Genetics of Some Beneficial Traits ◾ 147
markers (such as resistance to an antibiotic or metabolizing a carbohydrate) and a site that can
be hydrolyzed, preferably with the same restriction enzymes, is selected. The plasmid is digested
with this enzyme, mixed with the donor DNA fragment, and incubated for the fragment to
align in the opening of the plasmid DNA (Figure 13.2). The open ends are then sealed by using
suitable enzymes and nucleotide bases. This plasmid carrying the genes from the donor can then
be introduced in a bacterial cell in several ways, most effectively by electroporation in lactic acid
bacteria as described previously. This method is now being used to transfer genes from different
sources into lactic acid bacteria.
Cloning Vectors
Many vectors have been constructed by combining nucleotide segments encoding desirable characteristics from different sources, such as the origin of replication, highly efficient promoters,
multiple cloning sites, selective markers, and inducible systems. A vector should be stable and
able to replicate in host cells. Generally, vectors that replicate by the theta mechanism are more
stable than those that replicate by the sigma mechanism. Many small vectors have been constructed from small cryptic plasmids of lactic acid bacteria, such as pGK12 from pWV01. They
Bsp501
Bsp501 and Sall restriction and isolation of
3478 bp Bsp501 fragment
Bsp501
Bsp501
Bsp501
(8849)
Bsp501
(3171)
BamHl
Bcll
Bcll
Bgll
Nccl
Hindlll
Hindlll
Hindlll
Hindlll
Hindlll
Sall
pSMB74
8.9 kb
pHPS9
5.7 kb
pMBR1
9.1 kb
ORF4
ORF4
ORF1
ORF1
Em
r
Em
r
Cm
r
Cm
r
ORF2
ORF2
ORF3
ORF3
ori
ori
pUC
pUC
P5g
P5g
rep
rep
pTA1060
pTA1060
Sall,Pstl
Sall,Pstl
Pstl,Sall,BamHl,
Sma1,EcoRl
Pstl,Sall,BamHl,
Sma1 restriction
T4 DNA ligase
EcoRl
Ncol
Figure 13.2 Construction of recombinant plasmid. A 3.5-kb fragment carrying four open reading frames from pSMB74 was derived by digesting with Bsp501 and cloned in the Sma1 site of
pHPS9 to produce the 9.1-kb pMBR1.0.
markers (such as resistance to an antibiotic or metabolizing a carbohydrate) and a site that can
be hydrolyzed, preferably with the same restriction enzymes, is selected. The plasmid is digested
with this enzyme, mixed with the donor DNA fragment, and incubated for the fragment to
align in the opening of the plasmid DNA (Figure 13.2). The open ends are then sealed by using
suitable enzymes and nucleotide bases. This plasmid carrying the genes from the donor can then
be introduced in a bacterial cell in several ways, most effectively by electroporation in lactic acid
bacteria as described previously. This method is now being used to transfer genes from different
sources into lactic acid bacteria.
Cloning Vectors
Many vectors have been constructed by combining nucleotide segments encoding desirable characteristics from different sources, such as the origin of replication, highly efficient promoters,
multiple cloning sites, selective markers, and inducible systems. A vector should be stable and
able to replicate in host cells. Generally, vectors that replicate by the theta mechanism are more
stable than those that replicate by the sigma mechanism. Many small vectors have been constructed from small cryptic plasmids of lactic acid bacteria, such as pGK12 from pWV01. They
Bsp501
Bsp501 and Sall restriction and isolation of
3478 bp Bsp501 fragment
Bsp501
Bsp501
Bsp501
(8849)
Bsp501
(3171)
BamHl
Bcll
Bcll
Bgll
Nccl
Hindlll
Hindlll
Hindlll
Hindlll
Hindlll
Sall
pSMB74
8.9 kb
pHPS9
5.7 kb
pMBR1
9.1 kb
ORF4
ORF4
ORF1
ORF1
Em
r
Em
r
Cm
r
Cm
r
ORF2
ORF2
ORF3
ORF3
ori
ori
pUC
pUC
P5g
P5g
rep
rep
pTA1060
pTA1060
Sall,Pstl
Sall,Pstl
Pstl,Sall,BamHl,
Sma1,EcoRl
Pstl,Sall,BamHl,
Sma1 restriction
T4 DNA ligase
EcoRl
Ncol
Figure 13.2 Construction of recombinant plasmid. A 3.5-kb fragment carrying four open reading frames from pSMB74 was derived by digesting with Bsp501 and cloned in the Sma1 site of
pHPS9 to produce the 9.1-kb pMBR1.0.