Timentin, encore

Bob Ludwig ludwig at biology.ucsc.edu
Tue Mar 10 13:08:22 EST 1992

Since I posted this message a month ago, I have been pummeled with the
same three queries. I'm repeating that message, along with the stock,
three answers...

Timentin, not carbenicillin, is the antibiotic of choice for
Agrobacterium counterselection. While it is a proprietary product of
SmithKline Beecham Pharmaceuticals and, in the U.S., is a prescription
drug, Timentin is readily available and cheap (US$ 5 per gram).

Timentin is a mixture of two antibiotics: ticarcillin, a semi-synthetic
beta-lactam (like carbenicillin) and clavulanic acid, a beta-lactamase
inhibitor. SKB mixes Timentin in two formulae, 30:1 and 15:1 (w/w)
ticarcillin:clavulanic acid. The latter is preferable. Because most wild
Agrobacterium isolates have beta-lactamase activity, clavulanic acid is
particularly efficacious. 

All Agrobacterium isolates we have tested, including both tumefaciens
and rhizogenes strains, are highly Timentin susceptible. Further,
Agrobacterium strains carrying the standard pBR beta-lactamase gene are
also Timentin (clavulanic acid) susceptible. Timentin killing of
Agrobacterium wild-type strains is three logs greater than with
comparable doses of carbenicillin. In plant transformation experiments,
0.1 mg/ml Timentin (15:1) is sufficient to counterselect Agrobacterium.

Stock answers:

1. SKB have changed the Timentin formulas I posted (100:1 and 50:1) to
30:1 and 15:1. The appropriate dose in tissue culture medium is indeed
0.1 mg/ml (not 0.1 mg/liter) of 15:1 mixture or 0.2 mg/ml of 30:1
mixture. To improve efficacy (of any antimicrobial), solid media are
best slightly dried before use.

2. At these concentrations, Timentin exhibits no phytotoxicity to
Arabidopsis root cultures. 

3. I don't know why you can't get Timentin from Sigma.

Bob Ludwig
University of California
Santa Cruz

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