1. Expression vectors have a) a gene for antibiotic resistance; 2) a replication origin; and 3) a promoter
2. The effciency with which plasmids can be put into cells is about 1 cell in 10000 at the best. Antibiotic resistance (carried in a gene on the plasmid) is a good way of screening for bacterial cells that get a plasmid. This is done by plating bacteria on a plate of agar that contains a specific antibiotic that the plasmid gene gives resistance to. Only cells with the plasmid will grow under these conditions.
3. When recombinants are made by ligating a foreign DNA into a plasmid, this process too is not very efficient. A method for determining not only which cells get plasmids, but also which cells get plasmids with DNA inserted into them is also desirable. This latter method involves what is called blue-white screeening and it employs the lac-z (beta-galactosidase) gene of E. coli.
4. In blue-white screening, a plasmid contains the following things – antibiotic resistance, origin or replication, and lac-z gene with at least one restriction site in the gene. Researchers cut DNA with the same restriction enzyme as the restriction enzyme site in lac-Z and then ligate the fragments in the presence of the plasmid cut with the same gene. If a plasmid gets an insert into the lac-z gene, the gene doesn’t function. If no insert goes into the gene and the plasmid simply re-forms the original gene, lac-z functions.
5. The ligation mixture is transformed into E. coli cells and plated on an agar plate that contains an antibiotic (to ensure that only cells with plasmids grow) and a compound known as X-gal. X-gal has the property that if the lac-z gene is present, it will cleave it and produce a blue color. If no lac-Z is present, a white color is produced. Thus, researchers can tell which cells have a plasmid with an insert by looking for which colonies are white. Blue colonies come from cells that plasmids with no insert (functional lac-Z gene), whereas white colonies come from cells with plasmids with inserts that interrupt the lac-Z gene (no functional lac-Z gene).
6. A histidine tag is a short sequence of histidine amino acids linked to either the amino end or the carboxyl end of a polypeptide. This can easily be put onto a gene fragment that codes for a protein of interest. When the protein is translated, the histidines are linked to the amino acids of protein. To purify the protein away from the other proteins of the cell, one busts open the cells and then pours the mixture onto a column with beads containing nickel atoms. Proteins with histidines on them will “stick” to the nickel, whereas those lacking histidines will pass through the column.
7. Adding nickel in solution after this is done allows the “stuck” histidine tagged proteins to become unstuck. A protease can then be used to cleave away the histidines leaving behind the desired protein.
8. The Polymerase Chain Reaction (PCR) was invented in the late 1980s. It provides a way to amplify DNA sequences (make more of them) starting with very tiny amounts of a target DNA. The process borrows from the mechanism of DNA replication. It requires a target DNA, two DNA primers (one for each strand – each primer is complementary to a sequence on one strand of the target DNA), 4 dNTPs, and a DNA polymerase (such as Taq DNA Polymerase) that is resistant to denaturation under high temperatures. A cycle of PCR consists of priming the DNA strands, DNA replication, and denaturation. Each cycle of PCR can theoretically double the amount of DNA present. After 30 cycles, a theoretical amplification of over 1 billion fold is possible.
9. Once one has amplified DNA or cut it with a restriction enzyme, it is useful to be able to determine its size. This involves separating DNA fragments by gel electrophoresis.