This document discusses plasmids, which are autonomously replicating genetic elements found in bacteria, archaea, and eukaryotes, and their use in recombinant DNA technology. It covers types of plasmids, such as F, Col, R, D, and V plasmids, alongside their functions, structures, and the significance of features like antibiotic resistance genes and cloning sites. The document also highlights recent experimental findings on optimizing plasmid structure to improve gene expression levels for therapeutic applications.

1. PLASMID AND ITS TYPES
Presenter:
Janani Balamurugan (M.Sc. Biotech)
Periyar University,
Salem, India
Email: jananibalamurugan1111@gmail.com

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PLASMID AND ITS TYPES
INTRODUCTION
Microbial genome has unique dynamics (Eg. Bacteria), as they have 2 genetic system –
1. Chromosomal genome
2. Extrachromosomal genome
• Plasmids first made their mark in the field of recombinant DNA technology in the 1970s,
being used as a tool to insert genes into bacteria to encourage their production of human
insulin (Therapeutic protein).
• The word “Plasmid” - (any extrachromosomal hereditary element) was coined by Joshua
Lederberg in 1952.
Joshua Lederberg

3. PLASMIDS - ANY EXTRACHROMOSOMAL HEREDITARY ELEMENT
PLASMIDS –
Plasmids are autonomously replicating genetic elements that generally consist of
covalently closed, circular, double-stranded DNA molecules ranging from less than 10
kilobase pairs (kbp) to more than 400 kb.
• Plasmids are different in sizes based on their purpose.
• SOURCE - Bacteria, Archaea, and Eukaryota.
STRUCTURE OF PLASMIDS
Plasmids are circular double stranded and closed DNA, the two ends of double strand
are joined together by covalent bond. Some of the plasmid are closed and some of them are
linear. Plasmids usually composed of several major components - an origin of replication,
antibiotic resistance gene promoter sequence and restriction digestion site.
.
Plasmid – recombination tool

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ORI [Origin of Replication] –
• These site carries specific sequence to initiate replication.
• Plasmid has its own replication and transcription mechanism.
• Usually microbial plasmid has few ORI as they has small genome.
• Replication of plasmids depending on the host cell's respiration machinery.
• Plasmid replication can be conveniently divided into
three stages: initiation, elongation, and termination.
• The self-replication property of plasmid makes it to use in different molecular genetic
research such as gene therapy, gene transfer and recombinant DNA technology.
ANTIBIOTIC RESISTANCE GENE – Plasmid has antibiotic resistance gene.
• It is the site where the bacteria protects them from the antibiotics.
• Interestingly, the plasmid itself originates different antibiotic genes when it comes in
contact with different antibiotics.
Components of plasmid

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CONTI.
• Recent research shows that antibiotic resistance genes more found in bacteria that infecting
humans than those present in environment.
RESTRICTION SITE OR MULTIPLE CLONING SITE (MCS) -
Multiple cloning sites Or RE site in plasmid has unique palindrome sequences for restriction digestion
by breaking phosphodiester bond with the help of restriction enzymes having single or multiple
digestion site. This site is used to insert the gene of interest.
PROMOTER REGION- The promoter region facilitates transcription of target DNA.
SELECTABLE MARKER - The selectable marker allows the selection of bacteria containing the gene of
interest.

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TYPES OF PLASMID
In 1960, number of plasmids were identified. Some type of plasmids are classified based on their
function, size and GC content. Some types of plasmid is to protect the host or to do the reproduction.
F- PLASMID –
 Fertility plasmid is a type of conjugative plasmid helps bacteria to transfer its genes through the process
of conjugation.
 The F plasmid is usually smaller in size and does not have its own replication set up thus it is an
episome that is inserted into the bacterial chromosome for doing replication and transcription.
 Scientist reported the occurrence of a peculiar infective inheritance mediated by an agent called F
controlling the system of sex compatibility in E. coli K-12 strain.
COL PLASMID-
The col plasmid is another smaller type of plasmid that encodes gene that makes colicins
(Bacteriocin). This proteins kills other bacteria. This type of plasmid requires DNA polymerase I for
replication and amplification and are amplified by chloramphenicol (cm) with exception of colE2.

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R PLASMID-
The resistance plasmid possesses the genes for the natural antibiotic resistance and
protects the host from the antibiotic effects. This plasmid transferred to other bacteria through
conjugation.
D PLASMID-
The degradative plasmid which helps bacteria to digest or destroy the harmful
unnatural compound that harms them. Such plasmid digests xylenes, salicylic acid and
toluenes.
V PLASMID-
A virulence plasmid developed when the bacterium into pathogen. This type of plasmid
contains a bacterial strain that spreads diseases. Eg. E.coli. salmonella enterica

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ADVANTAGE OF PLASMID - Plasmid Play A Leading Role Among Non-Viral Vector.
Reducing plasmid DNA backbone length increases transgene expression levels in vitro in rat tenocytes
and in vivo in rat myocardium.
 OVERVIEW – Now a days, the area of Gene therapy has wide significance with clinical advantage - to cure
cancer, genetic diseases, cardiovascular disease, neurological disorders, etc.
 The gene therapy clinical trials - use viral vectors as an efficient method for gene delivery, because it
increases transgene expression.
 Due to Many safety concerns viral vectors not appreciable, so they aim to improve transgene expression
in non-viral delivery methods (plasmid).

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EXPERIMENTAL WORK - Using smaller pDNA backbone that is specifically designed to induce high
gene expression. pDNA consists of two main parts:
1. an expression cassette,
2. Bacterial backbone used largely in the production of pDNA.
This pDNA contains restriction sites and an antibiotic resistance marker.
• Components of both the expression cassette and the bacterial backbone can affect gene
expression.
•The expression cassette - regulatory sequences such as the promoter, intron(s), and
terminator can affect gene expression
•The bacterial backbone (Cloning site) in pDNA has been seen to negatively affect expression.
•The length of the spacer region found to be particularly important, as spacer regions over 1000
base pairs (bp) have been found to induce transgene silencing.
immunofluorescence analysis

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In vivo experiments use GET as a method of non-viral gene delivery to myocardium, skin and tenocyte.
 This transgene was inserted into two different vectors: gWiz and NTC9385R with tag
(immunofluorescence analysis).
 Immunofluorescence was used to determine the transfection efficiency of the plasmids.
RESULT – They found decreasing the decreasing the plasmid backbone length enhanced gene expression
levels and transfection efficiency in vitro when transfected using both equal mass and equal.
 Improving transgene expression levels has a variety of applications in the area of gene therapy.
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11. THANK YOU
1. Reference: Boye, C., Arpag, S., Francis, M., DeClemente, S., West, A.,
Heller, R., & Bulysheva, A. (2022). Reduction of plasmid vector backbone
length enhances reporter gene expression. Bioelectrochemistry, 144,
107981.
2. Shintani, M., Sanchez, Z. K., & Kimbara, K. (2015). Genomics of
microbial plasmids: classification and identification based on replication
and transfer systems and host taxonomy. Frontiers in microbiology, 6,
242. https://doi.org/10.3389/fmicb.2015.00242.