KIT 5A

Tn 5 Delivery Systems for Construction of Transposition Strains in S. typhimurium

 

Kit 5A.1. P22 Generalized Transduction of Tn5 - Construction of New Insertions

Utilizes P22 to construct insertional mutants with the transposon Tn5. Generalized transduction by P22 delivers a segment of E. coli. DNA containing Tn5 (Kan (R)); Kanamycin resistant transductants in S. typhimurium arise only from the transposition.

Roth and colleagues (Menzel and Roth, 1981, J. Mol. Biol. 148:21-48.) have made transposition mutants by P22 HT105/1int transduction of Tn5 into a recipient background with no homology for standard recombination. Homologous recombination has been prevented by using a donor strain (TT1780) which carries Tn5 inserted in the E. coli K-12 lac operon of an F' lac plasmid. An F(-) Salmonella spp. recipient strain has no sequences homologous to the site of the donor Tn5 so integration of Kanamycin resistance can only occur due to transposition of Tn5. Alternatively, one can use a recA(-) recipient to block recombination; in this case any Tn5 insertion mutant can be used as a donor.

 

Strain

Number

SGSC

Number

Plasmid

Chromosomal

Mutations

Lab of

Origin

TT1780

512

F' proB(+)

lacZ::Tn5

(E. coli genes)

pyrC7 put(+) leuD798 fol-101 supQ1238 (proAB) / F'proB(+) lacZ::Tn5

John Roth

SA1970

-

-

metA22 trpC2

hisF1009 rps120

xy1R1 recA1

srl-202::Tn10

Ken Sanderson

The Tn10 insertion in srl is 50% linked to recA by P22 mediated joint transduction. Thus recA strains can be selected by transduction and selection for tetracycline resistance, using SA1970 as donor, then screening transductants for recA(-), recognized as UV-sensitive.

A P22 phage has been constructed by N. Kleckner that carries Tn5; the phage genome carries mutations that prevents lysogeny and growth in the recipient such that the Tn5 element can only be stably inherited if it transposes from the phage to the host chromosome (Shanabruch et al., 1981, J. Bacteriol. 147:827-835).

NOTE: One problem with the use of Tn5 is its frequent transposition when first introduced into a new recipient (see Beck and Roth, 1980, Proc. Natl. Acad. Sci. USA 77:6047-6051). For example, if trp::Tn10 is transduced into a new recipient selecting for Km(R), about 10% of the Km(+) transductants will occur due to transposition of the Tn5 to new sites in the recipient. The tendency of Tn5 to transpose can be greatly reduced if the cross is performed at 40 degrees C since Tn5 transposes poorly at high temperature. Once a Tn5 mutation is established in a strain the transposition activity is inhibited; this transposition problem is only associated with initial introduction of Tn5 into a new strain.

 

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KIT 5A

Tn 5 Delivery Systems for Construction of Transposition Strains in S. typhimurium

 

Kit 5A.2. Delivery System for Lambda-sensitive Tn5 using lambda into Salmonella

Use of phage transfer by Lambda to deliver a Lambda::Tn5 (Kan (R)) into Salmonella. Kanamycin resistant recombinants arise only from the transposition.

 

Genotype of Strains Included with this Kit:

Strain

Number

Filing

Number

Genotype

LE392

SGSC422b
F(-) hsdR514(r(-)k, m(-)k) supE44 supF58 lacY1 galK2 galT22 metB1 trpR55 lambda(-)

M26

SAB2906
galE15 relA1 rpsL150 lambda(-)

TS736

SGSC436
hisG165 ilv-452 metA22 metE551 trpC2 galE496 xyl-404 rpsL120 fla-66 hsdL6 hsdSA29 malB/F112 mal(+) (of E. coli)

SAB2516

SAB2516
F(-) hsdR514(r(-)k, m(-)k) supE44 supF58 lacY1 galK2 galT22 metB1 trpR55 lambda(-) / lambda cI857 lysogen

 

Materials:

A. Strains used For phage propagation: any lambda-sens, Sup(+) (permissive) strain such as LE392 (SGSC422b) which is E. coli supE44.
  As transduction recipient: any lambda-sens, Sup(-) (restrictive) strain such as M26 (SAB2906) (E. coli K-12) or TS736 (SGSC 436) (malB (+) S. typhimurium).
B. Phages used B305 lambda::Tn5:b221 cI857 rex::Tn5 (Received from D. Weiner, Univ. of Alberta, propagated on LE392.
B297 lambda cI857, induced at 45 degrees C from SAB2516, a lambda cI857 lysogen.
  B299 lambda vir, propagated on LE392.

Methods:

A. Propagation of B305 (lambda Tn5 ) - LE392 was grown in lambda broth supplemented with 0.1 % maltose and 10 mM MgSO4 at 37 degrees C overnight with shaking. 0.1 ml of B305 phage (diluted to give an MOI of between 1 and 5) was added to 0.1 ml of the bacterial cells and adsorbed at room temperature for 30 minutes. The mixture was added to 3 ml of molten L-soft agar and overlaid on L-plates preheated to 40 degrees C. The plates were incubated at 30 degrees C until plaques appeared (4-5 hours) at which time 5ml of L-broth was added and the soft agar removed to a centrifuge. After centrifugation at 10,000 rpm for 10 minutes the supernatant was removed and stored in a sterile glass vial with a small amount of chloroform added.

The phage lysate was titered by dropping increasing dilutions of phage onto a lawn of LE392 cells on an L-agar plate. Plaque-forming units could be counted after incubation at 37 degrees C for 4-5 hours.

B. Transduction methods - Strains to be used for transduction were grown in lambda broth supplemented with 0.1% maltose and 10 mM MgSO4 and tested for sensitivity to lambda cI857, lambda vir and lambda Tn5. Strains which displayed lambda sensitivity but were not lysed by lambda Tn5 (ie, Sup (-) strains) were grown to mid-log phase (1.5 to 2 hours with shaking) in lambda broth plus 0.1 % maltose and 10 mM MgSO4 at 30°C, centrifuged to pellet the cells and resuspended in 0.1 ml of 10 mM MgSO4. The B305 phage was diluted to give an MOI of 1 in 0.1 ml (assume 2 times 10 cfu/ml in mid log phase culture). 0.1 ml of phage and 0.1 ml of cells were added and adsorbed at room temperature for 30 minutes. The mixture was then added to 10 ml of L-broth and grown for 2 hours at 30 degrees C. The cells were then pelleted and plated on L-plates containing 0.05 mg/ml kanamycin sulfate. Kanamycin-resistant colonies appeared after overnight incubation at 37 degrees C.

NOTES:

S. typhimurium does not adsorb lambda, but it can serve as a recipient for lambda if the malB (lam) genes are added. This can be done in either of two ways.

1/ The strain TS736 is an S. typhimurium strain carrying F'112 malB (+) (lam) of E. coli. This is filed under SGSC436. However, the F'mal does not seem to be capable of further transfer.

2/ Palva has constructed a pBR322-derived plasmid which carries malB(+) (lam). It is thus easier to make a strain sensitive to lambda, because this plasmid can be transferred with selection for Ap(R) It has the disadvantage that subsequent analysis may require the use of an independent pBR322-derived plasmid, which cannot co-exist in the same cell because of incompatibility.

 

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