Difference between revisions of "Madhadron:ChromosomeMarker"

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This protocol is a port of the system described in Nielsen, Ottesen, etc. (2006) in Molecular Microbiology to mycobacteria.
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 [http://www.citeulike.org/user/madhadron/article/977068 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 [http://www.citeulike.org/user/madhadron/article/992756 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.
LexA DNA binding domain fused to GFP apparently works in yeast.  See [http://www.citeulike.org/user/madhadron/article/1002084 Abedi et al, 2001].  I can't find anyone who's done it with ''E. coli'' LexA.
Start with pND-UV-GFP, a plasmid from Neeraj based on pMV361 which has a strong promoter and a GFP in position to make a fusion protein.  Insert the multimerized binding site at BspHI site upstream of the promoter, and the LexA fragment at the PacI site between promoter and GFP.
Binding site: 6bp of noise -- BspHI -- NarI -- SOS box -- ClaI -- EcoRV -- 6bp of noise
LexA 5': PacI -- 20bp of homology, 3': PacI -- 20bp of homology
Patrick says lysine, serine, aspartic acid, and asparagine generally produce unstructured linkers.  There's some paid of amino acids which yields rigid, rodlike linkers.  Ask a structural biologist.

Latest revision as of 03:14, 19 October 2007