MadSci Network: Genetics
Query:

Re: regarding with plasmids MCS, promoter, priming site

Date: Fri Oct 6 10:51:57 2006
Posted By: Billy Carver, Grad student, Biomedical Sciences, Vanderbilt University
Area of science: Genetics
ID: 1160065340.Ge
Message:

Hi Amy!

That's a lot of questions you have there, but here goes. The multiple cloning sites in plasmid are usually artificially introduced into that plasmid so geneticists don't have to work quite so hard to introduce a gene of interest. T7 is a bacteriophage (a virus that infects bacteria) element that is usefully because it can act like a promoter for a gene you place downstream of it - this is called promoter fusion. For instance, if I am interested in purifying a protein like insulin, I want to create a great deal of it in a cell that might not normally make it, so I would put a promoter upstream of the insulin gene and then expose the cell to conditions that activate that promoter. Often the T7 promoter is fused with a lac operator so that you can control when the protein of interest is expressed. Another advantage of T7 is that when it is transformed into a bacterial or mammalian cell, the T7 RNA Polymerase gene must also be integrated into the cell because host RNA Polymerases can't recognize the T7 promoter sequence. Neat, huh? Also, in certain circumstance you might want to amplify a portion of your completed plasmid to make sure you have inserted things correctly. T7 occurs in many, many plasmids and thus a primer in the T7 promoter region can be quite handy when checking sizes. So, let's say I have inserted my insulin open reading frame (ORF) after the T7 promoter. If I use a primer in the T7 region, and another primer from within the insulin ORF, I should only get amplification in the inserted plasmid, and not the unsuccessfully ligated plasmid. This will come in handy later.

Now, about that Xho1 and BamH1 thing - Without knowing exactly what is going on, here is my best guess. If you have a plasmid with a MCS that has Xho1 and BamH1 occurring in the 5' to 3' direction, and your gene of interest also has Xho1 and BamH1 in the 5' to 3' direction, everything is great! That means you can double digest both your gene (after you amplify it with PCR) and your plasmid with Xho1 and BamH1. They will get "sticky ends" that match each other, and you can mix them together, along with some DNA ligase, and they will come together. If you use two

different sets of restriction enzymes for these cuts, the sticky ends wouldn't match and there would be trouble. If you had just one enzyme in the plasmid, and then had a restriction enzyme site at both ends of the gene of interest and tried to ligate that, the gene could insert either 5' to 3', or 3' to 5'. Can you guess which one of those is bad news? If you INSIST on using a single enzyme, though, we can refer to our old friend T7. In a non-inserted ligant, there will be no amplification if there is no insert. Conversely, if we use two enzymes, there is a small chance that multiple copies of the fragment will ligate into the plasmid. If that happens, we will get several different sized PCR products. New England Biolabs has a great website that covers how restriction enzymes work, and how to use them in cloning experiments like this. I think you might get some good information from there. Finally, as far as the other components in the plasmid are concerned, other than the genes under control of the T7 (or in the case of the pcDNA3.1 plasmid, SV40) promoter, they are all expressed. If you were a researcher and you were really interested in using a plasmid without a certain gene, you could always switch plasmids. In the pcDNA3.1 plasmid you have neomycin and ampicillin resistance genes. These are both important antibiotic markers because ostensibly you are going to eventually going to transform your ligated construct pcDNA3.1:cIAP2 into competent E. coli to amplify it and then transfect that into mammalian cells to look at phenotypic changes. Because transformation and transfection are relatively inefficient processes, it is important to be able to kill off cells that don't take up your DNA. Ampicillin added to media will kill any E. coli cell that does not take up the plasmid and express it. Neomycin will kill any mammalian cells that don't take up the transfected plasmid and express it.

I hope that helped!

For further reading, I suggest you check out Invitrogen's pamphlet on the pcDNA3.1 (+/-) plasmids. It has a great overview of the plasmid's constituents and their usefulness in experiments. Goodluck!

You might check:
www.promega.com www.invitrogen.com www.neb.com

for more information on proprietary plasmids and their contents. A lot of these companies offer kits that include plasmids with variable components, and include detailed descriptions of their use and abilities.


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