mar 28;380(6572):364-6. Media:Pasqualini.pdf
Environmentally controlled invasion of cancer cells by engineered bacteria. J Mol Biol. 2006 Jan 27;355(4):619-27. Anderson JC, Clarke EJ, Arkin AP, Voigt CA. Media:Anderson.pdf
A bacterial protein enhances the release and efficacy of liposomal cancer drugs. Science. 2006 Nov 24;314(5803):1308-11. Cheong I, Huang X, Bettegowda C, Diaz LA Jr, Kinzler KW, Zhou S, Vogelstein B. Media:Cheong.pdf
This would be a really exciting project that would be feasible for the summer timeframe. We could contribute many new BioBricks to iGEM at the end (membrane proteins and all the peptides we identified as binders), and potentially create a system that many other scientists could use in their own future projects. The project also employs the power of combinatorial constructions (random peptide library), selections (select for binding to specific tissue types and cell types), and much needed advancements and modularity of cellular surface expression. We can also build on work done by Perry, myself, and some of the MCB100 students this past quarter on related surface expression projects.
Please add comments or let me know if I can provide any more information on this. I think it would be a great project to pursue, that would utilize all your talents, and help you learn many important experimental methods. We also have some members, postdocs, and TFs with experience and interest in this area, which will help the project move quicker this summer. Thanks, Mike (post 5/2)
Wow. Mike, I really like the idea, and the way you presented it was very clear and helpful. Aside from reading over the references that you've provided, what are the most important things for us to do between now and the next meeting?
I really think this sounds good too. It seems like there's a solid experimental plan that we could actually follow and manage to accomplish a lot. I have not read the papers yet, but I plan on doing so tomorrow (Thursday) or by Friday at the latest. I will post back my thoughts within that time frame. But for now, I'm pretty excited. --Shaunak (5/2)
Some great ideas here. I wanted to follow-up on some discussions of gut flora. I recently learned that E.coli is actually not one of the dominant inhabitants of the gut --- just one of the most easily cultured (see http://en.wikipedia.org/wiki/Gut_flora and references therein). I'm not usually a big wikipedia citer, but in this case is does an excellent job of summarizing and referencing specific citations.
Re a suicide program. A number of groups have developed methods for this. Check out work by Arnold and colleagues on "population control". Death genes abound, so putting them under the control of some external "switch" could be one possible course of action. See: You L, Cox RS 3rd, Weiss R, Arnold FH. Programmed population control by cell-cell communication and regulated killing. Nature. 2004 Apr 22;428(6985):868-71.
Great work, Jagesh
Also regarding gut flora: our friendly laboratory E. coli is apparently at a great competitive disadvantage vis-a-vis wildtype E. coli (wildtype E. coli secrete biofilms that protect themselves from our immune system). So, anything we do along the lines of vitamin biosynthesis is only a proof of concept.
After thinking about the legal and practical implications of using bacteria for jobs such as detoxification, biosynthesis of drugs, and detection of toxins, I realized that it might be useful to have some sort of "off" switch - a way to quickly and efficiently kill the engineered bacteria without using antibiotics or anti-septics. It might not be useful in the strict sense since engineered bacteria are likely to be maladapted for natural environments, but it might put people at ease.
There are any number of methods of doing this, but the easiest is probably adapting a bacteriophage that integrates its genome into the E. coli genome - perhaps the lamba phage. From there, we would need to play with the promoters so that we are able to switch the virus to the lytic part of its life cycle at will. We could either switch out the wildtype promoters and install an entirely new promoter, or we could tamper with the lambda promoters. More on this later
I hope you'll consider presenting more on this on Monday. Even if not, the thought is intriguing. I understand the hesitation to use antibiotics (presumably, resistance?), but my presentation on adaptamers tomorrow might have lightly-related ideas - since the potential uses of adaptamers are quite broad, we could potentially target drugs or bacteria-killing substrates to the cells. As you said, the legal and practical implications would probably cause me to discard the idea, but at least it's tangentially related.
I wanted to put forth an idea -- vitamin production & microbial therapy. The idea comes from the fact that vit B producing microbes inhabit the ruminant gut so these animals almost never have B-deficiency. But for humans, without a proper diet, we end up getting diseases like Beriberi.
- the other thing is the bioavailability of these vitamins whether we ingest them as food or as pills -- 'microbial therapy' could solve this by coupling these production loops in our engineered bacteria
One way to put an end to this could be synthesizing bacteria that can inhabit the human stomach and produce vitamins of all kinds.
...that's the idea
Response (Stephanie): I really like this idea. I wonder what potential limitations there are, etc. Did you find any interesting readings on this?
Response (Alex): The great convenience of this is that E. Coli is not only a well-understood model organism, but also one of the most abundant gut flora. If we can actually introduce genes for vitamin synthesis, this is actually feasible. The only problem is that the genes which would do our vitamin synthesis would leach energy from the bacteria and make them less competitive than the native E. coli. Hence the native bacteria would eventually crowd out the modified ones.h
Project Ideas 3/19/07
Brainstorming with Sammy, Alex, Shaunak, Stephanie, Mingming, Perry, and George. TFs and Advisors in attendance Nick, Mike, Harris, Tamara, George Church, Jagesh Shah, William Shih, and Alain Viel.
Biological Based Fuel Cells
Bacterial that Respond to (by fluorescence) or Degrade Plaque
Viruses as a Transfer Mechanism
Engineering E. coli to Resist Mutations
--The intention is prevention of evolution that would ruin biological parts; however, we recognize that directed evolution is a useful tool in 'discovering' potentially useful parts and mutations. http://www.seas.harvard.edu/projects/weitzlab/Jeremy%20web%20page.htm
Cellulose to EtOH in Algae or other system
""~~Some Papers on this Subject (added by SAV, feel free to add more)11:22, 2 April 2007 (EDT)""
Lynd, Zyl, et al. "Consolidated bioprocessing of cellulosic biomass: an update" Current Opinion in Biotechnology 2005, 16:577-583
---This paper gives a pretty good overview of research into consolidated bioprocessing of cellulose into ethanol, and some of the main problems as well. If we're interested in looking at fuels, this is definitely a good paper to look at.
Jeffries, Thomas. "Engineering yeasts for xylose metabolism" Current opinion in biotechnology 2006, 17:320-326.
---This paper looks at turning xylose into ethanol via yeasts, and recent results in this field of research. If we think we might not want to use bacteria, this is a good overview.
Sticklen, Mariam. "Plant genetic engineering to improve biomass characteristics for biofuels." Current Opinion in Biotechnology 2006, 17:315-319
--Looks at problems from biomass cellulose, such as lignin, and current research into ways to treat it. Also looks at other ways to engineer plants. I think less relevant for us, but still interesting as a way for getting a feel of some of the issues surrounding biomass cellulose
Fatty Acid Production and Degradation for Energy
Sequestration of Toxic Compounds by Bacteria (arsenic)
Bacterial Surface Expression
Artificial Vascularization in Bacterial Biofilms
Bacterial Biosensors (Detection in the Environment) (Water Samples)
Project Ideas from Second Meeting (04/05/07) Additional notes added by Stephanie; please contact her with questions.
- Selection mechanisms for key/lock riboregulators (see 2006 Berkeley Project)
- Though sequence complementary is necessary, which allows little variation in that region, the rest of the RNA might differ
- The RNA acts as a "key" to release the lock; only when both are present, allows for expression
- This can allow for creation of networks, if the expression "unlocked" is for another key
- Monitored by Red Fluorescent Protein, experimentally
- Advantage = fast response
- Can be used for either activation or inhibition
- Suggestion: look into the Duke group: human encryption
- Biofuel & light sensitive proton pump (see background reading #3) (Pseudomonas Putida for exportation of short chain alkanes)
- Powering medical devices
- Bacteria that can extract energy from sugars in blood and convert these to electricity
- Question posed: how often do the devices need energy? A: Depends on specific devices
- Related idea: implantable devices that release, or even synthesize, drugs
- Artificial cells
- Use of psuedomonas putida? (bacterial strain)
- High tolerance to many saturated alkanes (can we get it to form octane?)
- Issue: export vs. metabolism
- Quorum sensing and biofilms
- Mirror image proteins
- Nonribosomal synthesis of proteins
- Radon sensor (practical considerations of working with Radon)
Short discussion of project logistics: rather than attempting to tackle multiple projects at once (as we will tend to be overambitious!), perhaps we can propose a sequence of experiments that we would like to attempt over the summer. We should treat these as 'checkpoints' and finish one before proceeding to the next.