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Plates for Bacterial Growth
LB Plates: Selection by Antibiotic
LB Agar plates with Tetracycline 15. 150mm, 20 plates, sterile Cat.No. L5033
1% Tryptone, 0.5% Yeast Extract, 1.0% Sodium Chloride, 1.5% agar 15µg/ml tetracycline.
Certificate of Analysis available upon request. To request
CAS: 60-54-8 (64-75-5 for hydrochloride)
Molecular Weight: 444.435g/mol (480.9g/mol for hydrochloride)
HCl for hydrochloride)
Stock Solutions: Dissolved in water at 20 mg/ml or in Ethanol at 5 mg/mL. Stock solutions should be filtered sterilized and stored at −20°C.
Working concentration: 10-12.5ug/mL
Stability: Tetracycline is light-sensitive and is degraded by exposure to UV light. Stock solutions are stable for 4 days, with a half-life of approximately 24 hours.
Mode of Action: Bacteriostatic. Inhibits bacterial synthesis by binding to 30S ribosome. Tet is used as genetic marker in mammalian cell culture since no 30S ribosome (antibiotic wont accumulate).
Mode of Resistance: Several mechanisms, often resulting in rapid efflux. A number of
genes confer resistance. In
, resistance is conferred by most often by transposon Tn10 (TetR).
Tetracycline is a light-sensitive broad spectrum antibiotic, effective against both gram-positive and gram-negative bacteria. The first tetracycline, chlortetracycline, was discovered in 1948 as a naturally occurring antibiotic synthesized by
that was active against a wide range of Gram-positive and Gram-negative bacteria and protozoa. By 1980, more than 1000 tetracycline derivatives had been isolated and/or synthesized. The estimated global production is in excess of 500 metric tons. A few species of bacteria display intrinsic resistance to tetracycline, including
. Acquired (as opposed to inherent) resistance has proliferated in many pathogenic organisms and greatly eroded the versatility of this group of antibiotics. Resistance amongst
, and members of the
is now quite common.
The chief mechanism by which
becomes resistant to high concentrations of tetracycline involves multimeric antiporter proteins, known as Tet proteins, embedded in the bacterial inner membrane and, in exchange for a proton, catalyze the outward transport of tetracycline-Mg2+ complexes from the cytosol. Tetracycline enters bacterial cells by passive diffusion across the outer membrane through porin channels, which are composed of the OmpF protein. Transport of the antibiotic across the cytoplasmic membrane and into the cytoplasm requires pH or electropotential gradients.
Tetracycline inhibits bacterial growth by disrupting codon-anticodon interactions at the ribosome, thus blocking protein synthesis. Specifically, tetracycline binds to a single site on the 30S ribosomal subunit and inhibit protein synthesis by blocking the attachment of charged aminoacyl-tRNA to the A site on the ribosome. Thus, they prevent introduction of new amino acids to the nascent peptide chain.
The action is usually inhibitory and reversible upon withdrawal of the drug. Resistance to tetracycline results from changes in permeability of the microbial cell envelope. In susceptible cells, the drug is concentrated from the environment and does not readily leave the cells. In resistant cells, the drug is not actively transported into the cells or leaves it so rapidly (via efflux) that inhibitory concentrations are not maintained.
Mammalian cells are not vulnerable to the effect of tetracycline as these cells contain no 30S ribosomal subunits so do not accumulate the drug. Several mechanisms of tetracycline resistance are known including: tetracycline efflux, ribosome protection and tetracycline modification. The resistance genes are controlled by an allosteric repressor protein TetR which together with the regulatory elements of the Tn10 tetracycline operon turn gene expression on and off in mammalian systems. TetR protein senses intracellular levels of tetracycline. Binding of the antibiotic causes an allosteric change and decreased binding of TetR to the elements that repress transcription of TetA, which encodes a membrane-bound protein that exports tetracycline out of the bacterial cell, rendering cells harboring this gene more resistant to the drug.
Tetracycline is used to study transcriptional activation. Knowledge of tetracycline led to the development of a popular inducible expression system in eukaryotic cells known as Tet-Off and Tet-On. This system has the advantages of being a conditional system that is both reversible and tightly controlled with a lower incidence of leaky (background) expression compared to other inducible systems. Tet-Off systems are also used in generating transgenic mice, which conditionally express a gene of interest. Since the 19bp TetO sequence is naturally absent from mammalian cells, pleiotropy is minimized compared to hormonal control used by other inducible expression systems. Today there are several popular systems including Tet-off, Tet-on and Tet autoregulatory systems which help to minimize leaky background in uninduced cells. When using the Tet system in mammalian cell culture, it is important to either use animal-free media or to test each batch of fetal bovine serum (FBS) to confirm that contaminating tetracyclines are absent or are too low to interfere with induction. Doxycycline (Dox) is a water-soluble tetracycline derivative that is preferred for almost all Tet-controlled gene expression systems.
1. Green and Sambrook. Molecular Cloning, A Laboratory Manual (2012).
2. Backman K, Boyer HW. Tetracycline resistance in Esherichia coli is mediated by one polypeptide. Gene 26: 197-203. (1983).
3. Gatz, C. Novel inducible/repressible gene expression systems. Methods Cell Biol. 50: 411-424. (1995)
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