P1 Transduction in Salmonella
Phage P1 will infect or act as a transducing phage in Salmonella. Infection requires removal of the Salmonella O-antigen which seems to block the P1 Absorption sites. The O-antigen is conveniently removed through the use of a galE (epimerase) mutation in the recipient or host cell. This mutation prevents the cell from converting UDP-glu to UDP-gal and therefore cells growing on glucose cannot make galactose for O-antigen synthesis. If one wants O-antigen to be synthesized, the galE mutant can be grown on .2% glucose plus .2% galactose. The exogenous galactose can be used for O-antigen synthesis. Since galE mutants accumulate galactose PO4 they are sensitive to galactose and are killed; they grow normally on galactose plus glucose. Strains carrying galE are P22SensP1Res when grown on glucose plus galactose; they are p22ResP1Sens when grown on glucose alone. Strain SL3684 is a galE- which carries a closely linked hut+ region. The hut+ determinants permit the strain to use histidine as sole nitrogen source (on succinate medium). Since LT-2 strains are all hut-, one can transduce any LT-2 strain to hut+galE- on succinate + histidine medium using LS3684 as donor. These transductions can be done using P22 phage grown on SL3684 in broth containing glucose and galactose. About 50% of the transductants are galE-. These transductants can be identified by the following criteria:
(a) On the original transduction plate they form hard colonies that "fracture" when touched.
(b) They are galactose sensitive. On EMB gal they form characteristic thin patches with fast-growing gal- (Kinase negative) colonies.
(c) They are P22Res - P1Sens.
The conditions we use are described on the following page. The general method and strain SL3684 were obtained from B.A.D. Stocker. P1 methods were obtained from A.J. Clark.
Methods for P1 Growth
Phage assay
Mix 0.1 ml phage (diluted appropriately)
0.1 ml log culture of SL3684 grown in LC Broth
0.1 ml salts (0.3M MgSO4, 0.015M CaCl2)
Incubate 30' at 37o for absorption.
Add 2.5 ml LCTG soft agar.
Plate on LC plates.
Incubate overnight at 42o (we find that high temp gives bigger plaques).
Lysate Preparation (Plate method)
Mix 106 phage
0.1 ml log cells of P1-sens. strains (eg. SL3684 grown on LC Broth)
0.1 ml salts (see above)
Incubate 30' at 37o for absoption
Add LCTG soft agar.
Plate on LC plates and incubate plates at 37o.
Harvest after 6 hrs.
Store phage in LC Broth
Transduction by P1
Mix 0.5 ml of overnight culture of P1 sensitive recipients (no galactose in med.)
0.5 ml of phage (multiplicity of infection ~0.05)
0.5 ml of salts mix (CaCl2/MgSO4)
Incubate 30' at 37o
Spin down cells
Suspend in E medium (3 ml)
Spin again
Suspend in 0.5 ml E medium
Plate 0.2 ml, 0.1 ml or dilution on selective medium.
For controls without phage; do the above moves using LC Broth in place of phage
Recipes (For all P1 work, fresh plates work best)
LC Plates (or slants) LCTG Soft Agar
10g Bacto tryptone 10g
10g NaCl 10g
5g Yeast Extract 5g
10g Difco Agar 7g
1 liter H20 1 liter
Autocalve; cool; then add the following sterile solutions
4ml 0.5 M CaCl2 4 ml
2.5 ml 40% Glucose 2.5 ml
4 ml 0.25% Thymidine 4 ml
P1 Transductions
Grow P1
1. Overnight culture of strain in LBH
0.5 ml of o/n into 5 ml LBH + 5 mM CaCl2
density should equal 1-2x 108 cells/ ml
2. Add 5x107 pfu of P1
3. Absorb 20' at 37o
4. Add 0.2 ml of cells + P1 to 2.5 ml top LT +10 mM CaCl2
5. Plate onto fresh LB + 5 mM CaCl2 plates
6. Overnight 37o
7. Harvest
Titer
1. Overnight culture in LBH
2. Dilute, grow 5 mls to 2x108 cells/ ml in LBH + 5mM CaCl2
3. Dilute P1 to appropriaate concontrations into LBH + 5mM CaCl2
4. 0.1 ml cells + 0.1 ml P1 dilution
5. Absorb 20' at 37o
6. Add 2.5 ml top LT agar + 10 mM CaCl2
7. Plate on fresh LB +5mM CaCl2 plates
Transduction
1. Fresh o/n of cells in LB +5mM Ca++
2. Add 2x109 P1 to 0.2 ml of fresh o/n
2x108 P1 to 0.2 ml of fresh o/n
2z107 P1 to 0.2 ml of fresh o/n
2x109 P1 - no bacteria
no phages - 0.2 ml fresh o/n
3. Absorb 20' at 37o
4. Spin down
Resuspend pellet in 2 ml LBH + 20mM sodium citrate
Incubate l hr at 37o
5. Wash with minimal media once
Resuspend to 0.1 ml in minimal media
Plate onto selection plate
Last Update: Thursday June 19 2014
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Eric Kofoid
eckofoid at ucdavis.edu