(Kluyveromyces lactis, Yarrowia Lipolytica [7], Schwanniomyces
occidentalis [22], Pichia pastoris [23]).
6. Alternatively cells can be treated with a LiAc–DTT–TE mix
(10 ml: 7.75 ml water, 1 ml 10Â Tris–HCl–EDTA, 1 ml lithium acetate 1 M, 0.25 ml DTT 1 M) final concentrations:
0·1 M lithium acetate, 10 mM DTT, 10 mM-Tris–HCl,
pH 7·5, 1 mM EDTA [10].
7. Washing should be carefully operated to make sure that the
conductivity of the pulsed sample is low. This is needed to avoid
an excessive Joule heating that can be associated with arcing
between the electrodes.
8. Control experiments where plasmids are omitted result in no
transformant.
9. Plasmid pTSUK is used in our study. The selective media is as
described but should be chosen specifically in agreement with
the plasmid used for transformation.
10. Commercial single use cuvettes with parallel aluminum electrodes (2 mm gap) can be used but the delay to recover the
pulsed sample is longer than with the open electrode configuration. A meniscus can be formed resulting in a nonhomogeneous field distribution on the sample.
11. The solution should build a conductive gap between the two
stainless steel electrodes.
12. Not all pulse generators are equipped with such a device. Select
carefully your pulse generator. Monitoring the shape of the
pulse that is delivered, is needed to make sure that your experimental settings are applied. A voltage decay can be present as
the pulse duration T is long and as a consequence the electrical
charge Q that is delivered (Q ¼ IT, I being the current between
the electrodes, I is presumed to be roughly constant as the
voltage is constant (square wave conditions)) is large. But Q is
limited by the specifications of the pulse generator [20].
13. Control experiments where no electric pulse is delivered result
in no transformants.
14. We observe that the optimal pulsing conditions result in a
50–80% viability.
Acknowledgments
This research was conducted with the support of the EBAM
European Associated Laboratory (LEA).
192
E. Meilhoc and J. Teissie
occidentalis [22], Pichia pastoris [23]).
6. Alternatively cells can be treated with a LiAc–DTT–TE mix
(10 ml: 7.75 ml water, 1 ml 10Â Tris–HCl–EDTA, 1 ml lithium acetate 1 M, 0.25 ml DTT 1 M) final concentrations:
0·1 M lithium acetate, 10 mM DTT, 10 mM-Tris–HCl,
pH 7·5, 1 mM EDTA [10].
7. Washing should be carefully operated to make sure that the
conductivity of the pulsed sample is low. This is needed to avoid
an excessive Joule heating that can be associated with arcing
between the electrodes.
8. Control experiments where plasmids are omitted result in no
transformant.
9. Plasmid pTSUK is used in our study. The selective media is as
described but should be chosen specifically in agreement with
the plasmid used for transformation.
10. Commercial single use cuvettes with parallel aluminum electrodes (2 mm gap) can be used but the delay to recover the
pulsed sample is longer than with the open electrode configuration. A meniscus can be formed resulting in a nonhomogeneous field distribution on the sample.
11. The solution should build a conductive gap between the two
stainless steel electrodes.
12. Not all pulse generators are equipped with such a device. Select
carefully your pulse generator. Monitoring the shape of the
pulse that is delivered, is needed to make sure that your experimental settings are applied. A voltage decay can be present as
the pulse duration T is long and as a consequence the electrical
charge Q that is delivered (Q ¼ IT, I being the current between
the electrodes, I is presumed to be roughly constant as the
voltage is constant (square wave conditions)) is large. But Q is
limited by the specifications of the pulse generator [20].
13. Control experiments where no electric pulse is delivered result
in no transformants.
14. We observe that the optimal pulsing conditions result in a
50–80% viability.
Acknowledgments
This research was conducted with the support of the EBAM
European Associated Laboratory (LEA).
192
E. Meilhoc and J. Teissie