Kit 5B

Tn10 Delivery Systems for Construction of Transposition Strains in S. typhimurium

 

Kit 5B.1 P22 Generalized Transduction of Tn10 - Construction of New Insertions

Utilizes P22 to construct insertional mutants with the transposon Tn10. Generalized transduction by P22 delivers a segment of E. coli. DNA containing Tn10 (Tet (R)); Tetracycline resistant transductants in S. typhimurium arise only from the transposition.

The system described here was constructed in the labs of N. Kleckner and J. Roth. The donor strain (S. typhimurium) carries a defective Tn10 (lacking transposase function) inserted into a F'lac of E. coli. The Tn10 d(Tet) refers to a transposon constructed by Way et al (1984, Gene 32:369-379) called Tn10 del16 del17. P22 HT is grown on this strain and used to transduce a recipient. The recipient strain carries the plasmid pNK972, which is a pBR322 derivative, Ap(R), carrying and expressing the transposase gene of Tn10 (Way et al, 1984). When the transducing fragments carrying Tn10 dTet enter the strain carrying PNK972, they cannot insert by homologous recombination because the lac DNA of E. coli lacks homology; Tc(R) recombinants occur due to transposition. Because the concentration of transposase from the plasmid-borne genes is very high, there may be more than one Tn10 insertion in the recipient. It is possible to put the pNK972 plasmid into any strain and do the transposition in that strain.

It is the experience of the Roth lab that it is better to make pools of P22 phage, harvested from pools of transduced cells. The pools can be kept for years, with EGTA (ethyleneglycol-bis-(b -amino-ethyl ether)n,n'-tetra-acetic acid) added to prevent lysogenization, and used to select Tn10 insertions with different properties. The following description is modified from the Roth lab.

Making mini-Tn10 Pools (Tn10 dTet, Tn10 dCam, and Tn10 d Kam)

To make these pools, a recipient is provided with a plasmid carrying the Tn10 transposase (pNK972). The donor carries the Tn10-derived transposon on an E. coli F', which has no homology to, and cannot recombine with, the Salmonella chromosome. When the transduction is done, the drug resistant colonies have had a transposition event to rescue the transposon off the transducing fragment. Each colony is an independent hop.

 

1. Move pNK972 (TT10427) into the recipient strain by transduction, selecting Ap(R), or if desired, use TT10427 itself as the recipient.

2. Grow a phage lysate on the donor. Use the high frequency transduction form of P22 such as P22 HT105 int- (included) or another HT mutant.

Tn10 d-Tet

TT10423

Tn10 d-Cam

TT10605

Tn10 d-Kam

TT10426

3. Use a dilution series of the lysate to determine the correct concentration of phage. Plate on NB (nutrient broth) or LB (Luria broth) with agar. 1000 - 3000 Drug(R) colonies per plate is good. Remember the problems of scaling up. Also remember that NB is not as rich as LB, and may need supplements if used in the following steps. (We prefer LB, but the broth choice is of little importance.)

4. Scale up. A good pool has between 50,000 and 100,000 independent hops in it. This is 50 to 100 plates.

5. Don't let the colonies get too big; pinhead-size is perfect.

6.Add 2 ml of 1x E salts plus 10 mM EGTA to each plate and use a spreader to suspend all the colonies. The EGTA is included to chelate the Ca(+2) and prevent the adsorption of free P22 to cells in suspension.

7. Tilt the petri dishes and transfer the cell suspension to tubes, or 50 ml centrifuge tubes, with a pasteur pipette. All the cells can be pooled at this point, or sub-pools of 10,000 each can be maintained.

8. Spin down the cells.

9. Resuspend in an equal volume of 1 x E salts plus 10 mM EGTA and spin down again.

10. Resuspend in an equal volume of LB + 10 mM EGTA. Most of these cells can be frozen away in DMSO vials as is. This will provide "cell pools" which can be examined directly for insertions, or treated in steps 11-13 at a later time.

11. Dilute above cells 1:20 with LB + 10 mM EGTA and grow overnight. 20 to 50 ml of final lysate is a good goal that requires 4-10 ml of o/n culture of cells.

12. Use these cells to make a transducing lysate, as follows:

(a) Spin over-night cells out of LB + EGTA and resuspend in an equal volume of LB + 1x E salts.

(b) Wash once in an equal volume of 1x E salts, and resuspend in an equal volume of LB + 1x E salts.

(c) Mix with P22 HT phage to a titer of 10(8) pfu/ml. Don't scale up (it doesn't work very well); use multiple 5 ml cultures in tubes. Incubate several hours or overnight with very vigorous shaking.

(d) Spin down the cells and debris and put the phage pool over CHCl3. This phage pool can be stored for several years.

13. Use the phage pool to transduce any strain to antibiotic resistance. If the pool is large and random, it will represent insertions at very many different sites on the chromosome.

References:

Elliot, T., and J.R. Roth. 1988 Characterization of Tn10 d-Cam: A transposition-defective Tn10 specifying chloramphenicol resistance. Mol. Gen. Genet. 213:332-338.

Way, J.C., M.A. Davis, D. Mevisato, D.E. Roberts, and N. Kleckner. 1984. New Tn10 derivatives for transposon mutagenesis and for constuction of lacZ operon fusions by transposition. Gene 32:369-379.

 

Strains Included in Kit 5B.1:

Strain

Number

SGSC

Number

Genotype

TT10427

925
LT2 / pNK972

TT10423

924
proAB47 / F' pro(+) lac(+) zzf-1831::Tn10(del)16(del)17

TT10605

1559
proAB47 / F' (pro(+) lac(+) zzf-1837::Tn10dCm(R))

TT10426

1732
proAB47 / F'128 pro(+) / lac(+) zzf-1834::Tn10(del)Km

P22 HT105/1-int

-

-

 

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Kit 5B

Tn10 Delivery Systems

 

Kit 5B.2. Pre-Constructed Pools of Tn10 Insertions in S. typhimurium LT2

Strains SA2637 to SA2641 are pools of S. typhimurium LT2 which were transduced to Tc(R) by P22::Tn10 in the laboratory of J.R. Roth. They must be grown in 10 mM EGTA (to prevent lysogenization with P22) and 10 ug/ml tetracycline to select for Tc(R).

 

Strains Included in Kit 5B.2:

Strain

Number

Filing

Number

Genotype

SA2637

SA2637

Tn10 pool 1

SA2638

SA2638

Tn10 pool 2

SA2639

SA2639

Tn10 pool 3

SA2640

SA2640

Tn10 pool 4

SA2641

SA2641

Tn10 pool 5

 

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