Biomedical Engineering Reference
In-Depth Information
The temperate phage
φ
C31 is the streptomycete
this reason, Sosio
et al
. (2000) generated bacterial
artificial chromosomes (BACs) that can accommodate
up to 100 kb of streptomycete DNA. These vectors
can be shuttled between
E. coli
, where they replic-
ate autonomously, and
Streptomyces
, where they
integrate site-specifically into the chromosome.
equivalent of phage
λ
and has been subjugated as a
vector.
C31-derived vectors have upper and lower
size limits for clonable fragments with an average
insert size of 8 kb. In contrast, there are no such size
constraints on plasmid cloning, although recombin-
ant plasmids of a size greater than 35 kb are rare with
the usual vectors. However, phage vectors do have
one important advantage: plaques can be obtained
overnight, whereas plasmid transformants can take
up to 1 week to sporulate. Plasmid-integrating vectors
can be generated by incorporating the integration
functions of
φ
Homoeologous recombination
Homoeologous recombination is the recombina-
tion between DNA sequences that are only partially
homologous. In most bacteria, homoeologous re-
combination fails to occur because of mismatch
repair, but it does occur in streptomycetes, although
the frequency of recombination is about 10
5
-fold
lower than for homologous recombination. The
significance of homoeologous recombination is that
it permits the formation of hybrid genes, gene clus-
ters or even species and can lead to the formation of
new antibiotics (Baltz 1998).
C31.
As noted earlier, a major reason for cloning in
streptomycetes is to analyse the genetics and regula-
tion of antibiotic synthesis. Although all the genes
for a few complete biosynthetic pathways have been
cloned (Malpartida & Hopwood 1985, Kao
et al
.
1994, Schwecke
et al
. 1995), some gene clusters may
be too large to be cloned in the standard vectors. For
φ
