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6/19 (Monday)

Morning presentations

Cyanobacteria oscillator

  • It will probably be infeasible to reconstitute the cyanobacteria oscillator in E. coli, since we don't have any way to make phosphorylated KaiC do work, without cyanobacteria's myriad transcription factors
  • Previous researchers have succesfully inserted luciferase in cyanobacteria to produce oscillating luminescence
  • Idea: replace luminescence with production of a signaling chemical (e.g. AHL), and engineer E. coli to react to it, so cyanobacteria is like an external oscillator for E. coli.

Afternoon brainstorming

Cyanobacteria oscillator

DNA nanostructures

  • Spend 90 minutes discussing container designs tomorrow


Streptavidin Refrences

  1. Sano T and Cantor CR. Expression of a cloned streptavidin gene in Escherichia coli. Proc Natl Acad Sci U S A. 1990 Jan;87(1):142-6. DOI:10.1073/pnas.87.1.142 | PubMed ID:2404273 | HubMed [Initial_Streptavidin_Cloning]
  2. Sano T and Cantor CR. Streptavidin-containing chimeric proteins: design and production. Methods Enzymol. 2000;326:305-11. DOI:10.1016/s0076-6879(00)26061-8 | PubMed ID:11036649 | HubMed [Chimeric_Streptavidin_Proteins]
  3. 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 | PubMed ID:16600968 | HubMed [Displaying_Proteins_on_the_Cell_Surface]
  4. de Wolf FA and Brett GM. Ligand-binding proteins: their potential for application in systems for controlled delivery and uptake of ligands. Pharmacol Rev. 2000 Jun;52(2):207-36. PubMed ID:10835100 | HubMed [Ligand_Binding_Review]
  5. Sano T and Cantor CR. Expression vectors for streptavidin-containing chimeric proteins. Biochem Biophys Res Commun. 1991 Apr 30;176(2):571-7. DOI:10.1016/s0006-291x(05)80222-0 | PubMed ID:2025272 | HubMed [Technical_Details]

All Medline abstracts: PubMed | HubMed

Transcription circuits compared to Post-Translational circuits

6/16 Morning with William Shih

See IGEM:Harvard/2006/Brainstorming with William.

6/15 Afternoon

  • Bacteria temperature regulation: HOT PLATE
  • Heavy metal sequestration
  • Myosin-chained moving nanostructures
  • Ion-based radiating signal
    • For BioBurglarDetection
    • Needs:
  1. Bacterial ion-channel
  2. Bacterial ion pump
  • XOR gate
  • Firing Squad problem
  • Jackson Pollack painting bacteria
  • Sudoku/Magic Square solving bacteria
  • Useful-compound producing bacteria (ie. hijacked)
  • Self-assembling bacteria

6/15 Morning

- telomeres

 timer/fuse/counter (what is the point and applicable/generalizable?)
    timed order of events, cascade of timers
 telomerase: telomere length regulation, involves mammalian cells
 biobricks: in context artificial chromosomes (~100 kb)
 CS analogies: while/until constructs
 probably in yeast rather than mice
 placing mini-circuits on the ends of telomeres 
 lit.: Vicki Lindblad, Liz Blackburn, Check (sp?), Murray & Szostak (Chromosome length controls  
       mitotic chromosome segregation in yeast)
 A. Murray, G. Church

- cyanobacteria oscillation

 applicable to global warming idea
 create an oscillator: biobricks? clock?
 how do you report oscillation (idea: add GFP domain at end, put GFP on protein which can 
    stabilize), codon usage?, antibodies?
 NOTE: On codon usage
  Two paper reviews
 Also see Hetmann and Katie for additional research/papers

- nanostructures

 container that opens
 practical uses: drug delivery? how does it act w/in body - circulating DNA may be targeted
 issues: very porous (what to put inside...), dynamic applications? 
 nanorobotics - building mini-machines
 guidelines: biobricks? new kind of part? combine w/ other ideas?

- fusion proteins

 making parts toward certain goal
 work with DNA nanostructures: restriction enzymes (box having lid w/ restriction enzyme site),
    DNA binding proteins (figure out binding sites)
 receptors: engineering outside protein to "plug in" to ligand pathway
 sensors: odors, sweat (better polygraph machine)
 synthetic biology 2.0 talks
 engineered small molecule recognition - mutation of maltose binding site

- biocryptography?

 alternative splicing
 reconstructin simple alternative splicing systems
 decode alternative splicing

Brainstorming Paper Summaries


Go Here


from Tiff

Further thoughts on the DNA-origami-delivery idea:

  • Containers
    • How can we get things to open and close?
      • Latch that closes by default, opens upon binding
      • Entire container that's "closed" normally, opens upon some sort of signal
        • Has anyone read/heard Roderick MacKinnon, Nobel Prize winner for sussing out potassium ion channel structure? He gave these great talks a year or two back, and he showed a pretty animation of how the channels gate, using large motions of the six helices involved in the channel. They kind of twist out to open, twist in tight to close - like when you pull of a piece of those Twizzler's Pull and Peels, only in reverse, and all six at once. I know the scale of the ion channel is pretty low (I mean, it's meant to delive IONs, and dehydrated ones at that), but would it be possible to engineer something on a much larger scale w/ DNA helices?

  • Sensibility
    • This idea of using origami for delivery only makes sense if:
      • The molecule we need to deliver can't be done more efficiently in some other way
      • The origami is big enough to hold a decent number of copies of the molecule
      • The big origami can get into the area it needs to be in in the first place
  • Let's do it!
  • Other Notes
    • In Chem 285, we spent a lot of time talking about problems with getting small molecules to get where they ought to get for efficacy. Maybe we could see if one of those molecules would make sense for delivery? (I didn't pay enough attention/don't have such a good long term memory to pull a molecule off the top of my head.)
    • My pet interest is small non-coding RNA - ex. microRNAs, artificial RNAs used in RNAi - and another thing we saw in Chem 285 was the difficulty of getting RNA or even DNA into cells, to do their magic. What if we could make a structure that porated cells and inserted RNA or DNA+transcription-components? In other words, a virus, but low-damage - without the massive lysis we see for lots of viruses, maybe one hole per cell, or do it in such a way that the cell pinocytotes/phagocytotes the contents. And it has to be unhijackable by bad DNA.
    • Last summer I was working on Yersinia enterocolitica, a relative of Yersinia pestis (ie. bubonic plague). The Yersinias and a few other types of bacteria use Type III secretion, which means they jab a hole into a host cell using their needle structure (which grows only when it gets close to the low-Mg2++ cell membrane environment) and import proteins (or, debatably, small non-coding RNAs) into the host. Could we use that? (Just an idea, I don't really know what it would be useful for, but it's kind of cool.)
    • Does double-stranded DNA (dsDNA) get immunologically-chewed-up when it's free-floating around the body? That might be a problem. (I remember that bacterial single-stranded DNA did - or I think I remember that, along with CpG islands... man, I did really poorly in Immunology.)

Hope everyone's exams are going well, see you all soon!

from Tiff

(From the mailing list email I sent out awhile ago)

I'd just like to suggest that we consider doing something non-cloning based, as in Willliam Shih's DNA structure stuff.

Reasons Why:

  • cloning is probably going to be a LOT slower (and more difficult to get working, from personal experience) than what he was talking about
  • he's one of the world's experts, so I'm sure that with his guidance we could do something quick and error-free
  • it'll be so different and so much cooler than anything else any other group could think of, because it won't be bacterial-based
  • it's cutting edge
  • drug delivery systems are hot, and necessary, and this is seriously stuff NO ONE ELSE has ever thought of or done before
  • if we're going to try to make something with human (ie. medical) importance, starting in bacteria is good, but the eventual transition to eukaryotes is no fun to actually do (ie. transfection)

Reasons Against:

  • it won't be BBa_, so it might not be so legit in the competition
  • none of us probably have any experience doing stuff like that, but some of us already know how to clone (if I'm wrong, please correct me :))

from Dave

Hey everyone, here are some very high-level computer science-ish ideas I came up with tonight. I have no idea how hard these would be to implement, but I'll just throw these out there to get the spherical fullerene (haha) rolling:

  1. I really liked the idea of containers built using scaffolded DNA origami, and exploring these might prove useful. Once we figure out how to seal the container and get it to open in response to certain stimuli, it might be interesting to create a set of these that respond to different stimuli. I assume that the lids would have to be different in each case, which might be hard to implement in some cases.
  2. I was looking at MIT's 2004 brainstorming and saw some interesting stuff concerning insulin production/regulation in the bloodstream. We could even combine this with the containers, constructing cell "factories" that produce containers of insulin. These containers could then open when the concentration of blood glucose gets too high. I'm not exactly sure what benefits (if any) this extended scheme might provide, except maybe a quicker response to rising blood sugar levels since the insulin is already produced.
  3. cell instant messaging: One cell can send a message that will propagate through the network and reach a unique destination cell. The important thing would be that the intermediate cells wouldn't interact with the message; they would simply know it was a message and pass it on. Sort of like passing the salt across the table without everyone using it on the way. Difficult? Possible? No idea. Although, it would be cool if a cell realized it had certain deficiencies and was able to "query" the cellular network to find a provider for what it needed. The part could then be passed back to the cell in need. Once again, the DNA containers could be used but I'm sure there are plenty of ways to do this. We would also need to figure out how to send queries across the network and send packages back, which could be difficult.

Once again, these are very high level ideas; it's pretty late and I haven't really researched anything in detail. I'm not sure what's feasible, but hopefully this leads to something useful. Feel free to comment/edit as you like. ~Dave

from Perry

Here are some ideas that were mentioned in the mcb100 group last fall, when we were considering trying to develop our own system. some are really bad/impractical, i know, but hopefully these start some brainstorming.

- alarm clock, reporter in response to time lapse or sunlight

- stop watch, reporter expressed over time

- thermometer. different reporters or different amounts of one reporter depending on temperature.

- chemical detector. one idea was ethanol, maybe detecting different proofs; another idea was carbon monoxide.

- chip. 8 colonies = 8 bits? [i think this was mentioned in yesterday's lecture as having been attempted with failure thusfar.]

- flashlight. bioluminescent reporter gene expressed in the absence of light, or in other words, light acts as a repressor.

- litmus paper. one of two different colored reporter genes expressed in acidic or basic pH.

- color-by-numbers. there are those books that have the outlines drawn with numbers in the spaces, and a legend tells you which color you should fill into which numbered spaces. so here we would have a lawn of bacteria, separated physically into sections, and you would induce different sections to show different colors according to the stimulus: basically a multiple chemical detector system. the stimuli could diffuse to activate all bacteria in a section, or the stimuli could activate one bacterium which would send out the signal.

- wire. there was a BioWire project last year which send a coloration signal down a line of bacteria. can an electric signal be carried, maybe through ions? i guess this would require membrane ion-gates.