Some light was shining in from the streetlights at university, still the glow was visible.  

  Petri disk alight hand Petri disk alight6

Here’s an actual map of pVIB that shows the Insert (from Vibrio Fischeri genomic DNA) between the two SalI sites. It was inserted into the SalI site from pBR322, disrupting the Tetracyclin resistance gene. BglII cuts within the luxI coding site (in the green line), so the Ribosome binding site of LuxC is still intact. Thus by restiction with SalI and BglII you get a functional LuxCDABEG casette. Just attach a constitutive promoter before it (BglII), to get constitutive expression. Or a chloroplast promoter, to get glowing chloroplasts. Only one thing is annoying – there is a transcription terminator directly after LuxG (between LuxG and SalI site). No restriction site inbetween to cut away the terminator.

The green fragment (~1700 bp) contains a cell density induced Promoter, and the LuxI gene, and LuxR in the opposite direction. The native Lux cassette constantly produces the LuxR protein, and small amonuts of LuxI (which is an enzyme that produces the autoinducer). The autoinducer diffuses throgh the cell wall into the medium, and to some extent also in other cells. At high cell densities, much autoinducer is in both the medium and in the cell. The autoinducer binds to LuxR protein which then activates strong expression (1000-fold the constitutive expression) of the LuxICDABEG operon. (LuxI is then also  expressed at higher levels).

pVIB restriction (2)

Someday I will retransform pVIB and then add myristic acid to the petri dish. That should enhance the glow significantly. pVIB on steroids 😀

Advertisements