Insercin de DNA en un vector Corta y pega (Cut and paste) (Fragmentos de PCR)

Recombinacin (Fragmentos de PCR)

Incorporacin de dianas de RE por PCR

YFG

Diana 1

Diana 2

3. Restriction-independent cloning techniques Cloning by recombination vs traditional cloning

re1 ORF re2 Pro re1 re2 MCS Restriction analysis prior cloning

PCR re1 ORF re2 Antir Digestion by Re1 and Re2

PCR

3 hours

Digestion by restriction enzymes Pro re1 Purification Ligation

1 hour

re1

ORF

re2

re2

Antir

Purification and ligation

1 hour 16 hours

re1 Pro

ORF re2 Transformation 16 hours

Total time = 2 days

Transformation Antir

3. Restriction-independent cloning techniques Lambda phage recombination in E.coli

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

Integration reaction mediated by: -Integrase (Int) -Integration host factor (IHF)

Excision reaction mediated by: -Integrase (Int) -Integration host factor (IHF) -Excisionase (Xis)

Landi A. (1989) Annu. Rev. Biochem. 58, 913

Sistema Gateway
PCR and purification 3 hours

Primer 1 attB1 ORF attB2 Primer 2 BP clonase attP1 suicide gene pDONR (Kanr) attP2

attR1
attL1 ORF attL2

suicide gene

attR2

BP/LR reaction
2 hours Transformation 16 hours

entry clone (Kanr)

attR1

suicide gene pDEST (Ampr)

attR2

LR clonase Total time = 1 day attP1 attB1 ORF attB2 suicide gene by product (Kanr) attP2

expression clone (Ampr)

Different expression systems: E. coli, baculovirus, yeast, SFV, mammalian cells...

Different fusions: N- or C- terminal, MBP, GST, Trx, NusA, GFP

Sistema Gateway
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.

Cloning without restriction/ligation

In Fusion / Creator technology (Clontech)

In-fusion
1rst step: PCR product cloning in donor vector with BD In-Fusion enzyme
Vector linearization by deletion of the region comprised between Sal I and Hind III

Primer 5: GAAGTTATCAGTCGAC Primer 3: ATGGTCTAGAAAGCTT

Select on Amp / Xgal plates

Creator
2nd step: recombination in expression vector with Cre protein

Cre-loxP recombination in bacteriophage P1

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 strand exchange 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.
loxP sequence
8-bp overlap region

Cre recombinase

Inverted region

Inverted region

34-bp recognition sequence 13-bp inverted repeats flanked by 8-bp spacer region

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.

Sternberg N. et al. (1981) J. Mol. Biol. 150, 467

TOPO
Overhang invades doublestranded DNA, displacing the bottom strand

Linearized vector with topoisomerase I covalently bound to each 3 phosphate

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-3 and forms a covalent bond with the phosphate group of the 3 thymidine. It cleaves one DNA strand, enabling the DNA to unwind. 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 3 phosphate. This enables the vectors to readily ligate DNA sequences with compatible ends.