© 2019 JETIR January 2019, Volume 6, Issue 1 Plasmids And ...

© 2019 JETIR January 2019, Volume 6, Issue 1 www.jetir.org (ISSN-2349-5162)

JETIRDW06101 Journal of Emerging Technologies and Innovative Research (JETIR) www.jetir.org 650

Plasmids And Their Uses In Recombinant DNA

Technology: A Review

Megha Bahuguna1, Arun Karnwal2*

1Department of Biotechnology, School of Bioengineering and Biosciences, Lovely Professional University,

Phagwara, India

2*Department of Microbiology, School of Bioengineering and Biosciences, Lovely Professional University,

Phagwara, India.

Abstract

Plasmids are circular, double stranded DNA(dsDNA) molecules that are separated from a cell’s chromosomal DNA

or we can say that they are extrachromosomal genetic elements basically the small DNA molecules in a cell which

are physically separated from chromosal DNA which can replicate independtly.As compare to chromosomes they are

non-essential. Only few genes are carried by each plasmid. Its sizes ranges from 1 to more than 1000 kbp. They also

differ in number of copies in the cell. A great number of plasmids are found in bacteria but they can also be found in

multicellular organisms and archaea. Plasmids can be removed from the cell by chemicals in which their

proliferation is stopped. Due to multiplication of bacterial cells the number of plasmids decreases till the free cells of

bacteria are obtained from the plasmids. They are utilized in genetic manipulation and gene therapy research via gene

transfer to bacterial cells.

Keyword: Plasmid; RDT; nucleotide; nucleic acid; antibiotic resistance

Introduction

Genes which are required for survival of an organism and those genes which are beneficial to host

organism like antibiotic resistance are found in plasmids [1, 2]. In genetic engineering plasmids are used to

make recombinant DNA.

Plasmids are also known as replicons that they are self-replicating molecules because they occupy within

the host cells. They do not comprise of cell genome due to two reasons [3, 4]:-

1. In two different species the same plasmid may exist and can be transferred between the species [5].

2. Some members of same species have plasmids while some don’t have. Plasmids have useful genes

but in most growth conditions they are not necessary [6].

Functions of Plasmids

Plasmids perform many functions. Plasmids enable the process of bacterial replication. There may be

many coexisting plasmids each with various roles in a single cell [7]. They may contain the genes that

enhance the survival of an organism either by killing other organisms or by defending the host cell by

producing toxins [8].

Types of plasmids

1. Fertility/F-plasmids

It is found in the E.Coli bacterium. The F plasmid contains the genes that allow the plasmids DNA

to be transferred between the cells. The E. coli which contains this F factor is F+ or male bacterial cells and

http://www.jetir.org/



which do not contain is F- [2, 9]. The F- plasmid can be inserted into chromosomal DNA and known as

episomes. Two of F+ bacteria result upon conjugation of an F+ bacterium with an F- bacterium.

2. Resistance plasmids

They contain antibiotics or poison resistance genes and they help in the production of pilli [10-12].

They have the ability to use conjugation to transfer themselves to provide the bacterial strain resistance to

antibiotics.

3. Virulence plasmids

They encode genes that promote host – pathogen interactions [13, 14]. The bacteria which cause

diseases and infection among individuals by replication in the new host i.e. Salmonella enterica containing

bacterium.

Figure 1: Factors that influence biology of virulence plasmids [15]

4. Degradative plasmids

Their function is to aid the host bacterium in compound digestion which are not commonly found in

nature like camphor, salicylic acid, toluene, and xylene. They can be described as conjugative [16].

5. Col plasmids

They contain genes which re responsible for the production of bacteriocins called as colicins. They

kill the other bacteria by defending the host bacterium [2].

In genetic engineering, plasmids are used to improve multiple copies of such genes [17-19]. They are used

in numerous science and gene therapy strategies via gene transfer to cells genetic engineering.

DNA cloning with plasmids vectors

From large genomes to obtain pure DNA samples has been done by recombinant DNA technology

[20]. In this method any gene can be purified, it’s sequence is determined. A DNA gene of interest is linked




through 3’ → 5’ phospodiester bond to a vector DNA molecule which replicates during entering in host

cells, this produce a large number of recombinant DNA molecules [21].

Usually two types of vectors are used-

Bacteriophage λ vectors and E. Coli plasmid vectors. Plasmid vectors are replicate along with their host

cells and λ vectors are replicate as lytic virus, host cell destruction and DNA processing into virus.

E.Coli Plasmids Can Be Used As Cloning Vectors [22]

The most commonly used plasmids in recombinant DNA technology is E. coli. While working with

plasmids their lengths get reduced many plasmid vectors are approximately 3kb in length [23]. Most

plasmid vectors contain an essential nucleotide sequences which are needed in DNA cloning: a replication

origin, a drug resistance gene and a region in which exogenous DNA fragments can be inserted.

Plasmid DNA Replication

The origin of replication is a sequence from where replication starts and any pieces of DNA when

linked to this sequence can be made to replicate inside the host cells [24]. This property is basis of

molecular DNA cloning.

Figure 2: Plasmid DNA Replication [25]

Selection of Transformed cells

Selectable marker to classify and remove non-transformants is needed and to allow the transformant

to expand selectively. The cycle of transformation is the insertion of a DNA component into host bacteria

[26]. Usually, the genes encoding resistance to antibiotics like ampicillin, tetracycline etc.

Plasmid cloning enables DNA fragments to be separated from complicated mixtures. The implanted DNA

is repeated with the remainder of the plasmid DNA and is divided into daughter cells as the colony grows

[18, 27]. A DNA fragment is placed into a plasmid vector. In the colony of cells a significant number of

duplicate versions of the original fragment of DNA are repeated [28]. Since all of the cells in a colony




come out of the same parental transformed cell, they create a cell clone. The initial fragment of DNA

inserted into the parental plasmid is referred to as cloned DNA.

A plasmid vector incorporates each DNA fragment independently. Incubated with E. coli cells, the

resultant mixture of recombinant plasmids is converted and grown on an antibiotic specific disk [29]. For a

colony that grows from a single plasmid cell carries the same form of plasmid into all the cells of a colony.

As a consequence, in the different bacterial populations, copies of DNA fragments are isolated from one

another.

Figure 3: General procedure for cloning a DNA fragment in a plasmid vector [30]




Figure 4: DNA fragments are cloned in a plasmid matrix from a mixture [31].

Restrictions Enzymes Cut DNA Molecule at Specific Sites

Restrictions enzymes belong to a larger class of nucleases. They are of two types-

Exonucleases remove nucleotides from the ends of the DNA.Endonucleases make cuts at specific positions

within the DNA. Each restriction endonuclease function by inspecting the length of a DNA sequence and it

recognizes a specific palindromic nucleotide sequences [32, 33].

Figure 5: (a) Eco RI, a restriction enzyme from E. coli, makes staggered cuts at the specific 6-bp inverted

repeat sequence shown.(b) Bacterial cells with restriction endonucleases also contain corresponding

modification enzymes that methylate bases in the restriction-recognition site [32].


https://www.ncbi.nlm.nih.gov/books/n/mcb/A7315/def-item/A7481/



Figure 6: Chunks developed by Eco RI and HindIII from the adenovirus 2 (Ad2) DNA genome cleavage.

Single black lines in this diagram indicate double-stranded DNA [33].

Restriction Fragments with Complementary “Sticky Ends” Are Ligated Easily

When cut by same restriction enzyme the resultant DNA fragments have the same kind of sticky

ends and these can be joined together using DNA ligases.




Figure 7: Ligation of restriction fragments with complementary sticky ends. In present illustration, DNA I

(left) EcoRI clusters are blended with many other restriction fragments, like EcoRI chunks, created from

DNA II (right) [33, 34].

Poly-linkers Facilitate Insertion of Restriction Fragments into Plasmid Vectors

Plasmid vectors containing a polylinker, or multiple-cloning-site sequence, commonly are used to

produce recombinant plasmids carrying exogenous DNA fragments.

Figure 8: (a) Sequence of a polylinker that includes one copy of the recognition site, indicated by brackets,

for each of the 10 restriction enzymes indicated. Polylinkers are chemically synthesized and then are

inserted into a plasmid vector. Only one strand is shown. (b) Insertion of genomic restriction fragments into

the pUC19 plasmid vector, which contains the polylinker shown in (a) [35-37].






Conclusion

As discussed in the above sections plasmid is circular double stranded extrachromosomal DNA

which is present in the bacterial cells. It is mostly used as a genetic tool by molecular biologists in

laboratory. It is used as a vector to clone different type of DNA fragment. They have the capacity to

multiply in bacterial cells irrespective of chromosomal DNA. If we can attach an alien DNA to plasmid

DNA, we can calculate the numbers equivalent to the plasmid copy number.

There are five types of plasmid i.e. fertility plasmid, resistance plasmid, virulent plasmid,

degradative plasmid and col plasmid. E.Coli plasmid can be used as cloning vector. Plasmids are used in

genetic engineering to amplify many copies of certain genes which is followed by the- Origin of

replication, selectable marker, selection of transformants, polylinking. DNA cloning is an effective yet

straightforward way to purify a single fragment of DNA from a complicated fragment mix and build a wide

number of interest groups.

Acknowledgment

I am very thankful to Lovely Professional University, India for technical support to complete this study and

unlimited help in all steps.

Competing Interest’s Statement

The author(s) declare(s) that there is no conflict of interest.

References

[1] J. C. Moores et al., “Cloning of genes involved in the biosynthesis of pseudobactin, a high-affinity

iron transport agent of a plant growth-promoting Pseudomonas strain,” J Bacteriol, vol. 157, no. 1, pp. 53-

8, Jan, 1984.

[2] J. C. Moores et al., “Cloning of genes involved in the biosynthesis of pseudobactin, a high-affinity

iron transport agent of a plant growth-promoting Pseudomonas strain,” J Bacteriol, vol. 157, pp. 53-58,

1984.

[3] M. D. van de Rhee et al., “Highly efficient homologous integration via tandem exo-beta-1, 3-

glucanase genes in the common mushroom, Agaricus bisporus,” Curr Genet, vol. 30, no. 2, pp. 166-73, Jul

31, 1996.

[4] S. S. Saleh, and B. R. Glick, “Involvement of gacS and rpoS in enhancement of the plant growth-

promoting capabilities of Enterobacter cloacae CAL2 and UW4,” Can J Microbiol, vol. 47, pp. 698-705,

2001.

[5] M. D. Magazin, J. C. Moores, and J. Leong, “Cloning of the gene coding for the outer membrane

receptor protein for ferric pseudobactin, a siderophore from a plant growth-promoting Pseudomonas

strain,” J Biol Chem, vol. 261, no. 2, pp. 795-9, Jan 15, 1986.




[6] Y. Degefu, and M. Hanif, “Agrobacterium- tumefaciens-mediated transformation of

Helminthosporium turcicum, the maize leaf-blight fungus,” Arch Microbiol, vol. 180, no. 4, pp.

279-84, Oct, 2003.

[7] M. J. de Groot et al., “Agrobacterium tumefaciens-mediated transformation of filamentous fungi,”

Nat Biotechnol, vol. 16, no. 9, pp. 839-42, Sep, 1998.

[8] Y. Hao, T. C. Charles, and B. R. Glick, “ACC deaminase from plant growth-promoting bacteria

affects crown gall development,” Can J Microbiol, vol. 53, no. 12, pp. 1291-9, Dec, 2007.

[9] A. de Groot et al., “Characterization of type IV pilus genes in plant growth-promoting

Pseudomonas putida WCS358,” J Bacteriol, vol. 176, no. 3, pp. 642-50, Feb, 1994.

[10] M. E. Singer, and W. R. Finnerty, “Physiology of biosurfactant synthesis by Rhodococcus species

H13-A,” Can J Microbiol, vol. 36, pp. 741-745, 1990.

[11] U. A. Ochsner et al., “Isolation and characterization of a regulatory gene affecting rhamnolipid

biosurfactant synthesis in Pseudomonas aeruginosa,” J Bacteriol, vol. 176, no. 7, pp. 2044-54, Apr, 1994.

[12] M. Skaugen, and I. F. Nes, “Transposition in Lactobacillus sake and its abolition of lactocin S

production by insertion of IS1163, a new member of the IS3 family,” Appl Environ Microbiol, vol. 60, no.

8, pp. 2818-25, Aug, 1994.

[13] L. Svensson et al., “Uropathogenic Escherichia coli and tolerance to nitric oxide: the role of

flavohemoglobin,” J Urol, vol. 175, no. 2, pp. 749-53, Feb, 2006.

[14] A. V. Shelud'ko et al., “Effect of genomic rearrangement on heavy metal tolerance in the plant-

growth-promoting rhizobacterium Azospirillum brasilense Sp245,” Folia Microbiol (Praha), vol. 57, no. 1,

pp. 5-10, Jan, 2012.

[15] P. S. Phale et al., “Metabolic diversity in bacterial degradation of aromatic compounds,” OMICS,

vol. 11, pp. 252-279, 2007.

[16] Q. Wang et al., “Engineering bacteria for production of rhamnolipid as an agent for enhanced oil

recovery,” Biotechnol Bioeng, vol. 98, no. 4, pp. 842-53, Nov 01, 2007.

[17] S. Kilaru et al., “Establishing molecular tools for genetic manipulation of the pleuromutilin-

producing fungus Clitopilus passeckerianus,” Appl Environ Microbiol, vol. 75, no. 22, pp. 7196-204, Nov,

2009.

[18] F. H. Sant'anna et al., “Tools for genetic manipulation of the plant growth-promoting

bacterium Azospirillum amazonense,” BMC Microbiol, vol. 11, pp. 107, 2011.

[19] S. Schneiker et al., “Genome sequence of the ubiquitous hydrocarbon-degrading marine bacterium

Alcanivorax borkumensis,” Nat Biotechnol, vol. 24, pp. 997-1004, 2006.




[20] K. Hiramoto et al., “DNA strand breaking by the carbon-centered radical generated from 4-

(hydroxymethyl) benzenediazonium salt, a carcinogen in mushroom Agaricus bisporus,” Chem Biol

Interact, vol. 94, no. 1, pp. 21-36, Jan, 1995.

[21] D. Linke et al., “Carotene-degrading activities from Bjerkandera adusta possess an application in

detergent industries,” Bioprocess Biosyst Eng, vol. 38, no. 6, pp. 1191-9, Jun, 2015.

[22] J. F. Pothier et al., “Duplication of plasmid-borne nitrite reductase gene nirK in the wheat-

associated plant growth-promoting rhizobacterium Azospirillum brasilense Sp245,” Mol Plant Microbe

Interact, vol. 21, no. 6, pp. 831-42, Jun, 2008.

[23] M. A. B. Haase, D. M. Truong, and J. D. Boeke, “Superloser: A Plasmid Shuffling Vector for

[24] M. A. Kersten et al., “Molecular characterization of the glnA gene encoding glutamine synthetase

from the edible mushroom Agaricus bisporus,” Mol Gen Genet, vol. 256, no. 2, pp. 179-86, Sep, 1997.

[25] M. J. Wagemaker et al., “The ornithine cycle enzyme arginase from Agaricus bisporus and its role

in urea accumulation in fruit bodies,” Biochim Biophys Acta, vol. 1681, no. 2-3, pp. 107-15, Jan 11, 2005.

[26] H. Tsuji et al., “Cloning and sequencing of cDNA encoding 4-aminobenzoate hydroxylase

from Agaricus bisporus,” Biochim Biophys Acta, vol. 1309, no. 1-2, pp. 31-6, Nov 11, 1996.

[27] R. D. Anand, O. Sertil, and C. V. Lowry, “Restriction digestion monitors facilitate plasmid

construction and PCR cloning,” Biotechniques, vol. 36, no. 6, pp. 982-5, Jun, 2004.

[28] L. Dziewit et al., “DIY series of genetic cassettes useful in construction of versatile vectors specific

for Alphaproteobacteria,” J Microbiol Methods, vol. 86, no. 2, pp. 166-74, Aug, 2011.

[29] M. K. Chee, and S. B. Haase, “New and Redesigned pRS Plasmid Shuttle Vectors for

Genetic Manipulation of Saccharomycescerevisiae,” G3 (Bethesda), vol. 2, no. 5, pp. 515-26, May, 2012.


© 2019 JETIR January 2019, Volume 6, Issue 1 Plasmids And ...

Documents

Transcript of © 2019 JETIR January 2019, Volume 6, Issue 1 Plasmids And ...