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Isolate luxCDABE and add the 4 BioBrick restriction sites to the ends of the gene.
- Lab time: Wednesdays
- Upcoming Meetings:
- Wednesday July 26th - 3pm Ingalls
Mutagenesis in order to remove XbaI site from middle of luxCDABE.
- Alex K.
- Lab time: Tuesdays
- Upcoming Meetings:
Assembly, testing, and characterization of the LacZ photography system.
- Lab time: Mondays
- Upcoming Meetings:
Assembly of the luxCDABE BioBrick system and testing.
- Alex P.
- Lab time: Fridays
- Upcoming Meetings:
So the main task, as I understand it, is this (and I might have some of the specifics a bit wrong, so take this with a grain of salt): we want to make a new Part that contains the Lux operon (for clarity I am going to capitalize "Part" when I use it in the sense of a genetic part from the Registry). The physical manifestation of a Part is a special DNA sequence called a "BioBrick standard biological part". There are variety of BioBrick plasmids that contain one or more Parts, but all have fundamentally the same design: all contain an antibiotic resistance gene (a selector), an origin of replication, perhaps some other things, and the actual Part that has "BioBrick ends." The BioBrick ends consist of specific patterns of restriction sites which bracket the Part in a standard way, so that this section of the plasmid is always organized in the following manner: EcoRI - XbaI - INSERT - SpeI - PstI, where INSERT designates the actual functional Part (e.g., the Lux operon). It is essential that these four particular restriction sites occur only once -- and in the given order -- in the plasmid because their standard organization and single occurrence in each BioBrick enables the modularity and composability of the Parts. Hence the INSERT - the Part - must not contain any of the four restriction sites. I haven't checked to see if the Lux operon contains any of them, but it's pretty big, so chances are it does, and thus our primary challenge will be to re-engineer the operon to remove these sites (via the introduction of silent point mutations).
- Where do we get the operon and which one do we use?
- Into which BioBrick chassis should we insert it?
- What would be the most generally useful design?
- a luxCDABE part and a luxI part, to facilitate reporter functions (where does luxR go?)
- "Proteins that affect the wavelength of the emitted light, lumazine and yellow fluorescent protein, have been isolated from Photobacterium and Vibrio species, respectively. The lumazine proteins shift the color of the light to wavelengths shorter than 490 nm..." (Meighen 1991) Perhaps we could build a circuit to modulate the emitted wavelength by periodically expressing a carefully-chosen fluoresent protein. Think FRET and BRET.
- Let's modify the lux operon so our bacteria can play Conway's Game of Life. In the game, discrete "cells" interact with one another according to four extremely simple rules, which essentially boil down to this: if a cell has too many or too few neighbors it turns off, otherwise it turns/stays on. These rules and the initial state of all the cells often produce systems of fascinating and lifelike complexity. Perhaps we could add a circuit such that LuxI would only be activated in response to a narrow "medium" range of concentrations of its autoinducer (3OC6HSL), not too much or too little. In fact, I think such a circuit has already been built by the Weiss lab and demonstrated with their infamous bullseye. Damn! It looks like the Weiss lab already beat us to the Game of Life idea. Daaaarn. Well, maybe we could do it better, or something. Actually initialize the system with an interesting pattern using something like the UTAustin biofilm system. I mean, we could run a gigantic board! In 3D! With bioluminesence! I wonder at what threshold of gamespace our massively parallel computation would begin to outperform the serial speed of a modern desktop computer? A cubic foot? A cubic meter? Besides which, it would be so ironic.
"Vibrio fischeri regulatory protein LuxR (luxR) gene, complete cds; luxICDABEG operon, complete sequence; and unknown gene." Check out who the first author is: one T. Knight, Artificial Intelligence Laboratory, MIT! We should check and see if he still has some! PubMed GI:5726577
"Plasmid pUCD615, containing the V. fisheri luxC, luxD, luxA, luxB, and luxE genes without a promoter, was the parent plasmid for two genetic constructions... These plasmids were placed by CaCl2 transformation into two E. coli strains." Van Dyk 1994
Illuminesence cassette on puc19
Tom Knight has streaked out two strains of E. coli for us on Amp+ plates. One strain contains a Vibrio fischeri MJ1 luminescence cassette on a puc19 vector and the other a Photorhabdus luminescens luminescence cassette on a puc19 vector. (They spent the night growing above my refrigerator.) The vectors page in OpenWetWare has a link to the sequence of puc19 in ncbi, as well as some other vector-related resources like plasmid-plotting software.
- He mentioned that we probably wouldn't want the V. fishcheri operon because its luminescent activity is heat-sensitive and only occurs at relatively low temperatures (below 30 C?), so we should start investigating P luminescens.
- He also mentioned that they would work best in liquid culture with some agitation to facilitate the diffusion of oxygen into solution, which is in high demand for the luminescence reaction.
I'm bringing the plates to Dr. Gardner's lab today and will get them incubating and then growing in TMB or something else appropriate.
The accession number for the V. fischeri cassette is AF170104, which I sequenced a while back. The accession number for the P. luminescens cassette is M90093, sequenced by Ed Meighan. If you need them, I have genomic DNA and living cells of both of these strains. You might want to grow some of them up just to see the native luminescence. The V. fischeri MJ1 strain requires marine medium, whose composition I can get for you. If you need help in designing BioBricks from these, I'm here for (cheap) consultation. Tom Knight Tk 20:26, 8 June 2006 (EDT)
Plasmids for you
I've been given to understand that you had no luck growing the cultures I streaked for you. I've redone the plates, and grown them up here, verifying that they are luminescent. Come and get them... Tk 12:21, 21 June 2006 (EDT)
According to Tom Knight, the Photorhabdus luminescens luxCDABE operon that he cloned is NCBI accession number M90093. I checked this sequence against the BioBrick restriction enzymes (EcoRI, XbaI, SpeI, PstI, NotI) using the Sequence Manipulation Suite. Results: EcoRI cuts at the ends of the sequence (+2 and -4; i.e., the original sequence is intended to be cut out of its vector with EcoRI); XbaI cuts in the middle (+2411); and SpeI, PstI, and NotI do not cut M00093. The question therefore becomes, did Tom Knight's group add or remove restriction sites? We have the DNA, we can test this in lab.
- could we print LB + bacteria onto paper with an inkjet?
- what about printing the AHL quorum signals? Or other chemicals that could spatially control E. coli or compel them to do something (i.e., cause them to "print" or digest a substrate)?
- If we are successful with our light-repressed luminescence system, to what civic uses could we put it? Pathway/sidewalk lighting?
- What about re-engineering E. coli to repair things... such as fading paint on a building? I guess that's just like bioremediation.
- This is really wild: what if we understood the process from stem cell to tooth so well that we could predict and remodel the final structure using computers and then reprogram the stem cells for biofabrication? It wouldn't exactly be rapid prototyping, but it would be massively cheap!
Re: from what I thought, the understanding of stem cell lineage is not very well documented, with lots of discrepancies, and laboratory techniques to recreate them are few and very difficult! I may be wrong tho...
- I know people have transformed the membrane oscillations of yeasts into audible sounds (using an atomic force microscopes) and I wonder if E. coli could be signalled or manipulated in some way with acoustic waves.
--Mac 00:35, 14 June 2006 (EDT)