biology question #3735



Helen, a 15 year old female from Toronto asks on December 8, 2006,

Q:

How can you extract the bacteriocins (colicins) from E. Coli? I would like to test the effectiveness of bacteriocins in destroying its own species. So I plan to first extract the bacteriocins and then put it among the cultured bacteria to see if the growth of those bacteria is still present. The problem I am having is how to extract the bacteriocins. Should I use centrifugation? If so, at what speed, time length, and how can I extract it afterwards?

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the answer

Wendy Hutchins answered on December 8, 2006, A:

I know from student projects that colicins have potential value in biocontrol, but this is not my best field of study. When I run into such a subject I look in two major places for information: First I google "colicins" and then review the hits, one stands out that should help you a lot - www.ucs.mun.ca/~cjackman/Prokaryotes.html

Second, I check Google Scholar, a database of scientific papers. Search on Colicin and you will find a lot of papers on the subject. You might need your teacher, or a university student to help you read and understand the scientific paper. Review articles are often easier to read, so search for "colicin review".

Now, some ideas for real experiments. It would appear that the type of colicin protein produced by a strain is not going to be made until the bacteria is under stress - then it not only will be made, but also exported outside the bacteria. That suggests that they are likely not present in high quantities. Then they would be found in the media away from the bacteria. To really show that any effect you see on your test bacteria is really due to colicins, you would need to purify them. And that leads to really big headaches.

From the first site I found, it seems that the colocin genes are on plasmids, not usually in the genome of E.coli. That means you should be able to find the plasmids pretty easy from "natural" or clinical strains of E.coli. Well, that means you need to have the strains - could take a while. Well, what if you can find some plasmids that others have already found and stored away. Then you would need to find the gene, clone the gene way from the native E.coli and move it into lab E.coli. When we do this, we take over control of turning the gene on and off, so we can make the protein anytime we want. It may be lethal to lab E.coli though! Once a gene is cloned, and you can make the protein, you have to then purify it, but it is usually much easier to do because we can flag the cloned protein.

Still, this seems like a lot of work for YOU! How about the easy way to get you going - why don't you just buy some.

if you go to Sigma-Aldrich Chemical company, and search colicin, you will find
C3026 Sigma Colicin E1 from Escherichia coli
lyophilized powder, 10,000-20,000 units/mg protein

Prepared by a modification of the method of Schwartz, S. and Helinski, D., Journal of Biololgical Chemistry., 246, 6318 (1971).

One unit per mL is the minimal concentration required to cause a zone of clearing on a lawn of E. coli ATCC9637 cells.

The last piece of information then is a bit about a susceptible E.coli, but not the one that produced it. I bet the reference from the Journal of Biological Chemistry will tell you that. Then you just need to find these two E.coli strains. THAT will be MUCH easier.

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