Transformation is widely used in the laboratory to introduce new or altered DNA molecules into a bacterial cell. This is done most commonly with a plasmid. In order to stably transform a bacterial strain with a plasmid, some method is needed to ensure that the plasmid has been transferred and is being maintained as the cells divide. The most common procedure is to include an antibiotic resistance gene on the plasmid. Escherichia coli is naturally sensitive to antibiotics such as ampicillin, tetracycline, and many more—resistance to which is conferred by genes that are often borne on naturally arising plasmids. Artificially constructed plasmids carrying the same resistance genes are used as tools in molecular biology research.
One of the most widely used antibiotics in molecular biology research is ampicillin. Ampicillin is a β-lactam antibiotic, closely related in structure and action to the antibiotics penicillin and amoxicillin. You may have heard the story of how Alexander Fleming fortuitously discovered penicillin as a chemical secreted from a mould that could kill bacteria in his laboratory. If you grew up in the United States and had an ear infection as a child, you were almost certainly treated with amoxicillin. Ampicillin and antibiotics like it can be destroyed by some bacteria that produce β-lactamase enzymes. By including a gene, such as ampR, which encodes a β-lactamase that confers ampicillin resistance, on a plasmid, it is possible to identify and select for those cells that have been transformed.
So how does this selection work in practice? Millions of competent cells are exposed to the plasmid, and a few of the cells will be transformed. The population of cells is grown in medium that includes ampicillin. Those cells that have not been transformed die, whereas the few individual cells that have taken up the plasmid with the ampR gene will grow into ampicillin-resistant colonies. Thus, cells that include the ampR gene have been selected for; the ampR gene is referred to as a selectable marker. Even if only a tiny fraction of the potential recipient cells are transformed, the powerful selection using ampicillin allows resistant cells to repopulate an entire culture.
The idea is that cells that have taken up the plasmid with the ampR gene on it can simultaneously receive other genes on the plasmid. Each plasmid consists of at least 2500 DNA base pairs, of which only about 900 base pairs comprise the ampR gene. The rest of the vector must contain genes and sequences that will allow it to replicate in a new cell. However, other genes and functional DNA sequences can then be added to the plasmid and will be transformed into the bacterial cells along with the ampR gene. As such, the plasmid is being used as a vector to carry other genes, and the selectable marker (in this case, the ampR gene) is being used to select for the transformants.
If the other gene or genes has the normal transcriptional regulatory sequences recognized by E. coli, it will be transcribed as if it is a bacterial gene. If it also has the signals needed for translation, the transcript can be translated into protein. This method is often used to produce large quantities of a protein; the gene encoding the protein is inserted on a plasmid that also includes the ampR gene, or another selectable marker, and is expressed in bacterial cells. For example, bacteria can be made to glow green because they have been transformed with a plasmid carrying an ampR selectable marker and a green fluorescent protein (GFP) gene from a jellyfish, as shown in Figure A.
Figure A E. coli expressing ampicillin resistance and green fluorescent protein (GFP). A plasmid that included an ampR gene and the GFP gene was transformed into E. coli, and the bacteria were plated on medium with ampicillin. Colonies that were able to grow because they were ampicillin-resistant also expressed GFP.Hundreds of different plasmid vectors are commercially available, with many different selectable markers and signals for expression. Usually, the sequence of the plasmid has been artificially manipulated to make the insertion of other genes or sequences easier. Typically, the plasmid will include one or more sites for insertion of other DNA, known as a poly-linker sequence or multiple cloning site (MCS). For example, the pUC18 and pUC19 plasmids have this sequence, as shown in Figure B. Vectors pUC18 and pUC19 replicate very efficiently in E. coli from their origin of replication (ori), with as many as 300 copies per cell being made. The plasmids carry an ampicillin resistance gene, which serves as a selectable marker, as well as a β-galactosidase gene (lacZ) that contains an MCS, which can be cut by several restriction enzymes. When a piece of extraneous DNA is cloned into the MCS site, the lacZ gene is inactivated. Therefore, clones containing inserts can easily be distinguished from the vector because they lack β-galactosidase activity.
Figure B Plasmids A plasmid used as a cloning vector. This map shows the commercially available cloning vectors pUC18/19, among the first widely used plasmid vectors. The locations of various restriction sites are shown. In addition to the origin of replication (ori), which allows the plasmid to replicate, and the ampR gene, which allows for selection of cells with the plasmid, the plasmid also includes the lacZ gene. lacZ encodes β-galactosidase, as described in Chapter 14, and is used here as an indicator. As discussed in Tool Box 15.1, if cells are grown on plates with a chemical known as X-gal, the cells expressing lacZ will cleave X-gal to make a blue substrate. Note that the multiple cloning site (MCS) is within the lacZ gene. Thus, if a sequence of interest has been inserted at the MCS, the lacZ gene is disrupted and not expressed, so the colonies are white. This allows for a procedure known as a blue–white selection. As shown below the plasmid, the MCSs for pUC18 and pUC19 have different sequences and different enzyme restriction sites.