Tetracycline: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(→Properties: molecular weight clarified) |
m (Advised on solvent for tetracycline stock) |
||
| (One intermediate revision by one other user not shown) | |||
| Line 8: | Line 8: | ||
*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. | *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. | *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. Add 1 ml per liter of cooled broth or agar. Solutions and plates are light sensitive. | *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 | *Tetracycline is incompatible with (chelated by) magnesium ions, and thus unsuitable for use in minimal media | ||
| Line 16: | Line 16: | ||
==Properties== | ==Properties== | ||
*'''Molecular Weight''' - 480. | *'''Molecular Weight''' - 480.9 g/mol (Hydrochloride salt), 444.4 g/mol (free base) | ||
==References== | ==References== | ||
Latest revision as of 07:31, 13 February 2013
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)
