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. This may be possible because mycobacteria have the gram-positive version of the lexA binding sequence, while E. coli has the gram negative. the gram-positive Cheo box is: TACATT (-10) and TTGGTG (-35). Gram negative is TATAAT (-10) and (TTGACA) -35. Apparently Mycobacterial lexA does not bind to the E. coli promoter strongly.

Ligate 25 sites one after another, with lexA-YFP fusion? Use pMV361 as the carrier so as to get nice integration.

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