Date:         Wed, 5 Jul 1995 09:18:08 EST
Reply-To:     Spirochete Research Discussion Group 
Sender:       Spirochete Research Discussion Group 
From:         Scott Samuels 
Subject:      Electrotransformation
To:           Multiple recipients of list SPIROC-L 
We offer some observations on, and improvements to, the electrotransformation protocol for Borrelia burgdorferi that was described by us (Samuels, Mach, and Garon) in J. Bacteriol. 176:6045-6049 (1994), and may be applicable to other spirochetes. We routinely obtain 1000 to 10,000 transformants per ug of DNA when introducing mutations into the gyrB gene of strain B31.

We have had success transforming B. burgdorferi strains HB19 and N40 in addition to B31, although we have not confirmed the N40 transformants by sequence analysis.

The most important factor for successful transformation by electroporation is probably cell density (or growth phase) at the time of harvesting. We consider 5x10^7 cells/ml to be ideal; we have had success with cultures harvested at 1-7x10^7 cells/ml, but the low density cultures require centrifugation at higher g forces and resuspension in smaller volumes (to obtain a final concentration of 1-5x10^10 cells/ml of EPS).

The two phosphate-buffered saline washes during the preparation of competent cells are not required for successful transformation. We have not quantitatively assessed the effect of omitting these washes, and we still routinely perform them.

The most useful refinement is the freezing of competent cells. We prepare competent cells as described previously, distribute them (50 ul each in EPS) into sterile 1.7 ml microfuge tubes (on ice), and transfer them directly into a -70C freezer. The cells are thawed on ice 10-30 min prior to electroporation. Frozen competent cells have a transformation efficiency only about 15% lower than that of fresh competent cells.

Our observations indicate that one electroporation pulse produces higher transformation efficiencies than two or three pulses and that varying the resistance from 100-400 ohms does not reproducibly affect the transformation efficiency. Arcing, caused by the presence of salts during electroporation, is detrimental: one arc decreases transformation efficiency by about ten-fold and two arcs by 100%.

Transformants are incubated in 10 ml of medium for 18-48 h prior to plating in selective medium. We usually plate 0.1-1 ml, but if all of the cells are needed, then the culture is pelleted at 8000 x g for 5 min, about 9-9.7 ml of supernatant is decanted off, and the pellet is resuspended in the remaining medium.

Novobiocin (at 5 ug/ml), which is 20-fold less expensive than coumermycin (at 0.2 ug/ml) per use, can be used to select gyrB R133I (CR10E) transformants. Novobiocin is labile upon exposure to light or freeze-thaw cycles; we prepare it fresh.

A detailed chapter describing the electrotransformation protocol step-by step for B. burgdorferi can be found in Methods in Molecular Biology, Vol. 47, Electroporation Protocols for Microorganisms, 1995, edited by J.A. Nickoloff, Humana Press (ISBN 0-89603-310-4).

      I will be moving on 30 August 1995 from RML to:
           Scott Samuels
           Division of Biological Sciences
           University of Montana
           Missoula MT 59812
           Email samuels@selway.umt.edu

Take care,
Scott
ssamuels@rml.niaid.nih.gov