Internship at

Centre of Excellence in Molecular Biology, University of the Punjab, Lahore

Bachelor of Science in Biotechnology

by
Uswa Maryam
0 0 3 0 - B H - B I O - T - 1 9

INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY

GOVERNMENT COLLEGE UNIVERSITY
LAHORE

1
 A REPORT TITLED

“Internship at Centre for Excellence in Molecular Biology, University of the Punjab,

Lahore”

SUBMITTED TO GOVERNMENT COLLEGE UNIVERSITY, LAHORE

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF

Bachelor of Science

IN THE SUBJECT OF
BIOTECHNOLOGY
by
Uswa Maryam

Roll No.
0030-BH-BIO-T-19

Session: 2019-2023

INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY

GOVERNMENT COLLEGE UNIVERSITY
LAHORE

2
 DECLARATION

I, Uswa Maryam with Roll No. 0030-BH-BIO-T-19, as student of bachelor’s in

science in the subject of Biotechnology session 2019-2023, hereby declared that the
matter printed in this report titled “Internship at CEMB, University of the Punjab,
Lahore” is my own work and has not been printed, published and submitted as
research work, thesis or publication in any form in any University, Research Institute
etc. in Pakistan and abroad.

Dated: Signature of deponent

Uswa Maryam

3
 INTERNSHIP COMPLETION CERTIFICATE
This is certified by the Institute of Industrial Biotechnology, Government College
University Lahore, that the contents and form of entitled “Internship at CEMB,
University of the Punjab, Lahore” submitted by Uswa Maryam, Roll No. 0030-BH-
BIO-T-19, have been found satisfactory for the requirement of the degree of BS
(Hons) Biotechnology.

Date: ______________________ ________________________

Supervisor
Dr. Ikram-ul-Haq
Distinguished Professor
(SI, FPAS, ISESCO Laureate)

Institute of Industrial Biotechnology,

GC University, Lahore
Submitted through

____________________ _______________________
Prof. Dr. Nauman Aftab Controller of Examination
Director GC University, Lahore
Institute of Industrial Biotechnology,
GC University, Lahore

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 TABLE OF CONTENTS

Sr. No. Contents Page No.

1 Dedication 9
2 Acknowledgment 10
3 Centre of Excellence in Molecular Biology 12
4 Introduction 19
5 Different types of media preparation 24
6 Streak plate quadrant streaking 36
7 Gram staining 38
8 Stab culture preparation 40
9 Polymerase chain reaction 42
10 Agarose gel electrophoresis 45
11 SDS PAGE 48
12 Transformation 52
13 Mini Prep for Isolation of Plasmid 55
14 Syringe Filtration 57
15 Discussion 58
16 Comments 59
17 Suggestions 60
18 References 62

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 LIST OF FIGURES

Figure No. Title Page No.

1 Biopharmaceuticals and proteomics laboratory overview 20
1.1 Reagents for preparing LB agar 25
1.3 Melting agar in LB solution 26
1.2.3 Weighing LB powder to prepare LB broth 27
1.3.4 Autoclaved YPD Broth 31
1.4.1 Reagents for preparing YPD agar 32
1.4.3 Autoclaved YPD agar 33
1.5.3 Autoclaved SSB media 35
2.4 (a) Incubating streaked plates 37
2.4 (b) Colonies (S. Cerevisiae) 38
3.3 Gram staining process 40
5.3 (a) PCR components 43
5.3 (b) PCR set up on thermal cycler 44
5.3 (c) PCR product analysis via gel electrophoresis 45
6.3 (a) Preparation of gel 46
6.3 (b) Running gel electrophoresis 47
6.3 (c) Observed DNA bands 48
7.3 (a) Gel preparation for SDS PAGE 50
7.3 (b) SDS PAGE set up 51
7.3 (c) Protein bands observed 52
8.1 (a) Calcium chloride 53
8.1 (b) Preparation of competent cells 53
8.1 (c) Incubation in shaking incubator 54
9.1 Centrifuged plasmid 55
10.2 Syringe filter 57

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 LIST OF TABLES
Table No. Title Page No.
1 Objectives of 20
Biopharmaceuticals and
Proteomics Laboratory

2 Concentration of Reagents 30
to Make YPD Broth

3 Concentration of Reagents 32
to Make YPD Agar

4 Observed Characteristics of 40
Bacteria

5 Reagents in PCR Master- 43

mix

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 List of Abbreviations
DNA Deoxyribonucleic acid µl Microliter
RNA Ribonucleic acid ml Milliliter
EMA European Medicines Agency g Gram
FDA Food and Drug Administration hrs Hours
LC Liquid Chromatography mins Minutes
2D-PAGE 2 Dimensional Polyacrylamide cm Centimeter
Gel
Electrophoresis
LB Luria Bertani nm Nanometer
YPD Yeast Extract Peptone Dextrose rpm Revolution per
minute
SSB Sucrose Soy Broth psi Pound per
square inch
HCL Hydrochloric acid V Volts
NaOH Sodium hydroxide M Molar
TAE Tris-acetate-EDTA
TBE Tris-borate-EDTA
UV Ultra violet
PCR Polymerase Chain Reaction
DNTPs Deoxynucleotide triphosphates
TBST Tris-buffered saline Tween
qPCR Quantitative Polymerase Chain
Reaction
RT-PCR Real-Time Polymerase Chain
Reaction
SDS- Sodium Dodecyl Sulphate
PAGE
Polyacrylamide Gel
Electrophoresis
APS Ammonium Persulphate
TEMED Tetramethylethylenediamine
DTT Dithiothreitol
BSA Bovine serum albumin

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 DEDICATION
This internship report is dedicated to my parents, who have continuously inspired and
supported me throughout the years.

After Allah Almighty, I'd like to thank my supervisor Dr. Ikram-ul-Haq for
encouraging me in achieving my goals and believing in me. It has been an incredible
help to have your constant affirmation, direction, and faith in my abilities.

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 ACKNOWLEDGEMENTS
I would like to express my sincere appreciation and gratitude to everyone who
has contributed in making my internship an accomplishment and the played a major
part in complete preparation of this report.

I want to start by heartily thanking Dr. Ikram-ul-Haq, who supervised my

work at Government College University (GCU), Lahore. His expertise, support,
and guidance have been extremely valuable to me throughout my internship. I
appreciate his constant guidance, his patience, and all the opportunities he offered me
to progress professionally.

Moreover, I would like to extend my heartfelt thanks and appreciation to Prof.

Dr. Nauman Aftab Director IIB who provided with me an opportunity work and learn
at one of the best research institute of Pakistan.

Also, I want to express my gratitude to Prof. Dr. Kausar Malik, Director

CEMB and the entire CEMB team for their cordial welcome and for fostering a
productive and cooperative work environment. I acknowledge my co-workers' help,
informative discussions, and their efforts to consider myself as a valuable team
member. Working with them has given me skills and insights that will surely benefit
my future career pursuits.

I would also like to extend my deepest thanks to Assistant Prof. Dr. Nadeem
Ahmed for his invaluable mentoring and direction throughout this internship. My
professional and personal development has benefited much from his constructive
feedback, expertise and willingness to pass on it.

I express my sincere appreciation to the employees and professionals of the

Biopharmaceuticals and Proteomics Laboratory (BPL) at CEMB for providing
me support and cooperation while I was an intern. Their eagerness to help, respond to
my queries, and include me in various projects has enhanced my learning experience.

I would like to thank my instructors and academic advisors at CEMB for their
ongoing support and for providing me with the education and training I needed to
succeed in my internship.
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 I'm thankful to my friends and family's consistent encouragement, inspiration,
and compassion along this journey. Their faith and confidence in my potential have
been an endless source of motivation.

At last, I would like to thank everyone who have helped making my internship
experience possible, whether or not they were specifically named. Your inputs,
critiques, and collaborations have helped me to shape my knowledge and skills in my
field.

I would like express profound gratitude to everyone who helped in making my

internship experience memorable. Your contributions and assistance have been
immeasurable, and I am extremely grateful of having the opportunity for working and
learning in the most remarkable environment.

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Center of Excellence in Molecular Biology (CEMB)
In order to improve national expertise in the developing field of bioscience, the
University of the Punjab established a prestigious institute Center of Excellence in
Molecular Biology. In 1986, the Ministry of Education recognized the University
Center's achievements by designating it as National Centre of Excellence in Molecular
Biology.

CEMB’s laboratory complex spans over a sizable 60-acre plot of land, including
a spacious building, laboratory block, and a hostel specifically for PhD research
scientists. A total of 20 research labs, four conference rooms, a manufacturing unit, and
a support facilities unit are located in the Laboratory Block dividing into four separate
research units. A well-equipped Lab-aid Section is part of the support facilities unit and
is used for necessary chores like cleaning, autoclaving, and media preparation.
Additionally, it features six roomy plant growth rooms, an insectary, an animal house,
as well as supply storage areas.

The Federal Government generously funds the Center's research and training
initiatives. Paid research efforts also get additional funds from international funding
agencies, allowing the Centre to carry out a variety of research studies. The overarching
objective is to contribute to the advancement of society through academic studies,
ground-breaking research, and novel findings with the vision to build CEMB as a
world-class molecular biology center. The Centre seeks to develop strong, enduring,
and globally renowned breakthroughs across diverse fields of molecular biology by
cultivating a hub of expertise.

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Goals and Objectives:

The aim and vision at the Centre of Excellence in Molecular Biology (CEMB) are
guided by a specific set of goals which are:

• Research Development: The main goal of CEMB is to carry out ground-breaking

molecular biology research. The center seeks to improve our comprehension of
the basic biological systems by digging into the complex molecular mechanisms
underlying life. This includes investigating the structure and function of biological
molecules as well as unravelling molecular relationships, signaling networks, gene
expression, and regulatory mechanisms.
• Research Practice: CEMB is dedicated to turning its scientific findings into
useful applications that contribute to society. The center seeks to work with
business partners and other academic institutions by exploring potential
applications in fields including medical treatment, biotechnology, and farming.
These joint initiatives seek to speed the creation of innovative molecular biology-
based diagnostics, therapies, and technology.
• Interdisciplinary Collaboration Efforts: CEMB actively promotes
interdisciplinary collaboration by assembling specialists from many domains like
genomics, genetics, cell biology, biochemistry. To tackle difficult scientific
problems and hasten molecular biology breakthroughs, CEMB fosters a
collaborative environment and promotes knowledge exchange both inside and
beyond the organization.
• Training and Knowledge: A key goal of CEMB is to provide beneficial learning
opportunities to researchers and students at various stages of their careers through
well-designed seminars and workshops. Through these initiatives, people are
given the tools and knowledge they need to succeed in the field of molecular
biology and become future leaders and professionals.
• Scientific Outreach and Participation: The importance of communicating with
the larger scientific community and the general population is acknowledged by
CEMB. The center actively disseminates scientific knowledge, increases public
awareness of molecular biology research, and motivates the next generation of

13
 scientists through conferences, symposiums, and outreach initiatives. CEMB aims
to increase interest and understanding of the subject of molecular biology by
promoting scientific literacy and a deeper awareness of the discipline's
importance.
• Collaboration and Networking: CEMB actively seeks out alliances and
collaborations with organizations on a national and worldwide scale to advance in
molecular biology by working with renowned scientists and organizations. These
strategic alliances allow for the sharing of knowledge, resources, and skills, which
elevates its institutional effect on a global scale.

Accomplishments:

1. With 206 MPhils and 40 PhDs generated by the Centre to date, it has helped
many academics advance their academic careers. 134 students are currently
enrolled in a variety of programs, reaffirming the Center's position as a regional
training center. Reputable researchers from Pakistan, SARC, OIC including
those from Turkey, Sri Lanka, Nepal, Jordan, Iran, India, and Egypt, have
visited the Centre to learn about various approaches and pursue sabbatical
ventures.
2. Bt transgenic rice has been created and is currently being tested in the field.
3. Developed Bt cotton that is presently undergoing field tests.
4. There has been the creation of a novel Bt pesticide formulation.
5. Designed plant expression vectors with two to three genes and four Bt pesticide
genes that make optimal use of codons.
6. Three patents are being worked on, and five have already been obtained.
7. Time-tested techniques used to grow disease-free gladiolus, sugarcane, potato,
and sugarloaf stock. established procedures for prenatal b-thalassemia
diagnosis.
8. DNA typing for human identity is utilized in criminal investigations and
parentage confirmation. [334 cases of murder, parentage, corpse, rape,
recognition and robbery].

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9. Lowering the cost and increasing the accessibility of PCR-based diagnostic tests
for the general public in order to diagnosis diseases like H. Pylori, S. typhi, HIV,
SARS, hepatitis, and tuberculosis, among others.
10. Genes for human pharmaceutical proteins that have been cloned for commercial
use.
11. 17 novel genes, 3 novel loci for visually impaired individuals, 11 novel deafness
loci, and 1 novel gene for auditory modifiers have all been found.
12. The institute created the first indigenous transgenic CEMB-Klean Cotton
variety, which has patented triple genes and has gotten excellent reviews. It
might cut the nation's annual cotton manufacturing expenses to billions
of dollars. Preceding this, the Punjab Seed Council (PSC) and National
Biosafety Committee (NBC) approved the first locally generated double Bt
genes cotton cultivars (CEMB-33 & CA-12) offered through Academia-
Industry Partnership.
13. The very first local transgenic CEMB-Klean Cotton variety was engineered by
the University of the Punjab, Lahore and contains patented triple genes
and received a high rating. It has the potential to lower the country's yearly
cotton production costs by billions of dollars. Prior to this, CEMB-33 & CA-
12, which are being sold by Academia-Industry Partnership, were the first
locally developed double Bt genes cotton cultivars to receive approval from the
Punjab Seed Council (PSC) and National Biosafety Committee (NBC).
14. The CEMB Biopharmaceutical Group developed locally licenced technology
for the commercial production of recombinant therapeutic proteins for humans,
including cancer therapies, peg-interferon, filgrastim and interleukins, and
interferon.
15. There is not a single pharmaceutical company in Pakistan that has the cGMP
technology, or the scientific expertise required to create "purified transgenic
human proteins" for use in medicine. CEMB seeks to offer cGMP compliance
facilities in order to meet the demands of the country's biopharmaceutical
industry.

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Research Activities:

In the laboratory, there are numerous research facilities and projects that all stem from
molecular biology. The subsequent are:

1. Plant Biosafety: The goal of this research program is to create transgenic cotton
cultivars with a variety of features that can improve fiber quality and offer
defense against insect pests, including both sucking and chewing insects.
2. Plant Transformation: The main goal of this program is to create cotton plants
that have been genetically engineered to have desired qualities and to come up
with efficient ways to express those traits. Resistance to herbicides and
resistance to insect pests, including chewing and sucking pests, are among the
features addressed.
3. Plant Genetics: The main goal of this program is to identify, isolate, and
classify genes in cotton and Agave sisalana that are tolerant to salinity and
drought stress.
4. Research on stem cells: This study examines the potential benefits of stem cells
for the field of organ regeneration.
5. Inherited Mental Retardation: In the population of Pakistan, the main goal of
this program is to find new genetic loci and genes linked to congenital mental
retardation.
6. Auditory Impairment: The goal of this program is to clarify the underlying
genetic causes of hearing loss in Pakistani society.
7. Vision Impairment: The main objective of this program is to uncover the
fundamental genetic determinants behind the prevalence of congenital cataracts,
retinitis pigmentosa, and glaucoma among people in Pakistan.
8. Seed Biotechnology: The goal of seed biotechnology is to find small interfering
RNAs (siRNAs) that are efficient against the PVY (Potato Virus Y) and SCMV
(Sugarcane Mosaic Virus). It also includes creating, synthesizing, and cloning
short hairpin RNA (shRNA) in the pCAMBIA (1301) vector that targets PVY.
The development of recombinant biopharmaceutical products,
the transformation of the SCMV and PVY shRNA constructs, Bt hybrid and

16
 hybrid maize development, hybrid tomato production, maintenance and in vitro
propagation of 11 potato-based breeds for the production of virus-free miniature
form of potatoes are additional goals.
9. Forensics: The Forensic Services Laboratory conducts DNA profiling for
paternity and criminal cases, helping law enforcement organizations and the
legal system. The lab also intends to instruct crime scene detectives on evidence
gathering methods.
10. Molecular Medicine: This study aims to create a staphylococcal antimicrobial
enzyme with potential medical uses that is resistant to newly emerging drug
resistance.
11. Functional Genomics: The aim of the Applied and Functional Genomics
program is to create on-site microarray slides that are affordable and to provide
knowledge of chip fabrication at the research center.
12. Molecular Diagnostics & Virology: The goal of this project is to boost
resources and manpower in order to advance research and development
activities in molecular biology, notably in the areas of immunology and
hepatitis. Applications of virology in medicine and on animals are the main
focus.
13. Research Laboratory of Forensics: Using a variety of research techniques and
guidelines, this lab specializes in forensic DNA analysis-based human
identification.
14. GMO Detection Group: This group's goals include conducting GMO
(Genetically Modified Organism) detection research and creating F1 cotton
genotypes for the advancement of cotton variants with high yield potential in
the future.
15. Bioinformatics: The Bioinformatics group is committed to fostering both
domestic and international scientific exchange and education. Their main areas
of concentration are computerized drug design and bioinformatics, which they
advance through collaborations with research groups at universities, hospitals,
and other organizations in Pakistan and around the world. Seven experts from
various fields, including software development, molecular modelling,

17
computational drug design, immuno-informatics, cheminformatics, and
bioinformatics, make up the group, with a cutting-edge 5-node High-
Performance Computing (HPC) cluster. Their knowledge encompasses the
analysis of protein mutations, protein-ligand protein-protein interactions,
protein structure and function prediction, structure and ligand-based drug
design, molecular modelling, phylogenetics, primer designing, genomic data
analysis, and sequence analysis. They provide thorough computational support
to multiple teams of researchers within the Centre.

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Introduction
CEMB (Centre of Excellence in Molecular Biology) is a renowned research
institute located in Pakistan, dedicated to advancing the field of molecular biology and
biotechnology. With a focus on high-quality research, CEMB conducts cutting-edge
studies in genetics, genomics, structural biology, bioinformatics, and synthetic biology.
This internship report aims to provide an overview of my experiences and acquired
skills during my time as an intern at the institute.

As a hub of molecular biology, CEMB focuses on investigating interactions

within and between molecules such as DNA, RNA, proteins, other micro and
macromolecules, etc. The field of molecular biology is extensive as it comprises of
studies revolving around DNA replication, its structural significance, gene expression,
regulation and analysis. Moreover, the production of unique molecules of DNA and
recombinant proteins via genetic engineering or recombinant DNA technology is
another area that falls under the umbrella of molecular biology.

I worked under the supervision of Dr. Nadeem Ahmad during my tenure as a

CEMB internee in the lab of Biopharmaceuticals and Proteomics. Biopharmaceuticals
and Proteomics are the interdisciplinary fields related to molecular biology.

Biopharmaceuticals and Proteomics Laboratory

The class of therapeutic drugs known as biopharmaceuticals is generated from
biological sources, such as living cells or organisms (Chen & Yeh, 2018). Proteomics,
on the other hand is the study of all proteins in a cell, tissue, or organism, focusing on
their interactions and functions (Aslam et al., 2017).

The CEMB biopharmaceuticals and proteomics laboratory is focused on the

development of indigenous technology for producing recombinant human proteins like
cytokines at a commercial level. These drugs have a potential for a great dynamic
progress on a therapeutic scale because of their tremendous market demand. In order to
create a bioactive and extremely pure form of the desired protein, the desired protein
must first be expressed in host cells and then purified using techniques like
chromatography and filtration. The CEMB BPL group is also making an effort to

19
develop an efficient nanoparticle-based drug delivery system for the evaluation of
anticancer effects hidden in the recombinant proteins.

Fig. 1 Biopharmaceuticals and Proteomics Laboratory Overview

The lab also encompasses working techniques such as protein separation (e.g.,
chromatography, PAGE) and mass spectrometry for large-scale protein analysis.
Protein identification is achieved through database searching, comparing experimental
data with known protein sequences. We gain insights into the intricate world of
pharmaceutics and proteins, understanding their functions and contributions to
biological processes.

Table 1: Objectives of biopharmaceuticals and proteomics laboratory

Biopharmaceuticals and Proteomics Laboratory Objectives

Recombinant Production Developing Human Algae and Yeast
of Proteins Therapeutics and Drug Biotechnology
Delivery System
• Process • Development and • Evaluating GM
development of research on human algae for the
protein purification therapeutic proteins

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 • Protein • Nanoparticle-based production of
characterization drug delivery therapeutic proteins
system • Evaluating GM
• Studying anticancer algae for the
potential of production of edible
recombinant human vaccines
therapeutic proteins • Developing GM
yeast for the
expression of
therapeutic proteins

At my two-month internship program at CEMB, I have worked with an

extensive variety of experimental tools and techniques to study various processes at a
molecular level. I have honed essential skills through this platform in the techniques
such as:

➢ Media Preparation
➢ Streaking
➢ Gram Staining
➢ Stab Culture Preparation
➢ Polymerase Chain Reaction
➢ Agarose Gel Electrophoresis
➢ Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE)
➢ Transformation
➢ Plasmid Mini Preparation
➢ Syringe Filtration

Significance and Scope

➢ Media Preparation

Several media preparation methods are important because they offer the
nutrients and environments required for development and proliferation of

21
microorganisms. Researchers can isolate and distinguish particular types of
microorganisms according to their growth traits and metabolic activities using these
media, which can be categorized into distinct groups like selective, differential,
or enriched media (Sandle, 2014).

➢ Streaking

Researchers frequently employ a technique called streaking to separate pure

cultures of bacteria from mixed populations. Individual colonies are created by strewing
a sample onto an agar plate in a certain pattern. Each colony results from a single
bacterial cell. Using this method, researchers can characterize certain microbes, study
them in controlled environments, do biochemical tests on them, and examine their
growth and behavior (Eyler, 2013).

➢ Gram Staining

Gram staining is a basic technique which facilitates the division of bacteria into
Gram-positive and Gram-negative groups. Based on the variations in the composition
of their cell walls, this staining technique aids in the identification and classification of
bacteria (Biswas et al., 1970). It is extremely important since it offers insightful
knowledge on the morphology and categorization of bacteria, assisting in the
identification and management of infectious disorders.

➢ Stab Culture Preparation

Stab culture creates a special habitat for the development of bacteria and fungi
by inoculating solid media with a straight needle, enabling scientists to see their
morphological traits and analyze their metabolic processes (Williams, 1936). Overall,
stab culture contributes to a variety of scientific fields and real-world applications by
expanding our knowledge of microbes and their interactions with the environment.

➢ Polymerase Chain Reaction

A cutting-edge molecular biology technique called polymerase chain reaction

(PCR) amplifies particular DNA sequences to make them detectable and analyzable.
PCR enables a rapid expansion of the target DNA, even from incredibly tiny portions
22
of starting material, by employing a heat-stable DNA polymerase and certain primers
that target the desired DNA region (Nagamine et al., 1989). Disease diagnosis, forensic
analysis, DNA cloning, and gene expression analysis are just a few of the many uses
for PCR that have considerably enhanced our knowledge of genetics and
biotechnology.

➢ Agarose Gel Electrophoresis & SDS PAGE

Essential molecular biology procedures include sodium dodecyl sulphate

polyacrylamide gel electrophoresis (SDS-PAGE) and agarose gel electrophoresis.
Applications including DNA profiling, genome sequencing, and genetic research are
made easier by the ability to separate and analyze DNA fragments according to their
size and charge using agarose gel electrophoresis (Rudge & Monnig, 2011). The
molecular weight of the proteins can be used to separate them using SDS-PAGE, which
helps with protein characterization, estimation, and classification (Manns, 2011). These
methods are essential in areas including genetic research, protein analysis, forensics,
and diagnosis illuminating the functions of DNA and protein molecules (Wittmeier &
Hummel, 2022).

➢ Plasmid Mini Prep

The critical procedure of plasmid micro preparation is utilized to separate and

purify plasmid genetic material from bacterial cells. In addition to the bacterial
chromosome, plasmids are circular fragments of DNA that frequently include genes
that impart beneficial features, such as resistance to antibiotics (Pronobis et al., 2016).
Researchers can alter and investigate certain genes of interest, carry out cloning trials,
and produce chimeric DNA molecules for diverse uses in biotechnology by obtaining
and purifying plasmid DNA using micro-preparation procedures.

➢ Transformation

Transformation, the process of absorbing and integrating foreign DNA into a

host organism is an essential procedure. The introduction of particular genes or genetic
alterations into an organism is rendered possible by this method, which permits the
investigation of gene function, protein expression, and other genetic phenomena (Aune
23
& Aachmann, 2010). Wide-ranging implications for transformation involve the
development of GM organisms, gene therapies, and transgenic animals and plants.

➢ Syringe Filtration

A crucial method for sterile liquid filtration in laboratories is syringe filtration.

It entails filtering the liquid using a syringe in order to remove impurities and produce
a sterile filtrate (Scheer, 2009). This approach ensures dependable and contaminant-
free procedures in molecular biology and microbiology by maintaining sample
integrity, preparing culture medium, sterilizing reagents, and purifying solutions.

1. Different types of media preparation

Different types of media are essential in molecular biology, microbiological

experiments and cell culture preparation. It can be referred to as “A nutrient-rich
medium (solid/semi-solid/liquid) providing controlled conditions for the in vitro
growth and maintenance microbes (Lagzeil et al., 2020).” The environment in growth
media is full of necessary growth factors, minerals, vitamins, and other important
constituents.

1.1 Lysogeny media

Lysogeny (LB media) is an all-purpose cultivation medium for bacteria,

specifically Escherichia coli (Williams & Liao, 2006). It gives bacteria the vital
elements they need to thrive, such as vitamins, carbohydrates, and amino acids.

100 ml of LB Agar was prepared by the following steps:

1.1.1 Materials & glassware

• LB agar powder/LB agar mixture

• Flasks
• Distilled water
• Magnetic stirrer/stir plate
• Sterile petri dishes
• Test Tubes

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1.1.2 Equipment

• pH/pH meter
• Autoclave

Fig. 1.1 Reagents for preparing LB agar

1..3 Procedure

We performed the following procedure to prepare LB Agar:

➢ Weigh the LB agar powder

1–1.5g of LB agar was weighed per 100 ml of water and added in a flask filled
with 20 ml of distilled water.

➢ Raise the volume

The flask was filled with 80 ml of distilled water to raise it to 100 ml. The agar
powder was then mixed in distilled water by gentle stirring.

➢ Heating and dissolving

The flask was then placed on a stir plate used for accelerating the dissolving
process. Magnetic stirrers can also be used. A microwave or a hotplate was utilized for

25
heating the mixture. To ensure that the agar is completely dissolved, bring the agar
mixture to a rolling boil for a few minutes.

Fig. 1.3 Melting agar in LB solution

➢ Adjustment of pH

The pH of the LB agar solution can be determined by using pH strip or a meter.

The optimal pH is close to 7.0. Maintain the pH while gradually adjusting it with small
quantity of an acid like HCl or a base like NaOH. The procedure should be finished
once the desired pH is attained.

➢ Sterilization

Flasks were autoclaved, leaving space in them for expansion during

autoclaving. LB agar was sterilized by autoclaving the containers at 121°C at 15 psi
(pounds per square inch) for around 15-20 minutes. Make sure to use autoclave-safe
containers and adhere to the recommended autoclaving procedures (Davis, 1920).

➢ Cooling & solidification

Autoclaved LB was cooled to a room temperature and solidified into a gel-like

consistency. In order to create slants, pour the mixture in test tubes, tilt them at an angle
of 45 degrees and place in a raised position for some time.

➢ Storing the solution:

The solution can be stored in a dark, cool, and dry place until further use. Ensure
to write valid information on the labels of flasks, petri plates or test tubes, including the
preparation date or any other relevant details. When preparing and pouring agar, follow
aseptic procedures to prevent contamination.

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1.2 Lysogeny Broth

Follow the following steps to prepare 100ml of LB Broth: (Williams & Liao, 2006):

1.2.1 Materials & glassware

• LB agar powder/LB agar mixture

• Flasks
• Distilled water
• Magnetic stirrer/stir plate
• Sterile petri dishes
• Test Tubes

1.2.2 Equipment

• pH/pH meter
• Pressure cooker or autoclave

1.2.3 Procedure

➢ Weigh the LB broth powder

2.5g of LB agar was weighed per 100 ml of water and added in a flask filled
with 20 ml of distilled water.

Fig. 1.2.3 Weighing LB powder to prepare LB broth

27
 ➢ Raise the volume

The flask was filled with 80 ml of distilled water to raise it to 100 ml. The agar
powder was then mixed in distilled water by gentle stirring.

➢ Heating and dissolving

The flask was then placed on a stir plate used for accelerating the dissolving
process. Magnetic stirrers can also be used. A microwave or a hotplate was utilized for
heating the mixture. To ensure that the agar is completely dissolved, bring the agar
mixture to a rolling boil for a few minutes.

➢ Adjustment of pH

The pH of the LB agar solution can be determined by using pH strip or a meter.

The optimal pH is close to 7.0. Maintain the pH while gradually adjusting it with small
quantity of an acid like HCl or a base like NaOH. The procedure should be finished
once the desired pH is attained.

➢ Sterilization

Flasks were autoclaved, leaving space in them for expansion during

autoclaving. LB agar was sterilized by autoclaving the containers at 121°C at 15 psi
(pounds per square inch) for around 15-20 minutes. Make sure to use autoclave-safe
containers and adhere to the recommended autoclaving procedures (Erkmen, 2021).

➢ Cooling & solidification

Autoclaved LB was cooled to a room temperature and solidified into a gel-like

consistency. In order to create slants, pour the mixture in test tubes, tilt them at an angle
of 45 degrees and place in a raised position for some time.

➢ Storing the solution

The solution was stored in a dark, cool, and dry place until further use. Ensure
to write valid information on the labels of flasks, petri plates or test tubes, including the
preparation date or any other relevant details. When preparing and pouring agar, follow
aseptic procedures to prevent contamination.
28
1.3 Yeast extract peptone dextrose media

The yeast cultures mainly Saccharomyces Cerevisiae grow on YEPD (Yeast

Extract Peptone Dextrose) media. Dextrose is a carbohydrate source, peptone offers
nitrogen, carbon, vitamins, and minerals, etc. and yeast gives vitamin B-complex and
amino acids, which are all essential nutrients facilitating the growth of yeast.

The general procedure to prepare 100 ml of YEPD broth is given as follows:

1.3.1 Materials & glassware

• Yeast extract
• Peptone
• Dextrose
• Double Distilled water
• Sterile test tubes/bottles
• Magnetic stirrer/stir plate
• Flask

1.3.2 Equipment

• pH meter
• Autoclave

1.3.4 Procedure

YEPD media was prepared by the following procedure:

➢ Weigh ingredients

All the ingredients were weighed as mentioned below in the table and were
added to a flask filled with 20 ml double distilled water.

29
 Table 2: Concentration of reagents to make YPD broth

Reagents Concentration (g/ml)

Yeast extract 1

Peptone 2

Dextrose 2

➢ Raise the volume

The flask was filled with 80 ml of distilled water. The components were
carefully stirred to dissolve them.

➢ Heating and dissolving:

The flask having the YPD media was heated dissolve the ingredients
completely. A stir plate or a magnetic stirrer can also be utilized to accelerate the
dissolving process. The mixture should have at least one short boil for the ingredients
to completely dissolve.

➢ Adjustment of pH

The pH of the YPD solution was determined by a pH meter or a strip. The

optimal pH range is between 5.5 and 6.0 for the YPD mix. The pH was adjusted by
adding a few acid drops such as of HCl, or a base such as NaOH. The solution was
gently stirred while the pH adjustment.

➢ Sterilization

The YEPD mix was poured into a flask for autoclaving. A small headspace was
left to avoid the overflow while the flask was in autoclave. Flasks should be tightly
sealed. The flask was autoclaved at 121°C at 15 psi for about 15 to 30 minutes.
Autoclave-safe vessels were utilized, and the flasks were autoclaved as per the proper
guidelines.
30
 Fig. 1.3.4 Autoclaved YPD broth

➢ Storing the solution

The autoclaved flasks with YEPD broth were left for some time at a room
temperature to let them cool down before storage. They were stored at a cool and dry
place until further use.

Every flask was labeled with essential details, such as the date of preparation
and name of the medium. Aseptic practices were carried out throughout the entire
process of YPD broth preparation.

1.4 Yeast extract peptone dextrose agar medium

100 ml of YPD agar (solid media) is utilized for promoting the growth and
preservation of yeast culture. Following steps were carried out to prepare the YPD agar:

1.4.1 Materials & glassware

• Yeast extract
• Peptone
• Dextrose
• Double Distilled water
• Sterile test tubes/bottles
• Magnetic stirrer/stir plate
• Flask

31
1.4.2 Equipment

• pH meter
• Autoclave

Fig. 1.4.1 Reagents for preparing YPD agar

1.4.3 Procedure

We prepared YEPD agar by the following procedure:

➢ Weigh ingredients

All the ingredients were weighed as mentioned below in the table and the
weighed amounts of these ingredients were added to a flask filled with 20 ml double
distilled water.

Table 3: Concentration of reagents to make YPD Agar

Reagents Concentration (g/ml)

Yeast extract 1

Peptone 2

Dextrose 2

Agar 1.5

➢ Raise the volume

The flask was filled with 80 ml of distilled water. The components were
carefully stirred to dissolve them.
32
 ➢ Heating and dissolving

The flask having the YPD media was heated dissolve the ingredients
completely. A stir plate or a magnetic stirrer can also be utilized to accelerate the
dissolving process. The mixture should have at least one short boil for the ingredients
to completely dissolve.

➢ Adjustment of pH

The pH of the YPD solution was determined by a pH meter or a strip. The

optimal pH range is between 5.5 and 6.0 for the YPD mix. The pH was adjusted by
adding a few acid drops such as HCl, or a base such as NaOH. The solution was gently
stirred while the pH adjustment.

➢ Sterilization

The YEPD mix was poured into a flask for autoclaving. A small headspace was
left to avoid the overflow while the flask was in autoclave. Flasks should be tightly
sealed. The flask was autoclaved at 121°C at 15 psi for about 15 to 30 minutes.
Autoclave-safe vessels were utilized, and the flasks were autoclaved as per the proper
guidelines.

Fig. 1.4.3 Autoclaved YPD broth

➢ Cooling and storage

The medium in autoclaved flasks was poured into test tubes and they were kept
at a 45 degrees elevated level to let the medium solidify. These slants were then stored
at a cool and dry place until further use. Each flask and test tubes were labeled with

33
essential details, such as the date of preparation and name of the medium. Aseptic
practices were carried out throughout the entire process of YPD agar preparation (Treco
and Lundblad, 1993).

1.5 Sucrose soy broth

A nutrient-rich liquid medium called sucrose soy broth is employed to cultivate

and develop various microbes, mainly bacteria & fungi. It is made by combining a
blend of soy peptone, a hydrolyzed form of soybean protein, and sucrose, a type of
disaccharide. These components work well together to create an environment that is
conducive to microbial development.

100 ml of sucrose soy broth was prepared by the following procedure:

1.5.1 Materials & glassware

• Sucrose
• Soy peptone
• Distilled water
• Flask
• Magnetic stirrer
• Test tubes/bottles

1.5.2 Equipment

Autoclave

1.5.3 Procedure

➢ Measure and dissolve ingredients

2 g/ml of sucrose and 2 g/ml soy peptone were weighed and added in a flask
filled with 20 ml of water.

➢ Raise the volume

The flask was filled with 80 ml of distilled water. The components were
carefully stirred to dissolve them.

34
 ➢ Heating and dissolving

The flask having the SSB media was heated dissolve the ingredients completely.
A stir plate or a magnetic stirrer can also be utilized to accelerate the dissolving process.
The mixture should have at least one short boil for the ingredients to completely
dissolve.

➢ Adjustment of pH

The pH of the SSB solution was determined by a pH meter or a strip. The

optimal pH range is between 5.5 and 6.0 for the YPD mix.

The pH was adjusted by adding a few acid drops such as of HCl, or a base such
as NaOH. The solution was gently stirred while the pH adjustment.

➢ Sterilization

The SSB mix was poured into a flask for autoclaving. A small headspace was
left to avoid the overflow while the flask was in autoclave. Flasks should be tightly
sealed. The flask was autoclaved at 121°C at 15 psi for about 15 to 30 minutes.
Autoclave-safe vessels were utilized, and the flasks were autoclaved as per the proper
guidelines.

➢ Cooling and storage

The autoclaved flasks with YEPD broth were left for some time at room
temperature to let them cool down before storage. They were stored at a cool and dry
place until further use. Each flask and test tubes was labeled with essential details, such
as the date of preparation and name of the medium. Aseptic practices were carried out
throughout the entire process.

Fig. 1.5.3 Autoclave SSB media

35
2 Streak plate quadrant streaking

Streaking is used to separate isolated microbial colonies or cells from a culture

by distributing the inoculum in a pattern across the outer layer of an agar plate. The
streak plate technique relies on the dilution theory. One way to define it is a quick
qualitative isolation method (Sanders, 2012). Obtaining fewer or reduced colonies as a
result is the key requirement for isolation. The method used for streaking
Saccharomyces cerevisiae (yeast) is the same as the one employed for streaking other
microbes.

To streak an agar plate with the desired microbe, we followed the steps
mentioned below:

2.1 Materials & glassware

• Petri plates with agar media

• Inoculating loops
• Alcohol
• Spirit lamp
• Match box
• Microbial culture

2.3 Equipment

• Laminar air flow

• Incubator

2.4 Procedure

➢ Preparation of materials
• Petri plates of sterile agar such as YPD agar for growth of yeast, LB agar for
growth of bacteria, SSB in case of both.
• Inoculating loops/needles (disposable and sterilized).
• Prepared petri plates with bacterial or yeast culture or a liquid medium
containing bacterial or yeast culture.

36
➢ Sterilization

The inoculating loop was sterilized via flame till it turned red-hot. The loop was
then cooled down by gently rubbing it against the agar medium. Distance should be
kept while brushing/rubbing from the place of streaking.

➢ Collection of sample

The loop was used to pick a colony from the previously prepared plate of the
desired culture.

➢ Streaking

The lid of the agar plate was lifted to slightly expose the agar surface for a
streak. The plate was quadrantally streaked by starting at one edge and gently moving
the loop in a zigzag pattern over the surface of agar medium. This step was repeated for
each quadrant by moving the petri plate and sliding loop gently over each of the
remaining three portions of the agar surface. Each quadrant was streaked with a
decreasing density to obtain isolated colonies.

➢ Plate incubation:

The lid of the agar plate was then closed and secured with tape. The plate was
placed in an incubator at a temperature suitable for the growth of desired microbe (30
°C for yeast for 48 hours and 37 °C for bacteria for 24 hours).

Fig. 2.4 (a) Incubating streaked plates

37
➢ Observation of results:

The isolated colonies of yeast/bacteria were observed on the plate after fixed
times of incubation. The yeast colonies were in the form of cells or small clusters. While
bacterial colonies were elevated clusters or small isolated cells.

Scientists can utilize these colonies for countless scientific experiments or other
research purposes. Aseptic practices were maintained throughout the procedure and
microbe specific growth media and incubation settings were employed (Ogodo et al.,
2022).

Fig. 2.4 (b) Colonies (S. cerevisiae)

3. Gram staining:

Gram staining is a technique that helps classify bacterial species into:

• Gram-positive
• Gram-negative

The procedure was created by Danish bacteriologist Hans Christian Gramme in

1884 and consists of a number of steps (Moyes et al., 2009). In this method, crystal violet
and iodine are employed. Gram-positive bacteria are colored bluish/purple as a
consequence, whereas Gram-negative bacteria are colored reddish/pink as a result of
the safranin. Bacteria may be recognized, categorized, and examined to learn more
about the makeup of their cell walls using Gram staining.

38
 We performed the gram staining for Escherichia coli, a Gram-negative
bacterium by the following steps (Elbing and Brent, 2019):

3.1 Reagents and materials

• E. coli
• Sterile loop
• Glass slides
• Spirit lamp/Bunsen’s burner
• Crystal violet
• Gram’s iodine
• Acetone/ethanol for decolorizing
• Safranin
• Water

3.2 Equipment

Microscope

3.3 Procedure

A single colony was picked from a petri plate and clean glass slide was prepared
with a smear of E. coli. The smear was left for some time to let it air dry. After the
smear was air-dried, it was heat-fixed by rapidly and repeatedly passing it through the
Bunsen burner. We performed this step to firmly attach the bacteria onto slide and
prevent it from being washed away during the staining process. Next, crystal violet was
applied to the smear in a way that thoroughly covered the cells. The stain was left on
the smear for about one minute. The slide was gently rinsed with water to remove the
extra stain if there is any. A few drops of Gram’s iodine were applied onto the smear
and left to rest for one minute. This step was performed to promote the complex
formation between the cells of bacteria and crystal violet stain. The glass slide was
again rinsed carefully for the removal of extra stain. A decolorizing agent such as
acetone/ethanol was applied dropwise until the runoff was clear. The slide was tilted
the entire procedure of applying reagents. The slide was quickly rinsed with water. A

39
few drops of safranin were added to cover the smear and left to rest for about one
minute. The slide was rinsed with water to remove extra stain. The slide was completely
air dried, and a paper towel was utilized to blot away the remaining water. The results
were observed under a microscope at a high magnification to visualize the E. coli cells.

1) Making a smear 2) Applying iodine 3) Applying iodine 4) Applying decolorizer 5) Applying safranin

Fig. 3.3 Gram staining procedure

3.4 Observation of results

Table 4: Observed Characteristics of Bacteria

Characteristic Observation
Size Medium
Shape Round
Color Off-white
Margin Entire
Opacity Opaque
Gram Nature Gram negative
Motility Non-motile
4. Stab culture preparation

Stab culture is a technique for growing microorganisms in a tube on a solid

medium, usually agar. To introduce the bacteria, an inoculating needle or loop is
inserted vertically into the agar. A line or column of microbial colonies may be seen as
the resultant growth. Stab cultures are frequently used to preserve microorganisms for
a long time. The solid medium offers the organisms a stable habitat that enables long-

40
term survival. Stab cultures make it possible to assess how well an organism can use
different nutrients.

4.1 Materials

• Inoculating loop
• Spirit lamp
• Match box
• Agar medium
• Desired Culture
• Test tubes
• Alcohol

4.2 Equipment:

Laminar Air Flow

4.3 Procedure:

Following steps were carried out for stab culture preparation:

➢ Preparation of the culture medium:

A detailed recipe or instructions for preparing the suitable agar medium was
followed, for example selectiveagar, blood agar, or nutrient agar.
➢ Sterilization:

To sterilize the agar medium and to assure the removal of any

contaminants, the media was properlyautoclaved.
➢ Inoculation:

The stab method was then performed once the agar has significantly cooled
down. The agar medium was gently stabbed vertically into the center of the plate or
test tube with a sterile inoculating loop or needle, penetrating to a specific level (often
around halfway). The loop or needle was sterilized by either using a disposable
needle that has already been sterilized or by blazing it until it is red hot.
➢ Incubation:
The plate or test tube was then placed in an incubator that has been set to the
41
 suitable temperature for the growth of the Saccharomyces cerevisiae after the agar
media has been inoculated. The microbe was allowed to grow on the plate by
incubating it for overnight.

For the production of bacteria that need anaerobic conditions or have different
growth patterns, the stab methodis frequently used. It creates an ideal condition for
the growth of microbes that might not grow sufficiently onthe agar surface alone by
deeply penetrating the inoculating needle into the agar.

The yeasts growth pattern was observed after incubation. Depending on the
features of the microorganism being investigated, a distinct development along the
line of the stab (looking like a column or a line) or dispersed growth across the media
was observed when using the stab method. It's especially important to remember that
in order to avoid contamination and guarantee accurate results, adequate aseptic
procedure mustbe used during the stab method.
5. Polymerase chain reaction (PCR)

Polymerase chain reaction amplifies a single or a few copies of a DNA across

several orders of magnitude (millions copies of a particular sequence) (Kuslich et al.,
2019). This technique is most commonly used in molecular biology and has far-reaching
applications in forensics, cloning, genetic analysis, research and diagnostics, etc. (Zhu
et al., 2020).

5.1 Required materials & reagents

• Template DNA
• Primers
• DNA polymerase (Taq polymerase)
• dNTPs (dCTP, dGTP, dATP, and dTTP)
• Buffer Solution
• Distilled Water
• Ice box
• PCR tubes
• Micropipette
42
 • Micropipette tips

5.2 Equipment

Thermal Cycler (PCR machine)

5.3 Procedure:

We followed the method mentioned below to run PCR experiment:

➢ Setting up the reaction:

To set up a PCR reaction, the following ingredients were combined in a PCR

tube (Green and Sambrook, 2018):

• 10 µl template DNA
• 10.5 µl Taq polymerase
• Forward and reverse primers
• 200M of dNTPs
• 5 µl buffer solution

Fig. 5.3 (a) PCR components

Table 5: Reagents in PCR master-mix

Reagents Volume (µl)

Template DNA 10 µl
Reverse primer 22.5 µl
Forward primer 22.5 µl
Buffer 30 µl
Taq polymerase 10.5 µl

43
 dNTPs 30 µl (200 M)
Double distilled water 154.5µl

➢ PCR set up:

The PCR machine was set to repeat denaturation, annealing, and extension
cycles.

• Denaturation:

The temperature was set between 94-98 °C for denaturing the hydrogen-bonded
double stranded DNA.

• Annealing:

Annealing temperature was set between 45-68 °C so that the primers could
anneal to the sequences complementary to it in the template.

• Extension:

The extension temperature was set at 72 °C. This step was performed to help
facilitate the enzymes to add the primers along the length of PCR to synthesize DNA.
Total of 20-25 cycles were needed for the copies to be amplified.

Fig. 5.3 (b) PCR setup on thermal cycler

44
 ➢ PCR product analysis

The products were separated and analyzed through agarose gel electrophoresis,
and they were visualized through UV illuminator (Akram, 2018). The sizes were
estimated by comparing them to the ladder.

Fig. 5.3 (c) PCR product analysis via gel electrophoresis

6. Agarose gel electrophoresis

The migration of charged molecules in an applied electric field through

solutions is called electrophoresis. Agarose gel electrophoresis separates the molecules
(DNA, RNA, proteins) on the basis of charge, molecular weight and size.

To perform agarose gel electrophoresis we followed the steps given below (Lee
et al., 2012):

6.1 Materials & Glassware:

• Agar
• TAE Buffer
• Micropipettes
• Bunsen burner
• Beaker
• Bromophenol blue
• Stirrer
• Sample to be tested

45
6.2 Equipment:

• Microwave
• Electrophoresis apparatus
6.3 Procedure:
➢ Preparing the gel

Based on the range of molecular sizes that required to be separated, an

appropriate amount of agarose gel was selected. Typically, 0.7 and 2% of agarose
concentrations are added. 100ml of TBE or TAE buffer was put to a flask before 1.5g
of agarose was added to it. The gel was heated in a microwave until the agarose
completely dissolved in the buffer (Sun et al., 2012).

Fig. 6.3 (a) Preparation of gel

➢ Setting up the apparatus

A gel box or gel casting tray was placed on a level surface. To make sample
wells, a comb was put into the gel casting tray. In order to completely cover the wells,
the chilled agarose mix was cautiously poured in the gel casting tray. The agarose was
left to harden for 20 to 30 minutes.

6.3 Sample preparation

The DNA samples were combined with an appropriate loading buffer. When
samples were loaded into the electrophoresis wells, the loading buffer gave them
density and helped with sample visualization. The sample combination was heated for

46
a brief period of time, often in a water bath at 65–70°C, to denature any secondary
structures found in the nucleic acids.

6.4 Sample loading

The comb was scooped out of the gel. Using a pipette or a micro-titer plate
loading equipment, the samples were carefully put into the agarose gel wells, making
sure that none of the surrounding wells were contaminated. A few of the wells were left
empty, while others had reference markers inserted into one or more of them.

6.5 Running the gel

After the gel was completely immersed in buffer and polarity was double
checked when the power supply was connected to electrodes, the gel was run at 120 V
for 45 minutes.

Fig. 6.3 (b) Running gel electrophoresis

6.6 Result visualization

The gel was carefully removed from the chamber after the electrophoresis was
finished and set on an ultraviolet (UV) trans-illuminator or any other appropriate
imaging apparatus.

The nucleic acids were stained with an appropriate DNA or RNA dye, such as
ethidium bromide or a fluorescent dye. The gel was stirred for an hour while it was
submerged in the staining solution and subsequently the de-staining solution. Under
UV light or using the appropriate imaging equipment, the gel was analyzed to see the

47
different bands that represented the nucleic acids. As necessary, photos were taken, or
the outcomes were recorded in writing.

Fig. 6.3 (c) Observed DNA bands

Proper safety procedures were always taken when working with electrical
equipment, ultraviolet (UV) radiation, or stains. The agarose gel and staining agents
were also appropriately disposed of in accordance with the established norms. Protein
separation required certain adjustments to the process and gel composition, but the
technique's core ideas remained constant.

7. SDS PAGE

For the separation and molecular weight-based analysis of proteins, SDS-PAGE

(sodium dodecyl sulphate-polyacrylamide gel electrophoresis) is an extensively used
technique in biochemistry and molecular biology. In this process, proteins move across
a gel matrix while being driven by an electric field.

The steps for the SDS-PAGE procedure are mentioned below:

7.1 Materials & glassware:

• Eppendorf tubes
• Micropipettes and tips
• Acrylamide
• Bis acrylamide
• Buffers
• Coomassie brilliant blue

48
 • Acetic acid
• Glycerol
• TEMED
• Ammonium per sulphate
• Spirit lamp

7.2 Equipment:

SDS apparatus

7.3 Procedure:

➢ Extraction and solubilization

In the first step of the SDS page, the protein sample to be analyzed is extracted.
In general, this can be done in numerous ways, such as:
• Rupturing the cell membrane
• Using high-frequency sound waves via sonication
• High-speed blender
• Osmotic shock
• Lysis buffer
• Tissue homogenizer
After extraction, the protein sample is solubilized in a buffer and centrifuged
for the removal of debris. We performed SDS on a previously extracted sample.
7.3 Sample preparation

A detergent (SDS) and a suitable buffer was used to denature and solubilize the
proteins. Tracking dye (bromophenol blue) was added to visualize the tracking
movement. The sample was heated at an elevated temperature of 95 °C to break the
covalent bonds.

49
 7.4 Gel preparation
• Stacking and resolving gel

Acrylamide, bis acrylamide, Ammonium persulfate, buffer solution, and water

were added in an Eppendorf with a micropipette in appropriate quantities (Wyckoff et
al., 1977). The acrylamide percentage was adjusted depending on the expected size
ranges of protein. A small amount of TEMED (tetramethyl ethylenediamine) was added
at the end to start the process of polymerization. Resolving gel is poured first followed
by stacking gel. A comb was placed at the top after the gel was poured between two
glass plates in a vertical gel casting tray. The gel was left for some time to solidify.

Fig. 7.3 (a) Gel preparation for SDS PAGE

Note: Small sized proteins require higher percentages of acrylamide and bis-acrylamide
resulting in smaller gel pores and vice versa.

7.5 Loading the gel and electrophoresis

The protein samples were carefully loaded into the wells with a micropipette.
The molecular weight markers were loaded in the first well for determination of
molecular weight. The gel was immersed in a running buffer and then connected to the
electrophoresis apparatus. The voltage was applied, and the sample moved through the

50
gel owing to the electrophoretic mobility. The larger size of the proteins indicates
greater friction, whereas smaller proteins move faster.

Fig. 7.3 (b) SDS PAGE setup

8. Staining and visualization

The protein sample after separation on the gel can be stained with two methods:
• Coomassie Brilliant Blue
• Silver Stain
We used the former method in which the gel was saturated with methanol,
water, and acetic acid having Coomassie brilliant blue R-250 dye. This ensured that the
dye attach to the proteins due to numerous amino acid residues, while methanol and
acetic acid bind the proteins to the matrix of the gel. However, in silver staining, the
gel is saturated with silver nitrate, and a reducing agent is introduced to reduce silver
ions to metallic silver. The silver precipitates on the proteins present in the gel. This
precipitation results in the appearance of black protein bands.

Note: We can choose the staining procedure based on the sensitivity of detection
required for our sample. Silver staining has more sensitivity.
9. Results analysis

A gel documentation system was used to analyze the SDS-PAGE gel. The
protein bands on comparison with the ladder gave an easy estimation for their molecular
weights. We can also compare the protein profiles of different samples treated in
different conditions.

51
 Fig. 7.3 (c) Protein bands observed

The method called SDS-PAGE is often used in protein research for protein
purification, protein-protein interaction investigations, and protein molecular weight
measurement. It offers useful information on the size, relative abundance, and purity
of the proteins in a sample (Roy and Kumar, 2014). However, due to the risk
associated with handling acrylamide solutions, care must be taken, and appropriate
safety precautions must be followed (Atkins, 1991).

8 Transformation

Transformation is the direct uptake of DNA by bacterial cells. We use this

procedure for the development of gene therapies, GM organisms, transgenic animals
and plants (Das et al., 2017).

The procedure for transforming in E. coli is given as follows (Hasegawa et al., 2018):

8.1 Procedure:

• Preparation of the materials

• Making competent cells

The cells which have undergone physical/chemical treatment to enhance their

ability for the direct uptake of DNA are known as competent cells. We used ice cold
calcium chloride for the treatment of cells to enhance the characteristic ability of DNA
binding to the bacterial cells (Asif et al., 2017).

52
 • Thawing

The previously prepared vial was taken out of the freezer for thawing purpose.
Care was taken not to damage the cells by shaking the vial too hardly.

Fig. 8.1 (a) Calcium chloride

➢ Mixing competent cells with DNA of interest

The required amount of DNA constituting the gene of interest was added in a
sterile microcentrifuge tube. The process was performed in an ice box to avoid cell
damage. The previously prepared competent cells were added in the same tube. The
components was gently mixed by moving the tube up and down repeatedly. The
mixture (DNA and competent cells) was left on ice for incubation for 30 minutes.

Fig. 8.1 (b) Preparation of competent cells

53
 ➢ Heat shock treatment

Then , a heat shock treatment was given at an elevated temperature of 4°C to

42°C for 2 minutes in a water bath. It enabled the DNA of interest to move into the
bacterial cells of E. coli.

➢ Cell recovery

The tube was placed on ice immediately for 3-5 minutes after the heat shock
process was completed. It was done for cells to stabilize the processed membranes.

➢ Incubation in growth medium

The tube containing the transformed cells was filled with sterile LB broth and
the tube was incubated at 37°C (as we worked with E. coli) in a shaking incubator. The
tube was incubated for 1-2 hours to facilitate the bacterial cells in the expression of the
DNA of interest inserted previously.

Fig. 8.1 (c) Incubation in a shaking incubator

➢ Selection of the transformed cells

After the required period of incubation, the transformed cells were spread in an
agar plate along with an antibiotic. As the transformed cells develop resistance to
antibiotic, so this reagent helped us identify the true transformed cells. The place was
incubated overnight at a temperature well-suited to E.coli.

➢ Observation of results

Observing the agar plate showed visible bacterial colonies. The colonies that
appeared were successfully transformed as they grew in the presence of an antibiotic
54
reagent. The transformed colonies can later be utilized for various applications in
scientific research (Tsen et al., 2002).

9 Mini Prep for plasmid isolation

For the rapid and simple separation of plasmid DNA from bacteria, use a mini-
prep. It enables the extraction of minute quantities of plasmid DNA for later uses such
as cloning, sequencing, and ongoing research, etc.

9.1 Procedure:

The procedure for plasmid isolation (mini prep) is given below (Pronobis et al.,
2016):

➢ Preparation of the materials

• Mini prep kit

We worked with a plasmid isolation kit (GeneJET Mini prep Kit) with all the
essential reagents and buffer solutions (lysis, neutralization, resuspension, wash buffer,
etc.) Get a plasmid isolation kit that has all the necessary protocols and reagents.

• Bacterial culture

We started with a transformed single colony of E. coli previously incubated in

a selective growth medium. It was centrifuged on 10,000 x g in microfuge tubes and
resuspended using sterile water.

Fig. 9.1 Centrifuge plasmid

55
 ➢ Preparation of bacterial culture

The transformed plasmid was inoculated into 3-5 ml of the relevant media in a
culture tube and incubated overnight in a shaking incubator at a suitable temperature.

➢ Harvesting bacterial cells

The culture was transferred into a microfuge tube, and was centrifuged at 3,000
to 5,000 x g for 120 seconds for pellet formation. The supernatant was discarded.

➢ Re-suspension of the pellet

250 microliters of resuspension buffer was added to the pellet. The cells were
completely resuspended by gentle pipetting or vortex.

➢ Plasmid extraction

250 microliters of lysis buffer was added, and cells were completely
resuspended by gentle pipetting or vortex. The tube was incubated for 10 minutes at a
room temperature to allow the lysis buffer for disruption of the bacterial cell membrane
to release the plasmid.

➢ Neutralization

350 microliters of neutralization buffer was added into the tube followed by a
high-speed centrifugation for about five minutes. This step separated the cell debris and
proteins from the DNA.

➢ Binding and washing

The supernatant having the plasmid DNA was transferred to a spin column
followed by 60 seconds of centrifugation. This allowed the plasmid DNA to bind firmly
with the spin column. The column was washed with 500 microliters wash buffer for the
removal of contaminants.

➢ Elution

The spin column was placed in a new microfuge tube and 50 microliters of
elution buffer was added to the column. The plasmid DNA was eluted from the column

56
by the use of centrifuge and was collected in a microfuge tube. We can also use sterile
water in place of elution buffer.

➢ Storage

The extracted plasmid DNA was stored at -20°C.

Note: We can analyze this plasmid DNA by a spectrophotometer to evaluate its purity
or concentration.

10 Syringe filtration

A typical method for removing particles and microbes from liquid samples is
syringe filtration. It includes utilizing a disposable filter device called a syringe filter,
that attaches to the end of a syringe.

10.1 Materials & Instruments:

• Sample
• Syringe
• Syringe filter
• Falcon tubes

10.2 Procedure:

The general procedure for syringe filtration is as follows (Scheer, 2009):

We used a syringe filter with a pore size that matched the targeted
microorganisms or particles to be eliminated. The usual range of porous sizes is
between 0.2 and 0.45 micrometers. Before being taken out of the box, the syringe filter's
filter membrane was checked to make sure it was sterile and undamaged. The syringe
filter was attached to the end of a sterile, clean syringe.

Fig. 10.2 Syringe filter

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 The syringe was filled with the liquid sample to be filtered, being careful not to
fill it past the syringe filter's maximum capacity. To get rid of air bubbles, the syringe
with the connected filter was held vertically. We carefully tapped the side of the syringe
to remove any air bubbles that had formed in the sample. The plunger of the syringe
was progressively depressed to drive the liquid sample through the filter. Consistent
and gentle pressure was used, and care was made to prevent rushing the material
through the filter. Under the syringe filter, a sterile vial or container was positioned to
catch the filtrate or filtered liquid.

The sample was filtered again in batches if the sample volume was greater than
the filter's capacity. A fresh, sterile syringe filter was used for every filtering cycle. The
spent syringe filter was carefully removed from the syringe once filtering was finished
and disposed of in accordance with laboratory waste management guidelines.

All the materials used were sterile to maintain the integrity of the sample. It is important
to keep in mind that syringe filtration is frequently employed for tiny liquid sample
amounts. For larger quantities or continuous filtering, other filtration arrangements,
such as vacuum filtration or filtration units are preferable

All the materials used were sterile to maintain the integrity of the sample. It is
important to keep in mind that syringe filtration is frequently employed for tiny liquid
sample amounts. For larger quantities or continuous filtering, other filtration
arrangements, such as vacuum filtration or filtration units are preferable.

Discussion:

CEMB provided me with an invaluable platform to immerse myself in the world

of molecular biology and biotechnology during my internship at the CEMB (Centre of
Excellence in Molecular Biology). I had the honor of working at Dr. Nadeem Ahmad's
Biopharmaceuticals and Proteomics Laboratory throughout the duration of my time at
CEMB. This multidisciplinary lab is devoted to researching proteomics and creating
domestically produced recombinant human proteins. I actively used a variety of
experimental instruments and methodologies to investigate various molecular processes
throughout my internship.

58
 Modern molecular biology procedures, such as PCR, SDS PAGE, gel
electrophoresis, dot blot, plasmid isolation, DNA extraction, and bacterial
transformation, were carried out at the BPL using cutting-edge technology. My
knowledge of biological systems operating at the molecular level has been completely
reshaped by the exposure to these techniques and tools.

Each approach has distinct benefits and uses, and they are combined in a variety
of research investigations. In addition to imparting me with knowledge of molecular
biology, the institute also taught me valuable lessons such as following lab procedures
and the significance of teamwork. I acquired understanding on how to act professionally
in a lab environment and work effectively alongside others.

Overall, I had a rewarding experience during my internship at CEMB in the

Biopharmaceutical and Proteomics Lab. It helped me better grasp molecular biology
and gave me excellent exposure to cutting-edge research methods.

Comments:

CEMB is a renowned organization devoted to expanding the boundaries of

molecular biology research and instruction. With a deep dedication to elucidating the
molecular mysteries of life, CEMB acts as a vibrant center that brings together students,
researchers, and scientists in the pursuit of scientific knowledge. Our understanding of
life's fundamental processes is constantly being expanded by the institution's
unwavering commitment to ground-breaking research, education, and beneficial
collaborations, which has important ramifications for biotechnology, agriculture, and
human health.

A scientific powerhouse inside CEMB, the Biopharmaceuticals and Proteomics

Research Laboratory is unmatched in Pakistan for its state-of-the-art infrastructure with
highly skilled experts. I, as an intern, benefitted from this unmatched learning
environment created by their combined knowledge and constant support. The lab is a
prime example of CEMB's overarching objective to support individuals who are eager
to learn by giving them unrivalled chances for professional advancement.

59
Suggestions:

I became aware of placing a high priority on workplace safety during my

internship at CEMB. I suggest performing regular safety audits, offering thorough
safety instruction, and guaranteeing the availability and appropriate use of personal
protective equipment (PPE) in order to improve safety measures. By putting these steps
in place, CEMB can establish a safe working environment that encourages the
wellbeing of interns and staff.

For optimum focus and productivity, a healthy work-life balance is essential.

To establish a balance between the demands of the job and the wellbeing of the interns,
I recommend reevaluating the working hours. Increased attentiveness and general
efficiency can be achieved by adopting flexible scheduling choices or reducing working
hours.

Furthermore, a planned follow-up strategy to give interns ongoing help and

direction would add value to the internship program offered at CEMB. This can entail
frequent check-ins with interns for feedback, addressing problems, and providing
direction for career advancement. Learning and development can be promoted by
creating a supportive environment that permits some flexibility in the intern's workload.

By implementing these suggestions, CEMB can optimize the internship

experience as they will enable interns to have a fulfilling and productive experience,
ensuring their success and progress within and outside of the institution.

Also, I would like to request to the respected higher authorities of the Institute
of Industrial Biotechnology (IIB) at GCU to give research facilities to individuals or
groups of students. As experimentation and new discoveries are at the heart of
biotechnology, students should be able to learn and develop with no restrictions
preventing them from reaching new heights. In addition, instead of the present two-
day-per-week arrangement, I suggest designating the entire summer break for
internships. This would make it easier for students to balance their academics and
practical work.

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 Lastly, I urge IIB to create more chances for the students to explore learning
opportunities at CEMB as this esteemed organization offers a transforming experience
that will eventually help future batches redefine their scientific knowledge.

61
References:
Akram, F. 2018. Polymerase Chain Reaction. In: Akram, F. (Ed.), Introduction
to Biochemistry & Biotechnology Techniques. Paramount Books Pvt. Ltd., Pakistan.
Aslam, B., M. Basit, M. A. Nisar, M. Khurshid and M. H. Rasool. 2017.
Proteomics: Technologies and Their Applications. J. Chrom. Sci., 55(2): 182-196.
Asif, A., H. Mohsin, R. Tanvir and Y. Rehman. 2017. Revisiting the
mechanisms involved in calcium chloride induced bacterial transformation. Front.
Microbiol. 8(1): 1-5.
Atkins, T. 1991. Protein Electrophoresis in the Biology Classroom Using
“Safe” Gels. American Biology Teacher., 53(8): 490-495.
Biswas, B. B., P. S. Basu and M. K. Pal. 1970. Gram Staining and its Molecular
Mechanism. Elsevier., 29: 1-27.
Chen, Y, C. and M. Yeh. 2018. Introductory Chapter: Biopharmaceuticals. In:
Chen, Y, C. and Ming Y. (Ed.), Biopharmaceuticals, InTech, Thailand, pp. 10-15.
Das, M., H. Raythata and S. Chatterjee. 2017. Bacterial transformation: What?
Why? How? and when? Anal. Res. Rev. Biol., 16(6): 1-11.
Elbing, K.L. and R. Brent. 2019. Recipes and Tools for Culture of Escherichia
coli. Curr Protoc Mol Biol., 125(1): 1-6.
Eyler, E. 2013. Pouring Agar Plates and Streaking or Spreading to Isolate
Individual Colonies. Elsevier., 55: 3-14.
Green, M.R. and J. Sambrook. 2018. The basic polymerase chain reaction
(PCR). Cold Spring Harb Protoc.,18(5): 1-4.
Hasegawa, H., E. Suzuki and S. Maeda. 2018. Horizontal plasmid transfer by
transformation in Escherichia coli: Environmental factors and possible mechanisms.
Front. Microbiol., 9(8): 2-6.
Kuslich, C.D., B. Chui and C.T. Yamashiro. 2019. Overview of PCR. Curr.
Protoc. Ess. Lab. Tech., 9(23): 1-6.
Lee, P.Y., J. Costumbrado, C.Y. Hsu and Y.H. Kim. 2012. Agarose gel
electrophoresis for the separation of DNA fragments. J. Vis. Exp., (62): 1-8.
Moyes, R.B., J. Reynolds and D.P. Breakwell. 2009. Differential staining of
bacteria: Gram stain. Curr. Protoc. Microbiol., 10(3): 1-5.
Manns, J. M. 2011, SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of
Proteins. Cur. Prot. Microb., 22: 1-13.

62
 Ogodo, A.C., D.I. Agwaranze, M. Daji and R.E. Aso. 2022. Microbial
techniques and methods: In: Ogodo, A.C., D.I. Agwaranze, M. Daji and R.E. Aso.
(Ed.), Basic techniques and microscopy. Analytical Techniques in Biosciences: from
Basics to Applications.
Pronobis, M.I., N. Deuitch and M. Peifer. 2016. The Miraprep: A protocol that
uses a Miniprep kit and provides Maxiprep yields. PLoS One 11.
Roy, S. and V. Kumar. 2014. A Practical Approach on SDS PAGE for
Separation of Protein. Intl. J. Sci. Res., 3(8) 2-6.
Rudge, S. R. and C. A. Monnig. 2000. ELECTROPHORESIS TECHNIQUES,
Sep. Purif. Meth., 29(1): 129-148.
Sanders, E.R. 2012. Aseptic laboratory techniques: Plating methods. J. Vis.
Exp., 63(17): 1-3.
Sandle, T. 2014. Assessment of Culture Media in Pharmaceutical Microbiology.
Am. Pharm. Rev., 1(1): 2.
Serghini, M. A., C. Ritzenthaler and L. Pinck. 1989. A rapid and efficient
‘miniprep’ for isolation of plasmid DNA. Nucleic. Acid. Res., 17(9): 3604.
Sonagra, A. D. and S. J. Dholariya. Electrophoresis. In: Sonagra, A. D and S. J.
Dholariya. (Ed.), National Library of Medicine. StatPearls, India.
Sun, Y., K. Sriramajayam, D. Luo and D.J. Liao. 2012. A Quick, cost-free
method of purification of DNA fragments from agarose gel. J. Cancer., 3: 93-95
Treco, D.A. and V. Lundblad. 1993. Preparation of Yeast Media. Curr.
Protoc. Mol. Biol., 23(6): 2-6.
Williams, J. W. 1936, Use of the Stab Culture in the Growth of Certain
Pathogenic Fungi. Am. J. Clin. Path., 55(6): 444-447.
Tsen, S. Der, S. Sen Fang, M.J. Chen, J.Y. Chien, C.C. Lee and D.H.L. Tsen.
2002. Natural plasmid transformation in Escherichia coli. J Biomed Sci., 9(3): 246-
252.
Wittmeier, P. and S. Hummel. 2022. Agarose gel electrophoresis to assess PCR
product yield: comparison with spectrophotometry, fluorometry and qPCR.
BioTechniques., 72(4): 1-2.
Wyckoff, M., D. Rodbard and A. Chrambach. 1977. Polyacrylamide gel
electrophoresis in sodium dodecyl sulfate-containing buffers using multiphasic buffer
systems: Properties of the stack, valid Rf- measurement, and optimized procedure.
Anal. Biochem., 78(2): 459-482.
Zhu, H., H. Zhang, Y. Xu, S. Laššáková, M. Korabečná and P. Neužil. 2020.
PCR past, present and future. Biotechniques., 69(4): 317-325.
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