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Electroporation
and marker exchange of Pseudomonas syringae pv. syringae. CLICK
HERE TO RETURN TO THE EXERCISE.
by Brenda K. Scholz-Schroeder.
Section I-Questions for discussion
1. How does plasmid size affect the electroporation
efficiency?
The larger the plasmid, the more difficult it is
to get into the bacterial cell. The plasmid pUCP26 is much smaller than
pRK415 and therefore it will be electroporated into Pseudomonas
more efficiently.
2. What are some alternatives to using electroporation
for the introduction of a broad host range plasmid into P. syringae pv.
syringae strain B301D?
Triparental mating is an alternative to electroporation
for the introduction of a broad host range plasmid into Pseudomonas
strains. The triparental matings are time consuming and are prone to
contamination by E. coli.
Section II-Questions and Discussion
1. What were the most effective conditions for
electroporation of each strain of P. syringae pv. syringae?
Efficiencies of transformation can vary but generally an electroporation efficiency of 1 x 105 transformants/mg DNA is attained for pUCP26 and an electroporation efficiency of 1 x 104 transformants/mg DNA is attained for pRK415 when electroporated into P. syringae pv. syringae strain B301D with a voltage of 1.8 kV/cm and a resistance of 200 W. The electroporation efficiencies attained for strain B728a are usually very similar to those of B301D at these conditions, however, the electroporation efficiencies for strain HS191 are usually 10 to 100 fold lower at these conditions. The electroporation efficiencies for strain HS191 tend to increase as the resistance increases and the voltage is lowered.
2. Which variable, the resistance or the voltage,
exhibits the greater effect on the efficiency of electroporation in P.
syringae pv. syringae strains?
Efficiencies of transformation can vary as discussed
above. This question is designed to prompt the students to discuss the
results of their experiments for this section.
3. How does plasmid size affect the electroporation
efficiency in each strain?
The larger the plasmid, the more difficult it is to get
into the bacterial cell. The plasmid pUCP26 is much smaller than pRK415
and therefore it will be electroporated into Pseudomonas more
efficiently.
Section III-Questions and Discussion
1. How do the marker exchange mutagenesis efficiency and
the efficiency of electroporation for the broad host range plasmid
compare?
2. Why is there such a difference between the efficiency
of transformation and the efficiency for marker exchange mutagenesis?
The efficiency of transformation for introduction of a
broad host range plasmid is significantly higher than the efficiency of
marker exchange mutagenesis. The reason for this is that the process of
marker exchange mutagenesis requires more events to occur. Not only does
the plasmid have to be introduced into the Pseudomonas strain but
also the homologous DNA of the plasmid and the genome must come in close
proximity to one another. Crossing over of the DNA strands must occur such
that the DNA from the plasmid exchanges with the DNA of the Pseudomonas
genome resulting in the introduction of a mutated gene into the Pseudomonas
genome. The number of events that must occur significantly reduces the
transformation efficiency of marker exchange mutagenesis.
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