Page 27 - Advances in Textile Biotechnology

6 Advances in textile biotechnology

There are several DNA ligases isolated from different sources but the
most commonly used in the laboratory is derived from the bacteriophage
T4 (Cozzarelli et al., 1967; Weiss et al., 1968; Weiss, 1971). T4 DNA ligase
can join, through a covalent ligation, two DNA fragments with both sticky
or blunt ends, although in the latter case the reaction is less effi cient and
requires higher enzyme concentrations and optimized reaction conditions
(Murray et al., 1979).
Ligation between the target DNA and the vector is a very ineffi cient
reaction because the two cleaved ends of a cloning vector (if compatible)
can self-ligate. Self-ligation can be avoided if the vector is treated with a
phosphatase, an enzyme that removes both terminal 5′-phosphate groups
from the vector. Without it, the vector cannot be recircularized by ligase
because the phosphodiester bond does not form. When the vector is joined
with an insert, the 5′-phosphate is provided by the insert. Alternatively, if
the vector is digested by two different restriction endonucleases non-
complementary sticky ends, which hinder the self-ligation reaction and
promote controlled ligation of the DNA insert in the desired orientation
within the vector, are produced.

1.1.2 Transformation of host cells
The ligation reaction mixture can be introduced into host cells in a process
called transformation. Chemical transformation and electroporation are
two methods used to transform Escherichia coli cells. Several chemical
methods have been established that induce bacterial cell transformation.
Treatment of bacterial cells with CaCl 2 is still the most widely used cloning
method in small laboratories (Cohen et al., 1972; Mandel and Higa, 1970;

Oishi and Cosloy, 1972) and over the years its efficiency has been greatly
improved (Dagert and Ehrlich, 1974; Chen et al., 2001; Fregel et al., 2008;
Huff et al., 1990; Nakata et al., 1997). The permeable ‘competent’ cells are
then mixed with DNA to allow the uptake of the exogenous DNA into the
bacterial cell. Electroporation can also be used to introduce DNA into cells
by subjecting them to a strong electric current, thus producing transient
pores in their membranes.
Multiplication of the vector DNA, either recombinant or non-
recombinant, occurs within each transformed bacterium. A single bacterial
cell cultivated in an appropriate medium, containing the necessary nutri-
ents, can multiply to form a visible colony made of millions of identical cells
(clones). As the host cell divides, the vectors, commonly plasmids, are passed
on to progeny, where they continue to replicate. Numerous cell divisions of
a single transformed bacteria result in a clone of cells, that, when cultivated
into an agar plate, produce a visible bacterial colony. The cloned DNA can
then be isolated from the bacterial cells.

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