de Cock, "Lipopolysaccharide transport to the bacterial outer membrane in spheroplasts
," Journal of Biological Chemistry, vol.
The spheroplasts were washed with 1 mL of 1M sorbitol and collected by centrifugation at 6000 rpm for 5 minutes at 30[degrees]C.
Thus, in order to perform the patch-clamp experiment inside ESEM, it is important to confirm the ability of ESEM to observe spheroplast cells successfully before further manipulation could be carried out.
The observation and comparison of W303 cells and spheroplast W303 were successfully performed using ESEM.
To examine the effect of these mutations on the gating threshold of MscS, the channel activities were measured while applying negative pressure through a patch pipette to the inside-out membrane patch of the giant spheroplasts
expressing the wild-type or mutant MscS.
The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts
. J Biol Chem 1965;240(9):3685-3692.
One of the labs working with spheroplasts
was led by Ching Kung, PhD, at the University of Wisconsin-Madison.
Briefly, fungal suspensions were centrifuged for 10 min at 3000g, and 1 mL of lyticase buffer was added to generate spheroplasts
[10 kU/L recombinant lyticase (Sigma), 50 mmol/L Tris,1 mmol/L EDTA, 2 mL/L ([beta]-mercaptoethanol].
Initial attempts to regenerate transformed spheroplasts were performed on CD plates supplemented with uracil, uridine, and 5-FOA.
CzapekDox (CD) medium composed of 0.2% (w/v) NaN[O.sub.3], 0.1% (w/v) [K.sub.2]HP[O.sub.4], 0.05% (w/v) MgS[O.sub.4] x 7[H.sub.2] O, 0.05% (w/v) KCl, 0.001% (w/v) FeS[O.sub.4] and 3.0% (w/v) sucrose at pH 5.5 (supplemented with 0.2% (w/v) uracil and 20 mM uridine) was used for spheroplast cell wall regeneration.
This was probably because multiple nuclei were present in each spheroplast, and therefore a single pyrG[DELTA]0 locus could not prevent production of the suicide inhibitor FdUMP.
Patch-clamp experiments on such giant spheroplasts
revealed two types of MS-channel activities, one with a very large conductance (2.5 nS) and one with a smaller conductance (0.8 nS).