Tetracycline

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
m (Properties: units added)
Current revision (09:31, 13 February 2013) (view source)
m (Advised on solvent for tetracycline stock)
 
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

Current revision

Contents

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)

References

Personal tools