Role of AI-2 in Biofilm Formation of L. monocytogenes

[Trader]

This is an article I am very interested in.

I was wondering how "feasible" it would be to go through the genetic manipulation/mutant strain synthesis process that is described in this article? (link below)

http://aem.asm.org/cgi/content/abstract ... type=HWFIG

Is it something involving really advanced biotechnology (something that I know in theory, but in practice no experience at all)?

Or else, I would be interested in seeing first of all the effects of suppressing LuxS in L. monocytogenes, compare it with a wild-type strain to see what LuxS does in L. monocytogenes. Then start from there.

[Trader]

And when I mean "feasible", I mean that it would be something that can be accomplished in a common university lab.

Do these strains require to be specially sent from a special source? (I'm not sure if that is the case judging from what I can understand in this research paper) Or are these strains just typical WT, only some which have gone through mutagenesis?

Would the restriction enzymes responsible for the transformation/creation of the transgenic organism (that really needs to be specific in cutting the exact part of the DNA) be something to be found in a common university lab?

Thank you for answering!

[Trader]

Exactly how complicated is engineering a mutant luxS strain?

I have some contacts with a local university but I'll have to explain exactly what I need.

Thanks

[aelin]

Hi,

Sorry for getting to your post so late. At any rate, this is a really interesting topic. Actually, my current organic chem professor, Martin Semmelhack, at Princeton has done a lot of important research in this field. Here's one of his more recent papers: http://www3.interscience.wiley.com/cgi- ... 2/PDFSTART.

1. Genetic manipulation - This should be very common at almost any university lab (at least procedure wise). In theory, you just need to be able to culture the WT bacteria, engineer a plasmid with the gene you want to introduce, and transform the plasmid into the bacteria. The procedure in the URL is actually a little more complex because a good deal of the first few methods deals with identification and amplification of the insertion site. The second main odd thing they do is allelic recombination between the plasmid and the bacterial chromosome, which can result in a few different strains of the mutants. They select one of those resulting strains via identification by RT-PCR toward the end and then culture that as a biofilm. So, the theory gets a little bit messy, but in terms of materials, I don't think there is anything in there that wouldn't be in a university lab. The enzymes (EcoRI, HindIII) equipment (thermal cycler, PCR, Southern, electroporation) are all very common in most biotech labs.

2. Bacterial strains - The L. monocytogenes bacteria might be a little more difficult to obtain (they got their WT stock from the Chinese CDC, but you should be able to find those strains somewhere in the US as well), and the vectors are common ones (they come from KSV, which I suspect utilize the promoter from a common herpesvirus; you will still have to do some preparatory work to insert the gene of interest though). They also have their own mutant L. monocytogenes, the LM-49 and LM-49RE, so you might have to contact them if you wish to obtain those mutant strains.

That being said, it would be much easier to work in a lab that is already doing similar work to what you want to conduct. They would already have most of the materials and equipment and would know the procedure that you need to follow.

Good luck!
Aaron Lin