Kloningsvektorer
Olika typer av vektorer
Plasmider
Fagmider
Fager
Cosmider
pET-systemet för expression
pBR322
Sid 89
Konstruktionen av pBR322
Sid 91
pUC plasmiden
Sid 92
PGEM3Z in vitro syntes
Sid 93
Bakteriofager
Sid 17
M13
Sid 21
Genkarta M13
Sid 94
Konstruktion av M13 varianter
Sid 95
Konstruktion av M13 varianter
Sid 95
Fagmiden
pEMBL8
Sid 96
Replikation λ-fag
Sid 22
Problem med λ-fager
Sid 97
Genkarta λ-fagen
Sid 21
Genkarta λ-fagen
Sid 98
Selektion av λ-fager utan EcoRI
restriktions sites
Sid 99
λ insertionsvektorer
Sid 99
λ replacement vektorer
Sid 100
Kloning med λ vektor
Sid 101
Kloning av stora DNA fragment
cosmider
Sid 102
N= ln(1-p)/ln(1-a/b)
N= antalet kloner
p=sannolikheten
a=storleken på fragmentet
b= storleken på totala genomet
Sid 103
pET systemet
pET system
pET system
•DE3 lysogen encodes T7 RNA
polymerase.
LacIQ gene is always expressed and
protein product binds to both
operators when IPTG is absent.
•Cloned gene is only transcribed by
T7 RNA polymerase enzyme in
presence of IPTG.
Absence of IPTG
•lacIQ protein binds to both operator
sites (T7RNA polymerase and your
cloned gene) both genes are silent.
IPTG added
•IPTG binds to lacIQ and LacIQ
protein leaves both operator sites.
•T7 polymerase gene is
transcribed/translated.
•T7 polymerase enzyme binds to T7
promoter site and your gene is
expressed.
Expression med IPTG
