Distinguishing stringent from relaxed?

Richard_Heath heath at mbcf.stjude.org
Wed Feb 1 16:12:37 EST 1995


SMG plates can be used to screen for relaxed mutants:  rel+ cells grow on this
medium, and rel- cells die, so make sure you make duplicate streaks of each
colony onto normal plates!  Whether this is actually the *best* method...?   It
is probably worth doing however, unless you are expecting *very* high
co-transduction frequencies if the Tn10 and relA allele.  I would also
demonstrate that the mutants you do isolate are in fact defficient in the
stringent response by labelling with 32P, starving for an amino acid, and
running the extracts on PEI-cellulose plates - mutants won't accumulate ppGpp. 
Any reveiwer will want to see this kind of demonstration...(at least, I would). 
You may need to also show these mutants still accumulate stable RNA after
starvation - this depends on what you are planning on doing with the cells!

Good luck

Richard Heath
   

In article <3g66v4$a6v at mserv1.dl.ac.uk>, mbpln at s-crim1.dl.ac.uk (M.J. Pallen) writes:
> I  am in the process of transduing a Tn10 relA mutation from one strain of 
> Salmonella into another. Does anyone have a handy way of scoring the 
> relA-negative phenotype, so that I can check that I really do have a relA 
> mutation in the second strain? Cahel & Rudd in Neidhardt's E. coli & S. 
> typhimurium book mention SMG medium. Is this the best way of 
> distinguishing stringent from relaxed strains?
> 
> Mark 
> ******************************************************************************
> Dr Mark Pallen, Senior Lecturer in Medical Microbiology, 
> St Bartholomew's Hospital Medical College, London, EC1A 7BE
> currently at the Dept of Biochemistry, Imperial College, London, SW7 2AY 
> email:m.pallen at ic.ac.uk  
> phone: day ++44(0)1715945254, eves ++44(0)1815057937, FAX ++44(0)1715945255
> ******************************************************************************
>                             lag                   ellum
>                              f   e               lag 
>        ecoliecoliecoliecoliecoli  l            mf 
>       cecoliecoliecoliecoliecolii  l          u 
>       eecoliecoliecoliecoliecolic   umf     ll      
>       coecoliecoliecoliecoliecoli      l  ge 
>        ecoliecoliecoliecoliecoli        a         
>          p                   f       fl g 
>         i                   l     lum   el
>        l                     agel         lu
>       u                                      mflagellum
>      s
> ******************************************************************************



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