DH5a genotype - phi80delM15 (constitutive or regulated)? - (Jun/19/2007 )
I am interested in using b-gal activity from lacZ as a reporter of promoter activation in e coli DH5a. Because lacZ is absurdly large, I would prefer to clone only the alpha fragment (AA 3-41) behind my promoter of interest on a plasmid. DH5a should produce lacZΔM15 (mutation removes aa 11-41) from a phi80 prophage for complementation.
My question is... What promoter controls lacZΔM15 in DH5a? Is it the wt lac promoter (with lacR and CAP binding sites?) or is it some constitutive promoter? I would like to be able to use IPTG to control expression of another protein on the plasmid (a transcription factor for my promoter of interest) and I do not want b-gal activity to be dependant on IPTG and cAMP levels. I know you need to plate on IPTG when doing blue/white screening, but I'm not sure if that's only to induce lacZalpha transcription in the plasmid vs M15lacZ in the host as well. Anyone know where I can get this information?
I found this paper http://www.pnas.org/cgi/reprint/87/12/4645.pdf describing the construction of DH5a but the references were a dead end for me.
I attempted to find nucleotide sequence information in Pubmed but came up empty.
This info would really help me out. Thanks.
I did a bit of a search on Google for "DH5 Alpha IPTG" and came across this link: http://www.ag.uidaho.edu/mmbb/kgustin/files/DH5alpha.pdf
On page 6 is contact info. Perhaps they can help you?
United States Headquarters:
1600 Faraday Avenue
Carlsbad, CA 92008 USA
Tel: 1 760 603 7200
Tel (Toll Free): 1 800 955 6288
Fax: 1 760 602 6500
E-mail: Fax: +44 (0) 141 814 6287
Invitrogen Japan K.K.
Nihonbashi Hama-Cho Park Bldg. 4F
2-35-4, Hama-Cho, Nihonbashi Inchinnan
Tel: 81 3 3663 7972
Fax: 81 3 3663 8242
3 Fountain Drive
Paisley PA4 9RF, UK
Tel (Free Phone Orders): 0800 269 210
Tel (General Enquiries): 0800 5345 5345