|
|
| (6 intermediate revisions by the same user not shown) |
| Line 1: |
Line 1: |
| 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.
| |
|
| |
| 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.
| |
|
| |
| Check for Gram negative binding sequences in the smeg genome first.
| |
