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