Upload
marc-castells-gomez
View
224
Download
0
Embed Size (px)
Citation preview
8/13/2019 Clonacion Por Recombinacion
1/13
Insercin de DNA en un vector
Corta y pega (Cut and paste)
(Fragmentos de PCR)
Recombinacin
(Fragmentos de PCR)
8/13/2019 Clonacion Por Recombinacion
2/13
Diana 1 Diana 2
Incorporacin de dianas
de RE por PCR
YFG
8/13/2019 Clonacion Por Recombinacion
3/13
ORF
ORFre1 re2
ORF
MCS
Pro
re1
re2
Antir
re1
re2
Antir Pro
PCR
Digestion by Re1 and Re2
+
Purification and ligation
Pro
Antir
PCR
Digestion by
restriction enzymes
Purification
Ligation
Restriction analysis
prior cloning
Transformation
3 hours
1 hour
1 hour
16 hours
16 hours
Total time = 2 days
ORF
Transformation
re2
re2
re1
re1
re1 re2
3. Restr ic t ion- independent clon ing techniques
Cloning by recombination vs traditional cloning
8/13/2019 Clonacion Por Recombinacion
4/13
Integration reaction mediated by:-Integrase (Int)
-Integration host factor (IHF)
Excision reaction mediated by:-Integrase (Int)
-Integration host factor (IHF)
-Excisionase (Xis)
Lysogenic pathway: recombination occurs between the phage circular DNA and the E. coli
chromosome via a short common sequence called "att. Att1 and att2 sites confer directionality and
specificity for recombination, so that only attB1 will react with attP1, and attB2 with attP2
3. Restriction-independent cloning techniques
Lambda phage recombination in E.coli
Landi A. (1989)Annu. Rev. Biochem. 58, 913
8/13/2019 Clonacion Por Recombinacion
5/13
8/13/2019 Clonacion Por Recombinacion
6/13
Different expression systems:
E. coli, baculovirus, yeast, SFV, mammalian
cells...
Different fusions:
N- or C- terminal, MBP, GST, Trx, NusA,
GFP
attB1 ORF attB2 attP2attP1
BP clonase
pDONR
(Kanr)
entry clone
(Kanr)
LR clonase
pDEST
(Amp
r
)
expression clone
(Ampr
)
attB1 ORF attB2
attL1 ORF attL2
by product
(Kanr)
attR2attR1 suicide gene
attR2attR1
suicide gene
suicide gene
Primer 1
Primer 2
attP2attP1 suicide gene
Sistema
Gateway
PCR and purification
BP/LR reaction
Transformation
3 hours
2 hours
16 hours
Total time = 1 day
8/13/2019 Clonacion Por Recombinacion
7/13
8/13/2019 Clonacion Por Recombinacion
8/13
8/13/2019 Clonacion Por Recombinacion
9/13
Advantages
1) No restriction analysis of ORF prior to cloning.
2) No restriction digestion of the vector.
3) Fast, parallel sub-cloning into different expression vectors.
4) ~100% sub-cloning efficiency (no background).
5) Flexibility, automation.6) Recombination sites may serve as linkers.
Disadvantages
1) Number of available expression vectors.2) Mandatory recombination sites.
Sistema Gateway
8/13/2019 Clonacion Por Recombinacion
10/13
In Fusion / Creatortechnology (Clontech)
Cloning without restriction/ligation
8/13/2019 Clonacion Por Recombinacion
11/13
Select on Amp / Xgal plates
Vector linearization by deletion of the regioncomprised between Sal I and Hind III
Primer 5:GAAGTTATCAGTCGAC
Primer 3:ATGGTCTAGAAAGCTTIn-fusion
1rst step: PCR product cloning in donor
vector with BD In-Fusion enzyme
8/13/2019 Clonacion Por Recombinacion
12/13
Transform E. coli and select on Chloramphenicol / sucrose plates:
SacB gene inhibits sucrose metabolism. In the presence of sucrose,
bacteria that carry the SacB gene swell and die due to osmotic shock.
Any bacteria that carry non-recombinant donor Clones are eliminated
when grown on sucrose-containing media.
Creator
Cre binds to the loxP sites on both
the donor vector and the acceptor
vector, cleaves the DNA, and
covalently attaches itself to the
DNA. Then Cre catalyzes strandexchange and ligation of the DNA
so that the gene is transferred
from the donor vector into the
acceptor vector without mutation
and in the correct orientation.
Cre recombinase
Cre-loxP recombination in bacteriophage P1
2nd step: recombination in
expression vector with Cre
protein
34-bp recognition sequence
13-bp inverted repeats flanked by 8-bp spacer region
8-bp overlap regionloxPsequence
Inverted region Inverted region
Sternberg N. et al.(1981) J. Mol. Biol. 150, 467
8/13/2019 Clonacion Por Recombinacion
13/13
TOPO
DNA topoisomerase I functions both as a restriction enzyme and as a ligase.
Vaccinia virus topoisomerase I specifically recognizes the pentameric
sequence 5-(C/T)CCTT-3and forms a covalent bond with the phosphate
group of the 3
thymidine. It cleaves one DNA strand, enabling the DNA tounwind. The enzyme then religates the ends of the cleaved strand and
releases itself from the DNA. To harness the religating activity of
topoisomerase, TOPO vectors are provided linearized with topoisomerase I
covalently bound to each 3phosphate. This enables the vectors to readily
ligate DNA sequences with compatible ends.
Linearized vector with topoisomerase I
covalently bound to each 3
phosphate
Overhang invades double-
stranded DNA, displacing the
bottom strand