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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 ([[Media: Brainstorming_Overview.ppt| 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)
== Quorum Sensing ==


------------------------------------------------------------------------------------------Final Project Ideas, Discussions, and Lobbying (post 4-23-07)<br>
Basically all of the parts that are available are derived from the [http://www.che.caltech.edu/groups/fha/quorum.html LuxI/LuxR system] found in ''V. fishcheri''. The Voigt paper that we read a long time ago used this system to demonstrate a cell-density dependent expression of invasin in E. Coli ([[Media:Voigt.pdf]]).  
<br>
I really like the idea of pathway engineering. I think we should stick to <i> E. coli </i> 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. [http://www.ibmb.uni.wroc.pl/prace2/praca12.pdf Commercial Production of Chemicals Using Pathway Engineering]. I particularly like the following excerpt: <br>
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.<br>[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.]<br>
... 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?


--Stephanie (4/25)
The basic idea that Perry and I discussed requires two basic parts which Perry transformed into three separate tubes of Top10 cells this afternoon:
# An HSL signal sender ([http://parts.mit.edu/registry/index.php/Part:BBa_F1610 BBa_F1610])
# An HSL signal receiver attached to a report
([http://parts.mit.edu/registry/index.php/Part:BBa_I13263 BBa_I13263] or
[http://parts.mit.edu/registry/index.php/Part:BBa_I13272 BBa_I13272])
The only difference between the two signal receivers is a different YFP reporter protein.


A quick rundown of how it will work: we will stick some sort of promoter in front of the [http://parts.mit.edu/registry/index.php/Part:BBa_F1610 BBa_F1610] in order to produce  constitutive expression of HSL. We can play around with which promoter we want to use in order to tweak the sensitivity of the system. This HSL will normally diffuse quickly. Meanwhile, there is constitutive expression of the protein produced after transcription and translation of luxR (called R from now on) is continually going on in the bacteria with the receiver. The HSL will bind to the R and these bound complexes will dimerize and activate transcription of the YFP reporter. However, normally the concentration of HSL is too low and the equilibrium highly favors unbound HSL and R. In areas of high cell concentration, the concentration of HSL will be great enough to shift the equilibrium toward the bound complex. This bound complex will then activate the transcription of the reporter gene. An interesting note is that the bound complex supposedly also represses the luxR gene according to the Biobricks parts list. However, I haven't found any confirmation of this. If it is true, then it means that along with the activation of transcription of the reporter gene the amount of luxR will decrease and transcription of the YFP reporter would probably decrease.


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:
There are a few ways we could approach this:
# Have both the signaller and receiver+reporter parts in the same plasmid
# Have the signaller part in one plasmid and the receiver+reporter part in another plasmid, both in the same bacterium
# Have two different bacteria: one with the signaller part and one with the receiver+reporter part


1. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Martin VJ, Pitera DJ, Withers ST, Newman JD, Keasling JD.    ([[Media: Martin.pdf| Nat Biotechnol. 2003 Jul;21(7):796-802.]])
Perry asked Mike about the first two possibilities and confirmed that both should be doable. The last possibility was demonstrated in the Voigt paper.


2. Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes.  Pfleger BF, Pitera DJ, Smolke CD, Keasling JD.  ([[Media: Pfleger.pdf| Nat Biotechnol. 2006 Aug;24(8):1027-32. ]])
I'm not completely sure what the advantages/disadvantages of each system is, but I think we should try all three possibilities.


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.
Also, if we can get this to work, we could potentially build more complex systems that involve logical gates. One example paper ([[Media:Pulse.pdf]]) used two bacteria and five separate parts controlled by inducible/repressible promoters two create a pulse of fluorescence.


--Mike (4/30)
== Signal Transduction ==


*Nick and I were talking over a paper on OmpR/PhoB and we thought of a possible method of making bacteria transduce a signal in response to binding our target.  The OmpR/PhoB family of membrane proteins dimerize and activate in response to binding a ligand.  Perhaps we can induce dimerization and activation of the proteins by holding them close together.


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.  
*We could do this by adding a library to the extramembrane portion of OmpR/PhoB and selecting for adhesion to a ligand.  Then we could co-express two different genes for OmpR+library in a single E. coli. Hopefully the two different target-binding portions will bind separate portions of the target, and will bring the two monomers of OmpR close enough together to activate them.


--Stephanie (5/1)
*Nick refined this idea by suggesting that we use a protein with clearly defined C-terminal and N-terminal domains as a target.  We could use a protease to snip the C-terminal and N-terminal domains apart, then separately select for libraries that bind to each.  Once we have sequences that bind to the C-terminal and N-terminal domains separately, we can combine those two sequences in the same plasmid. This would ensure that the two OmpR genes target different portions of the bacteria, and hopefully that they'll be brought close enough to dimerize. 
-Alex


*Papers of Interest
** [[http://www.hhmi.org/research/investigators/stock.html OmpR/PhoB Overview See Figure 1]
** A common dimerization interface in bacterial response regulators KdpE and TorR (OmpR family members) PMID: 16322582 Protein Sci. 2005 Dec;14(12):3077-88.
== Adhesion Targets ==


'''Bacterial Surface Expression and Cellular Targeting''' (compiled 5/2/07 by Mike)
Brainstorming of Possible Adhesion Targets (feel free to add/reorganize/whatever--I just thought I'd get this started):


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.
*Keratins -- was thinking of new lines of "hair conditioners" --> these tend to add body to the hair shaft by using bonding polymers; bacteria could be the new line of "bonding polyproteinecious entities" etc etc




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). 
'''textiles'''  
*silk
*wool
*polyester
*nylon fibres
*Could clone E coli that secrete pleasant smells and similarly bind to these fibers -- a living deodorant!




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.
'''eukaryotic cells'''
*Mosquito gut epithelial cells as targets




An outline of the strategy is as follows:
'''other mammalian'''
*Laminins
*fibronectin




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.
'''Mammalian cell surface proteins'''


''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]]


'''Protozoan membrane proteins'''
*By the way, I was thinking about expressing plasmodium falciparum proteins on the surface of E coli and maybe showing somehow that an immune rxn can be elicited in mice or something like that e.g.  in humans, "High levels of plasma chitotriosidase are a marker of macrophage activation..."
*Speaking on targets, heregoes:
*MSP-1 --> well characterized
*CSP
*EBA-175, a 175 kDa 'erythrocyte binding antigen' from P. falciparum
*DBP, Duffy-binding protein from P. vivax and P. knowlesi
*SSP2, Plasmodium sporozoite surface protein-2. Also known as TRAP (thrombospondin-related adhesive protein).
*Proteins with homology to SSP2/TRAP from Toxoplasma (MIC2), Eimeria (Etp100), and Cryptosporidium
*CTRP, circumsporozoite- and TRAP-related protein of Plasmodium found in the ookinete stage
*(see http://www.tulane.edu/~wiser/malaria/cmb.html#junction)
*MSP-1 polypeptide fragments


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.


'''Viral Membrane Proteins'''
*Hepatitis C surface proteins
**we could use the adhesion to these proteins to adapt the ELISA assay to the detection of such proteins '''in vitro'''
*[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=3882238&dopt=Abstract Influenza A M2 Protein]
*Neuraminidase
*Hemaglutinin
*Semliki Forest membrane proteins
*[http://jvi.asm.org/cgi/content/full/79/7/4415 Epstein Barr LMP1]
*[http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=249393 Epstein Barr LMP2]
*HIV gp120


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.


'''Cancer-Related Membrane Proteins'''
*[http://www.molecular-cancer.com/content/5/1/41 Eag1 Potassium Channels]
*[http://bioinformatics.org/pcgdb/Genes/muc1/muc1.htm Muc1]
*[http://cancerres.aacrjournals.org/cgi/content/abstract/67/4/1594 NGEP-L]
*[http://clincancerres.aacrjournals.org/cgi/content/abstract/7/12/4182?ck=nck HP59]
*[http://clincancerres.aacrjournals.org/cgi/content/abstract/2/11/1837 gp75] - Surface expression of gp75 on mouse melanoma cells correlates with the ability of a monoclonal antibody against gp75 to reject melanomas in syngeneic mice


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.
'''Assorted'''
*Presenilin


== 2 Component Systems ==


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).
* We should start researching these as well as the tagets. 
*Organ targeting in vivo using phage display peptide libraries. Pasqualini R, Ruoslahti E.
* Perhaps The TF's (myself included, of course) can help point out some papers with which to start.
Nature. 1996 Mar 28;380(6572):364-6.  [[Media:Pasqualini.pdf]]
- Nick
*How about arsenic sensor using an ''ars'' regulator, ''ars'' promoter and ''GFP'' gene -- we can have it just warn one if the arsenical concentration is greater than 5 ppb.


''Environmentally controlled invasion of cancer cells by engineered bacteria.
* Maybe look over the light sensitive bacteria paper again, they use a 2 component system there.
J Mol Biol. 2006 Jan 27;355(4):619-27.  Anderson JC, Clarke EJ, Arkin AP, Voigt CA.'' [[Media:Anderson.pdf]]
<biblio>
# sb1 pmid=16306980
</biblio>


''A bacterial protein enhances the release and efficacy of liposomal cancer drugs.
*Controlled spider-silk production in the presence of calcium ions or vitamin K
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]]
**Could have the bacteria attach to skin (keratin, using LppOmpA+appropriate peptide sequence) and then , since Ca2+ ions increase when one bleeds, could have the bacteria respond to this by spritzing out spider silk protein (antimicrobial). It will matter less that monocytes will attack these bacteria since we just want them to play a role early on during the wound-healing process (they may very well contribute to debris used to create a wound plug upon their death.
In case we still want to tackle the macrophage activity headon, then I suggest we immunoisolate the bacteria using alginate beads.
**A second application would be to select bacteria that have peptides with binding affinity to silks and then have them attach to individual silk yarns --> Thus, when the yarns are soaked in culture media, the bacterial secretions (silk) increase the thickness of the yarns --> conversely, when dried out, the bacteria are inactivated and subsequent washing reduces yarn thickness through shrinkage and wear. In effect, you'd have clothes that replenish themselves. Additionally, we could use the research by MIT's group last time on sweetsmelling ''E.coli'' to make yarns innately sweet-smelling.


== Cascade Network Structure ==


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.
* Some loose brainstorming on a possible reporter network that can be activated from the membrane protein as we discussed would be beneficial as well, if we plan to do something like that.
* May be good to start with something similar to what has been done previously and hook it up to GFP.
* Some TF direction would be warranted here as well.
- Nick


== Previous Brainstorming Ideas ==


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)
Btw, can we reorganize the brainstorming page to put the older brainstorming into folders or something and only have the stuff relevant to our project still on the front page? I have no idea how to do this...thanks. -Shaunak


Hope you like the formatting.<br>
--Stephanie (5/12)


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?
[http://openwetware.org/wiki/IGEM:Harvard/2007/Brainstorming/423to59 Brainstorming, 4/23 to 5/9]<br>
 
[http://openwetware.org/wiki/IGEM:Harvard/2007/Brainstorming/miscpre59 Misc Brainstorming, pre-5/9]<br>
--Stephanie (5/2)
[http://openwetware.org/wiki/IGEM:Harvard/2007/Brainstorming/405 Brainstorming from Second Meeting, 4/05]<br>
[http://openwetware.org/wiki/IGEM:Harvard/2007/Brainstorming/319 Brainstorming from First Meeting, 3/19]<br>




------------------------------------------------------------------------------------------
------------------------------------------------------------------------------------------
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.
Alexander
--------------------------------------------------
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
Alexander
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.
Stephanie
--------------
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
<br>
<br>
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
<br>    --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)""
<br>
Lynd, Zyl, et al. "Consolidated bioprocessing of cellulosic biomass: an update" Current Opinion in Biotechnology 2005, 16:577-583
<br>
---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.
<br>
Jeffries, Thomas. "Engineering yeasts for xylose metabolism" Current opinion in biotechnology 2006, 17:320-326.
<br>
---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.
<br>
Sticklen, Mariam. "Plant genetic engineering to improve biomass characteristics for biofuels." Current Opinion in Biotechnology 2006, 17:315-319
<br>
--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
Molecular Motors
Sequestration of Toxic Compounds by Bacteria (arsenic)
Bacterial Surface Expression
Vascular Tissues
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
<br> - The RNA acts as a "key" to release the lock; only when both are present, allows for expression
<br> - This can allow for creation of networks, if the expression "unlocked" is for another key
<br> - Monitored by Red Fluorescent Protein, experimentally
<br> - Advantage = fast response
<br> - Can be used for either activation or inhibition
<br> - Suggestion: look into the Duke group: human encryption<br>
* Biofuel & light sensitive proton pump ([[Background_Reading|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
<br> - Question posed: how often do the devices need energy? A: Depends on specific devices
<br> - Related idea: implantable devices that release, or even synthesize, drugs
<br>
* Artificial cells
* Use of psuedomonas putida? (bacterial strain)
- High tolerance to many saturated alkanes (can we get it to form octane?)
<br> - Issue: export vs. metabolism
* Quorum sensing and biofilms
* Mirror image proteins
* Nonribosomal synthesis of proteins
* Radon sensor (practical considerations of working with Radon)
<br> 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.

Latest revision as of 19:56, 25 June 2007

Quorum Sensing

Basically all of the parts that are available are derived from the LuxI/LuxR system found in V. fishcheri. The Voigt paper that we read a long time ago used this system to demonstrate a cell-density dependent expression of invasin in E. Coli (Media:Voigt.pdf).

The basic idea that Perry and I discussed requires two basic parts which Perry transformed into three separate tubes of Top10 cells this afternoon:

  1. An HSL signal sender (BBa_F1610)
  2. An HSL signal receiver attached to a report

(BBa_I13263 or BBa_I13272) The only difference between the two signal receivers is a different YFP reporter protein.

A quick rundown of how it will work: we will stick some sort of promoter in front of the BBa_F1610 in order to produce constitutive expression of HSL. We can play around with which promoter we want to use in order to tweak the sensitivity of the system. This HSL will normally diffuse quickly. Meanwhile, there is constitutive expression of the protein produced after transcription and translation of luxR (called R from now on) is continually going on in the bacteria with the receiver. The HSL will bind to the R and these bound complexes will dimerize and activate transcription of the YFP reporter. However, normally the concentration of HSL is too low and the equilibrium highly favors unbound HSL and R. In areas of high cell concentration, the concentration of HSL will be great enough to shift the equilibrium toward the bound complex. This bound complex will then activate the transcription of the reporter gene. An interesting note is that the bound complex supposedly also represses the luxR gene according to the Biobricks parts list. However, I haven't found any confirmation of this. If it is true, then it means that along with the activation of transcription of the reporter gene the amount of luxR will decrease and transcription of the YFP reporter would probably decrease.

There are a few ways we could approach this:

  1. Have both the signaller and receiver+reporter parts in the same plasmid
  2. Have the signaller part in one plasmid and the receiver+reporter part in another plasmid, both in the same bacterium
  3. Have two different bacteria: one with the signaller part and one with the receiver+reporter part

Perry asked Mike about the first two possibilities and confirmed that both should be doable. The last possibility was demonstrated in the Voigt paper.

I'm not completely sure what the advantages/disadvantages of each system is, but I think we should try all three possibilities.

Also, if we can get this to work, we could potentially build more complex systems that involve logical gates. One example paper (Media:Pulse.pdf) used two bacteria and five separate parts controlled by inducible/repressible promoters two create a pulse of fluorescence.

Signal Transduction

  • Nick and I were talking over a paper on OmpR/PhoB and we thought of a possible method of making bacteria transduce a signal in response to binding our target. The OmpR/PhoB family of membrane proteins dimerize and activate in response to binding a ligand. Perhaps we can induce dimerization and activation of the proteins by holding them close together.
  • We could do this by adding a library to the extramembrane portion of OmpR/PhoB and selecting for adhesion to a ligand. Then we could co-express two different genes for OmpR+library in a single E. coli. Hopefully the two different target-binding portions will bind separate portions of the target, and will bring the two monomers of OmpR close enough together to activate them.
  • Nick refined this idea by suggesting that we use a protein with clearly defined C-terminal and N-terminal domains as a target. We could use a protease to snip the C-terminal and N-terminal domains apart, then separately select for libraries that bind to each. Once we have sequences that bind to the C-terminal and N-terminal domains separately, we can combine those two sequences in the same plasmid. This would ensure that the two OmpR genes target different portions of the bacteria, and hopefully that they'll be brought close enough to dimerize.

-Alex

  • Papers of Interest
    • [OmpR/PhoB Overview See Figure 1
    • A common dimerization interface in bacterial response regulators KdpE and TorR (OmpR family members) PMID: 16322582 Protein Sci. 2005 Dec;14(12):3077-88.

Adhesion Targets

Brainstorming of Possible Adhesion Targets (feel free to add/reorganize/whatever--I just thought I'd get this started):

  • Keratins -- was thinking of new lines of "hair conditioners" --> these tend to add body to the hair shaft by using bonding polymers; bacteria could be the new line of "bonding polyproteinecious entities" etc etc


textiles

  • silk
  • wool
  • polyester
  • nylon fibres
  • Could clone E coli that secrete pleasant smells and similarly bind to these fibers -- a living deodorant!


eukaryotic cells

  • Mosquito gut epithelial cells as targets


other mammalian

  • Laminins
  • fibronectin


Mammalian cell surface proteins


Protozoan membrane proteins

  • By the way, I was thinking about expressing plasmodium falciparum proteins on the surface of E coli and maybe showing somehow that an immune rxn can be elicited in mice or something like that e.g. in humans, "High levels of plasma chitotriosidase are a marker of macrophage activation..."
  • Speaking on targets, heregoes:
  • MSP-1 --> well characterized
  • CSP
  • EBA-175, a 175 kDa 'erythrocyte binding antigen' from P. falciparum
  • DBP, Duffy-binding protein from P. vivax and P. knowlesi
  • SSP2, Plasmodium sporozoite surface protein-2. Also known as TRAP (thrombospondin-related adhesive protein).
  • Proteins with homology to SSP2/TRAP from Toxoplasma (MIC2), Eimeria (Etp100), and Cryptosporidium
  • CTRP, circumsporozoite- and TRAP-related protein of Plasmodium found in the ookinete stage
  • (see http://www.tulane.edu/~wiser/malaria/cmb.html#junction)
  • MSP-1 polypeptide fragments


Viral Membrane Proteins


Cancer-Related Membrane Proteins


Assorted

  • Presenilin

2 Component Systems

  • We should start researching these as well as the tagets.
  • Perhaps The TF's (myself included, of course) can help point out some papers with which to start.

- Nick

  • How about arsenic sensor using an ars regulator, ars promoter and GFP gene -- we can have it just warn one if the arsenical concentration is greater than 5 ppb.
  • Maybe look over the light sensitive bacteria paper again, they use a 2 component system there.
  1. Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, Davidson EA, Scouras A, Ellington AD, Marcotte EM, and Voigt CA. Synthetic biology: engineering Escherichia coli to see light. Nature. 2005 Nov 24;438(7067):441-2. DOI:10.1038/nature04405 | PubMed ID:16306980 | HubMed [sb1]
  • Controlled spider-silk production in the presence of calcium ions or vitamin K
    • Could have the bacteria attach to skin (keratin, using LppOmpA+appropriate peptide sequence) and then , since Ca2+ ions increase when one bleeds, could have the bacteria respond to this by spritzing out spider silk protein (antimicrobial). It will matter less that monocytes will attack these bacteria since we just want them to play a role early on during the wound-healing process (they may very well contribute to debris used to create a wound plug upon their death.

In case we still want to tackle the macrophage activity headon, then I suggest we immunoisolate the bacteria using alginate beads.

    • A second application would be to select bacteria that have peptides with binding affinity to silks and then have them attach to individual silk yarns --> Thus, when the yarns are soaked in culture media, the bacterial secretions (silk) increase the thickness of the yarns --> conversely, when dried out, the bacteria are inactivated and subsequent washing reduces yarn thickness through shrinkage and wear. In effect, you'd have clothes that replenish themselves. Additionally, we could use the research by MIT's group last time on sweetsmelling E.coli to make yarns innately sweet-smelling.

Cascade Network Structure

  • Some loose brainstorming on a possible reporter network that can be activated from the membrane protein as we discussed would be beneficial as well, if we plan to do something like that.
  • May be good to start with something similar to what has been done previously and hook it up to GFP.
  • Some TF direction would be warranted here as well.

- Nick

Previous Brainstorming Ideas

Btw, can we reorganize the brainstorming page to put the older brainstorming into folders or something and only have the stuff relevant to our project still on the front page? I have no idea how to do this...thanks. -Shaunak

Hope you like the formatting.
--Stephanie (5/12)

Brainstorming, 4/23 to 5/9
Misc Brainstorming, pre-5/9
Brainstorming from Second Meeting, 4/05
Brainstorming from First Meeting, 3/19


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