GENE CLONING AND PLASMID VECTORS

LECTURE-3

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The cloning of a gene by the method described byStanley Cohen and coworkers (1973) requires the useof a suitable gene cloning vector.

A gene cloning vector should have capability ofautonomous replication, small size, origin ofreplication, presence of a selectable marker gene(s)and presence of unique restriction enzyme site(s).

In the first gene cloning experiment plasmid pSC101was used for cloning of E. coli kanamycin resistantgene. Natural plasmids are not suitable vectors forgene cloning.

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•Several plasmid vectors like Col E1, pBR322, pUC series and

pGEMR series have been developed to suit the various requirements.

•It is not easy to clone a DNA fragment larger than 10 kb using a

plasmid vector as the transformation frequency decreases

considerably with the increased size of the recombinant plasmid. To

overcome this difficulty three research groups independently reported

the construction of bacteriophage lambda cloning vectors in 1974.

•Subsequently, to meet the various requirements of cloning, the

following vectors were constructed in the respective years mentioned

in the brackets: Bacteriophage M13 derivatives (1977), cosmids

(1978), yeast artificial chromosomes (1987), bacteriophage P1

(1990), bacterial artificial chromosomes (1992), P1 artificial

chromosomes (1994), human artificial chromosomes (1997) and

maize mini chromosomes (2007).

Properties and construction of a vector DNA molecule

1. Capability of autonomous replication• Bacterial and viral genomes contain only one origin of

replication while eukaryotes contain multiple origins

2. Small size• In small molecules the chances of occurrence of unique sites

for restriction enzymes increases• Efficiency of gene transfer is high with small vector

molecules

3. Presence of selectable marker gene(s)• For easy detection of recombinants• E.g.: antibiotic resistant genes, lac z or resistant to toxins, etc

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4. Presence of unique restriction enzyme sites or multiple cloning sites for inserting the target DNA• Position of these restriction sites should be such that theinsertion of a segment of DNA in any of these restriction sitesbring about a phenotypic change in the characteristic of a vectormolecule e.g.: loss of gene expression or loss of resistance to anantibiotic

5.Ease of purification

6. No effect on the replicative ability of vector due to insertion of target DNA

7. Ease of reintroduction into host cell with high efficiency

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8. Biological containment•Vectors should be biologically contained with no possibility of gene escape•This can be achieved by non-conjugative and non-mobilized plasmid vectors

9.Presence of promoters and ribosome binding sites

10. Presence of two different origins of replication or broad host range origin of replicationE.g.: shuttle vectors that contain two different origins of replication

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• 1973 Plasmid• 1974 Bacteriophage lambda• 1977 Plasmid pBR322

Bacteriophage M13 (M13 mp1)• 1978 Cosmid• 1987 Yeast artificial chromosome-YAC• 1990 Bacteriophage P1

• 1992 Bacterial artificial chromosome-BAC

• 1994 P1 artificial chromosome- PAC• 1997 Human artificial chromosome-HAC• 2007 Maize mini-chromosomes

Cloning Vectors Used in Genetic Engineering

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1. Generation of the DNA fragment

2. Construction of the recombinant DNA molecule by joining of DNA fragment with a vector

3. Introduction of the recombinant vector into host cell, multiplication of recombinant DNA molecule along with host cell

Steps involved in gene cloning

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Plasmid characteristics1. Capability of autonomous replication

2. Small size

3. Presence of selectable marker gene(s)

4. Presence of unique restriction enzyme sites

5. Non-conjugative and non-mobilizable

6. Replicon under relaxed control

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Natural plasmid vectors for E. coli

pSC101• First used for in vitro cloning of eukaryotic DNA• 9kbp in size• Low copy number(1-2 copies)• Has advantage of a single Eco RI site at which DNA can be

inserted• Has selectable marker for tetracycline resistance • Derived from the conjugative plasmid R6-5Disadvantages are• Large size• Stringent replicative control• Low copy number• Low insert capability

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pSC10111

pSF 2124( RSF2124)

• Produced by transfer of the ampicillin resistance gene•Has ability for colicin biosynthesis•Has high copy number •Has single sites for Bam H1 and EcoR1

• Not currently used as vector as it does not provide easy selection by insertional inactivation•Mobilizable plasmid

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Col E1 Plasmid

• Small, circular colicingenic plasmid•Codes for 57kDa protein toxin•Size is 6,466bp•Has cea gene for colicin production• imm for immunity against colicin•kill for killer or lysis protein•mob for mobilization

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Col E1 Plasmid 14

Artificially constructed plasmid vectors for E. coli

pBR322

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•pBR322 is a plasmid and was the first widely-used E.coli cloning vector•Created in 1977, it was named after its Mexicancreators, p standing for plasmid, and BR for Bolivarand Rodriguez

•pBR322 is 4361 base pairs in length and contains areplicon region (source plasmid pMB1)

•The ampR gene, encoding the ampicillinresistance protein (source plasmid RSF2124)

•The tetR gene, encoding the tetracycline resistanceprotein (source plasmid pSC101 )

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•Has unique restriction sites for more than 40restriction enzymes•11 of these 40 sites lie within the tetR gene•Has 2 sites for restriction enzymesHindIII and ClaI within the promoter of the tetR

gene•Six key restriction sites inside the ampR gene•The origin of replication or ori site in this plasmidis pMB1 (a close relative of ColE1)•The ori encodes two RNAs (RNAI and RNAII) andone protein (called Rom or Rop )

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DERIVATIVES OF pBR322

pBR324

pBR325

pBR327

pBR328

pBR329

pAT15318

pUC series

•pUC19 is one of a series of plasmid cloning vectors created by Messing and co-workers in the University of California..

•The p in its name stands for plasmid and UC represents the University in which it was created. It is a circular double stranded DNA and has 2686

base pairs

•Series include pUC 18 pUC19, pUC12 and pUC 13, etc

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•It has one ampR gene (ampicillin resistance gene)

•Has an N-terminal fragment of β-galactosidase (lac Z ) gene of E.coli

•The multiple cloning site (MCS) region is split into the lac Z gene(codons 6–7 of lac Z are replaced by MCS), wherevarious restriction sites for many restriction endonucleases arepresent

•The ori site or replicon, rep is derived from pMB1 vector

•pUC vector is small but has a high copy number. The high copynumber of pUC plasmids is a result of the lack of the rop geneand a single point mutation in rep of pMB1

•The lac Z gene codes for β-galactosidase20

pGEMR series

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•The pGEM-3Z Vector is intended for use as a standard cloning vector, as well as for the highly efficient synthesis of RNA in vitro

•The vector carries the lacZ α-peptide and the multiple cloning region arrangement from pUC18 allowing selection of recombinants by blue/white screening

•In addition, the vector contains both the SP6 and T7 RNA polymerase promoters flanking the multiple cloning region

•The pGEM-3Z and pGEM-4Z Vectors are essentially identical except for the orientation of the SP6 and T7 promoters

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• The pET-28a-c(+) vectors are most powerful for cloning and expression of recombinant proteins in E. coli.

• The pET-28a-c(+) vectors carry an N-terminal His•Tag®/thrombin/T7•Tag® configuration plus an optional C-terminal His•Tag sequence.

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