Stable nuclear transformation of Gonium pectorale

BMC Biotechnology

Table 1 Influence of different parameters on transformation efficiency.

Parameter and parameter specification	Efficiency
Transformation method
glassbeads (untreated cells)	-
glassbeads (pretreated with protease)	-
particle gun (homemade, without vacuum chamber)	+ +
particle gun (PDS-1000/He biolistic device, with vacuum chamber)	+ + + + +
Material of microprojectiles
gold	+ + + + +
tungsten	+ +
Size of microprojectiles
0.6 μm in diameter	+ + + + +
1.0 μm in diameter	+
1.6 μm in diameter	-
Selectable marker plasmid
paphG	+ +
pPmr3	+ + + + +
promoterless aphVIII	(+)
Coating of microprojectiles
plasmid-DNA/microcarrier/NaAc/EtOH-precipitation	+ + +
plasmid-DNA/microcarrier/CaCl₂/spermidine/EtOH-precipitation	+ + + + +
Target cells
resuspended in as less liquid as possible; spread in an empty Petri dish	-
immobilized on moist filter paper; more or less free of liquid	+ + +
immobilized on cellulose acetate membrane filter; almost free of liquid	+ + + + +
Burst pressure of rupture disks
450 psi	-
650 psi	-
900 psi	+
1100 psi	+ + + + +
1350 psi	+ +
Rupture disk-macrocarrier distance
8 mm	+ + + + +
16 mm	+ +
Macrocarrier-stopping screen distance
7 mm	+ + + + +
12 mm	+ + + +
18 mm	+ + +
Stopping screen-target cell distance
6 cm	+ + + + +
11 cm	+ +
18 cm	-
Chamber evacuation
(almost) no evacuation	+ +
15 inch Hg	+ +
27 inch Hg	+ + + + +
Cultivation after particle bombardment
on agar plates	-
in liquid medium	+ + + + +

Within each subgroup, the condition that gave the largest number of transformants was set to "+ + + + +"; there is a "-" when no transformants were recovered. The data allow just a qualitative estimation, because a) they were obtained under varying other conditions in the course of optimization, b) poor results disqualified a condition without rechecking, and c) some of the transformants might be false-positives as only a few were analyzed in detail.

ISSN: 1472-6750