University of Texas at San Antonio
SOP No.: UTSA 4 ver 1.0
Effective Date: 9/23/2008
Page 1 of 5
Title: Conjugation of a plasmid into Francisella tularensis LVS
Approved:
Karl Klose
1
Date: 09/23/2008
Introduction
This SOP details the procedure for conjugation of the plasmid carrying the upstream,
antibiotic marker and downstream cassette into F. tularensis LVS to mutate a specific gene
in F. tularensis LVS.
2
Purpose
The purpose of this SOP is to describe how to perform the conjugation procedure between
the conjugative plasmid and F.tularensis LVS to disrupt a specific gene in F. LVS.
3
Responsibility
Research Technologist: performs the SOP
Technical Specialist: reviews the data, calculations and interpretation
Principal Investigator: reviews the data and interpretations
4
Scope
This procedure is used by the UTSA team under the direction of Dr. Karl Klose.
5
Precautions
Sterility and the correct ratio of the mixture between the plasmid/E.Coli and LVS are critical
steps in successfully conjugating the plasmid into LVS. Be sure to use sterile media and
tubes during the entire procedure.
6
6.1
Materials
Supplies
6.1.1 (Tryptic Soy Agar) TSA++(0.1% L-cysteine, 250ug/ml FeSO4, 250ug/ml Sodium
Pyruvate and 250ug/ml Sodium Metabisulfite) agar plate with or without a specific antibiotic
6.1.2 (Tryptic Soy Broth) TSB++ liquid medium
6.1.3 (Luria Broth) LB agar plate with desired antibiotic
6.1.4 1XPBS, sterile
6.1.5 Disposable cell spreader L shape (Biolink, BLM5102)
6.1.6 Inoculating loop (Fisher,13-075-2)
6.1.7 Sterile 1.5 ml centrifuge tubes (Biolink, BL3150LR)
6.1.8 Sterile filter membrane (Millipore, 0.22um, Cat No# GVWP 025 00)
6.1.9 Petri Dishes 100mm x 15mm (ISC BioExpress, D-2550-2)
6.1.10 Nichipet EX Plus pepettes, calibrated annually (p10, p20, p200, p1000)
SOP No.: UTSA3
Page 2 of 5
Title: Conjugation of the plasmid into Francisella tularensis LVS
6.2 Equipment
6.2.1 37ºC Incubator (VWR, model 1545)
6.2.2 Spectrophotometer (Beckman, DU530)
6.2.3 Eppendorf table top centrifuge (Model 5415 D)
7 Procedure for conjugation of the plasmid/E.coli into LVS
 Wild type LVS strain is resistant to Ampicillin naturally
 The conjugative plasmid is resistant to antibiotic-1 (in parent plasmid) and antibiotic-2 (in
upstream, antibiotic-2 and downstream cassette)
7.1 Preparation of medium and agar plates
7.1.1 Luria Broth (LB) plate with antibiotic-2 preparation
Dissolve 5g NaCl, 10g Tryptone, 5g Yeast extract and 15g bacto-agar in 1 L double
deionized water. Autoclave. Cool down to 55C and add antibiotic-2. Pour onto Petri Dishes.
7.1.2 TSA++ agar plate preparation.
Dissolve 40g Tryptic Soy Agar in 1L double deionized water. Autoclave and cool down to
55C. Add the ingredients at final concentration of 0.1% L-cysteine, 250ug/ml FeSO4,
250ug/ml Sodium Pyruvate and 250ug/ml Sodium Metabisulfite. Mix and pour onto Petri
Dishes.
7.1.3 TSB++ liquid medium preparation.
Dissolve 30g Tryptic Soy Broth in 1L double deionized water. Autoclave and cool down. Add
the ingredients at final concentration of 0.1% L-cysteine, 250ug/ml FeSO4, 250ug/ml Sodium
Pyruvate and 250ug/ml Sodium Metabisulfite. Mix.
7.1.4 TSA++ agar plate with antibiotic-2 preparation
The same recipe as Step7.1.2 except adding a specific amount of antibiotic-2.
7.1.5 TSA++ agar plate with Ampicillin and antibiotic-1
The same recipe as Step7.1.2 except adding certain amount of antibiotic-1 and 100ug/ml
Ampicillin at final concentration.
7.1.6 Place the prepared medium and agar plates in the refrigerator for storage.
7.2 Growth of F. tularensis LVS and the plasmid / E.coli on agar plates.
7.2.1 Inoculate LVS from the frozen stock onto TSA++ plate and E.coli/plasmid from the frozen
stock onto LB/antibiotic-2 plate. Use an inoculating loop to streak at least half full loop of LVS
frozen stock or E.Coli./plasmid frozen stock (about half volume of LVS) on the corresponding
plate.
7.2.2 Incubate the bacteria on the plates in 37C incubator for overnight (E.coli) or 1-2 days
(LVS).
7.3 Growth of LVS and E.coli onto the corresponding plate at 37C for 4-6 hours (LVS) or 23hours (E.coli). .
7.3.1Scrape E.coli or LVS off from overnight incubated agar plate and resuspend them in 0.1ml
1xPBS (for both bacterium) or TSB++ liquid medium (for LVS), respectively
7.3.2 Spread all of the suspension of LVS or E.coli onto the corresponding fresh plate.
7.3.3 Incubate LVS at 37C for 7-8 hours and E.coli at 37C for 3-4 hours.
7.4 Mix LVS and E.Coli at ratio LVS: E.coli =10:1
SOP No.: UTSA3
Page 3 of 5
Title: Conjugation of the plasmid into Francisella tularensis LVS
7.4.1The bacterium are scraped off from the corresponding plate and resuspended in about 2ml
of 1XPBS or TSB++ liquid medium.
7.4.2 Measure OD600 for both LVS and E.Coli to get OD value.
7.4.3 Make the mixture of LVS and E.coli at ratio of LVS:E.coli. =10:1 based on OD600 value.
7.5 Incubate the mixture of LVS and E.Coli at 37C on TSA++ plate to conjugate the plasmid
into LVS.
7.5.1 Place the sterile filter membrane onto TSA++ plate.
7.5.2 Add the mixture of LVS and E.coli onto the filter membrane.
7.5.3 Incubate the mixture of bacterium at 37C for overnight.
7.6 Transfer the mixed bacterium onto TSA++/antibiotic-1/Ampicillin plate to select LVS
carrying the plasmid.
7.6.1 Scrape the mixture off from the filter membrane on TSA++ plate after overnight
incubation.
7.6.2 Resuspend the scraped cells in 0.1ml 1XPBS or TSB++ liquid medium.
7.6.3 Make serial 1:10 fold dilution in 1xPBS or TSB++ liquid medium.
7.6.4 Spread the diluted suspension onto TSA++ /antibiotic-1/Ampicillin plate.
7.6.5 Incubate the bacterium at 37C for at least 4-6 days.
7.7 Patch the colonies from TSA++/antibiotic-1/Ampicillin plate onto TSA++/antibiotic-2 agar
plate and incubate at 37C for 48 hours.
7.8 Screen the colonies by PCR.
8 Quality Control
Temperatures of incubators are noted with each usage
Media sterility is checked with each incubation. An aliquot of the liquid media or an agar plate
without any bacteria inoculated was set up as the control to monitor the media sterility.
Antibiotics are checked with the strain sensitive to this antibiotic.
Spectrophotometer is tested automatically for the memory, voltage, systems, offset correction,
lamp alignment and the wavelength calibration each time it is powered up.
9
9.1
9.2
References
DU Series 500 Spectrophotometer Operating Instructions
Jeffery H. Miller. 1992. A Short Course in Bacterial Genetics—A Laboratory Manual and
Handbook for Escherichia Coli and Related Bacteria. CSHL PRESS.
10 Calculations and Formulas
Not Applicable
11 Glossary
 TSA – Tryptic Soy Agar
 TSB – Tryptic Soy Broth
 LB- Luria Broth
SOP No.: UTSA3
Page 4 of 5
Title: Conjugation of the plasmid into Francisella tularensis LVS

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LVS – live vaccine strain
mL – milliliter
µm- micron
PBS – phosphate buffered saline
RPM- revolutions per minute
E.Coli.-Escherichia coli
g-gram
Mg-milligram
12 Appendices
Media Preparation/QC Log
Appendix
Media Preparation/QC Log
Preparation
Media: _______TSA++ agar plate________ Amount Prepared:_____1000ml___
Date Prepared: __03/29/2007___
Prepared by: PING CHU_______
Expiration Date: __04/29/2007______________
Ingredients
Manufacturer
Catalog #
Lot #
EXP Date
Amount
Tryptic Soy Agar
Bio-link
7100A
101,479 B
08/11
40g
10% L-Cysteine
Sigma
C7880100G
067K0763
Not
Applicable
10ml
250mg/ml FeSO4
Sigma
F-7002
106H1205
Not
Applicable
1.0ml
250mg/ml Sodium
Pyruvate
Sigma
P5280100G
105K07232
Not
Applicable
1.0ml
250mg/ml Sodium
Metabisulfite
Sigma
055556100G
11419EE
Not
Applicable
1.0ml
Sterilization Method:
_____________Autoclaving________________________________________
Comments: N/A
SOP No.: UTSA3
Page 5 of 5
Title: Conjugation of the plasmid into Francisella tularensis LVS
Quality Control
QC start date: ____ 04/01/2007____
QC end date: ____04/07/2007______
QC completed by: _____Ping Chu_________________
Room Temperature Results: No Growth
If growth, corrective action taken: _____________________________________
Incubated Temperature Results: No Growth
If growth, corrective action taken: _____________________________________
Reviewed by: ________Dr. Karl Klose____Date: ___04/07/2007_