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Laboratory Exercise Instructor Notes
Electroporation and marker exchange of
Pseudomonas syringae pv. syringae.
Section I. Introduction of a broad host range plasmid into Pseudomonas syringae pv. syringae strain B301D.
Section
I, Note 1:
The ability to
vary the resistance on the electroporation machine is recommended for
electroporation of P. syringae pv. syringae strains. Several of
the strains exhibit greater electroporation efficiencies at higher
resistances. For many P. syringae pv. syringae strains, standard
E. coli conditions can be used for electroporation (although they
will be less efficient) and, therefore, any brand of electroporator will
work.
Section
I, Note 2:
The purity of the water is very important for efficient
electroporation. It is important to use bottled deionized water if it is
not readily available in the laboratory.
Section
I, Note 3:
Electroporation
efficiency is cell density dependent up to 1 x 1010 CFU/ml. As
long as the cell concentrations do not go below 1 x 1010 CFU/ml,
the variations in electroporation efficiencies are usually dependent on
other factors such as bacterial strain, resistance, voltage, and DNA
concentration (3).
Section
I, Note 4:
During
electroporation, there is a possibility of the sample
"arcing". This is characterized by a loud "pop"
sound. Arcing occurs when the sample is too conductive. There are
several reasons that a sample will arc including: not washing all of the
salt from the growth medium out of the bacterial suspension; placing too
much DNA in the electroporation mix; using DNA dissolved in a high salt
buffer; using a bacterial suspension that is too concentrated; using a
bacterial suspension containing lysed bacteria; and trying to
electroporate using electroporation cuvettes that are too warm. If
arcing occurs, dilute the DNA and try again, this is usually the most
common problem. If arcing continues, perform an electroporation without
DNA to see if the cells are too concentrated. Expect a time constant
over 4 seconds; electroporation at time constants below 4 seconds will
not produce good efficiencies of transformation and should be repeated
with less DNA.
Section
I, Note 5:
A good protocol
for plasmid mini-preps can be found in Sambrook et al. (10).
Section
I, Note 6:
All
bacterial and fungal strains listed in this exercise, as well as plasmid
constructs and vectors may be obtained from Brenda K. Scholz-Schroeder,
POB 646430, Department of Plant Pathology, Washington State University,
Pullman, WA 99163.
Section II. Introduction of a broad host range plasmid into various Pseudomonas syringae pv. syringae strains.
Section
II, Note 1:
The ability to
vary the resistance on the electroporation machine is recommended for
electroporation of P. syringae pv. syringae strains. Several of
the strains exhibit greater electroporation efficiencies at higher
resistances. For many P. syringae pv. syringae strains, standard
E. coli conditions can be used for electroporation (although they
will be less efficient) and, therefore, any brand of electroporator will
work.
Section II, Note 2:
The purity of the water is very important for efficient
electroporation. It is important to use bottled deionized water if it is
not readily available in the laboratory.
Section
II, Note 3:
Electroporation
efficiency is cell density dependent up to 1 x 1010 CFU/ml. As
long as the cell concentrations do not go below 1 x 1010 CFU/ml,
the variations in electroporation efficiencies are usually dependent on
other factors such as bacterial strain, resistance, voltage, and DNA
concentration (3).
Section
II, Note 4:
For some P.
syringae pv. syringae strains an increase in electroporation efficiency can
be observed with an increase in resistance. When using new bacterial
strains, a control experiment testing various settings is recommended
using a broad host range plasmid such as pRK415.
Section
II, Note 5:
During
electroporation, there is a possibility of the sample
"arcing". This is characterized by a loud "pop"
sound. Arcing occurs when the sample is too conductive. There are
several reasons that a sample will arc including: not washing all of the
salt from the growth medium out of the bacterial suspension; placing too
much DNA in the electroporation mix; using DNA dissolved in a high salt
buffer; using a bacterial suspension that is too concentrated; using a
bacterial suspension containing lysed bacteria; and trying to
electroporate using electroporation cuvettes that are too warm. If
arcing occurs, dilute the DNA and try again, this is usually the most
common problem. If arcing continues, perform an electroporation without
DNA to see if the cells are too concentrated. Expect a time constant
over 4 seconds; electroporation at time constants below 4 seconds will
not produce good efficiencies of transformation and should be repeated
with less DNA.
Section
II, Note 6:
A good protocol
for plasmid mini-preps can be found in Sambrook et al. (10).
Section
II, Note 7:
All
bacterial and fungal strains listed in this exercise, as well as plasmid
constructs and vectors may be obtained from Brenda K. Scholz-Schroeder,
POB 646430, Department of Plant Pathology, Washington State University,
Pullman, WA 99163.
Section III. Introduction of mutated genes into Pseudomonas syringae pv. syringae strain B301D, selection for marker exchange, and screening strains for phenotypic changes.
Section
III, Note 1:
The ability to
vary the resistance on the electroporation machine is recommended for
electroporation of P. syringae pv. syringae strains. Several of
the strains exhibit greater electroporation efficiencies at higher
resistances. For many P. syringae pv. syringae strains, standard
E. coli conditions can be used for electroporation (although they
will be less efficient) and, therefore, any brand of electroporator will
work.
Section
III, Note 2:
The purity of the water is very important for efficient
electroporation. It is important to use bottled deionized water if it is
not readily available in the laboratory.
Section
III, Note 3:
Electroporation
efficiency is cell density dependent up to 1 x 1010 CFU/ml. As
long as the cell concentrations do not go below 1 x 1010
CFU/ml,
the variations in electroporation efficiencies are usually dependent on
other factors such as bacterial strain, resistance, voltage, and DNA
concentration (3).
Section
III, Note 4:
During
electroporation, there is a possibility of the sample
"arcing". This is characterized by a loud "pop"
sound. Arcing occurs when the sample is too conductive. There are
several reasons that a sample will arc including: not washing all of the
salt from the growth medium out of the bacterial suspension; placing too
much DNA in the electroporation mix; using DNA dissolved in a high salt
buffer; using a bacterial suspension that is too concentrated; using a
bacterial suspension containing lysed bacteria; and trying to
electroporate using electroporation cuvettes that are too warm. If
arcing occurs, dilute the DNA and try again, this is usually the most
common problem. If arcing continues, perform an electroporation without
DNA to see if the cells are too concentrated. Expect a time constant
over 4 seconds; electroporation at time constants below 4 seconds will
not produce good efficiencies of transformation and should be repeated
with less DNA.
Section
III, Note 5:
Syringomycin bioassay
(5)
1. Inoculate 5 ml of NBY cultures with P. syringae pv. syringae
mutant strains using a sterile loop and incubate for 16 h at 25°C with
shaking.
2. Harvest 1 ml of the overnight culture by centrifugation (14,000 rpm for
1 min).
3. Resuspend the cells in 1 ml of sterile distilled water (SDW) and spin
the cells down by centrifugation (14,000 rpm for 1 minute).
4. Resuspend the cells in 1 ml of SDW and then dilute the sample to an
O.D.420=0.3 (~2 x 108
cfu/ml).
5. Inoculate a 5 µl droplet of the cell suspension to potato dextrose
agar (20 ml/plate) supplemented with 0.4% casamino acids and 1.5% glucose
and incubate at 25°C for 3-5 days.
6.
Overspray the plates lightly with an arthrospore suspension of the
bioassay fungus, Geotrichum candidum strain F-260, which is
sensitive to syringomycin, and incubate for 16 h at 25°C.
7. A zone of inhibition
around the bacterial colony is indicative of the production of
syringomycin.
Section
III, Note 6:
The instructor could have the students perform a complementation test. A
complementation experiment is a test to confirm that a mutation within a
particular gene is responsible for the observed phenotype. In this
experiment, the wild type syrB1 gene carried on a broad host range
plasmid was introduced into the newly constructed syringomycin mutant
strain by electroporation. The mutant strain carrying the wild type syrB1
gene was then screened for syringomycin production. Recovery of the
ability to produce syringomycin by the complemented strain confirmed that
introduction of the mutated syrB1 gene in the genome of P.
syringae pv. syringae was responsible for loss of the ability
to produce syringomycin.
Section
III, Note 7:
A good protocol for
isolation of bacterial genomic DNA can be found in Ausubel et al. (1)
and a protocol for Southern hybridization can be found in Sambrook et al. (10).
Section
III, Note 8:
All
bacterial and fungal strains listed in this exercise, as well as plasmid
constructs and vectors may be obtained from Brenda K. Scholz-Schroeder,
POB 646430, Department of Plant Pathology, Washington State University,
Pullman, WA 99163.
© Copyright 2001 by The American Phytopathological Society
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