Assurance of correct conjugal strains (was Re: Microbial diagnostic kits)

Sat Feb 4 08:18:56 EST 1995

In article <3gume5$csv at nntp.Stanford.EDU> "Barry Lifland" <blifland at> writes:

>To:  "benedik at" <benedik at> 
>In-Reply-To: Your message of 1 Feb 1995 19:00:22 GMT.<3golo6$mbv at>
>Encoding:  37 TEXT , 4 TEXT , 4 TEXT 
>*Subject: Microbial diagnostic kits

>*strains on a repeatable basis. In other words we are doing some
>*genetics and transferring plasmids between strains and I want to be
>*sure that my putative strain from a cross is really a new exconjugant
>*and not a contaminant or revertant. There are no useful phenotypes in
>*hand (except on the plasmid). 
>*Are there some diagnostic strips or microtiter dish systems that I can
>*utilize that have lots of different tests so that I most likely can
>*distinguish between these strains. 
>*Suggestions or pointers would be quite welcome.

>*Michael Benedik               benedik at
>*Biochemical Sciences>*University of Houston
>Barry Lifland - Stanford Univ.,  Dept. Comparative Medicine
>Diagnostic lab - Microbiology section

>You could use "BIOLOG", a 95 test system in a 96 well plate. It works
>reasonably well for our I.D.'s.
> BIOLOG , Hayward, CA (510)785-2591 Sales; 785-2585 Technical.

You could label your recipient strain with resistance to *two* antibiotics, 
such as streptomycin (25-50 ug/ml) and rifamycin (10-20 ug/ml) which produce 
stable chromosomal mutations.  With a number of bacteria I work with, such 
mutations arise at ~ 1 per 10*-6/10*-8 (just plate 100 ul of overnight culture 
onto the abc-containing agar).  With streptomycin, I believe, you need to be 
careful since one class of Sm/r mutants requires the presence of streptomycin 
for good growth.  Anyway, you can consecutively acquire one mutant, then do 
the same selection on the other abc to obtain the other.   Your 
recipient strain is now constructed and selection of transconjugants can be 
obtained by adding both abc's into the selection media (i.e., two abc's for 
contraselection against the donor) and the plasmid-encoded abc as well for 
contrasection against recipient-only cells.  Therefore the likelihood of donor 
cells coming up against 2 abc's would be ~ 10*-12/10*-14 - very unlikely 
(obviously, transformation controls should include donor- & recipient-only 
controls).  The effort to go to double abc-contraselection, in my experience, 
was only necessitated by low conjugation frequencies.
Regards, Peter
*  Peter M. Muriana, Ph.D.             317-494-8284   TEL            *
*  Dept. of Food Science               317-494-7953   FAX            *
*  Purdue University                   murianap at   *
*  Smith Hall                                                        *
*  W. Lafayette, IN  47907-1160                                      *

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