Lux operon

From 2006.igem.org

This is a page about the Lux operon ([http://www.citeulike.org/user/BUiGEM/tag/gene_luxcdabe CiteULike]) and the technicalities of making it into a Part in the [http://partsregistry.org Registry].

Background

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).

Questions

• 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?)

Progress

"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 [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=5726577 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." [http://www.citeulike.org/user/BUiGEM/article/683483 Van Dyk 1994]