Cloning in bacteria other than Escherichia coli - Principles of Gene Manipulation - page 141

Box 8.1 continued

rescue. In practice, foreign DNA is ligated to monomeric
vector DNA and the in vitro recombinants are used to
transform B. subtilis cells carrying a homologous
plasmid. Using such a 'plasmid-rescue' system,
Gryczan et al . (1980) were able to clone various
genes from B. licheniformis in B. subtilis .
One disadvantage of the plasmid-rescue method
is that transformants contain both the recombinant
molecule and the resident plasmid. Incompatibility
will result in segregation of the two plasmids. This
may require several subculture steps, although
Haima et al . (1990) observed very rapid segregation.
Alternatively, the recombinant plasmids can be
transformed into plasmid-free cells.

for the introduction even of cryptic plasmids. In
addition to its much higher yield of plasmid-containing
transformants, the protoplast transformation system
differs in two respects from the 'traditional' system
using physiologically competent cells. First, linear
plasmid DNA and non-supercoiled circular plasmid
DNA molecules constructed by ligation in vitro can
be introduced at high efficiency into B. subtilis by
the protoplast transformation system, albeit at a
frequency 10-1000 lower than the frequency
observed for CCC plasmid DNA. However, the
efficiency of shotgun cloning is much lower with
protoplasts than with competent cells (Haima et al.
1988). Secondly, while competent cells can be
transformed easily for genetic determinants located
on the B. subtilis chromosome, no detectable
transformation with chromosomal DNA is seen using
the protoplast assay. Until recently, a disadvantage
of the protoplast system was that the regeneration
medium was nutritionally complex, necessitating
a two-step selection procedure for auxotrophic
markers. Details have been presented of a defined
regeneration medium by Puyet et al. 1987 .

Transformation of protoplasts
A third method for plasmid DNA transformation in
B. subtilis involves polyethylene glycol (PEG) induction
of DNA uptake in protoplasts and subsequent
regeneration of the bacterial cell wall (Chang & Cohen
1979). The procedure is highly efficient and yields up
to 80% transformants, making the method suitable

Table B8.1 Comparison of the different methods of transforming B. subtilis.

Efficiency

System(transform ants/ m g DNA) Advantages Disadvantages

Competent Unfractionated plasmid 2 × 10 4 Competent cells readily prepared Requires plasmid oligomers or

cells Linear 0 Transformants can be selected internally duplicated plasmids,

CCC monomer 4 × 10 4 readily on any medium which makes shotgun experiments

CCC dimer 8 × 10 3 Recipient can be Rec − difficult unless high DNA

CCC multimer 2.6 × 10 5 concentrations and high vector/

donor DNA ratios are used
Not possible to use phosphatase-
treated vector

Plasmid Unfractionated plasmid 2 × 10 6 Oligomers not required Transformants contain resident

rescue Can transform with linear DNA plasmid and incoming plasmid and

Transformants can be selected on these have to be separated by

any medium segregation or retransformation

Recipient must be Rec +

Protoplasts Unfractionated plasmid 3.8 × 10 6 Most efficient system Efficiency lower with molecules

Linear 2 × 10 4 Gives up to 80% transformants which have been cut and religated

CCC monomer 3 × 10 6 Does not require competent cells Efficiency also very size-dependent,

CCC dimer 2 × 10 6 Can transform with linear DNA and and declines steeply as size increases

CCC multimer 2 × 10 6 can use phosphatase-treated vector