How do you express a human gene in E. coli?

Answer 1

E.coli is used to express human genes because it can be easily grown in the lab. The gene is extracted from the DNA (by doing a partial digest with a restriction enzyme), and given a cohesive sticky end with a linker or adapter. It is then ligated to a plasmid vector, which had a restriction site compatible with the ends on the gene, eg if the plasmid contains a BamH1 site then you would add a linker or adapter which is compatible with the 5'GGATCC3' BamH1 recognition sequence. The cells are transformed (made to take up the plasmid vector) by chemical treatment; they are mixed with the plasmid, then a strong concentration of calcium (Ca2+) ions is added to the mixture to make the E.coli's membranes porous. The mixture is then heated to heat-shock the cells, to approx 50 degrees C for one minute. They are then cooled and allowed to recover in a nutrient rich broth at optimum temperature. This is a very inefficient process - only about 1 cell in every million is transformed. The pUC18 plasmid vector is useful because it contains the gene for ampicillin resistance. Any cells which subsequently grow on a medium containing ampicillin, therefore, have been transformed with the plasmid vector. It is also useful because it contains a beta-galactosidase gene, which itself contains the recognition site for a number of restriction enzymes, including BamH1. This is good because you can tell if the vector has taken up the gene you are trying to express when the vector no longer codes for the beta-galactosidase protein product. If the vector has been ligated with the gene, the gene will have disrupted the beta-galactosidase gene. This can be tested with IPTG (an auto-inducer) and X-gal, which will turn colonies of E.coli with the beta-galactosidase gene intact blue (ie, those without the gene of interest). Colonies which have had their beta-galactosidase gene destroyed by the ligation of the gene of interest will be colourless in the presence of X-gal and IPTG. These colonies are all clonal, so all cells in colourless colonies contain copies of the pUC18 plasmid vector which has been ligated with the human gene.