Genetic engineering and recombinant DNA technology ...

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Genetic engineering and recombinant DNA technology

Plasmids, Lambda based vectors and derivatives

Paper No. : 04 Genetic engineering and recombinant DNA technology

Module : 18 Plasmids, Lambda based vectors and derivatives

Principal Investigator: Dr Vibha Dhawan, Distinguished Fellow and Sr. Director

The Energy and Resouurces Institute (TERI), New Delhi

Paper Coordinator: Dr Mohan Chandra Joshi, Assistant Professor, Jamia Millia Islamia, New Delhi

Content Writer: Dr Ashutosh Rai, SERB-National Post Doctoral Fellow, ICAR- Indian Institute of Vegetable Research, Varanasi-221305

Content Reviwer: Dr Mohan Chandra Joshi, Assistant Professor, Jamia Millia

Islamia, New Delhi

Co-Principal Investigator: Prof S K Jain, Professor, of Medical Biochemistry Jamia Hamdard University, New Delhi

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Description of Module

Subject Name Biotechnology

Paper Name Genetic engineering and recombinant DNA technology

Module Name/Title Plasmids, Lambda based vectors and derivatives

Module Id 18

Pre-requisites

Objectives Plasmids, Plasmid vectors, Lambda based vectors, Cosmids , Summary

Keywords Plasmids, Cloning vectors, Lambda based vectors, Bacteriophage, Lytic cycle, Lysogenic

cycle, Cosmids

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A. Plasmids, Lambda based vectors and derivatives

Plasmids:

Plasmids may carry from half dozen to several hundreds of functional genes. The limiting

features of plasmids are that they can multiply only within a host cell. Mostly plasmids are

carried by bacteria and are essential part of 50% of bacteria found on earth. In some higher

organisms also these plasmids are found as extra chromosomal segments like yeast and

fungi. One of the best example of higher organism's plasmid is 2m circle, a well known

cloning vector of yeast origin. The number of plasmids found in bacteria per chromosome is

known as the copy number of that plasmid, mostly bacteria have one or two copy number,

but in some cases they may carry a copy number of 50 to hundreds, these are called high

copy number plasmids. Similarly the size of plasmids are also enormously variable and it

varies from few hundred base pairs to thousands of base pairs. Mostly plasmids carry genes

which are responsible for their own maintenance, but some plasmids attribute characters to

the host cells. In molecular biology, we use a number of genetically engineered plasmids to

carry the genes for tailoring of genetic information, and other purposes.

Plasmids are circular, double stranded DNA (dsDNA) molecules, found free in host cells,

and these are additional DNA than chromosomal DNA. The occurrence of these extra

chromosomal DNAs naturally as parasitic or symbiotic relationship in bacteria and some

lower eukaryotic cells like yeast. The distribution of plasmids in each daughter cells are

performed just like chromosomal DNA, these plasmids replicate and segregate themselves

to distribute equally. Plasmids are naturally occurring extra chromosomal DNA found in

various groups of bacteria. These plasmids have capability to self replicated due to presence

of origin of replication site in it. The size of plasmid DNA ranges from 1 kb to 250 kilo base

pairs. In nature plasmids have very important role in bacterial evolution. Almost all plasmids

are double stranded circular DNA, conferring an extra phenotypic character to the bacteria.

There are various functions which are served by these independent molecules. These

characters may include antibiotics resistance (ampicillin, tetracycline, kanamycin etc.),

abiotic stress tolerance (heat, cold, salt, toxic etc.) or may responsible for synthesis of some

special polypeptides (toxins, metabolites). The closed-circular DNA of plasmid are cross

coiled over its own axis in three-dimensional space to form a Super coil. Plasmids can be

classified on the basis of various features found in it.

Some of these are being summarized here-

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1. Fertility F-plasmids, having tra genes which enables bacterium to conjugation through

sex pili.

2. Resistance (R) plasmids, containing antibiotics or poisons resistant genes, conferring

antibiotics resistance to the host bacterium also known as R-factors.

3. Col plasmids, are genes that transcribed and translated for bacteriocins, these proteins

are responsible for killing of other bacteria.

4. Degradative plasmids, provide the capability to bacterium for degradation of various

organic/inorganic substances for harmful for bacteria, e.g. toluene and salicylic acid.

5. Virulence plasmids, confers pathogenicity to the bacterium.

Plasmids having resistance and Defense related mechanisms:

Antibiotic resistance for aminoglycosides, b-lactams, chloramphenicol, sulfonamides,

trimethoprim, fusidic acid, tetracyclines, macrolides, fosfomycin

Heavy metal ions resistance for Ni, Co, Pb, Cd, Cr, Bi, Sb, Zn, Cu and Ag

Tolerance to mercury and other mercury compounds

Toxic anions resistance such as chromate, selenate, tellurite, arsenate, arsenite,

borate, etc

Intercalating agent resistance such as acridines and EtBr

Radiation like UV and X-rays damage protecting plasmids

Bacteriophage DNA restriction systems

Resistance towards some bacteriophages

Virulence related plasmids:

Bacteriocins synthesizing plasmids

Antibiotics synthesizing plasmids

Crown gall tumors and hairy root inducing plasmids

Nodulation in legumes related plasmids

Metabolic Pathways:

Solbulization of sugars like lactose, raffinose, sucrose related plasmids

Biodegradation of aliphatic and aromatic hydrocarbons

Biodegradation of halogenated hydrocarbons like polychlorinated biphenyls

Bioegradation of proteins

Hydrogen sulfide Synthesising Plasmids

Alcaligenes Denitrification

Pigment synthesising plasmids

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Fertility F-plasmids: With the discovery that genetic recombinants can be done by mixing

particular Escherichia coli K 1 2 strains together, it was realized that the reason behind

getting recombination is an iniderectional transfer of DNA segments from F- donor strains

that contained an infectious "fertility factor," F. The F was found to replicate independently

and inducts its DNA crossing the cell envelopes of bacteria which comes in contact. The

injected DNA gets recombined with the chromosomal DNA at various locations. The F

plasmid is covalently closed circular plasmid having approximately 60 genes with a total

length of 100 kilo bases.

Resistance (R) plasmids: The resistance in a bacterium attributed to various mechanisms

such as chromosomal mutations, in chromosomal alterations the resistance is most

commonly associated with extra chromosomal elements which are commonly acquired from

other bacteria. These moving transposing elements may be plasmids, transposons, and/or

integrons. The intrinsic mechanisms that evolves efflux pumps to out multiple kinds of

antibiotics, are supposed to be major contributors to multidrug resistance. Bacteria can

acquire antibiotic resistance by any of the two mechanisms whether intrinsic or acquired

mechanisms. The bacteria can have naturally occurring genes such as, AmpC, β-lactamase

of gram-negative bacteria may have Intrinsic mechanisms in combination with efflux

systems.

Col plasmids: Escherichia coli and some other bacteria produce Colicins, a toxic protein.

The production of colicin involved in bacteria to bacteria competition and virulence

determination. These colins are different in their activity to kill other bacteria though they

share some common features like lethal colicin release, smililar genetic sequences having

genes like a colicin, lysis and immunity related genes producing peptides that interacting

with a specific locus in the colicin protein seuqence. These have immunity towards colicin

but when colicin is produced by a cell, it dies. These colicin related gene clusters are carried

by some special plasmids.

Degradative Plasmids: In our daily life microorganisms play a great role in degradation of

various products and by products like sewage, oil waste, agricultural byproducts, various

pesticides, toxic substances etc. Many microorganisms have capacity to break down

complex organic molecules and ability to recycle them by including these in to their meabolic

cycles exhibiting a variety of degradative functions. Bacteria like Pseudomonas, Alcaligenes,

Chromobacterium, have plasmids which have different genes responsible for degradation of

a variey of inorganic and organic substances, these are known as biodegrading bacteria.

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Virulence plasmids: The bacteria can be subdivided into many groups on the basis of their

pathagenicity, the pathogenicity of a bacterium mainly depends upon the presence or

absence of pathogenic DNA sequences which are frequently associated with various

pathotypes. In these bacteria the genetic information for pathogenicity have been acquired

horizontally through plasmids, bacteriophages and genomic islands. These genomic

rearrangements are utilized by bacteria for their evolution though variants efficient in

rearrangements, excision and transfer for affinity to additional DNA for creation of new

(pathogenic) variants.

Plasmid vectors

To carry and replicate the DNA fragment for various purposes, a molecular vehicle is needed

in the process of molecular cloning. The transfer of DNA fragments with the help of

molecular cloning vectors became possible due their ability to self-replication in E. coli or any

other host cell, autonomous replication are responsible for this feature. Thus the origin of

replication plays an important role in the molecular cloning. Most cloning vectors were

engineered originally from extra chromosomal elements found in nature such as

bacteriophage and plasmids. Plasmids are the DNA molecules that can replicate itself inside

a host cell. These molecular vectors are used for carrying cloned fragments of DNA. The

vectors may be a small multi-copy plasmid or a designed and engineered virus. Almost all

plasmid vectors are the engineered extra chromosomal naturally found plasmids, isolated

from different types of bacteria. Naturally found plasmids have several limitations; for

example, some are stringent and not relaxed (pSC101), some has poor marker genes

(ColE1), and some are too large (RSF2124). To overcome the limitations of natural vectors,

artificial plasmid are designed and engineered by combining different elements. Artificial

plasmids vectors are classified into two broad types based on their use:

1. Cloning vector

2. Expression vector

Apart from these two, there is another class of vectors known as shuttle vector. Shuttle

vectors can be propagated in two or more different host species (both in prokaryotes and

eukaryotes). Hence, inserted DNA can be manipulated and replicated in two different cellular

systems.

Cloning vectors are designed for efficient transfer of foreign DNA into the host. Expression

vectors have efficient machinery for cloning and expression of foreign gene in the host

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system. Selection of a vector depends upon various criteria decided by the experimental

goal.

Plasmid Expression vector:

Once a recombinant DNA has been made into a vector, the expression of that gene may or

may not take place. The proper expression of a gene the structural gene and the

corresponding promoter should be cloned on the same segment. In specialized expression

vectors these promoter genes are provided by the vectors, only the structural genes have to

be inserted on proper places. The best example of it is the vectors having capability of blue

white screening have lac promoter, in these the multiple cloning site lies just in the promoter

region.

The most important and exploit use of genetic engineering is to use recombinant

microorganism for production of various biochemicals. These biochemicals are being used

widely in different industries for well being of the human used in pharmaceutical industries,

cosmetics, bio-plastics etc. These biochemical have different origins, but due to advantage

of recombinant DNA technology we are able to produce in bacteria. The basic of

phenomenon of this is, the over expression of genes by some modifications or

rearrangements in the natural sequences. This technique of over expression of genes is also

being used in various structural studies, in determining biological functions etc. As

convenient Escherichia coli have been widely used as the prokaryotic host for expression

and overproducing various proteins. Its advantages are

(1) The genetics and physiology or E. coli is well characterized and arrays of expression

vectors are present

(2) The manipulation in E. coli is easily and cost efficient;

(3) The production of foreign protein can be achieved up to the level of 5%-30% or more of

the total protein of the cells.

In many instances, however, we cannot use E. coli. like, for proteins that requires post

translational modification. In the case of heterologous expression, it fails to produce

polypeptide to assume its native configuration.

Plasmid vectors: Origin of replication

In Plasmids genes related to replication are often clustered at a place which is called ori

region. Most of the bacteria like E. coli, have a single origin of replication (called OriC).

Saccharomyces cerevisiae has been estimated to have about 300 replication origins. Human

cells utilize over 20000 origins during the replication of the genome. pBR322, are based on

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the ColE1 origin. pUC18 has the a mutated ColE1 origin that enables it to be in high copy

number.

Multiple cloning sites:

MCS is a short DNA sequence consisting of restriction sites for many different restriction

endonucleases. MCS escalates the number of potential cloning strategies available by

extending the range of enzymes that can be used to generate a restriction fragment suitable

for cloning. By combining them within a MCS, the sites are made contiguous, so that any two

sites within it can be cleaved simultaneously without excising vector sequences. The MCS is

inserted into the lacZ sequence, which encodes the promoter and the α-peptide of β-

galactosidase. Insertion of the MCS into the lacZ fragment does not affect the ability of the

α-peptide to mediate complementation, while cloning DNA fragments into the MCS does.

Therefore, recombinants can be detected by blue/white screening on growth medium

containing X-gal in presence of IPTG as an inducer.

Antibiotic resistance:

Selection of the transformed cells from the non-transformed population is done by using

selectable marker genes that confers resistance to antibiotics. Hence, cells only having the

vector with the resistance gene for the antibiotic would grow in the selection media

containing the antibiotic (ampicillin, tetracycline etc.); while the non-transformed cells would

die.

Size and Copy number:

Relaxed plasmids are maintained at multiple copies per cell (10–200). Stringent plasmids

are present at a single copy, or a low number of copies (1–2) per cell. This difference is due

to separate mechanisms employed by plasmids in order to replicate themselves. In general,

relaxed plasmids replicate using host derived proteins, while stringent plasmids encode

protein factors that are necessary for their own replication.

Plasmid Size (kb) Copy Number Ori

pBR345 0.7 18-20 pMB1

pBR322 4.361 15-20 pMB1

ColE1 6.36 20 ColE1

F 95 1 f1

pUC18 2.686 >200 pMB1

pUC19 2.686 >200 pMB1

pGEMt 3.0 >500 pMB1 and f1

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This table represents the common reporter gene markers, which are used in day to day

cloning activities.

Gene Protein Size Source

cat Chroloamphenicol acetyltransferase 219 E.coli Tn9 transposon

lacZ β-galactosidase 1024 E.coli

gusA Β-glucuronidase 603 E.coli

luc Luciferase 550 Firefly

GFP Green fluorescent protein 238 Jellyfish

Plasmid vector: pBR322

pBR322 is a widely-used E. coli

cloning vector. It was created in

1977 in the laboratory of Herbert

Boyer at the University of California

San Francisco. The p stands for

"plasmid" and BR for "Bolivar" and

"Rodriguez", researchers who

constructed it.

pBR322 plasmid vector has the

following elements:

“rep” replicon from plasmid pMB1 which is responsible for replication of the plasmid.

“rop” gene encoding Rop protein. Rop proteins are associated with stability of RNAI-

RNAII complex and also decrease copy number. The source of “rop” gene is

pMB1plasmid.

“tet” gene encoding tetracycline resistance derived from pSC101 plasmid.

“bla” gene encoding β lactamase which provide ampicillin resistance (source:

transposon Tn3).

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Plasmid vector: pUC18

pUC18 plasmids are small, high copy number plasmids of size 2686bp. This series of

cloning vectors were developed by Messing and co-workers in the University of California.

The p in its name stands for plasmid and UC represents the University of California. pUC

vectors contain a lacZ sequence and multiple cloning site (MCS) within lacZ. This helps in

use of broad spectrum of restriction endonucleases and permits rapid visual detection of an

insert. pUC18 and pUC19 vectors are identical apart from the fact that the MCS is arranged

in opposite orientation. pUC vectors consists of following elements:

pMB1 “rep” replicon region derived from plasmid pBR322 with single point mutation

(to increase copy number).

“bla” gene encoding β lactamase which provide ampicillin resistance which is derived

from pBR322. This site is different from pBR322 by two point mutations.

E.coli lac operon system.

Fig: Key steps for cloning in plasmid vectors

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Bacteriophage λ and derived vectors:

Bacteriophage λ

A temperate bacteriophage of the family Styloviridae;

host: Escherichia coli K12. Bacteriophage λ is the

most studied bacteriophage with a fully sequenced

linear double stranded DNA genome of 49 kb.

Bacteriophage λ have 50 genes and 12 bp

complimentary over hangs at both ends, known as

cos sites. Only half number of genes are essential for

it to infect, replicate and package its DNA in to viral

capsid. The infection of bacteriophage λ starts with

absorption of the phage DNA by bacterium. After

interring in to host cell, the linear double-stranded

DNA molecule cyclizes through the cos sites at its ends to form a circular DNA like plasmid.

The virion has an icosahedral head (55 nm diameter) and a non-contractile tail (~150 × 10

nm).

Bacteriophage λ: Host interaction

A bacteriophase labda just adheres to to cell membrane of its host and injects its DNA in to

the bacterium. After interring in to host cell, the linear double-stranded DNA molecule

cyclized through the cos sites at its ends to form a circular DNA like plasmid. After

circularization it opts one of the two life cycles present in it. In isogenic cycle it get integrated

in to bacterial genome and replicates and transferred along with bacterial genome. While in

lytic cycle it replicates using host cell machinery and continue upto the death of bacterium

due to over production of virons.

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Bacteriophage λ genome organization in capsid:

λ DNA exists in a linear form in the bacteriophage and in a circular form upon entering the

bacterium. The switch from the linear to the circular form occurs through complementation of

the overhanging DNA ends at the cos sites. Many of the genes required for the integration of

λ into the host chromosome, or for new phage replication and assembly, are grouped

together on the λ chromosome.

Bacteriophage λ genome organization in host

It is the circular form of the lambda in host cell. After entering into circular form the lambda

starts replication by rolling boll manner. Here in fig the cos site can be shown with its

complementary sequence.

Bacteriophage λ : Insertion vectors

These are the simplest λ vectors, similar to the concept of plasmid vectors. To prepare an

artificial insertion λ vector, the restriction sites present for any RE in the λ vector is minimized

to one. To achieve it, restriction sites are deleted or a phage is searched for mutation in the

restriction site. The packaging limit for any insertion λ vector is between 37 kb to 51 kb,

accommodating maximum of 14 kb and minimum 4.3 kb foreign DNA .

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Bacteriophage λ : Replacement vectors

Full length λ molecule having two identical restriction sites flanked by “stuffer fragment”.

Stuffer fragment is replaced by foreign DNA during restriction cloning. The vector without the

foreign insert cannot be packaged due to the size limitation (smaller than the required).

Insert size ranges between 10-23 kb. Eg. λ EMBL 3, λ EMBL 4, λ DASH etc. Here in the

figure you can see the Stuffer segment of 14 kb that can be replaced by a foreign DNA of

almost same size.

The pEMBL4 replacement vectors have capability to carry upto 8-24 kb size insert. The

bacteriophage λ cloning vector has a middle segment responsible for insertion/excision (I/E

Region) and this region can be replaced with the foreign DNA with the help of two BamHI

sites present on the either side of I/E region. Hence, in a cloning strategy described in

Figure, foreign DNA is put into the vector and then allowed to infect the bacteria. In the

presence of I/E region, phage will integrate into the bacterial chromosome and continue

lysogeny cycle. But when I/E region is disrupted or replaced with the foreign DNA, it will

continue lytic cycle and form plaque.

Replication in λ-Bacteriophage

Lambda (λ) bacteriophage replicates by a rolling circle mechanism before lytic cycle and the

cos sites helps in recognition by RE, resulting in to concatameric molecules composed of

several linearly arranged recombinants. Just before packaging of the lambda DNA in to

capsid, it cleaved at cos site and a single Lambda DNA packaged into capsid.

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Lysogenic pathway: After entering in to cell the phage DNA integrates into the host

bacterial genome (it takes place with the help of homologous recombination between attP

and the bacterial genomic attB site) and it starts replicating along with the bacterial DNA. It is

knowns as prophage DNA and it remains integrated in the bacterial genome until it is

induced to enter the lytic pathway.

Lytic pathway: In this pathway large-scale synthesis and release of bacteriophage particles

(proteins and DNA) takes place that commonly leads to the killing or lysis of the host cell.

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In-vivo packaging

In the in vivo packaging of λ DNA, first the pre-heads are made, these preheads are the

major capsid protein encoded by gene E. After synthesis of preheads the single λ DNA

molecules are inserted into each pre-heads. These single λ DNA molecules are prepared by

cutting of concatamerized λ genomes at each cos sites. The maturation of preheads are

done by insertion of a minor protein named D to complete the head and the products of other

genes serve as assembly proteins, ensuring joining of the completed tails to the completed

heads.

In-vitro packaging of λ with the use of helper phage

The in-vitro packaging of λ takes place by utilizing two E. coli strains having λ lysogens that

have several defects in the genes of pathway responsible for packaging. Due to mutation in

gene responsible for production of protein E, prevents preheads being produced in strain

BHB2688 (helper phage). In strain BHB2690 (helper phage), mutation in gene D prevent

maturation of the preheads, with packaged DNA, into complete heads. The functional parts

of the BHB2688/BHB2690 mixed lysate having all the components and provide all the

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products for correct packaging, complementing each other’s deficiencies. Accordingly,

recombinant λ genomes is being constructed in vitro and enclosed into mature λ phage

particles before being propagated and replicated in host E. coli cells.

Bacteriophage λ derived cloning vectors

Phage vector is used to carry and replicate foreign DNA inside the bacterial host system.

The phage DNA inserts into the host chromosome by recombination. Phage λ had short

regions of single-stranded DNA with complementary base sequences called “cohesive” (cos)

sites. Base pairing between the complementary cos sites allows the linear genome to form a

circle within the host bacterium. Circularized viral genome can be integrated into the

bacterial genome by homologous recombination between attP site of viral genome and attB

site of bacterial genome.

Cosmids

Cosmids vectors provides additional benefit

over bacteriophage λ based cloning vector

due to their bacterial origin of replication.

They are chimeric cloning vectors and

consist of segments from a bacterial

plasmid and bacteriophage λ. They contains

adjoining cos site that helps in

circularization after entering in to host cell.

As cosmids contain bacterial origin of

replication, it can be maintained in bacteria

as such. In addition, it has antibiotic

resistance gene (tetracycline) and allow

selection of transformed host cells. The cloning strategy follows the similar mechanism as

discussed before for bacteriophage λ based vector and it is outlined. The example of cosmid

vector is pJB-8. As plasmid vectors, cosmids contain an origin of replication as well as a

selectable marker. Cosmids also have a exclusive restriction enzyme recognition site into

which DNA fragments can be added. The new recombinant λ particles are used to inter the

host bacterial cell for multiplication. The DNA is injected into the bacterium like normal λ

DNA and circularizes by complementation of the cos ends. The selection of transformants is

made on the basis of antibiotic resistance and bacterial colonies (rather than plaques) will

form that contain the recombinant cosmid. Since λ phage particles can accept between 37

and 51 kbp of DNA, and most cosmids are about 5 kbp in size, between 32 and 47 kbp of

DNA can cloned into these vectors. This represents considerably more than could be cloned

into a λ vector itself.

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B. Summary:

From this module we learned that the molecular vectors are used for carrying cloned

fragments of DNA. The vectors may be a small multi-copy plasmid or a designed and

engineered virus. We also learned about various. Key steps for cloning in plasmid vectors,

Bacteriophage λ and derived vectors, Insertion vectors, Replacement vectors, Lysogenic

pathway, Lytic pathway, In-vitro packaging of λ DNA with the use of helper phage, and about

various Bacteriophage λ derived cloning vectors.

Bibliography:

Brown T.A. 2010. Gene cloning and DNA analysis: an introduction (6th edition); Willey

Blackwell Ltd.

Clark, David P., and Nanette J. Pazdernik. Molecular biology. Elsevier, 2012.

Griffiths A.J.F, Miller J.H., Suzuki D.T, Lewontin R.C., Gelbert W.M. 2000. An Introduction to

Genetic Analysis (7th edition); New York: W. H. Freeman.

Robertis E.D.P.De, Robertis E.M.F. De. 2010. Cell and Molecular biology (8th edition);

Lippincott Williams and Wilkins.

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