University of Texas at San Antonio
SOP No.: 2 ver 1.0
Effective Date: 5/9/2008
Page 1 of 4
Title: Generation of Electrocompetent Francisella and transformation procedure
Approved:
Karl Klose
UTSA PI
Author: Jeff Barker
Date: 6/17/2008
1. INTRODUCTION
The generation of Electrocompetant Francisella tularensis is critical for the efficient
transfer of foreign DNA into the bacteria.
2. PURPOSE
This protocol describes the process of generating competent F. tularensis and the
electroporation of foreign DNA into the competent cells. Plasmid and linear DNA
can be successfully transformed into competent bacteria. This is useful when it is
required to express a given gene from plasmid DNA. This protocol can be used for
subspecies of F. tularensis, including novicida and holartica (LVS).
3. RESPONSIBILITY
Research Technologist: performs the SOP
Technical Specialist: reviews the data, calculations and interpretations
Principal Investigator: reviews the data and interpretations
4. SCOPE
This procedure is used by the UTSA team under the direction of Dr. Karl Klose for
preparing Francisella tularensis to receive foreign DNA through a transformation
procedure.
5. PRECAUTIONS
Sterility and temperature maintenance are critical steps in successfully generating
competent bacteria for transformation. Be sure to use sterile media and tubes during
the entire procedure.
6. MATERIALS AND METHODS
6.1 Eppendorf table top centrifuge (Model 5415 D)
6.2 Sterile 1.5 ml centrifuge tube (Biolink, BL3150LR)
6.3 Chamberlains Medium Dry Premix (Teknova, Cat. No. C0711)
6.4 Fisher Scientific pH meter (Model AB15)
6.5 0.5 M Sucrose (Fisher)
6.6 Corning 250 ml Filter bottle system (0.22 um filter)
6.7 Nichipet EX Plus pipettes, calibrated annually (p10, p20, p200, p1000)
6.8 37C incubator (VWR, model 1545)
University of Texas at San Antonio
SOP No.: 2 ver 1.0
Effective Date: 5/9/2008
Page 2 of 4
Title: Generation of Electrocompetent Francisella and transformation procedure
Approved:
Karl Klose
UTSA PI
Author: Jeff Barker
Date: 6/17/2008
6.9 Sterile 5 ml glass tubes (VWR, 89000-496)
6.10 Sterile 1 ml glass tubes (VWR, 89000-494)
6.11 Bio-Rad 0.2 cm electrode gap cuvette (Cat #: 165-2093)
6.12 Bio-Rad Gene Pulsar II
7. PROCEDURE
The following procedure is designed to generate 1 sample of electrocompetant
Francisella tularensis from a 5 ml overnight culture. If more electoporations are
required, upscale the number of overnight cultures proportionately.
7.1 Reagent Preparation
7.1.1 0.5 M SUCROSE
7.1.1.1.1 FW = 342.30
7.1.1.1.2 Dissolve 42.78 grams of sucrose in 250 ml double deionized
H2O for a 0.5 M concentration. Filter Sterilize in a Corning
250 ml bottle (0.22um).
7.1.2 Chamberlain’s media
7.1.2.1 Dissolve 12.1 grams of Chamberlains medium dry premix powder in
500 ml double deionized H2O. pH to 6.4. Filter sterilize in 2 250 ml
Corning bottles (0.22 um). Use the sterilized, liquid media within three
months.
7.1.2.2 Note: the Chamberlain’s powdered media has a manufacturer’s
expiration date. The powedered Chamberlain’s media has been
observed to darken with age; however, LVS and Francisella
tularensis, subspecies novicida have grown successfully in the
darkened media at UTSA
7.2 Generate overnight liquid culture of Francisella tularensis
7.2.1 Inoculate 5 ml of Chamberlains media with F. tularensis from a colony on
a fresh TSA++ plate.
7.2.2 Incubate at 37C in incubator overnight
7.3 Preparation of Electrocompetant Francisella tularensis
7.3.1 Inoculate 4 ml of fresh Chamberlains media with 1 ml of overnight culture
and incubate at 37C for 3-4 hours.
University of Texas at San Antonio
SOP No.: 2 ver 1.0
Effective Date: 5/9/2008
Page 3 of 4
Title: Generation of Electrocompetent Francisella and transformation procedure
Approved:
Author: Jeff Barker
Karl Klose
UTSA PI
Date: 6/17/2008
7.3.2
7.3.3
7.3.4
7.3.5
7.3.6
To concentrate down the 5ml into a single tube, perform five consecutive
spins of approximately 1 ml/spin in a single sterile 1.5ml eppendorf tube
. Each spin is in a tabletop centrifuge at 10,000 RPM for 1 minute. After
each spin, discard the supernatant and after the fifth spin, proceed to next
step in this protocol.
Discard supernatant and resuspend pellet in 1 ml 0.5 M sucrose
Spin at 10,000 RPM for 1 min in an eppendorf microfuge.
Repeat sucrose wash and spin 2 more times.
Final resuspension should be done with 50 ul of 0.5 M sucrose; these are
your electrocompetant cells and must be used immediately.
7.4 Transforming electrocompetant Francisella tularensis
7.4.1 Add DNA of interest to tube containing 50 ul electrocompetant cells.
Amount of DNA varies from plasmid DNA (~1 ug) to linear DNA (~10
ug).
7.4.2 Incubate at room temperature for 10 minutes.
7.4.3 Transfer cells to a 0.2 cm electrocuvette.
7.4.4 Electroporate using the Gene pulsar with the settings of 2.5 kV, 25 uF
capacitance, 600 (ohms) resistance.
7.4.5 Add 1 ml of Chamberlains media to the cells and transfer to a sterile 5 ml
glass tube.
7.4.6 Incubate at 37C for 2 hours (Francisella novicida) or 3 hours
(Francisella tularensis or holarctica).
7.4.7 Spin cells down in 1.5 ml eppendorf tube at 10,000 RPM in a table top
centrifuge for 1 minute.
7.4.8 Resuspend pellet in 100 ul Chamberlains media and plate on agar media
containing the appropriate antibiotic.
7.4.9 Keep plate in 37C incubator for ~2-3 days.
7.4.10 Methods to assess the success of the electroporation and selection of the
desired inserted DNA into FT depend on the application.
8. QUALITY CONTROL
8.1. Sterility checks on media are performed by incubating an aliquot of the media
overnight at 37C ; if no growth occurs, then the media is sterile.
8.2. PCR or restriction digests are used to verify that the transformation occurred
successfully with the correct foreign DNA finally transformed into the
Francisella tularensis subspecies novicida or holartica (LVS).
University of Texas at San Antonio
SOP No.: 2 ver 1.0
Effective Date: 5/9/2008
Page 4 of 4
Title: Generation of Electrocompetent Francisella and transformation procedure
Approved:
Karl Klose
UTSA PI
Author: Jeff Barker
Date: 6/17/2008
9. REFERENCES
Genetics and genetic manipulation in Francisella tularensis. Frank DW, Zhart TC.
Ann NY Acad Sci. 2007
10. CALCULATIONS AND FORMULAS
None
11. GLOSSARY
 ug- microgram
 ul- micro liter
 ml- milliliter
 cm- centimeter
 LB- Luria Broth
 Kv- kilovolts
 RPM- revolutions per minute
 C- centigrade
12. APPENDICES
None