P1 transduction problems

Rafael Maldonado rafamaldo at geocities.com
Fri Feb 26 14:35:11 EST 1999


, David Alexander escribió:
> 
> Hello Netters,
> 
> The P1 vir lysate made on the MG1655 strain with my gene/TetR
> is of very high titer (believe it or not, around 10 ^ 16)
> However, I have yet to see a TetR transductant.

I agree with Chris, it is far too high. Beter check your calculation on
the dilution and count. Then, if you are not adding the correct amount
of phage, forget it.

> I am using the tried and true procedure from Miller:
> 5 mL over night of MC4100 + 5 mL MC Buffer (Mg, Ca)
> incubate 37C for 15 min with shaking
> mix 0.1 mL cells with 0.1 mL diluted phage lysate from MG1655
> incubate at 37C for 20 min to let phage attach
> then, add 0.2 mL 1M NaCitrate and plate onto LB + Tet.

I guess that Tet resistance is from Tn10. Normally, you needn't any
expression for that resistance. What I would check is the viability of
your phage. Have you had the same problems with other strains? Maybe
some detergent in the solutions is killing the phage.

Since you have following Miller protocol, I guess you have following
correctly. Maybe it could help if you add citrate to the Tet plates. One
possibility is that your MC4100 is not sentive to P1. Can you get
plaques on that strain? It is easy to check that the phage can get
inside the cell and kill it. Because if for some reason, the strain is
resistant to the phage, there is no possibility for getting
recombinants.

Another possibility is that your resistance marker is in the big
deletion MC4100 carries, del(argF-lac)U169. There is no chance to get
recombinants in a deletion!

And finally, I don't remember the genotype of MG1655, but IF it has got
a F plasmid, and if your resistance is in the F plasmid, you won't get
recombinants either, since MC4100 is F-.

> Initially I thought that my multiplicity of infection was too high
> (i.e. too many phage - no surviving E. coli)
> but I have attempted the transduction over a wide range
> of phage concentrations (10 ^-2 through 10^ -24)
> and still no TetR clones.

Well, you won't kill the cells by putting too many phages. On the
contrary, you kill them when the multiplicity of infection is low. So if
you are underestimating the number of phages, there you have the
mistake.

I hope this help,

-Rafa



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