8 replies to this topic

[Ro-85]

I'm co-infecting my bacteria with e.coli to observe competitive growth and I'm planning on using Lambda phage to remove the e.coli later.
Does anyone know of a protocol to do this? What amounts, what conditions etc?
  Thank you very much for your help.

[phage434]

If your goal is complete removal of E. coli, this is unlikely to work.  Lambda will chromosomally integrate and protect some E. coli cells from lysis.  T7 might be a better choice, but even with a lytic phage you are unlikely to kill all of the E. coli.

[Ro-85]

phage434, on Jul 8 2009, 06:39 AM, said:

If your goal is complete removal of E. coli, this is unlikely to work.  Lambda will chromosomally integrate and protect some E. coli cells from lysis.  T7 might be a better choice, but even with a lytic phage you are unlikely to kill all of the E. coli.

  I see, that might be a problem. Do you perchance have any ideas on how to selectively remove a bacteria from a mixed culture?

[gfischer]

What about a selective media?  If E. coli needs something to survive that your bacteria of interest doesn't (i.e. a nutrient) or if it can survive in a condition like high salt concentrations that E. coli can't, you should be able to plate the mixed culture and get a pure population out.  The easiest would probably be to transform your bacteria with an antibiotic resistance gene, and use that to select.  Flow cytometry also comes to mind if you have antibodies to some antigen on one of the bugs, though I don't actually know whether you can do FACS on bacteria.

[swanny]

gfischer, on Jul 9 2009, 04:33 AM, said:

What about a selective media?  If E. coli needs something to survive that your bacteria of interest doesn't (i.e. a nutrient) or if it can survive in a condition like high salt concentrations that E. coli can't, you should be able to plate the mixed culture and get a pure population out.  The easiest would probably be to transform your bacteria with an antibiotic resistance gene, and use that to select.  Flow cytometry also comes to mind if you have antibodies to some antigen on one of the bugs, though I don't actually know whether you can do FACS on bacteria.

Whatever selection criterion you choose, it is essential it cannot enter the bacterial conjugation pathway: that is, the bugs cannot have the capacity to transfer plasmid DNA. That probably argues against using an antibiotic selection. In addition, the presence of plasmids has been shown to affect normal cell growth because of metabolic stress.

A better way might be to use a modified E. coli that is an auxotroph. That way, the bugs can be killed by removing the essential compound.

[Ro-85]

gfischer, on Jul 9 2009, 04:33 AM, said:

A better way might be to use a modified E. coli that is an auxotroph. That way, the bugs can be killed by removing the essential compound.

I like this idea, I'll see if I can track it down.
  My PI is rather attached to the Phage idea so perhaps I will try both, either way, just for future reference, how does one go about getting their hands on some Phage? I would think that it would be sold from some biotech company but I have yet to track some down.
Am I missing an obvious resource?

[fishdoc]

Add 10 ug/ml of colistin to agar plates. Unless it carries a resistance plasmid E. coli doesn't survive in that.

We use E. coli to conjugate plasmids to our bacterium (another enteric, but not E. coli), and to select conjugates, we plate on 10 ug/ml colistin to remove E. coli. Works very well, and it's quite easy.

The only caveat would be whether or not your species is colistin resistant.

Edited by fishdoc, 09 July 2009 - 07:22 AM.

[HomeBrew]

I did a competitive growth experiment, plated diluted samples in duplicate, and did colony lifts.  I then probed the blocked membranes with antibodies that recognized one or the other bacteria.  Worked beautifully.

[phage434]

You can get phage samples cheaply from Carolina Biological.