Madhadron:ChromosomeMarker

This protocol is a port of the system described in Nielsen, Ottesen, etc. (2006) in Molecular Microbiology to mycobacteria.

Background

Two component system from phage: parS is a binding site for the ParB protein. parS is inserted in the chromosome (a single copy appears to be sufficient). ParB is expressed inducibly as a fusion protein with a GFP on a plasmid.

Two organisms already adapted thus: P1 phage, and pMT phage (from Y. pestis; uses a deletion of the the 23 N-terminal amino acids of ParB instead of the whole thing).

The system depends on ihf genes in E. coli, and there is the HU homolog.

Alternately, make long strings of lexA sites. Mycobacterial LexA binds to Gram-positive style Cheo box. According to Movahedzadeh et al, 1997 the binding sequences are:

Gram negative SOS box: taCTGTatatananaCAGta

Gram positive Cheo box: GAACNNNNGTTC

Ligate 25 sites one after another, with lexA-YFP fusion? Use pMV361 as the carrier so as to get nice integration. First 83 residues of LexA encode the DNA binding domain. 84-85 is the autocleavage site, so stay before that. Need to get antibodies against both that piece of LexA and the fluorescent protein, so can check that we aren't getting cleavage by purifying with one, and blotting against the other. LexA (in E. coli) structure in Fogh et al, 1994.

Check for Gram negative binding sequences in the smeg genome first.

Probably won't get total penetrance (if there are ten chromosomes, plasmid may only integrate into one or two), but rtPCR or DNA blotting is sensitive enough to give full number. Just use primers or restriction sites that contain the area with the insert.