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 plasmid called a "biobrick". There are variety of biobricks, 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 + "biobrick ends." The biobrick ends are a specific sequence 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 Part ( i.e. the Lux operon). It is essential that these four particular restriction sites occur only once, in the given order, in the plasmid, for their standard organization and single occurance in each biobrick enables their modularity. Hence the INSERT - the Part - cannot contain any of the four restriction sites. I haven't checked 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 rengineering the operon to lack these sites (via the introduction of silent point mutations).
- Where do we get the operon / which one do we use?
- Into which biobrick chassis should we insert it?
- What would be the most generally-useful design?
- a luxCDABE-part & a luxI part, to facilitate reporter functions (where does luxR go?)
- "Proteins that affect the wavelength of the emitted light, lumazine and yellow fluorescence protein have been isolated from Photobacterium and Vibrio species, respectively. The lumazine proteins shift the color of the light to shorter wavelengths than 490 nm..." (Meighen 1991) Perhaps we could build a circuit to modulate the emitted wavelength by periodically expressing a carefully-chosen fluoresence 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 only 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 illuminescent 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 in 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, which I can get you the composition of. If you need help in designing biobricks from these, I'm here for (cheap) consultation. Tom Knight Tk 20:26, 8 June 2006 (EDT)
Also see the LuxCDABE listing in prodoric.
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)