Vibrio fischeri isolation

[Mega]

Hi, two days ago I bought a piece of Salmon, cut away the skin. I placed the skin on a plate and put some salty water (30g/L) on the plate, yet leaving the salmon-skin unwetted. 

Left it in our basement (some 10-15°C maybe). 

As I had a look today, it was amazing. They are very bright. (They are nearly as bright as the pVIB E Coli were, althought the Colis had the operon ~20 times (medium copy based on pBR322), while V. Fischeri has just a single copy of the operon).

You just walk into the room, switch of the light and it glows amazingly (i.e. visibly without eye adaption). 

[Daniel C.]

I may have missed an earlier discussion on this, but... can you give
more information on what's going on here? It sounds like you just put
fish skin on a plate with saltwater and then it started glowing.

-Dan

[Andreas Sturm]

Hi.

Basically that's right.

On squids and herings from the sea there always are some bacteria on their skin.
A professor of mine told me that it would also work with salmon.

So I went to the "Nordsee" fish shop where I bought a piece of salmon (flesh+skin)

I put the skin in salty water and the glowing bacteria grew.


Now I have a source for a PCR of the lux Operon at least ;)

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[Andreas Sturm]

http://tequals0.wordpress.com/2011/09/29/isolation-of-vibrio-fischeri-from-seafish/

Here a link how to do it ;)

Btw., there are nearly no instructions in English available, in German there are plenty.

[Cathal Garvey]

Yea, copy number of the DNA isn't the limiting factor in
bioluminescence: quantity of inputs is much more limiting.
You can get great luminescence from natural Vibrio species, and they can
be easier to grow in pure culture if you isolate Vibrio phosphoreum
rather than Vibrio fischeri (if it's bright blue and can grow at 4C,
odds are it's phosphoreum).

Sadly, my cultures of phosphoreum don't seem to have survived long-term
freezer storage, but they're satisfying to re-isolate when lost!

Unfortunately, they smell pretty bad during growth.. :P
According to some guides I've seen, you can maximise their brightness by
feeding them glycerol rather than glucose/sucrose. This might be due to
whether or not they produce acid from glycerol or not; acid would
probably screw up bioluminescence.

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[Andreas Sturm]

Ah, ok... So the substrate that lux CDE uses, right? So adding those genes which make the precursor will enhance luminescence much more than gene copy?

Well, I hope it's V.fischeri, because the operon is very compact. So one PCR with two primers can ghet you all the lux genes needed.

The structure of P.Phosphoreum is more seperated. IIRC, the flavin reductase gene is not even in the operon -> 2 PCRs needed