Streptomyces integrating vector
ori
e.g. pSET152
E. coli
Res
int
PP = attP
Streptomyces chromosome
BB = attB
transformation
or conjugation
BP
PB
Do we always obtain a single copy of the vector integrated into BB?
slide 1
E. coli plasmids from rec- strains are mostly dimers.
1
BP
2
PP
PB
ori
E. coli
Res
int
PP
How can we find out?
slide 2
Some streptomycetes have several inefficient bb
bb
bP
bb
Pb
bP
How can we find out?
slide 3
Pb
Sequence the unknown flanking regions
bP
Restriction digest
R
R
R
R
R
bP
slide 4
Pb
Pb
R
R
R
R
bP
Pb
ori
E. coli
LM-PCR
Res
Ligation-mediated PCR
int
PP
b
slide 5
R
b
Summary
1. Integrating vectors often for tandem inserts
2. Some streptomycetes lack a perfect BB, and
insertion occurs into multiple, secondary bbs
This can be important if experiments need to be
reproducible.
slide 6
Complementation: testing whether a mutation of a particular
gene causes the observed phenotype.
E.g. does a gene knockout have a polar effect on downstream
genes in an operon?
mRNA
slide 7
Mutant phenotype
Wild-type phenotype ?
integrated or on plasmid
Can we be sure that the downstream gene is not involved ?
slide 8
homogenotization
homo-geno-tization
up to 5% !!
and
How can we prove that complementation was correct?
slide 9
homogenotization
homo-geno-tization
up to 5% !!
and
1.
2.
slide 10
How can we prove that complementation was correct?
Is there another possible gene arrangement?
1. Integrating plasmids, dimers and bbs
2. Homogenotization
slide 11
Kleinia
a species of Mangrove
slide 12
slide 13
slide 14