Gene Cloning

Gene Identification and DNA Libraries 87
Q4.2. What are the basic steps you would need to construct a library containing
genomic DNA from a bacterium? Hint.This is just a variation of the
techniques outlined in Chapter 3. Instead of cloning a particular DNA fragment,
you want all the DNA from an organism.
4.2 Genomic Library
A genomic library is a collection of recombinant clones representing the
entire genome of an organism. Each clone is like a book in that library containing
part of the information contained in the genome. Thus a genomic
library does not only contain the coding regions of the genes but also the
regions between genes which may include important sequences involved
in gene regulation. If the library is made from a eukaryotic organism, a
genomic library would also contain introns, repetitive and junk DNA
(Section 2.3).
To see how to construct a genomic library we will use a relatively
straightforward example. Soil organisms such as species of Pseudomonas
possess enzymes that enable them to use a wide range of compounds as
sources of carbon and energy. One such enzyme, catechol 2,3-oxygenase,
carries out one of the steps involved in the breakdown of xylene; it is
encoded by the xylE gene. The presence of a functioning xylE gene can be
detected by spraying colonies that express the xylE gene with catechol: as
the catechol is broken down the colonies turn yellow. We can use this as a
simple and direct way of selecting our clones from the library. Alternative
approaches to screening DNA libraries are discussed in Chapter 5.
4.3 Constructing a Genomic Library
Genomic DNA extracted from P. aeruginosa (Box 4.1) is cut with a restriction
enzyme such as BamHI. The P. aeruginosa genome is 6.3 Mb, larger
than that of E. coli, and BamHI will cut it into about 1500 fragments with an
average size of 4000 bp. The restriction fragments are then ligated into the
BamHI site of a suitable vector, such as pUC18, introduced into E. coli by
transformation (Figure 4.1), and plated onto agar containing ampicillin.
The colonies that grow on the agar plate make up the library. Remember
that each individual colony is made up of identical bacteria containing the
same recombinant plasmid. Different colonies will contain different recombinants
each consisting of pUC18 with a different piece of Pseudomonas
DNA in it. As the average size of a bacterial gene is about 1 kb and the average
size of the inserts in this example is about 4 kb it is likely that each clone
in the genomic library will contain the DNA for more than one gene.
Having constructed our library the next step is to find out which of the
clones in our library encode the xylE gene. If we spray the plate with
catechol, the colonies that are expressing catechol 2,3-oxygenase will turn
yellow. These are the colonies we are interested in. Plasmid DNA can now