Recombinant Bacterial Technologies

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Tony Rook
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Recombinant Bacterial Technologies

Is there any good general references (books, articles, etc.) that describe recombinant technologies? I would like to add flourescent markers to specific bacterial species so that at specific phases of growth a flourescent event occurs. Any references would be appreciated.

itaylor
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I don't know of any specific

I don't know of any specific text, but have quite a bit of experience with, the ever popular, gfp in gram positives and gram negatives. Getting the gfp to cooperate can be a task generally, it can be toxic at mid to high levels in the cell (depending on species).

For your applications it may not be the best because its always on and has a fairly long half life. From your brief description, you might require a flash type of flourescents or luminescence, but measurements would then be the issue and not the marker.

Tony Rook
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vasussci:

vasussci:

Thank you for you response! I am currently investigating the use of gfp for this application. The long half life of gfp is actually beneficial to my application (that is once the event occurs, I would like to detect it for a long period of time). You seem to have some experience with this type of application. We are looking at using a gram positive organism. Would you happen to have some good reference that sheds light on some of the pitfalls to look out for?

thanks in advance,

trook

itaylor
itaylor's picture
Unfortunately, I don't have

Unfortunately, I don't have any specific text reference. We were working in Staph. The GFP we used was subcloned from a commercial plasmid, but I can't recall the company. We were using GFP to assay promoter activity. It worked well, however, it hindered strain growth in liquid and on solid media at higher levels of expression.
We transferred to using firefly luciferase mainly because of licensing issues. The luciferase which we subcloned from a promega plasmid we used extensively. We also used their BrightLight reagent to assay, which was also very dependable, but expensive.
ian

Tony Rook
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Vasussci:

Vasussci:

Thanks! I have experience with luciferase assays for bacterial detection but had not thought of using it for a recombinant application. I will look into the feasibility of this.

trook