LacZ Xsomal deletions

Rafael Maldonado rafael at corona.med.utah.edu
Mon Mar 27 19:57:00 EST 1995


On Sat, 25 Mar 1995, Duncan Clark wrote:

> Hi Folks,
> 
> I want to delete lacZ in E.coli. Now there are a few strains I can 
> transduce a deletion from but I'm unsure of what the best selection 
> method is.
[edited]
> I would prefer not to have a transposon linked deletion, selection 
> may be easy but it then limits the plasmids one can subsequently use.

Duncan:

The easiest way is still using a Tn10 (tet) lBinked to a lac deletion. You 
can cure the Tn10 using Bochner-Maloy medium, afterwards. This medium 
selects for tet(s). You can get a Tn10 linked to lac from the E. coli GSC.
For selection of tet(s) strains, check:

	Maloy and Nunn (1981) J.Bacteriol. 145; 1110-1112

	Bochner et al. (1980) J.Bacteriol 143; 926-933

The only problem is that your strain may keep a IS10 around, but it is 
not very important: E. coli has too many IS's to be a new trouble.

If you are interested in other direct methods for selection of lac-, more 
time-consuming and less clean, check Methods in Enzymology 204 (1991), 
Beckwith's chapter called "Genetic fusions". You can use a galE mutation 
(you were using it few days ago, aren't you?), or TONPG, metabolic poison in 
the presence of lacY. A very good review of the lac system, including all 
the selection methods used to date, can be found in the new Miller's 
Book: A short course in bacterial genetics. J.H. Miller, 1992, CSHL Press.

But I strongly recomend the Tn10 and Bochner-Maloy system.

I hope this helps

Rafa

___________________________________________________

Rafael Maldonado			  
room 6160 Eccles Institute of Human Genetics
Department of Human Genetics		 
University of Utah			
Salt Lake City, Utah 84112. USA.	                  
Rafael at genetics.med.utah.edu
Rafael at corona.med.utah.edu	
Tlf: 801-581-4429			
Fax: 801-585-3910			
					                  





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