Plasmids Selection and Detection of Recombinant Plasmids PDF

Cloning vectors for E. coli Plasmids selection and detection of recombinant plasmids Introduction of DNA into Living Cells The next step in a gene cloning experiment is to introduce these molecules into living cells, usually bacteria, which then grow and divide to produce clones. Cloning serves two...

Cloning vectors for E. coli Plasmids selection and detection of recombinant plasmids Introduction of DNA into Living Cells The next step in a gene cloning experiment is to introduce these molecules into living cells, usually bacteria, which then grow and divide to produce clones. Cloning serves two main purposes. 1) First, it allows a large number of recombinant DNA molecules to be produced from a limited amount of starting material. 2) The second important function of cloning can be described as purification. The manipulations that result in a recombinant DNA molecule can only rarely be controlled to the extent that no other DNA molecules are present at the end of the procedure. The ligation mixture may contain, in addition to the desired recombinant molecule, any number of the following (Figure 5.2a): -Unligated vector molecules -Unligated DNA fragments -Vector molecules that have recircularized without new DNA being inserted (“self- ligated” vector) -Recombinant DNA molecules that carry the wrong inserted DNA fragment. Of course, different colonies contain different molecules: some contain the desired recombinant DNA molecule, some have different recombinant molecules, and some contain self-ligated vector. The problem therefore becomes a question of identifying the colonies that contain the correct recombinant plasmids. Transformation—the uptake of DNA by bacterial cells Most species of bacteria are able to take up DNA molecules from the medium in which they grow. Often a DNA molecule taken up in this way will be degraded, but occasionally it is able to survive and replicate in the host cell. In particular this happens if the DNA molecule is a plasmid with an origin of replication recognized by the host. Not all species of bacteria are equally efficient at DNA uptake Most species of bacteria, including E. coli, take up only limited amounts of DNA under normal circumstances. In order to transform these species efficiently, the bacteria have to undergo some form of physical and/or chemical treatment that enhances their ability to take up DNA. Cells that have undergone this treatment are said to be competent. Preparation of competent E. coli cells As with many breakthroughs in recombinant DNA technology, the key development as far as transformation is concerned occurred in the early 1970s, when it was observed that E. coli cells that had been soaked in an ice cold salt solution were more efficient at DNA uptake than unsoaked cells. A solution of 50 mM calcium chloride (CaCl2) is traditionally used, although other salts, notably rubidium chloride, are also effective. Exactly why this treatment works is not understood. Possibly CaCl 2 causes the DNA to precipitate onto the outside of the cells, or perhaps the salt is responsible for some kind of change in the cell wall that improves DNA binding. In any case, soaking in CaCl 2 affects only DNA binding, and not the actual uptake into the cell. When DNA is added to treated cells, it remains attached to the cell exterior, and is not at this stage transported into the cytoplasm (Figure 5.3). The actual movement of DNA into competent cells is stimulated by briefly raising the temperature to 42°C. Once again, the exact reason why this heat shock is effective is not understood. Selection for transformed cells Uptake and stable retention of a plasmid is usually detected by looking for expression of the genes carried by the plasmid. For example, E. coli cells are normally sensitive to the growth inhibitory effects of the antibiotics ampicillin and tetracycline. However, cells that contain the plasmid pBR322, which was one of the first cloning vectors to be developed back in the 1970s, are resistant to these antibiotics. This is because pBR322 carries two sets of genes, one gene that codes for a b-lactamase enzyme that modifies ampicillin into a form that is non-toxic to the bacterium, and a second set of genes that code for enzymes that detoxify tetracycline. After a transformation experiment with pBR322, only those E. coli cells that have taken up a plasmid are ampRtetR and able to form colonies on an agar medium that contains ampicillin or tetracycline (Figure 5.4); non-transformants, which are still ampStetS, do not produce colonies on the selective medium. Transformants and non-transformants are therefore easily distinguished. -Most plasmid cloning vectors carry at least one gene that confers antibiotic resistance on the host cells, with selection of transformants being achieved by plating onto an agar medium that contains the relevant antibiotic. -Bear in mind, however, that resistance to the antibiotic is not due merely to the presence of the plasmid in the transformed cells. -The resistance gene on the plasmid must also be expressed, so that the enzyme that detoxifies the antibiotic is synthesized. Expression of the resistance gene begins immediately after transformation, but it will be a few minutes before the cell contains enough of the enzyme to be able to withstand the toxic effects of the antibiotic. -For this reason the transformed bacteria should not be plated onto the selective medium immediately after the heat shock treatment, but first placed in a small volume of liquid medium, in the absence of antibiotic, and incubated for a short time. Plasmid replication and expression can then get started, so that when the cells are plated out and encounter the antibiotic, they will already have synthesized sufficient resistance enzymes to be able to survive (Figure 5.5). Identification of recombinants The next problem is to determine which of the transformed colonies comprise cells that contain recombinant DNA molecules, and which contain selfligated vector molecules. With most cloning vectors, insertion of a DNA fragment into the plasmid destroys the integrity of one of the genes present on the molecule. Recombinants can therefore be identified because the characteristic coded by the inactivated gene is no longer displayed by the host cells (Figure 5.6). We will explore the general principles of insertional inactivation by looking at the different methods used with the two cloning vectors mentioned in the previous section—pBR322 and pUC8. Recombinant selection with pBR322—insertional inactivation of an antibiotic resistance gene pBR322 has several unique restriction sites that can be used to open up the vector before insertion of a new DNA fragment (Figure 5.7a). BamHI, for example, cuts pBR322 at just one position, within the cluster of genes that code for resistance to tetracycline. A recombinant pBR322 molecule, one that carries an extra piece of DNA in the BamHI site (Figure 5.7b), is no longer able to confer tetracycline resistance on its host, as one of the necessary genes is now disrupted by the inserted DNA. Cells containing this recombinant pBR322 molecule are still resistant to ampicillin, but sensitive to tetracycline (ampRtetS ). Screening for pBR322 recombinants is performed in the following way. After transformation the cells are plated onto ampicillin medium and incubated until coloniesappear (Figure 5.8a). All of these colonies are transformants (remember, untransformed cells are ampS and so do not produce colonies on the selective medium), but only a few contain recombinant pBR322 molecules: most contain the normal, self- ligated plasmid. To identify the recombinants the colonies are replica plated onto agar medium that contains tetracycline (Figure 5.8b). After incubation, some of the original colonies regrow, but others do not (Figure 5.8c). Those that do grow consist of cells that carry the normal pBR322 with no inserted DNA and therefore a functional tetracycline resistance gene cluster (ampRtetR). The colonies that do not grow on tetracycline agar are recombinants (ampRtetS); once their positions are known, samples for further study can be recovered from the original ampicillin agar plate. Insertional inactivation does not always involve antibiotic resistance Although insertional inactivation of an antibiotic resistance gene provides an effective means of recombinant identification, the method is made inconvenient by the need to carry out two screenings, one with the antibiotic that selects for transformants, followed by the second screen, after replica-plating, with the antibiotic that distinguishes recombinants. Most modern plasmid vectors therefore make use of a different system. An example is pUC8 (Figure 5.9a), which carries the ampicillin resistance gene and a gene called lacZ′, which codes for part of the enzyme b-galactosidase. Cloning with pUC8 involves insertional inactivation of the lacZ′gene, with recombinants identified because of their inability to synthesize b-galactosidase (Figure 5.9b). Beta-Galactosidase is one of a series of enzymes involved in the breakdown of lactose to glucose plus galactose. It is normally coded by the gene lacZ, which resides on the E. coli chromosome. Some strains of E. coli have a modified lacZ gene, one that lacks the segment referred to as lacZ′and coding for the a-peptide portion of b-galactosidase (Figure 5.10a). These mutants can synthesize the enzyme only when they harbor a plasmid, such as pUC8, that carries the missing lacZ′segment of the gene. A cloning experiment with pUC8 involves selection of transformants on ampicillin agar followed by screening for b-galactosidase activity to identify recombinants. Cells that harbor a normal pUC8 plasmid are ampR and able to synthesize b-galactosidase (Figure 5.9a); recombinants are also ampR but unable to make b-galactosidase (Figure 5.9b). Screening for b-galactosidase presence or absence is in fact quite easy. Rather than assay for lactose being split to glucose and galactose, we test for a slightly different reaction that is also catalyzed by b-galactosidase. This involves a lactose analog called X- gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside) which is broken down by b- galactosidase to a product that is colored deep blue.  If X-gal (plus an inducer of the enzyme such as isopropylthiogalactoside, IPTG) is added to the agar, along with ampicillin, then non-recombinant colonies, the cells of which synthesize b-galactosidase, will be colored blue, whereas recombinants with a disrupted lacZ′gene and unable to make b-galactosidase, will be white. This system, which is called Lac selection, is summarized in Figure 5.10b. Note that both ampicillin resistance and the presence or absence of b-galactosidase are tested for on a single agar plate. The two screenings are therefore carried out together and there is no need for the time-consuming replica- plating step that is necessary with plasmids such as pBR322.