streptomyces integrating vector e.g. pset152
DESCRIPTION
Streptomyces integrating vector e.g. pSET152. ori E. coli. Res. Streptomyces chromosome. int. PP = att P. BB = att B. transformation or conjugation. B P. P B. Do we always obtain a single copy of the vector integrated into BB?. - PowerPoint PPT PresentationTRANSCRIPT
slide 1
Streptomyces integrating vectore.g. pSET152
PP = attP
int
ori E. coli
Res
BB = attB
Streptomyces chromosome
BP PB
transformation or conjugation
Do we always obtain a single copy of the vector integrated into BB?
slide 2
E. coli plasmids from rec- strains are mostly dimers.
PP PBBP
1 2
PP
int
ori E. coli
Res
How can we find out?
slide 3
Some streptomycetes have several inefficient bb
bbbb
How can we find out?
bP PbbP Pb
slide 4
bP Pb
Sequence the unknown flanking regions
RRestriction digest
bP PbR R R R R R
slide 5
bP PbR R
PP
int
ori E. coli
Res
b bR
LM-PCRLigation-mediated PCR
slide 6
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 7
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 8
Mutant phenotype
Wild-type phenotype ?
Can we be sure that the downstream gene is not involved ?
integrated or on plasmid
slide 9
homogenotizationhomo-geno-tization up to 5% !!
and
How can we prove that complementation was correct?
slide 10
homogenotizationhomo-geno-tization up to 5% !!
and
1. How can we prove that complementation was correct?2. Is there another possible gene arrangement?
slide 11
1. Integrating plasmids, dimers and bbs
2. Homogenotization
slide 12
Kleiniaa species of Mangrove
slide 13
slide 14