Gene cloning vectors and their charecteristics

Faculty of Basic Sciences and Humanities Dr. R.P.C.A.U. , SAMASTIPUR, PUSA, Bihar

BIHAR

PRESENTED BY : SIMPAL KUMAR SUMAN B.Tech(Biotech.)

Roll no. : BT/18/15

Broad classification of vectors• Two broad categories on the basis of objective /purpose of

RDT(recombinant DNA technology)A. Cloning vectors – These vectors are only useful for storing a genetic sequence . By themselves , they are incapable of allowing for transcription and

translation of the gene into a functional protein product. High copy no. within cells. Examples- pBR322 A. Expression vectors – It is a specialized type of cloning vector. Expression vectors are designed to allow transcription of the cloned gene

and translation into protein. Low copy no. within cells. Example-vector for Human insulin(humulin) production .

What is gene cloning vectors?• Gene cloning Vectors- A cloning vector is a genome that can accept the

target DNA(gene) and increase the number of copies through its own autonomous replication( from Elsevier’s integrated Biochemistry, 2007).

• www.sciencedirect.com• Examples- plasmids, phages or virus• Obtainig millions of copies• First artificial cloning vector pBR322 (1977) constructed by Bolivar &

Rodriguez from E.coli plasmids.

Structure of E.Coli plasmid cloning vector pBR322

Gene cloning vectors

• Uses :a. Genomic librairies b. Preparing probesc. Genetic engineering research/experiment• Selection of cloning vector depends

on - a. Objective of cloning experiment b. Ease of workingc. Knowledge existing about the vectord. Suitabilitye. Reliability

DNA cloning /gene cloning strategies

• Cloning vectors have three common properties :

a. a selectable marker, which is almost always antibiotic resistance ;

b. an origin of DNA replication to allow vector propagation ; and

c. MCS (multiple cloning sites) or polylinker that contains a no. of

restriction sites to clone foreign DNA.

Choosing a cloning vector • Criteria for choosing a cloning vectora. Insert size/ molecular weightb. Copy numberc. Incompatibilityd. Selectable markere. Cloning sites

Candidate used as cloning vectors

• Cloning vectors can be –

a. Plasmid vectorsb. Hybrid vectors/Cosmid vectorsc. Bacteriophage /phage vectorsd. Yeast plasmid vectors e. Artificial chrmosome vectors

Three Phases of the development of plasmid cloning vectors

A. First phase – (1973-1976)Examples- Psc101,Col E1 , PCR1Drawbacks :a. They replicated poorly b. They carried unstable selectable markersc. They carried not more than two restriction sites for cloning. Because of the above drawbacks another plasmid pBR313 was constructed but it

was unnecessary large and half of its DNA being non-essential The plasmid pBR 313 was then reduced in size and it gives rise to to the plasmid

pBR322 .B. Second phase -(after1977)Examples – pAT153 , pXF3 , pBR327, etca. The size of plasmid vectors was greatly reduced .b. These plasmids made use of antibiotic resistance for selecting

recombinant clones.

Third phase • The third phase of the development of plasmid vectors

involved-a. Incorporation of HincII fragment of beta-galactosidase

gene to permit selection through blue /white colonies due to alpha complementation ,e.g.,PUC vectors

b. Incorporation of DNA sequences from single stranded M13 phage for generation of single stranded DNA templates for sequencing ,e.g., phagemid vectors

c. Incorporation of promoter DNA sequence ,e.g., transcription in vitro , e.g., PGEM vectors

d. Incorporation of high expression promoters for expression of large amounts of foreign protein ,e.g., a variety of expression vectors

Plasmid vectors Introduction- Plasmids are self-replicating ,double-stranded , circular,

extra-chromosomal DNA molecule that are found in all bacterial genera (both gram positive and gram negative) and also in some yeast but not in higher eukaryotes.

The word plasmid was introduced by Joshua Lederberg in 1952. Chang and Cohen first proved the use of plasmid as gene cloning

vectors.

First artificial cloning vector pBR322 (1977)

Structure of E.Coli plasmid cloning vector pBR322

Desirable properties of plasmid cloning vehicles

An ideal cloning vehicle would have the followingthree properties:• low molecular weight;• ability to confer readily selectable phenotypic

traitson host cells;• single sites for a large number of restrictionendonucleases, preferably in genes with a readilyscorable phenotype.

Properties of plasmid

• They exist as super coiled(closed circle) , nicked(open circle) , linear. Though supercoiled in most bacteria , they are linear in some species.

• They are found in both gram – positive and gram – negative bacteria.

• They show very little or no homology to host chromosomal DNA sequences.

• Plasmids are widely distributed throughout the prokaryotes, vary in size from less than 1 × 106 Daltons to greater than 200 × 106, and are generally dispensable.

• Size range- 1-200kb• Relaxed replication control• Two suitable markers for identification

Plasmids in gene technology (artificial plasmids) Plasmid designed for rDNA technology possess the following

properties :a. They must be able to replicate autonomously in bacterial cellb. They must possess one or more than one markers , such as

resistance to antibiotics.c. Plasmid molecules should contain the cleavage site for the

restriction enzymes.d. The molecular weight of plasmids should be as low as possible

because the efficiency of transformation decreases with increasing molecular weight.

e. Plasmid should be maintained as multiple copies per cell.

Classification of plasmid vectors • According to their conjugative properties• There are two groups of naturally occurring plasmids known as

conjugative and non- conjugative .A. Conjugative plasmids – Those plasmids that mediate DNA

transfer from a donor into a recipient cell.• This transfer is mediated by two groups of plasmid –coded genes

which are the transfer gene (tra gene) and mobilizing gene (mob gene).

• Example- F-plasmdB. Non- conjugative plasmids - • The plasmid which lack the transfer function are non-

conjugative but can be mobilized by another conjugative plasmid present in the same cell( tra-, mob-) , e.g., Col plasmids

Types of plasmid The most useful classification of naturally occurring plasmids is

based on the main characteristic coded by the plasmid genes. The five major types of plasmid according to this classification are

as follows:

a. Fertility or F plasmidsb. Resistance or R plasmidsc. Col plasmidsd. Degradative plasmidse. Virulence plasmids

Examples of plasmid vectors pBR322

pBR327

pBR325

pBR328

pUC18pUC 8pUC19pUC12

pUC13

Pgem3zpUC- plasmid , university of columbia

pBR322 Cloning Vector. 15 copies. Reconstructed plasmid. Derived from Ecoli plasmid- ColE1. pBR322 – 4362 base pairs P – denotes Plasmid B – Boliver R - Rodriguez 322 – number given to distinguish. Ampilicin resistance gene derived from RSF2124. Tetracycline resistance gene from PSC101. Origin of replication from PMB1. Two selectable markers - ampr ,tetr

pUC8 Popular Ecoli cloning vector. Derivative of pBR322. Two parts derived:- Ampicillin resistance gene. ColEI – origin of replication. 2700 base pairs. lac Z gene derived from E.coli. Polylinker sequence having unique restriction siteslies in lac region.

Hybid /cosmid vector

Component from both plasmid & phage chromosomes. Helper phage provided. Developed in 1978 by Barbara Hohn & John Collins. 30 – 40 Kb Origin of replication, cloning site, marker gene, DNA cos site. Smaller than plasmid. Use – construction of genomic libraries of eukaryotes. e.g. Cosmid

Cosmid Combine parts of the lambda chromosome with parts

of plasmids. Contain the cos sites of λ and plasmid origin of

replication. Behave both as plasmids and as phages. Cosmids can carry up to 50 kb of inserted DNA.Structure of Cosmid Origin of replication (ori). Restriction sites for cleavage and insertion of foreign

DNA. Selectable marker from plasmid. A cos site - a sequence yield cohesive end (12 bases). Ampicillin resistance gene (amp).

Continued……..

• The advantages of cosmids are that-a. relatively large size of insert DNA (upto 45kb)

can be clonedb. DNA can be introduced into the host using

bacteriophages derived by in vitro packegeing • The disadvantages are that-a. It is difficult to store bacterial host as glycerol

stock; andb. In vitro packegeing is needed to maintain

cosmids inside the viral heads.

Bacteriophage cloning/phage vectors

Cloning large DNA fragmance. Linear Phage molecule. Efficient than plasmid. Used in storage of recombinant DNA. Cloning Vectors. It infects bacteria Bacteriophage cloning vectors based on λ phage are λgt10, λgt11,

EMBL3(European molecular biology lab), EMBL4, Charon etc. Commonly used E.coli phages :-

λ phage M13 Phage

Lambda phage vector Genome size is 48,503 bp. High transformation efficiency. 1000 times more efficient than the plasmid vector. Origin of replication. Genome linear in head. Single- stranded protruding cohesive ends of 12 bases. Cos site – site of cleavage of phage DNA.• Advantages-a. The size of cloned DNA is relatively large(9-25kb);b. The multiplication of cloned DNA is very easy by infecting a suitable host

with bacteriophages containing rDNA;c. There is direct selection of clones containing rDNA during in vitro

packeging ;d. Storage of clones is easy.

Phage M13 Vectors

Filamentous bacteriophage of Ecoli. Used for obtaining single stranded copies. ds DNA sequencing using Sanger method Single stranded. Inside host cell become double stranded Genome size-DNA of 6407nt

Artificial plasmids Linear or Circular. 1 or 2 copies per cell. Different types – Bacterial Artificial Chromosome (BAC) Yeast Artificial Chromosome (YAC) P1 derived artificial chromosome (PAC) Mammalian Artificial Chromosome (MAC) Human Artificial Chromosome. (HAC) YAC – Cloning in yeast BAC & PAC – Bacteria MAC & HAC – Mammalian & Human cells.

Bacterial Artificial Chromosome (BAC)• Cloning vector system • Developed by- mel simon & his colleagueus • Insert size- 50-300kb• BAC vectors contain F-plasmid origin of replication(oriS). • F-plasmid genes control plasmid replication & plasmid copy

no. (parA,parB,parC)• The methods used to construct a BAC library.• Cloning of large regions of eukaryotic genome.• 1st BAC Vector – PBAC108L.• Used in analysis of genomes.• Example- pBeloBAC11 (Developed in Simon lab)

Yeast Artificial Chromosome (YAC)• Very simple gene cloning vectors.• Insert size- 1000-2000kb• Linear Plasmid Vector.• Developed by- David Burke et.al. (1987)• pYAC3 - first YAC developed• It contains- an origin of replication, centromere

element(CEN4), two telomere sequences ,one or more growth (URA3).

• Use – mapping complex eukaryotic chromosome Occurring two forms:- Circular – grows in bacteria. Linear – multiplies in yeast cells It contains :- ARS sequence – replication CEN4 sequence – centromeric function TRP1 & URA3 – 2 selectable markers

P1 derived artificial chromosome (PAC)

• This cloning system was developed by Loannou et.al.(1994).

• Insert size- 100-300kb• It is devoid of problems such as chimarism

and instability of cloned DNA.

REFERENCES

1. John W. Pelley , Elsevier’s Integrated Biochemistry,2007(

www.sciencedirect.com/topics/page/loning vector

2. www.biologyexams4u.com

3. www.microrao.com>plasmids

4. www.majordiffrences.com

5. Introduction to plant Biotechnology, second edition, H.S. Chawla

6. Gene cloning and DNA analysis ,sixth edition, T.A. Brown

7. Priciples of gene manipulation , sixth edition, Sandy B. Primrose, Richard M.Twyman and

Robert W. Old.

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