Cloning Vectors and Restriction Enzymes

So, it turns out that this online test is actually an in-class test next week. Which means I might actually have to pay attention to details and commit them to memory just in case they ask some horrible questions. Yikes. (I'm one of those people who does better at understanding general concepts rather than remembering details, so this will be fun.)

To understand the types of cuts to DNA made by restriction enzymes.

See my previous post- Introduction to Cloning.

To be familiar with some of the applications of restriction enzymes in recombinant DNA technology.

This is something that's probably going to crop up again and again over the next few posts in more detail. In a nutshell, though: recombinant DNA technology is all about "cutting and pasting" bits of DNA together. Restriction enzymes act as "scissors" to allow the cutting to be done. (DNA ligase acts as the paste.)

To be aware of the essential characteristics of plasmid vectors.

There are several different kinds of plasmid vectors, but they all have some essential features:

1. An origin of replication (ori) site to allow the plasmid to be replicated.
2. Genes that confer antibiotic resistance. This way they can be "isolated out" by growing a bunch of plasmids on a plate with a particular antibiotic. Only the plasmid vectors with the antibiotic resistance genes will survive.
3. A cutting site where the plasmid can be "cut open" and the DNA fragment inserted.

There are many modern engineered plasmids which have a "polylinker" at the cutting site. A "polylinker," in a nutshell, is essentially a section of DNA which contains several cutting sites for several different restriction enzymes. This way, scientists don't have to be limited to just using EcoRI (a restriction enzyme in E. coli).

To be familiar with other cloning vectors including BACs and YACs.

BACs, or Bacterial Artificial Chromosomes, are plasmids that can be used to copy fairly large genomic fragments (100 000-300 000 base pairs). They are based on the F plasmid of E. coli (yeah, I get the feeling that we're going to be hearing a helluva lot about E. coli for the rest of my degree). Here are some of the essential characteristics in a list, because lists are great:

1. It has a low copy number (i.e. few copies per cell), which apparently makes the insert more stable. Not sure how this works, though I would assume that fewer replications leads to a lower propensity for mutations.
2. It contains par genes which couple plasmid replication to chromosomal replication. This ensures that every daughter cell gets at least one plasmid.
3. It has resistance to chloramphenicol, so this can be used as a selectable marker. Unfortunately the plasmid without the inserted DNA also has resistance to chloramphenicol, so this can't be used to tell the recombinant DNA plasmid apart from the plain old normal plasmid. Good thing there's another built-in selectable marker- see my next point:
4. The plasmid contains the lacZ gene which codes for beta-galactosidase (see my previous post on the lactose operon) AND the restriction site is located in the middle of the lacZ gene. The implications of this is that when the restriction site is cut and a fragment added, the lacZ gene ceases to function and beta-galactosidase is no longer produced. When colonies are cultured on plates containing X-gal, those colonies with a functioning lacZ gene and thus sufficient amounts of beta-galactosidase will react with X-gal to form a blue product. The recombinant DNA plasmids that do not have a functioning lacZ gene, however, will remain white.

YACs, or Yeast Artificial Chromosomes, are cloning vectors that can be used for eukaryotes. They have two TEL (telomere) sites and a CEN (centromere) site which are required for stability and cell division. YACs can exist in a circular form in bacteria. They can be turned into a linear form by digestion with BamHI- this form is required to insert the YAC into a yeast cell. As for the actual insertion process (which is actually called "transformation")- the yeast cell wall is first digested with enzymes, and then electroporation (the use of an electric current) is used to insert the YAC into the cell.

To know the important characteristics of expression vectors and how they can be used to express the protein encoded by a foreign gene. 

Expression vectors are used to stimulate the production of a protein. The most important characteristic of an expression vector is that it has an inducible promoter (i.e. a promoter that you can switch on and off). For example, the lactose operon, which I've written about before, has a promoter which can be induced by allolactose (although in the lab IPTG, a lactose analogue, is more commonly used). In order to get this vector to produce something else, however, the rest of the gene is simply switched out with the gene of interest.