plasmid, restriction enzyme and dna...

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PLASMID, RESTRICTION ENZYME AND 

DNA TRANSFORMATION

Lucia Dhiantika Witasari

http://dhiantika.staff.ugm.ac.id

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DNA cloning

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3/14/2011

What is Plasmids ?• Extrachromosomal DNA • Having ori (origin of replication)• Very small• Carry genetic information• Can be isolated from bacterial cells• Important in a genetic engineering

http://dhiantika.staff.ugm.ac.id

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Using a plasmid vector, it would be possible to recover a single colony from an agar plate and to use this to produce a bacterial culture in which each cell carries a copy of the original 

DNA fragment

http://dhiantika.staff.ugm.ac.id

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Origin of replication The first requirement is that the plasmid must be able to replicate in the chosen host (in this case, E. coli).

Selectable markerit is necessary to be able to select those cells which have received the plasmid (transformants) :•one or more antibiotic resistance genes•b‐lactamase gene  resistance to ampicillin

Cloning siteit must contain suitable recognition sites for cleavage by one or more restriction endonucleases.  the circular plasmid can be opened up at that point and the ends ligated with the ends of the DNA fragment to be cloned

http://dhiantika.staff.ugm.ac.id

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3/14/2011Endang S. Rahayu                                                      

Fak.Teknologi Pertanian UGM

Antibiotic resistance genens and replicator genehttp://dhiantika.staff.ugm.ac.id

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Plasmid pBR322

pBR322• One of the original plasmids 

used• Two selectable markers (Amp 

and Tet resistance)• Several unique restriction sites 

scattered throughout plasmid (some lie within antibiotic resistance genes = means of screening for inserts)

• ColE1 ORI

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pUC18• Derivative of pBR322• Advantages over pBR322:

• Smaller – so can accommodate larger DNA fragments during cloning (5‐10kbp)

• Higher copy # per cell (500 per cell = 5‐10x more than pBR322)

• Multiple cloning sites clustered in same location = “polylinker”

http://dhiantika.staff.ugm.ac.id

3/14/2011

POLYLINKER

http://dhiantika.staff.ugm.ac.id

3/14/2011 Praktikum Biomol, 2008

pUC18

• Interruptable gene encoding for enzyme beta galactosidase (lacZ)

– Polylinker resides in the middle

– Enzyme activity can be used as marker for gene insertion

– Disrupted gene = nonfunctional

– Intact gene = functional

– Media containing XGAL chromagenic substrate used (blue colonies = intact; white colonies = disrupted)

• Amp resistance gene still present (= beta lactamase), Tet resistance gene omitted

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CLONING IN PLASMID VECTORS

IMPORTANT

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Restriction Enzymes

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DNA double helixes are cut at the axis of symmetry:

Although less efficient, blunt‐end ligation can be useful because it does not require the fragments to have been generated with the same enzyme

http://dhiantika.staff.ugm.ac.id

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CLONING BLUNT‐END MOLECULES

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DNA double helixes are cut : sticky end

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EcoR1 Recognition Site

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3/14/2011 http://dhiantika.staff.ugm.ac.id

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3/14/2011Endang S. Rahayu                                                      

Fak.Teknologi Pertanian UGMhttp://dhiantika.staff.ugm.ac.id

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TRANSFORMATION

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Basic procedure for plasmid transformation of E. coli

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http://dhiantika.staff.ugm.ac.id

Transformasi

3/14/2011

3/14/2011 http://dhiantika.staff.ugm.ac.id

http://dhiantika.staff.ugm.ac.id3/14/2011