Hi all,
What I have to ask to this newsgroup is probably kind of silly but here it goes.
I am not a microbiologist but for some reasons (I don't want to go into
details but will if you want me to) I need to make an E.coli strain with a
modified functional lacZ gene (that I have almost made already) inserted in
its genome.
I don't know exactly how to proceed since my knowledge in the field is
close to nil.
What I have is the modified lacZ' gene (coding for alpha peptide) on the
pGEM7Z vector. The modification is a short in-frame insertion in the
N-terminal part (polylinker of pGEM7Z) of the gene.
I have two alternatives from here:
1) insert this lacZ' gene in a DH5alpha-like strain (i.e. carrying a
delta(lacZ)M15 insertion or episome) under the 'native' pGEM promoter
(lacUV5?)
2) rebuild a full lenght lacZ gene with our modification and insert into a
lacZ- strain.
If someone has hints about what is best, thanks for your help.
But regarding of what choice I make, my main question is :
- How do I insert the lacZ into E. coli genome ?
Do I need to clone my lacZ gene into lambda DNA (I don't like this
idea)?
Are there vectors easier to handle and carrying everything one
needs for insertion into the genome ?
P.S.:Someone here in the lab indicated to me the work of Simons et al.
(1987, Gene 53:85-96) who made a lacZ vector for insertion into E.coli.
Does someone have this (these) vectors around or other related vectors ?
Thanks for your help
Gilles Vachon
Laboratoire de Genetique Moleculaire des Plantes,
UMR 5755, CERMO
Universite J. Fourier, BP 53X
38041 GRENOBLE CEDEX
FRANCE
Tel: (33) 76 63 56 58
fax: (33) 76 51 43 36
e-mail: vachon at bio.grenet.fr
