Thu May 15 12:22:00 EST 1997


I have constructed a transcriptional lac fusion on a
high copy, ColE1-based vector.  This fusion was supposed
to allow me to monitor transcriptional regulation of my gene.
Unfortunately,  the high copy number appears to be interfering
with regulation.   I would like to know if it possible to use this
construct to create a chromosomal fusion.

The fusion looks something like this:

 upstream - lacZYA - tet - dowstream

The upstream portion is approximately 600 bp and contains
the promoter region (ie -35, -10, start codon and putative regulatory 
regions) for my gene and some upstream, flanking sequence.
The downstream region is 500 bp and contains the 5' end of 
the gene and additional, downstream flanking sequence.
The upstream and downstream regions are homologous to
sequences on the chromosome.
The promoterless lacZYA reporter and tet marker region is
about 8 kbp.

Is it possible for the intact (ie circular) plasmid to undergo 
recombination with the chromosomal copy of the gene and
thus create a chromosomal lac fusion?

Would recombination frequency be better with a linearized plasmid
even if linearization reduces the length up upstream DNA to 300 bp?
(my only unique restriction site is in the upstream region)

How might recombination frequency differ in  recA(-),
recD(-) and wild type [recA(+) rec D(+)]  E. coli?

Thanks in advance for any help


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