Plasmid cloning vectors

Two main types of vector system are used in cloning, those that use plasmids and those that use bacteriophages (revisit Chapters 3 and 10, respectively, for a reminder of the main features of these). Naturally occurring examples of these are manipulated so that they possess the above properties. A popular vector in the early days of gene cloning was the plasmid pBR322; Figure 12.4 shows how it contains the features described above.

Let us consider now what happens, at a molecular level, when donor DNA is ligated into a plasmid vector (Figure 12.5). Sticky ends of the donor fragment form hydrogen bonds with the exposed compatible ends of the opened up plasmid by complementary base pairing, and DNA ligase consolidates the join. Many plasmids contain engineered sequences called multiple cloning sites (MCS); these provide additional flexibility with respect to the restriction fragments that may be accommodated.

The ligation of insert DNA into the cloning site of the plasmid is not the only possible outcome of the procedure described above, however. Unless experimental conditions are carefully controlled (there are ways of doing this), a more likely outcome is that the two compatible ends of the plasmid will simply 'find' each other

£coRl H/ndlll

£coRl H/ndlll

Figure 12.4 Plasmid pBR322. One of the earliest plasmid vectors, pBR322 illustrates the major features required for use in gene cloning: an origin of replication (ori), selectable markers (genes for resistance to ampicillin and tetracycline), and single recognition sites for a number of REs

A multiple cloning site or polylinker is a region of a cloning vector designed to contain recognition sequences for several REs.

The lacZ' gene actually only encodes a part of the ^-galactosidase enzyme, called the a-peptide. The strain of E. coli used as host makes an incomplete version of the enzyme, which lacks this portion. Only if the cells contain the plasmid with the lac Z' gene can they produce functional ^-galactosidase, by a-complementation.

! G !

! GATCC !

! CCTAG : 1 1

: g : 1 1

Compatible 'sticky ends'

Compatible 'sticky ends'

Formation of recombinant vector molecule. DNA ligase consolidates the join by repairing the phosphate-sugar backbone.

Figure 12.5 Formation of a recombinant plasmid. A recombinant plasmid is formed when a fragment of foreign DNA is taken up and ligated into the plasmid. By cleaving both plasmid and foreign DNA with the same RE, compatible 'sticky' ends are created, facilitating the join. Treatment with the enzyme alkaline phosphatase prevents the cut plasmid ends rejoining together, thereby favouring the formation of recombinant molecules

BamHI

recognition site amp" —I__x lacZ

pUC19

BamHI

recognition site

Fragment with BamHI sticky ends

Inserted fragment

Transform into E. coli cells and plate onto medium containing ampicillin and Xgal (artificial substrate for p-galactosidase)

Recombinant colony (white)

Non-recombinant colony (blue)

Figure 12.6 Recombinant plasmids can be detected by insertional inactivation. Insertion of foreign DNA is carried out at a site within one of the selectable markers, thus interrupting its gene sequence. Here, a fragment has been inserted into the BamHI site situated within the gene that codes for f-galactosidase. Bacteria transformed with such a plasmid will not produce the functional enzyme, and so can be distinguished from those carrying plasmids with no inserted DNA

Recombinant colony (white)

Non-recombinant colony (blue)

again, and rejoin. Since a certain amount of this is inevitable, how are we able to tell the difference between those bacteria that contain a recombinant plasmid (one containing a piece of donor DNA) and those that have taken up a recircularised 'native' plasmid? Since both types will contain the gene for ampicillin resistance, we cannot distinguish them by this means. A strategy commonly used to get around the problem is insertional inactivation (Figure 12.6). This clever ploy exploits the fact that we can manipulate DNA, and, for example, insert RE recognition sequences at desired points. If a recognition site occurs in the middle of a gene sequence, and a piece of foreign DNA is inserted at this position, the gene will be interrupted, and unable to produce a functional gene product. In the example shown, the gene is lacZ', necessary for the successful expression of the enzyme f -galactosidase. This will only be expressed in those bacteria that contain plasmids in which the gene has remained uninterrupted, i.e. those that have not taken up an insert. Expression of the j-galactosidase can be detected by growing the bacteria on an artificial substrate, which is converted to a coloured (usually blue) product when acted on by the enzyme. Those cells that contain recombinant plasmids are easily identified because disruption of the lacZ' gene means that no j -galactosidase is produced, resulting in non-pigmented (white) colonies.

One problem remains. Remember that our inserted DNA was derived from the digestion of total (genomic) DNA from the donor organism; this means that our DNA library will contain recombinant plasmids with a whole range of fragments from that digestion, and not just the specific one that interests us. How are we able to distinguish this fragment from the others?

A technique called nucleic acid hybridisation is used to solve the problem. This once again depends on complementary base pairing, and involves the creation of a probe, a short length of single-stranded DNA that is complementary to part of the desired 'target' sequence, and therefore able to seek it out. If searching for the right clone can be likened to looking for a needle in a haystack, then the probe is a powerful 'magnet' that makes the task much easier. The probe carries a tag or label, so that its location can be identified (Figure 12.7).

Once we have identified the clone of bacteria containing plasmids with the insert that interests us, we can grow a pure culture of it and then isolate plasmid DNA. Using the same RE as before, the inserted donor DNA can be removed and purified. We now have enough of this specific DNA sequence (a tiny proportion of the donor organism's total genome) to analyse and manipulate.

. Nitrocellulose

. Nitrocellulose

Peel sheet to produce replica of colonies

Petri dish with colonies of bacteria containing recombinant plasmids

Peel sheet to produce replica of colonies b)

Lyse bacteria and denature DNA

DNA bound

Lyse bacteria and denature DNA

DNA bound

Petri dish with colonies of bacteria containing recombinant plasmids c)

Incubate with probe and wash

Incubate with probe and wash d)

Expose to film d)

Expose to film

Figure 12.7 Colony probing. (a) A replica of the bacterial colonies is made using a nitrocellulose membrane. (b) Alkali treatment lyses the cells and denatures the DNA, making it single stranded. (c) Following a period of incubation with a radiolabelled probe to allow hybridisation to take place, the membrane is washed to remove any unbound radioactivity. (d) Position of bound radioactivity revealed by autoradiography. Comparison with original master plate reveals the location of colonies carrying specific target sequence. Alternative detection systems such as biotin-streptavidin that avoid the use of radioactivity have become much more commonly used in recent years. From Reece, RJ: Analysis of Genes and Genomes, John Wiley & Sons, 2003. Reproduced by permission of the publishers

Although plasmids are easily isolated and manipulated, their use as cloning vectors is limited by the fact that they tend to become unstable if we attempt to insert much more than about 5kb of foreign DNA. For inserts larger than this, we must turn to other vector systems.

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