Tetracycline
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Mode of Action
Tetracyclines (there are many) enter the cell by diffusion through OmpF porins. It inhibits growth by preventing codon-anticodon interactions during translation.
Resistance Mechanism
There are a number of resistance mechanisms, most require tetracycline antiporters. The most commonly used is the tetA(C) gene. The gene product of tetA(C) forms a pore in the inner membrane and exports one tet molecule for every proton imported. Molecular Cloning states that tet resistance has a number of adverse affects on the cell, chief among those being a reduction in growth rate and viability.
Working concentration and Stock Solutions
- Using a plasmid with copy number 15-20, 20μg/ml tet has proved a successful selector. The reduction in growth rate, described above was significant.
- The RajBhandary lab uses 5μg/ml and see a minimal reduction in growth rate and never lose plasmid from their cells.
- Stock solution is 15 mg/ml in 50% ethanol (note: using 95-100% ethanol is much better for dissolving stock powder). Add 1 ml per liter of cooled broth or agar. Solutions and plates are light sensitive.
- Tetracycline is incompatible with (chelated by) magnesium ions, and thus unsuitable for use in minimal media
Counterselection techniques
- Fusaric acid can be used to select against tetracycline resistant colonies. See PMID 6259126, and modifications PMID 7007341.
- Nickel ions can also be used for tetR counterselection. See PMID 8954882.
Properties
- Molecular Weight - 480.9 g/mol (Hydrochloride salt), 444.4 g/mol (free base)
