INTERNSHIP REPORT ON LABORATORY

TECHNIQUES IN BIOTECHNOLOGY, HBD

NIBGE
BY
IQRA KHALID

DEPARTMENT OF PHYSIOLOGY
GOVERNMENT COLLEGE UNIVERSITY, FAISALABAD
(SESSION 2014-2018)

1
 LABORATORY TECHNIQUES IN BIOTECHNOLOGY

BY
IQRA KHALID
Registration no: 2014-GCUF-03824

Internship report submitted to Govt.College University, Faisalabad, in partial

fulfilment of the requirement for the degree of
BACHELOR OF STUDIES (4 YEARS PROGRAME)
IN
PHYSIOLOGY

DEPARTMENT OF PHYSIOLOGY
GOVT.COLLEGE UNIVERSITY, FAISALABAD
(SESSION 2014-2018)

2
 INTERNSHIP COMPLETION CERTIFICATE

It is certified that internship work in this report has been carried out and observed by
Iqra Khalid D/O Khalid Hussain, in duration from 14-March-2018 to 25-May-2018, Roll
#15364 under my supervision, as per requirement of her BS (Hons) Physiology as a student of
Government College University, Faisalabad.

Signature: _________________ Signature: _________________

Dr. Shahid M. baig Dr.M.Tariq Khan

Head of Health Biotechnology Division Senior Scientist

Deputy Chief Scientist NIBGE, Faisalabad
NIBGE, Faisalabad

Signature: _________________ Signature: _________________

Dr. Haseeb Anwaar Dr.Ghulam Hussain
Head of Department of Physiology Assistant Professor
Govt.College University, Faisalabad Department of Physiology
Govt.College University, Faisalabad

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Dedication

I dedicate this humble effort to my dear Almighty Allah,

my honourable Holy Prophet ‫ﷺ‬, my beloved, loving and
caring parents, without them I am nothing and my
respected teachers.

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And do good; indeed, Allah loves the doers of good
Al-Quran (2:195)

5
 Table of content

1: NIBGE Introduction
2: Human Molecular Genetics lab
 DNA Extraction from Blood…………………...……………… 12
 Nano drop ……………………………………..………………..14
 Routein PCR……………………………………..………………15
 DNA Extraction from CVS…………………………………...…17
 ARMS PCR…………………………………………………..…17
 Horizontal Gel electrophoresis……………………………….…19
3: Molecular and Cytogenetics lab
 Karyotyping……………………………………………………….23
4: Diabetic and Metabolic Disorder lab
 Biochemical analysis of blood samples……………………………28
5: Human Enteric Pathogen lab
 Preparation of TSB for culturing…………………………………..35
 Preparation of TSI slants………….………………………………..37
 Bacterial DNA extraction…………..………………………………39
 Gene specific PCR…………………………….……………………40
 Antibiotic sensitivity test……………………………………………42
 Bacterial stock preparation……………………………….…………44
6: Structural Biology lab
 Gene cloning and expression in E.coli...............................................46
 Competent cell preparation…………………………………….……47
 Transformation ……………………………………………………...48
 Miniprep of plasmid DNA…………………………………………...49
 Cell lysis with beads…………………………………………………55
 SDS-Polyacrylamide gel electrophoresis…………………………….56
 Ni-NTA chromatography……………………………………………60
 Minimum Inhibitory Assay…………………………………………..61
7: National Probiotics lab
 Bile assay………………………………………………………...…63
 Preparation of MRS broth………………………………………..…63

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 Riboprinter…………………………………………………………65
 Anaerobic chamber…………………………………………………65
 Colour Q-count………………………………………………….….66
 Spectramax plate reader………………………………………..…..67
 Bio flux…………………………………………………………..…67
 Safety cabinet…………………………………………………..…..68
 Autoclave…………………………………………………….…….69
 Gram staining………………………………………………………69

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 Acknowledgements

All glories and praises to my dear Almighty Allah, for His strengths and blessings, the One
who has no beginning and no end and millions of DAROOD-O-SALAM upon HAZRAT

MUHAMMAD ‫ ﷺ‬who is perfect model of guidance and knowledge for humanity.

I would like to pay my deepest gratitude to Dr.Shahid Mansoor Director, NIBGE for giving
me the opportunity to do work in labs. I extend my sincere thanks to my internship supervisor
Dr.Shahid Mehmood Baig (SI HOD), for his scientific guidance and providing a good
learning environment. My appreciation also extend to Dr. Muhammad Tariq for his
overwhelming attitude to help in every possible way. I am deeply grateful to Dr.Fazli Raabi,
Dr.Arsalan Zaidi, Dr.MoaazurRahman, Dr.Aamir Ali, Dr.Abdul Ghaffar for their
encouragement, scientific feedback, continuous advice and best wishes for bright future.

A massive thanks to Dr. Haseeb Anwaar, HOD Department of Physiology, GC University,

who motivate me and guided me in every step. His moral values and extraordinary scientific
knowledge will remain a source of inspiration for me. My warmest and most sincere gratitude
to Dr.Ghulam Hussain, Assistant professor ,Department of Physiology GCUF, for providing
me his guidance, consistent encouragement and constructive criticism.

I am especially grateful to all M.phill and Phd scholars Miss. Ghazal ,Tooba, Iqra, Sherish,
Anam, Zahra, Khanwal, Samra, Aimen, Noor, Quratulain and Mr. Majid, Ayaz, Zubair,
Imran, Raheem ullah, Omer and Farooq for their guidance and support.

I am extremely fortunate and blessed in my life to have the most supportive and loving family.
None of these would have been possible without the unconditional love, help and support of
my dear, honourable, loving, caring and respected parents.

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NATIONAL INSTITUTE OF BIOTECHNOLOGY AND GENETIC ENGINEERING

Introduction
NIBGE is the biotechnology institute under PAEC (Pakistan Atomic Energy Commission) and
was formally inaugurated by the President of Pakistan in 1994. Also affiliate center of
International Centre for Genetic Engineering and Biotechnology. Through applications of
modern biotechnology and genetic engineering NIBGE provides technology units to help the
country in better way. The research programs which are under process are mainly aimed at
improving agriculture, health, environment and industry. These projects are funded by national
and international financial grants. The institute research facilities include IT facility and a
Library for Biological Sciences. NIBGE also offers several services and marketable products.

Research Departments
There are five research divisions at NIBGE
 Agricultural Biotechnology Division
 Health Biotechnology Division
 Industrial Biotechnology Division
 Environmental Biotechnology Division

Research is being carried out in these divisions pertaining to the problems prevalent in Pakistan.
Also new research and studies on the upcoming techniques and technologies is being carried
out. The researchers strive towards creating a better future for Pakistan.

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 Health Biotechnology Division
I worked in Health Biotechnology Division under the supervision of Dr. Shahid Baig (Chief
Scientist). Around the globe human health is the most concern topic of scientific research and
every human wants better quality life.

The molecular basis of infectious, genetic and metabolic disorders are investigate by using
modern techniques of biotechnology to establish nucleic acid based diagnostics and to design
improved cost effective therapeutic strategies for quality life in HBD division. The major
ongoing research projects are development of modern vaccines, discovery of drugs, structure
and function studies of membrane and viral proteins.

Overall aim of Health Biotechnology Division is to do research and development for diagnosis
and treatment of genetic, metabolic and infectious diseases and to discover, identify and
develop different drug targets and probiotics.

Disease gene mapping for various phenotypes to diagnose prenatal affected births, molecular
basis of drug resistance and virulence of bacterial pathogens causing tuberculosis &typhoid
and production of conjugate vaccines for typhoid and recombinant vaccines for hepatitis B and
C is underway.

There are following laboratories in HBD

 Human molecular genetics lab
 Cytogenetic lab
 Diabetes and metabolic disorder lab
 Enteric pathogen lab
 Structural and biological lab
 Probiotics

Dr.Shahid Baig
Deputy Chief Scientist

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 Human Molecular Genetics Lab

I worked in HMG under the supervision of Dr. Muhammad Tariq Khan (Senior
Scientist).
The reason of genetic disorders in Pakistani population is tradition of cousin’s
marriages and relative high frequency of disease alleles. Awareness seminars are
conducted by scientists of this department on genetic counselling and
characterization of the mutations causing β-thalassemia, deafness, microcephaly,
obesity and other disorders in Faisalabad and D.G. Khan to establish prenatal
diagnosis. Samples of patients are analysed, mutations and variants are detect by
using the ARMS-PCR. Chorionic villus sampling (CVS) is drawn for prenatal
diagnosis. In Pakistan 150 million people are carrier of β-thalassemia. The state
of β-thalassemia is alarming as consanguinity is very high and the literacy rate is
low in South Punjab.
In the families with affected children and in the general population a positive
attitude and cooperative trend towards prevention of as β-thalassemia is currently
being developed.

Dr. Muhammad Tariq

Senior Scientist

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 Human Genomic DNA extraction from Blood

 Phenol-chloroform or organic method

Principle

Several chemicals were added for organic DNA extraction. Sodium dodecylsulfate (SDS) and
proteinase K breaks the histone proteins that bind the DNA molecule in compact form.
Phenol/chloroform mixture separates the proteins from the DNA. The organic–aqueous
mixture dissolves the DNA molecule efficiently. After centrifugation, the unwanted proteins
and cellular debris are separated away from the aqueous phase and DNA molecules are
precipitated in eppendrof.

Protocol

 I took 0.75 ml blood from vacutainer and mixed with 0.75mL of solution A in a 1.5mL
micro centrifuge tube and wait for 10 min.
 Then I centrifuged the tube for 1 min at the speed of 13,000 rpm and discard the
supernatant.
 Resuspended the pallet in 400 µL of solution A and then centrifuged for 1 min at same
speed.
 Discarded the supernatant and the nuclear pellet was resuspend in 400 µL of solution
B, 25 µL of SDS and 8 µL of Proteinase K.
 Tube was left at 65 °C for 3 hours.
 Freshly prepared 0.5mL of solution C and D were added and mixed gently.
 Vortex the tube for 50 seconds then centrifugation at 13, 000 rpm for 15 mins.
 In new 1.5mL tube upper aqueous phase was collected and same volume of solution D
was added, gave 10 minutes centrifugation at 13,000rpm speed and collected the
aqueous layer in separate tube.
 55 µL of Na-acetate and 500µL of isopropanol (chilled) were added and tube remains
inverted to precipitate DNA.

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  Centrifuged at 13,000 rounds per minute for 15 mins.
 Removed the solvent phase carefully.
 Centrifuged again after adding 350µL of 70% ethanol at 13,000 rpm for 15 mins.
 Removed the ethanol and dried the DNA pallet and kept at 65 °C for 10 mins.
 Lastly dissolved the DNA in DNA dissolving solution (200-250 µL) or PCR water.
 Quality was checked on agarose gel electrophoresis.
 Store at -20 °C
Solutions Composition

Solution A Sucrose , Tris (pH 7.5), MgCl2, Triton X-

(Cell lysis buffer) 100
Solution B Tris (pH 7.5), NaCl, EDTA (pH 8.0)
(Nuclear lysis buffer)
Solution C Phenol , Tris

Solution D Chloroform: Isoamyl alcohol

(24:1)
Proteinase K Stock solution: 20 mg/mL in dH2O,
SDS (pH 7.2)

Na-acetate 3 M (24.6102 g in 60 ml of dist.)

Table 1: Solutions compositions used for DNA extraction

Extracted DNA

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 Nano Drop for DNA Quantification

Principle

Spectrophotometric analysis is based on the principles that nucleic acids absorb

ultraviolet light in a specific pattern. In the case of DNA and RNA, a sample is exposed
to ultraviolet light at a wavelength of 260 nanometres (nm) and a photo-detector
measures the light that passes through the sample. Some of the ultraviolet light will
pass through and some will be absorbed by the DNA / RNA. The more light absorbed
by the sample, the higher the nucleic acid concentration in the sample. The resulting
effect is that less light will strike the photo detector and this will produce a
higher optical density (OD).

Protocol

 Turn
on
the
computer
attached
to the
Nano Drop and washed the both pedestal with

washing buffer
 Purified 
water (2 µL) was added
to
the
lower
pedestal.
 Then placed upper arm on
lower. Click“Ok”on
the software and
wait
for 20
secs.
 Lifted the upper arm
and dried
the
pedestal with
a
wipe.
 Added the 2 µL sample on lower pedestal, then
lower
the
upper
arm.
 Entered the sample id and click
the
“Measure”
and
wait for result. Then upper
arm
was lifted
and
dry
the both
pedestal.
 Checked
the
absorbance
number
at
various wavelengths.
 After all, pedestal was wiped and the programme was shut down carefully.

Nano drop and software

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 Polymerase Chain Reaction
PCR used to amplifies a particular region of DNA strand for different purposes.

Principle

Polymerase Chain Reaction is an in vitro technique based on the principle of DNA

polymerization reaction. It relies on thermal cycling consisting of repeated cycles of
heating and cooling of the reaction for DNA melting and enzymatic replication of the
DNA using thermo stable DNA polymerase, primer sequence (complementary to target
region) and dNTPs. It can amplify a specific sequence of DNA by as many as one
billion times.

Protocol

 First of all, I took PCR tubes and one eppendrof, labelled them as sample id or master
mix.
 For master mix preparation following ingredients were added in eppendrof. This is for
one sample, for multiple samples multiply following quantities with number of
samples.
Ingredients Quantity
Dream Taq master mix 10 µL
(DNA Polymerase, Dream Taq Green
buffer, dNTPs, and MgCl2)

Forward primer 0.5 µL

Reverse primer 0.5 µL
DNA template 2 µL
PCR water 7 µL

Total 20 µL

Table 2: Ingredients and quantities used for routine PCR

 Total reaction volume was 20 μL performed in 0.2 mL PCR tubes.

 All PCR ingredients were added in tubes vortexed briefly for proper mixing.
 Tubes were set into thermal block holes.
 PCR was carried out for 30 cycles with certain thermal cycling guidelines.

15
  Amplification product was then visualized on gel by gel electrophoresis

PCR tube containing ingredients

Thermal Cycler Profile

1. Initial denaturation 95 °C for 5 min

2. Amplification
Denaturation 95 °C for 35 sec
Annealing 55 °C for 30 sec
Elongation 72 °C for 45 sec
3. Final extension 72 °C for 10 mins

PCR Thermal Cycler

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 DNA Extraction from CVS
Chorionic villus sampling (CVS) was done to diagnose prenatal chromosomal or genetic
disorders in the fetus. Samples was taken from the chorionic villus (placental tissue) and tested
it for chromosomal abnormalities via ARMS PCR. CVS was done at 10–12 weeks gestation.

Protocol
 After separation from normal saline CVS sample was lysed with 500 μL of CVS lysis
buffer in eppendrof.
 Incubated for 2 hrs at 60 ˚C after adding 50 μl of proteinase K and 5.0 µL of SDS until
solution become clear.
 Then 500 μL of solution C+D was added, vortexed briefly and centrifuged for 5 minutes
at 13,000 rpm.
 Separated the supernatant in new eppendrof.
 Added 250 µL of solution D and centrifuged it for 4 mins at 13,000 rpm, separate the
aqueous layer in a new eppendrof.
 Added 250 μL of chloroform and centrifuged for 4 mins at 13,000 rpm and separate the
aqueous layer.
 Then 250 μL of Na-acetate and 500 μL of isopropanol (stored at -20˚C) was added and
kept the tubes inverted to precipitate DNA again centrifuged for 10 mins.
 Removed the supernatant and washed DNA pellet with 350 μL of 70% ethanol and
dried in incubator at 37˚C.
 DNA was dissolved in 30-50 μL of double distilled PCR water after evaporation of
ethanol.
 This DNA sample was then used for ARMS PCR to detect point mutations which was
visualized by doing gel electrophoresis.

Amplification Refractory Mutation System PCR

The Amplification Refractory Mutation System (ARMS) is an application of PCR in which
allele specific primers are used to amplify DNA. Point mutations or polymorphisms are identify
by using this. It also tells the heterozygous or homozygous change in DNA and differentiated
by using ARMS primers for the mutant and the normal alleles. Both reactions are usually
carried out for mutant or normal alleles in separate tubes. If two primers with different
fluorescent dyes are labelled then reaction may be done in same tube.

17
Principle
Two primers identical in sequence except for the terminal 3’ nucleotides are required. One of
which is complimentary to the changed sequence (Mt ARMS primer) and other to the normal
DNA sequence (N ARMS primer), both used as reverse primers. For both mutant and normal
Common C was used as a complimentary Forward primer to screen a specific mutation. To test
the success of ARMS reaction amplification 2nd pair of primers added to amplify unrelated
DNA sequence which is used as internal control.

Protocol
 20 μL reaction mixture was prepared for analyses.
 Recipe for reaction mixture, PCR profile and primers used for ARMS reactions are
given here.

Reagents Quantity per reaction

Genomic DNA 2µL

Buffer A 2µL

MgCl2 0.8µL

Primers(N,Mt & com C) 0.3µL

2nd pair of primers 0.8µL

dNTPs 1µL

Taq DNA polymerase enzyme 0.3µL

PCR water 3.8µL

Table 3: ARMS PCR profile

Thermal cyclic conditions

 Denaturation (initial) 94°C for 1 min
Cycles: 25
 Denaturation 94°C for 1 min
 Annealing 65°C for 1 min
 Extension 72°C for 1 min
 Final extension 72°C for 3 min

18
 Horizontal Agarose Gel Electrophoresis

Principle

Agarose gel electrophoresis used to separate DNA or RNA and sometimes purify them.
Nucleic acid contains a specific charge(-ve) and when placed in electric field it moves towards
opposite pole which contain opposite charge(+ve) and movement depend on size or molecular
weight of molecules as small molecules migrate faster than large.

Protocol

2% agarose gel in 100ml TBE buffer

 2 gram agarose was added in 100 mL TBE buffer in a flask.
 Heated in microwave oven until solution becomes transparent.
 Allowed to cool down till the temperature reach 50 °C.
 Carefully added 4 µL of ethidium bromide and mixed gently.
 Prepared the casting tray and combs were set at appropriate position.
 Agarose solution was then poured into tray carefully to avoid bubble formation.
 Gel become solidify after 30 mins.
 Combs was removed and placed the gel in electrophoresis tank containing TBE
buffer.
 Loaded the samples carefully into wells along with DNA ladder without breaking
them.
 The gel was run at 80-150V until the dye line was approximately 75-80% of the way
down the gel.
 Turned off power supply, and removed the gel from casting tray.
 Seen under gel documentation system which has UV light, DNA bands were visualized
on computer screen by using software.

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 Agarose Gel and Gel Electrophoresis

Solutions Reagents

10 X TBE Tris base, Boric acid, EDTA

(pH 8) stored at 4˚C (Make up volume up to 1 liter with distilled
water)
Autoclaved for 20 mins

0.5 X TBE 50 ml 10 X TB in 950ml of d.H2O

Gel Loading Dye Bromophenol Blue , Sucrose

(stored at 4˚C)

Ethidium Bromide 10 mg/mL in d.H2O

Ivs I-5
Table 4: Composition of solutions used in gel electrophoresis

-ve homo M F CVS CVS

ARMS PCR product on agarose gel

20
 DNA
Ladder

DNA band under gel documentation system

Gel Documentation System

21
 Molecular and Cytogenetic lab
I worked under the supervision of Dr.Abdul Ghaffar. Blood samples was taken from
patients who arrived from different regions and check their chromosome status by
following protocol of karyotyping under standard condition.

Dr. Abdul Ghaffar

Principal Scientific Assistant

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 Karyotyping

Karyotyping is a lab procedure for chromosomal examination. It is the study of inheritance in

relation to chromosome. The chromosome number increase or decrease, insertions or deletions
can result in the loss of large amount of data carried by the DNA. This can give rise to various
anomalies in the body like, the Turner’s syndrome, Klienfelter syndrome, Pseudo organs and
Down syndrome. These are a few of the diseases diagnosed in the cytogenetics lab along with
determine the sex of a person through visualizing and studying the chromosomes.
During cell division, cells divides and equal number of chromosomes in case of mitosis and
half number in case of meiosis pass into new cells. Normally, when a cell is at rest the
chromosomes are unorganized form in cell but during division chromosomes starts line up and
moves towards each pole. In a karyotype test, which examines dividing cells, these pairs are
arranged by their size and appearance, allowing to easily determine if any chromosomes are
missing or damaged karyotype identify them by their number, size and appearance.

Principle
Blood cells were incubated for 71 hours after adding in nutrient media at 37 °C and stopped
cell division at metaphase after added colcemid. Slides were made after treated with different
solutions and seen under microscope.

Protocol

 Sample collection
 3 mL blood was taken from patient in lithium heparin vacutainer and process
within 24 hours.
 Setting lymphocyte culture
 Tube was allowed to stand for half an hour for sedimentation of blood cells.
 10 ml of RPM1640 media was poured in 25 cm cell culture flask then 1ml of
whitish portion and few RBCs was added in this flask.
 Incubation
 Gently mixed the media and blood and 100 uL of PHA-P solution was added.
 Placed the flask in preheated incubator at 37 °C.
 Incubate for 71 hours and during incubation mix the solution gently twice a
day.

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 Stopping the cell division at metaphase
 After 71 hours incubation pre-warmed 22 uL of Colcemid (37 °C) was added.
 Again mixed the culture gently and incubate for further 30 to 40 minutes.
 Harvesting lymphocyte culture
 Blood culture was removed from incubator and mixed gently.
 All the contents were transferred into 15ml centrifuge tube and centrifuged at
3000 – 4000rpm foe 10 mins.
 Supernatant was removed with Pasteur pipette carefully without disturbing the
cells on bottom.
 Pre-warmed 10 mL of 0.56 M KCl was added and mixed gently.
 Placed the tube into 37 °C water bath for 20 mins.
 Added 2-3 drops of fixative (methanol and glacial acetic acid with 3:1) and
mixed, after this add more fixative up to 8 mL and centrifuge for 10 mins at
3000-4000 rpm.
 Supernatant was removed left the cells at bottom.
 Added 10 mL of fixative solution again and centrifuged at 3000-4000 rpm for
10 mins.
 Supernatant was removed carefully.
 These two steps was repeated until all the RBCs are removed from solution,
leaving only white blood cells.
 Preserved the cells in 2-3ml fixative and placed at 4 °C.
 Chromosome slide preparation
 Glass slides were labelled and cleaned with methanol and HCl (4:1).
 Add 30 uL of fixed cells on respective labelled slide.
 Fixed by placing slide on hot plate for few seconds.
 Placed the slides for 1 hour at 90 °C in incubator.
 Geimsa banding
 Slides were incubated in phosphate buffer which is pre-warmed at 56 °C for
13 mins.
 Wash slides with cold distilled water and air dry.
 2-3 mL giemsa staining was poured on slides and incubate at room
temperature for 5-10 mins.
 Wash slides with distilled water, air dry and see under microscope.

24
 Slides flooded with geimsa stain

 Microscopic analysis
The metaphase was selected under microscope and mark that points on slides.
Then these slides were observed under Chromoscan CytoVision 4.02 workstation.
About 30 metaphases were analysed under this and all the chromosomes arranged
automatically or manually at their respective positions.

Chromoscan CytoVision 4.02 workstation software

25
Report

Karyotyping report of Down’s syndrome patient

26
 Diabetes and Metabolic Disorder Lab
I worked under the supervision of Dr. Fazli Rabbi Awan (Principal Scientist)
Abnormality in insulin secretion or action cause diabetes which is a metabolic
disorder. Increased level of blood glucose level is harmful for vital organs of body
like eyes, brain, kidneys etc. or in chronic condition this may lead to death.
Basically, there are different types of diabetes and in this department scientists
interested in Type 2 Diabetes, which is the mostly result of obesity in Pakistan.

They used human and mouse as models which suffers from type 2 diabetes. Main
objective is to identify biomarkers for novel genomic, proteomics, adipokine,
metabolomics, transcriptomics, microRNA based disease to diagnose early,
monitoring response to therapies in the Pakistani diabetics and its
management. They are also interested to discover novel drug targets for diabetes
and develop molecular medicines for insulin resistance and type 2 diabetes in
future.

They also work on Wilson disease which is a rare autosomal recessive inherited
disorder due to abnormal copper metabolism that cause excessive deposition of
copper in the brain, liver, and other tissues.

Dr.Fazli Rabbi Awaan

Principle scientist

27
 Biochemical analysis of blood samples
Serum was separated from blood for biochemical analyses from kits and results were
analysed by using Micro lab 300 analyzer.

Bio chemistry Analyser

Creatinine Test

Creatinine is a waste product of protein breakdown that forms creatine this test tells how
well kidneys are working because creatinine is secreted through kidneys.

Protocol

 Turn on the machine and start flushing for 2 mins from distilled water
 Select creatinine from menu and sip distilled water
 I took 250 µL reagent no.1 and 250 µL reagent no.2 in a test tube and incubate for 5
min at 370C in water bath
 Then took 50µL standard reading
 Added 50µL sample in tube and sip by microlab 300
 Wait approx. 10 sec for result

28
 Reagents Chemicals
Standard Creatinine
Reagent no. 1 Picric acid
Reagent no.2 Sodium hydroxide,Disodium
phosphate

Cholesterol Test

Cholesterol plays very important role in cell membrane maintenance and regulation. Increased
level cause severe health issues.

Protocol
 I mixed 650 uL of Reagent with 6.5 µL of standard
 Added 650 uL Reagent and mixed with 6.5µL of sample
 Tubes were placed in water bath for 5-10 minutes at 370C
 Standard and then sample was sipped by machine
 Wait for results

Reagents Chemicals
Standard Cholesterol
Reagent Pipes buffer
Phenol
4-aminoantipyrine
Cholesterol esterase
Cholesterol oxidase
Peroxidase

Triglyceride Test

Triglyceride is a type of fat present in body. Coronary artery disease is the major disease due
to its high level. This test tells the triglyceride level in blood.

Protocol
 I took 650 uL Reagent and 6.5 µL of standard in tube
 Then added 650 µl Reagent and 6.5 µL sample in tube
 Tubes were incubated for 5-10 minutes at 370C

29
  After that reading was taken by microlab 300

Reagents Chemicals
Standard Triglycerides

Reagent Pipes buffer

р-chlorophenol
ATP , Mg2+
Glycerol kinase
Peroxidase
Lipoprotein lipase
4-aminoantipyrine
Glycerol-3-phosphate-oxidase
Potassium ferrocyanide

ALAT Test

An alanine aminotransferase (ALT) is an enzyme synthesized by cells in liver and normally

present inside liver cells. Any trauma to liver can released ALT into bloods and causes serum
ALT levels to rise.

Protocol
 I took 400µL Reagent no.1 and added 100µL reagent no.2
 Tubes were incubated for 5 min at 370C in water bath
 Then added 50µL sample and sipped by microlab 300
 Took values on the microlab 300

Reagents Chemicals
Reagent no.1 Tris buffer
L-Alanine

Reagent no.2 Lactate dehydrogenase

Α-ketoglutarate
NADH

30
 Uric acid Test

Uric acid blood test tells about uric acid present in blood. It helps to determine the
function of kidneys.

Protocol
 I took 250µL Reagent no.1 and added 250µL Reagent no.2 in the tubes then took 10µL
standard in a tube
 Then I took 250µL Reagent no.1, 250µl Reagent no.2 and 10µL sample in test tubes
 Incubate tubes for 5-10 minutes at 370C
 Readings were taken from microlab 300

Reagents Chemicals
Standard Uric acid
Reagent no.1 Phosphate
2-4 dichlorophenolsulfonate (DCPS)
Reagent no. 2 Uricase
Peroxidase
Ascorbate oxidase
4-aminophenazone

Urea Test

Urea is the waste product of protein breakdown and converted by liver in non-toxic form and
entered in blood and ultimately eliminated through kidneys. Level of urea in blood tells the
kidney function.
Protocol
 Mixed 800µL Reagent No.1 and 200µL Reagent No.2 in tubes
 Left the tubes for 5 minute
 Then added 10µL standard in one tube
 Added 10µL sample in another tube
 Sipped by microlab 300 and result was noted

31
 Reagent Chemical
Standard Urea
Reagent no.1 2-oxoglutarate
ADP, Urease , GLDH,
NADH

Reagent no.2 Tris

Protein Test

Albumin and globulin are identify through total protein test in blood. This test is very helpful
in diagnoses of liver and kidney diseases.

Protocol
 500µL Reagent and 10µL standard was mixed in the test tubes
 500µl Reagent and 10µl sample was mixed in another tube
 Incubated tubes for 5-10 minutes at 370C
 Then took the values on the micro lab 300

Reagent Chemical
Standard Protein
Reagent Potassium iodide
Potassium sodium tartrate
Copper sulphate
Sodium hydroxide

Albumin Test

Albumin is made by the liver and about 60% of the total protein in the blood and plays
important role. Albumin prevent fluid leakage from blood vessels; nourishes tissues; and
transports hormones, vitamins, drugs, and substances like calcium throughout the body. This
test tells about liver function.

32
Protocol

 I took 500µL Reagent and 10µL standard in the tube.

 Then took 500µL Reagent and added 10µL sample in another tube.
 Incubated tubes for 5-10 minutes at 370C
 Took the values on the microlab 300

Reagent Chemical
Standard Bovine albumin
Reagent Succinate buffer
Bromocresol green

Test kits

33
 Human Enteric Pathogen Lab

In this lab I worked under the supervision of Dr. Aamir Ali.

Diabetic foot samples were taken from patients and bacterial strains
were isolated then checked the drug resistance of staph. aureus.

Typhoid is the major disease in Pakistan due to lack of suitable

vaccine because polysaccharide antigens are poorly immunogenic.
They are worked to conjugate vaccine with a carrier protein.
They conjugate vaccine against local isolates of Salmonella Typhi
which they have shown to be naturally existing.
Bacterial outer membrane proteins (OMP) are used for conjugation.
PCR-based diagnosis of typhoid was also done by scientists which
was followed by development of multiplex PCR for drug resistance
and discrimination of typhoidal pathogens. Typhoidal pathogens
and Shigella species virulence and drug resistant studies also
ongoing.

Dr. Aamir Ali

Senior Scientist

34
Preparation of Tryptic Soy Broth

TSB is a culture broth which is used for growth of aerobic pathogenic bacteria.
Protocol
 I weighed 6 grams of TSB and added in 200 mL of d.H2O to make a volume up
to 200 mL.
 Solution was heated in microwave oven until all the powder dissolved and
solution become clear.
 Added 3 mL in 15 test tubes and put cotton stopper on tubes.
 Tubes were autoclaved for 15 mins at 121°C

Culturing
 This procedure was performed in bio safety cabinet
 TSB media was used for culturing of bacteria

Protocol
 I pipetted 40 µL of bacterial glycerol stock sample and added in 3 mL TSB
tube and mix gently.
 Placed the test tubes in incubator at 37 C for 24 hours.

Left:TSB broth with bacterial growth , Right: TSB broth without growth

35
Preparation of MacConkey agar for sub –culturing

Protocol
 I took 7.5gram of MacConkey agar and dissolved in 150 mL of distilled
water and heated in microwave oven.
 This media was then autoclaved.
 Then agar was poured into petri plates and allowed to solidify

Streaking on MacConkey agar

Protocol
 When agar become solidify streaking was done by taking cultured TSB
with the help of loop.
 End to end streaking was done to minimize the number of colonies at
specific point
 Then plates were incubated at 37°C for 24 hours.

Agar plate with bacterial colonies

36
Pure culture
Protocol
 After seeing the growth on agar plate after 24 hrs incubation, I selected
the different colonies which was different in size, shape or colour and
marked on plate.
 I picked one of colony from plate and streaked in new agar plate to get
pure culture.
 Plates were incubated at 37 C.

Triple Sugar Iron slant for biochemical testing

The Triple Sugar Iron (TSI) test was used to test the ability of bacteria to ferment
sugars and to produce H2S. Enteric bacteria including Salmonella and Shigella are
basically identified from this. The TSI slant is a test tube which contains agar, a pH-
sensitive dye (phenol red), lactose, sucrose, glucose, and sodium
thiosulfate & ferrous sulfate or ferrous ammonium sulfate.

 After getting pure culture biochemical testing was done to confirm bacterial
species. TSI slants was used for this purpose.

Protocol
 Dissolved 1.95 g TSI in 30 mL of distilled water.
 Heated the solution in microwave oven to dissolve completely.
 Added 2 mL in test tubes and autoclaved.
 After this, I made slants by placing the tubes in tilted position until slants
becomes solidify.

Stabbing on Triple Sugar Iron Slants

Protocol
 Transparent colony was picked from pure culture with sterilized loop and
inoculate TSI agar by stabbing through the middle of slant to the bottom of
the tube and then streaked on the surface of the agar slant.
 These slants were placed in incubator at 37°C for 24 hours.

➢ After overnight incubation I examined the streaked colony on the slants and
interpret the result by seeing morphology of butt and slant and organisms were
identify.

37
Interpretation

Name of Slant/Butt (colour) Gas production Hydrogen sulphide

organism
Escherichia, Yellow/Yellow Pos (+) Neg (-)
Klebsiella,
Enterobacter
Shigella, Serratia Red/Yellow Neg (-) Neg (-)
Salmonella, Red/Yellow
Proteus Pos (+) Pos (+)
Pseudomonas Red/Red Neg (-) Neg (-)
Staph aureus Yellow/Yellow Neg (-) Neg (-)

Table 5: TSI slants results and interpretation

TSI Slants tubes after stabbing

38
Culturing for DNA extraction
 After confirmation of bacterial species from slants DNA was extracted to
confirm at genetic level.

Protocol
 TSB tubes were prepared and inoculated with growth from TSI slant with
the help of loop.
 Then these tubes were incubated at 37°C and next day growth was used for
DNA extraction.

Bacterial DNA Extraction

Protocol
 I inoculated 3 mL of TSB (Tryptic soy broth) with typical growth on triple sugar
iron slant and incubated for 24 hours at 370C.
 Transferred culture in 2 eppendorf tube and centrifuged at 10,000 rpm speed for
10 mins.
 Removed supernatant and suspended the pellet in 1 ml of Tris buffer. The
suspension was centrifuged at 10,000 rpm for 10 mins.
 Removed the supernatant again and pellet was thoroughly suspended in 720 µL
of sodium dodecyl sulphate.
 I kept the suspension at 650C for two hrs in heat blocks and then brought to
room temperature.
 chloroform: isoamyl alcohol (24:1) of same volume was added and mixed by
rapid inversion several times until mixture become milky and centrifugation
was done for 10 mins and transferred aqueous layer to another eppendorf tube
without disturbing central protein layer.
 Repeat the above step three times and transferred the aqueous phase from the
last step to another eppendorf tube and added 100 µl of potassium acetate and
700 µl of chilled (40C) isopropanol. After gentle mixing by inversion it was
kept at -200C overnight.
 Next day,I centrifuged the tubes at 10,000 rpm speed for 5 mins and removed
the supernatant.
 Suspended the pellet in 200 µl of 70% ethanol and repeated the above step.
 Removed the supernatant; air dried the pellet for 30 mins and then dissolved in
20-30 µL of Tris buffer.
 DNA was stored and checked the quality on 1% agarose gel electrophoresis and
visualized under gel documentation system.

39
 Polymerase chain reaction

Gene specific PCR was done after DNA extraction to see the bands on agarose gel

PCR reagents

Ingredients Quantity per sample

PCR buffer 2.5 µL

Mgcl2 4 µL

dNTPs 1µL

Forward & reverse primer 1µL each

Taq polymerase 0.2 µL

Distilled water 11.3 µL

DNA template 2 µL
Total 25 µL

Table 6: Ingredients used for PCR

 All the ingredients were added in PCR tubes and gave a short spin.
 After that all the tubes were placed in PCR blocks and start the machine.

Thermal cyclic conditions

1. Denaturation (initial) 95 °C for 5 min

2. Amplification
Denaturation 94 °C for 30 sec
Annealing 55 °C for 30 sec
Elongation 72 °C for 60 sec
3. Final extension 72 °C for 5 min

40
 PCR Profile presentation on machine LCD

Gel electrophoresis for PCR product

Preparation of 1.5% agarose gel

 I added 0.45g of agarose in 30ml of TBE buffer. Mixed and heated it in oven
until it started boiling. Then set the comb and poured the gel on gel rack and
allowed it to solidify.

Loading of PCR product on gel

 I Added 2 µL of bromophenol blue dye in 3 µL of DNA sample and mixed it

gently.
 Then loaded these samples and DNA ladder into respective well carefully one
by one. Then turned ON the electrophoresis apparatus and charged it 90V for 1
hour.
 Gel was seen under gel doc.

41
 PCR product bands on agarose gel

Antibiotic Sensitivity Testing

AST was done to check which antibiotic is most effective to treat specific bacterial infections
in the body.

Principle
Placed the antibiotic disks agar plate which has inoculated with bacterial culture to be tested.
During incubation drug release from disks and create concentration gradient in agar and makes
a clear zone around if bacteria are sensitive for this drug.

Protocol

Preparation of TSB (Tryptic soya broth) tubes

 I prepared TSB tube by adding TSB powder in distilled water and heated in
microwave oven and autoclaved for 15 min at 1210 C.

42
Inoculation

 I inoculated the pure colonies of bacteria into TSB tubes.

 Then placed these tubes in incubator for 2-4 hours until turbidity appear.

Muller Hinton Agar plates

 I added 1.9g of Muller Hinton agar in 30ml of distilled water then autoclaved it
and finally poured it into Petri plates and allowed to solidify.
 Then I took the sample of pure colonies from TSB tube with the help of cotton
swab and spread the surface of MHA (Muller Hinton agar).
 Placed the antibiotic discs of Gentamycin, Chloramphenicol and
Sulfamethoxazole on it and pressed them gently. Incubated the plates for 24 hrs.
 Next day, I checked the plates and observed the zone of inhibition around the
antibiotic disc then measured the zone of inhibition with the help of ‘mm’ scale.

Measurement
The diameters of the zone of inhibition was measured (including disk) using a
metric scale. The measured zone diameter was compared with a standard chart
for obtaining the susceptible and resistant values. There are zone of intermediate
resistance which means that the antibiotic may not be sufficient enough to
eradicate the organism from the body.
Results
The absence of growth of the organism around the antibiotic disks indicates
that, the respected organism is susceptible to that antibiotic and the presence of
growth around the antibiotic disk indicates the organism is resistant to that
particular antibiotic.

43
 Agar plate with drugs sensitivity zones

Bacterial stock preparation

Pure culture of bacteria was stored for future use as a glycerol stock whereas glycerol
prevents the ice crystal formation in bacterial cells.

Protocol

 I took 3 mL TSB in test tube and inoculated with pure colony of bacteria then
incubated at 37 °C for 24 hours.
 Next day test tube was removed and 1.5 ml poured in eppendrof then
centrifuged at 13,000 rpm for 5 mins.
 Supernatant was discarded and the other 1.5 ml poured in same eppendrof then
centrifugation at 13,000 rpm speed for 5 mins.
 Discarded the supernatant again and saved the pallet.
 Resuspended
 the pellet in 1 mL of 3:7 (30 mL glycerol & 70 mL TSB) glycerol stoke and
freeze at -20 C.
 When need of same bacteria for studies in future just revive the stock and use it.

44
 Structural and Biological Lab

I worked under the supervision of Dr. Moazur Rahman (Principle Scientist).

Major objectives
 Designing, synthesis and biological screening of anti-viral agents that is
against Hepatitis B and C viruses.
 Two classes of organic compounds, Thiazolides and Cyclopentenoids,
have already been identified that are used to inhibiting HBV and HCV
viruses with particular emphasis on HCV genotype-3, the widely prevalent
genotype in Pakistan
 Identify cell membrane protein structure and their homologues in bacteria
and yeast and viral proteins for therapeutic importance.

Research interests

 antiviral and anti-cancer agents synthesis

 2nd generation drug’s candidates synthesis
 Drug target identification.
 Membrane and viral proteins evalution

Dr. Moazur Rahman

Principal Scientist

45
 Gene Cloning and Expression in E. coli
Gene cloning is a molecular biological technique in which our gene of interest is added into
plasmid (a self-replicating genetical structure usually present in bacteria used as a vector),
which is then introduced into a host organism (mostly bacteria)in which plasmid replicates and
generates a large number of identical copies of that particular gene.

 A typical gene cloning protocol includes the following steps:

 Host and vector organism selection
 Isolation of bacterial plasmids and foreign DNA which contains gene of
interest
 Treatment of both foreign DNA and bacterial plasmids with restriction
enzymes in order to produce restricted ends or sticky ends.
 Ligation in which DNA fragment containing our gene of interest and the
vector are allowed to be ligate with each other.
 Transformation in which recombinant plasmid is transferred into the bacterial
cells.
 Then bacteria are grown on growth media (containing specific antibiotics that
allow only plasmid containing bacteria to grow). The bacteria are grown in
large numbers, and each of these plasmids would carry the gene of interest as
the insert, which can be isolated if needed using the same restriction enzyme
that was initially used to insert them into the vector.
 Gene cloning was done in SBL by following these steps.

Nucleotide sequence of target gene

By using online database NCBI nucleotide sequence of target gene was retrieved. Target gene
was already cloned in cloning vector for PCR amplification.pET28a cloned vector was stored
at -20 °C until transformed into Escherichia coli competent cells for vector DNA propagation.

Primers designing

By using “amplify sequence” program of “Vector NTI software PCR primers were designed to
flank a protein gene fragments of DNA. Restriction sites were added to the forward and reverse
primer in the cloning vector.

46
 E. coli competent cells preparation
E. coli cells are incorporate foreign DNA if their cell walls are altered so that DNA can pass
through more easily. These cells are said to be "competent" and made competent by using
calcium chloride and heat shock.

Protocol

 TOP 10 bacterial culture was revived which is stored at – 80 °C.

 LB media agar was prepared by adding (Tryptone , yeast extract, NaCl, agar ) in
distilled water and autoclaved the media.
 Agar was then poured into plates and allowed to solidify. These plates were spread with
sterile loop from the culture stock and placed in incubator at 37°C for 20 hours.
 Pre culture was prepared by taking single colonies from plates and transferred to 5mL
LB broth then placed at 37 °C in shaking incubator with continues agitation at 225 rpm
overnight.
 Next day 5 ml culture was transferred into 200 mL LB broth in flask for large culture
growth and placed at 37 C in shaking incubator until OD at 600 nm reached at 0.4-0.5.
 Then culture flask was immediately transferred from 37°C incubator to ice and
centrifuged at 4,000 rpm speed at 4°C for 10 mins.
 Supernatant was discarded and resuspended cell pellet in 20mL chilled CaCl2. Cells
were kept for 30 mins on ice and again centrifuged at 2000 rpm at 4°C for 10 minutes.
 Resuspended the pellet again in 20mL chilled MgCl2 and kept on ice again for 30 mins
and afterwards centrifugation at 2500 rpm speed for 5 mins.
 Supernatant was discarded and pelleted cells were resuspended in 2mL CaCl2
containing 15% glycerol.
 For the future use, aliquots of 200μL were prepared in eppendorfs and stored in freezer
at -70°C.

47
 Spreading on LB agar plate

Transformation of Plasmid DNA into E. coli

Heat shock method was used for transformation of plasmid DNA into E. coli competent
cells.

Protocol

 Plasmid DNA (1-2μL) was added into one labelled eppendorf containing 200μL
competent cells while transformation in second eppendorf was set up for negative
control where no DNA was added.
 Then both eppendrof were kept on ice for 30 minutes and then transferred to water bath
operating at 42°C for 1.5 minutes. Cells were then immediately transferred on ice and
incubated for 5 minutes.

48
  200μL of pre-warmed LB medium was added in eppendrofs and incubated at 37°C with
225 rpm shaking.
 Cells were finally centrifuged at 3000 rpm for 3 minutes and supernatant was discarded.
Cells were re-suspended in 200μL of sterilized LB media.
 100μL of transformation mixture from each vial was spread on a pre-warmed selective
LB agar plate. Plates were incubated at 37°C overnight.

Heat shock at 42°C

Miniprep of Plasmid DNA

Miniprep was performed to isolate plasmid by using AxyPrep Plasmid Miniprep Spin kit

Protocol
 Single colonies were picked up from the plate spread with transformation mix and
transferred into 5mL LB media in 50 mL falcon. Cells were cultured at 37°C for 12-16
hours with shaking at 250 rpm.
 1-4 mL of overnight LB culture was collected and centrifuged at 12,000 rpm for 1
minute to pellet the bacteria then pipette off the supernatant.
 Resuspended the bacterial pellet in 250 µL of buffer S1 with RNase A and vortex.
 Added 250 µl of buffer S2 and mixed by gently inverting the tube for 4-6 times.
 Within 5 minutes 350 µl of buffer S3 was added and mixed by gently inverting 6-8
times then centrifugation at 12,000 rpm speed for 10 mins to clarify the lysate.

49
  Miniprep column was then placed into an open 2 mL microfuge tube and transferred
the clarified supernatant into this column.
 Miniprep tube and microfuge were transferred to microcentrifuge and spin at 12,000
rpm speed for 1 mins.
 700 µL of buffer W2 was pipetted into miniprep column and centrifuged at 12,000 rpm
for 1 minute.
 Filtarate was discarded from 2 mL microfuge tube and miniprep volume was placed
back into the 2 mL microfuge tube then centrifuged at 12,000 rpm for 1 minute.
 Miniprep column was transferred into a clean 1.5 mL microfuge tube and 60-80 µL of
eluent or deionized water was added to elute the plasmid DNA to the center of
membrane.
 Allowed to stand for 1 min at room temperature and centrifuged at 12,000 rpm for 1
min.

Buffer Composition

Buffer S1 Resuspension buffer

Buffer S2 Lysis buffer contain SDS

Buffer S3 Neutralization buffer

Buffer W2 Desalting buffer contain ethanol

Eluent 2.5 Mm Tris-HCl, pH 8.5

Table 7 : Buffer composition used for miniprep

Preparations of Glycerol Stocks

In order to preserve bacterial cultures, 800μL of culture was mixed with 200μL of autoclaved
glycerol (80%) in 1.5mL eppendorfs. Eppendorfs were stored at -20°C and used as a source for
revival of bacterial cultures in future.

50
 Agarose Gel Electrophoresis
For determination of plasmid size
Protocol
 Mixed 2μL of DNA with 2μL loading dye and loaded into the wells of 1 % agarose gel.
 Gels was prepared in 1X TAE buffer.
 After loading of DNA samples into the gel, it was electrophoresed at 90 volts and run
for 30-40 minutes.
 DNA stained with ethidium was viewed by exposing to ultraviolet (UV) in a
transilluminator. The length of the plasmid was determined by comparing it with 1 Kb
DNA ladder.

PCR Amplification of gene of interest

For determination of size of gene of interest
Protocol
 PCR of GOI was performed from plasmid DNA with gene specific primers having extra
sequences of restriction enzymes.
 Reaction mixture of 100μL was prepared containing Dream-Taq mix 50μL and 10μM
of each primer and 10pg of plasmid DNA as template in 1.5mL eppendorfs tube. 25μL
was distributed in four thinned walled 0.25 mL PCR tubes.
 Thermal cycler was programmed for preheat treatment at 95°C for 3 min followed by
35-40 cycles of 95°C for 1 min, 55°C for 30 sec and 72°C for 2.5 min.
 Once the reaction was completed, the amplification of gene was checked by loading the
3μL of PCR reaction into the wells of 1 % agarose gel.
 After loading of DNA samples into the gel, it was electrophoresed at 90 volts and run
for 30-40 minutes. DNA stained with ethidium was viewed by exposing to ultraviolet
(UV) in a transilluminator.
 The size of amplified target gene was determined by comparing it with 1 Kb DNA
ladder.

51
 Digestion of Plasmid and Amplified Gene

Plasmid and amplified PCR product of the amplified gene was digested in eppendorfs,
separately, using specific restriction enzymes and their corresponding buffers. Buffers were
calculated using Double Digest Calculator available at Thermoscientific website.
Protocol
 For restriction of plasmid, a total 100μL reaction mixture was prepared containing
60μL (500 ng) DNA, 2.5μL each restriction enzyme, 20µL Tango buffer (10X) having
50-100% efficiency for both enzymes and 15μL water.
 For the amplified gene restriction reaction mixture was prepared using restriction
enzymes, 20µL Tango buffer (10X) having 50-100% efficiency for both enzymes and
46μL water.
 Eppendorfs containing reaction mixture were placed at temperature of 37°C
overnight.
 Restricted DNA fragments were separated by agarose gel electrophoresis.
 DNA fragments stained with ethidium bromide were visualized under UV light and
their size was determined by comparing to standard molecular weight DNA 1Kb
marker.

Plasmid cutting with restriction enzymes seen on gel

52
 Purification of vector and gene from agarose Gel
Restriction products were purified with wizardR SV Gel and PCR Clean-Up system.

Protocol

 Gel slices containing gene were cut close to their band positions on the gel under UV
light and transferred to 1.5mL microcentrifuge tube.
 10µL membrane binding solution was added for 10 mg of gel and incubated at 50-
65°C and vortexing.
 SV minicolumn was inserted into collection tube and gel mixture was then transferred
to Minicolumn assembly and incubated at room temperature for 1 minute.
 Afterwards minicolumn assembly was centrifuged at 16,000 rpm for 1 min then flow
through was discarded and minicolumn was reinserted in the collection tube.
 To wash bound DNA to the matrix, 700μL membrane wash solution (ethanol added)
was added to the minicolumn and centrifuged at 16,000 rpm for 1 minute. Collection
tube was emptied and minicolumn was inserted back into it.
 Washing step is repeated by the addition of 500μL of membrane wash solution and
centrifuged at 16,000 rpm for 5 minute.
 Empty column was centrifuged for 1 min at 16000 rpm to remove remaining ethanol
and keeping lid of microcentrifuge open.
 Carefully transferred this minicolumn to a clean 1.5 ml microcentrifuge tube and was
added 50 µl of Nuclease free water to minicolumn then left at room temperature for 1
minute.
 Then centrifuge at 16,000 rpm speed for 1 minute.
 Purified DNA stored at -20°C

Cloning of amplified DNA in pET28a expression vector

For construction of pET28a expression vector, restricted Plasmid DNA and amplified gene
were ligated. For ligation various molar ratio of 5’ and 3’ ends of DNA was maintained between
vector and gene DNA concentration in the reaction mixture.

53
 Ligation of amplified gene into pET28a expression vector
For ligation of gene into vector, conversion of microgram of linear DNA to picomoles of ends
was done using bio-math calculator available at Promega website. Quantity of insert and vector
DNA and other parameters used in bio-math calculator are as follow:

 Quantity of insert DNA in ng = 33ng/μL

 Size of insert = 3.3 Kb
 Quantity of vector in ng = 80ng
 Size of Vector DNA = 5.3 kb
 Generally ligations are tested at 1:3, 1:1, and 3:1 insert: vector molar ratios.

For 1:3 insert to vector ratio

Vector 3.6μL

Insert 1.78μL

Buffer 2.5μL

Ligase enzyme 1μL

Water 15.89μL

Total 25μL

Table 8: quantities of solutions used for ligation

 Incubated reaction mixture overnight at 16 °C in 1.5mL micro centrifuge tube.

 Transformed the ligation mixture into competent E. coli cells by heat-shock method
next day
 These cells were then spread on solid LB media plate containing 50μg/mL ampicillin
and incubated at 37°C for 16 hours.
 Single colonies on these plates were touched with yellow tips. Tips were touched on
the surface of fresh LB agar plate having gratings from 1-20 and then dipped in the
PCR reaction mixture in order to confirm the cloned construct.

54
 Confirmation of recombinant clones by colony PCR

Colony PCR is a method for determining the presence or absence of insert DNA in plasmid
constructs. Transformants are lysed firstly in water with a short heating step or added directly
to the PCR reaction because it lysed during the initial heating step.

Protocol

 For this, reaction mixture of 25μL was prepared containing single ampicillin resistant
colony from LB agar plate.
 Dream-Taq mix 12.5μL and 10μM of each primer in a thin walled 0.25mL tube.
 Thermal cycler was programmed for preheat treatment at 95°C for 3 min followed by
35 cycles of 95°C for 1 min, 52°C for 30 sec and 72°C for 3 min.
 Once the reaction was completed, the presence of gene insert cloned into vector was
verified using agarose gel electrophoresis following staining with ethidium bromide.

Transformation of cloned gene in pET28 expression vector into E. coli competent Cells
By using heat shock method cloned vector was transformed into E. coli.

Protocol

 25mL LB agar plates were spread with cells and placed at 37°C in incubator for
overnight.
 3-4 colonies were picked from each plate and transferred each to the 10mL LB media
in 50mL tubes. Then tubes were incubated at 37°C with continuous shaking at 225rpm,
overnight.
 For expression, 2 mL of overnight culture was transferred to 250mL flasks containing
50mL LB media and placed in incubator with continuous shaking at 225rpm until
OD600 reaches to 0.4-0.5.
 After induction, cultures were again incubated with shaking for further 3 hours and
final OD600 was measured before centrifuged at 4000rpm for 15 min at 4°C.
 Supernatant was discarded and dissolved the pellet in 5mL of chilled resuspension
buffer. Lysozyme (0.75mg) was added in each sample.
 Each sample was sonicated. The sonicator was operated for 12 cycles (each cycle
having 30 second sonication and 20 second rest). After this, each sample was
centrifuge at 11,000 rpm for 30 minutes at 4°C.

55
  Supernatant from each sample was separated in 15mL tubes and pellet was re-
suspended again in 1mL chilled re-suspension buffer, both supernatant and pellet were
stored at -20°C for onward protein analysis through SDS-Polyacrylamide gel
electrophoresis (SDS-PAGE).

Cell lysis with Glass beads

This step was done for lysis of bacterial cells and getting desired protein by using glass
beads.

Protocol

 Pellet was resuspended from induced culture of E. coli transformed with desired
vector, in pre-chilled lysis buffer (Tris HCl, NaCl, and β-ME) by vortex.
 In separate falcons, glass beads was taken and washsed with distilled water, then
with absolute ethanol and again with distilled water.
 Resuspended pellet was added to respective falcon.
 Falcons were vortex for 30 seconds and then placed on ice for 30 seconds (repeat
this cycle 15 times).
 After cycles, these falcons was left on ice for further 15 minutes to settle down the
beads.
 Supernatant was aspirated from these beads.
 Centrifuged each supernatant at 8000- 12,000 rpm for 20 minutes at 4 °C.
 Supernatant was transferred in individual Eppendorf tubes.
 Pellet was resuspended in Lysis buffer (Tris HCl, NaCl, and β-ME) in separate
Eppendorf tube (volume of lysis buffer should be equal to that of supernatant).
 Cell lysate, supernatant and pellet was run on 12% SDS PAGE to check the
expression of matrix protein.

Separation of protein by SDS-Polyacrylamide gel electrophoresis

Principle

Protein samples and ladder are loaded into wells in the gel and electric voltage is applied.
Mercaptoethanol or dithiothreitol which are reducing agent (in the presence of SDS) breaks
down the disulfide bridges that are responsible for protein folding, and a detergent such as SDS

56
imparts a negative charge to the proteins thereby linearizing them into polypeptides.
Polyacrylamide provides a matrix for the polypeptides to run. Polypeptides run towards the
positive electrode (anode) through the gel when an electric field is applied. Electrophoretic
mobility of the proteins depends upon shape, charge and size.

Protocol

 Poured 10-12% of separating gel (Table 9) into the casting assembly and very thin
layer of ddH2O was added over the gel to level it.
 Allowed the gel to polymerize then removed the layer of ddH2O.
 Poured 5% stacking gel (Table 10) immediately over the separating gel and comb was
inserted in it.
 After polymerization of stacking gel, the gel was transferred to the gel tank containing
the running buffer (Table 11) and removed the comb.
 Protein sample with 4% loading dye (Table 12), and protein ladder was loaded in each
well and connected the apparatus to a constant DC source of 120 V.
 After completion, recovered the gel from the assembly and dipped in staining solution
(Table 13) at room temperature for 5-7 hours and then destained with 10% acetic acid.
 The separation of protein sample was analyzed under light source.

57
 SDS-Page Gel Tank loaded with samples

Components Volume required for one gel (5mL)

dH2O 1-1.67 mL

Acrylamide 2-2.67mL

Tris pH 8.8 1.25 mL

SDS 25 µL

Ammonium persulfate 50 µL

TEMED 2.5 µL

Table 9: Composition of 10-12% separating gel

Components Volume required for one gel (3mL)

dH2O 1.702 mL

Acrylamide 0.5 mL

Tris pH 6.8 0.75 mL

SDS 15 µL

Ammonium persulfate 30 µL

TEMED 3 µL

Table 10: Composition of stacking gel

Components Quantity (in grams) for 1 L buffer

Glycine 144

Tris 30

SDS 1

Table 11: Composition of Running buffer for SDS-PAGE

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Components Quantity Final concentration

Glycerol 5g 5.4 M

SDS 1g 10%

EDTA 37.2 mg 10 mM

Table 12: Composition 4X sample loading buffer (10 mL)

 Dissolved in 0.5 M Tris HCl pH 6.8, bromophenol blue was added to color
deep blue

Components Quantity Percentage

Acetic acid 50 mL 10

Isopropanol 125 mL 25

Coomassie brilliant blue

125 mg 0.025
R-325

dH2O 325 mL -

Table 13: Composition of 500 mL staining solution

SDS Page after destaining showing protein bands

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 Purification through Ni-NTA Chromatography

Recombinant proteins contain polyhistadine tag on either terminus are purified by using Nickel
column. A 6xHis tag containing recombinant proteins has a high affinity for nickel, others bind
loosely.

Protocol
 Pack 1 mL of nickle-nitrilotriacetate (Ni-NTA) affinity resin in column.
 Purification was done at 4 °C.
 Column was washed with distilled water.
 Equilibrated the column with 10x column volume (5 mL) equilibration buffer
(Tris-HCl, NaCl, and β-ME).
 Sample protein added to Ni-NTA resin and this ultimately pass through the
column.
 Column was washed with 20x column volume (10 mL) wash buffer (Tris-HCl,
NaCl, imidazole, and β-ME) to remove loosely bound non-target proteins and
collect the wash in fractions.
 Protein was eluted with elution buffer (Tris-HCl, NaCl, imidazole, and β-ME)
in fractions of 0.5 mL in 1.5 mL eppendorf tubes.
 Concentration of protein was measured in all fractions using Nanodrop.
 Run 12 % SDS-PAGE for analyses.

Ni-NTA column with resin

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 Minimum inhibitory concentration (MIC) assay
Minimal inhibition concentration (MIC) is the lowest concentration of an antimicrobial
agent that required to cease the visible growth of microorganism after incubation. MIC
is used as diagnostic tool and also in research methods to evaluate the efficacy of drug.
Protocol
 LB broth and antibiotic stock solution was prepared.
 LB media was inoculated with bacterial cells and placed at 37°C on shaking at
220 rpm for 24 hours.
 Optical density (OD600) of overnight grown culture was measured at 100-fold
dilution.
 Fresh LB media was inoculated with the overnight grown culture so that it’s
OD600 become equal to 0.02.
 Diluted culture was then placed at 37°C for about 2 hours on shaking at 220 rpm
to raise the OD600 up to 0.2±0.02.
 OD600 was checked again and 200µL culture was added in each well of
microtiter plate.
 Varying concentrations (µg/mL) of different drugs were added in the wells
containing culture.
 The micro titer plate was read in Micro titer plate reader immediately after
adding drug.
 Incubated the micro titer plate containing culture at 37°C overnight and then
read it again in microtiter plate reader.

Interpretation

Note the reading as lowest concentration at which no visible growth of bacteria in solution and
the difference of measured and background OD600 is must less than 0.01.

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 National Probiotics Lab

I worked under the supervision of Dr. Arsalan Zaidi (Principal Scientist).

Probiotic bacteria are beneficial for human consumption such as lactic acid bacteria (e.g.
lactococci and bifidobacteria).clinical infections, new LAB strains and antibiotic resistance
genes are most public concern. The lab scientists focus on LAB biosafety. Probiotics isolates
from different sources are studied and check their pathogenic potential by using different
microbiological test.

OBJECTIVES

Their main objective is to check the biosafety of probiotics before marketing by guidelines,
make standard and proposing criteria.

Dr.Arsalan Zaidi
Principal scientist

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 Bile assay
Bile assay was done to check the pathogenic potential of probiotics

Protocol
 Bacterial culture was inoculated from glycerol stocks into 10 ml sterile broth media and
incubated for 24 hours at 37 °C.
 Bacterial culture was then centrifuged at 5000 rpm for 3 min at 15 °C.
 Pellet was re suspended in 1 mL PBS and centrifuge again and re-suspended the pellet
in 5 mL PBS.
 Bacterial cell suspension was took in cuvette and OD600 at 600nm taken using
spectrophotometer.
 Bacterial culture was diluted to give the final OD600 0.2-0.8 using sterile PBS.
 100 µL of bacterial culture was added in new falcon tube containing 10 mL broth media
and incubated at 37 °C, time depends on exponential growth/log phase of isolate.

Bile solution preparation

For 20% stock solution prepration 1 gram of ox bile was mixed in 5 mL sterile broth media
and filtered using 0.2 µm syringe filter.

 From stock solution different concentrations of bile solution was made which is 0%,
0.15%, 0.30%
 Glass tubes were labelled with bile concentrations and incubated at 37°C.
 OD was measured by using spectrophotometer after 6-8 hours.
 Readings were measured and bar graph was ploted.

MRS broth
MRS broth is used for the cultivation of lactobacilli in a laboratory setting and supports
luxuriant growth of lactobacilli from dairy and other sources.

Preparation
 26.1 gram of media powder was weighted to prepared 500 mL media broth in d.H2O
and gently mixed.
 Autoclaved the media at 121 °C for 15 minutes.
 Checked the pH which should be 5.7 at 25 °C after autoclaving.
Preparation of MRS supplement L-cysteine stock solution
 The media was cooled immediately at 50-55 °C.
 Powdered L-cysteine was weight and dissolved in distilled water

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  Filter sterilized L-cysteine solution using a syringe filter and filtrate was
collected in sterile falcon tube.

Syringe filtration of L-cysteine

 Added measured amount of L-cysteine in media that has been cooled to 48 °C.
 Samples were inoculated directly into MRS broth and incubated at 35 °C for 3 days.
 Sub cultured growth in broth tubes to solid media and streak specific strain of
lactobacillus, sealed with paraffin film and incubated.
 Growth on plates were seen next day.

Lactobacillus colonies on MRS plate

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 Riboprinter
Application
 The riboprinter system provides an automated genetic snapshot, or RiboPrint patterns
of any bacterium in less than eight hours.

 This fragment-based technology utilizes restriction enzymes to target and cut regions
of the ribosomal RNA genes, generating a DNA fingerprint that is unique to the
organism at the strain level.
 This system generates genetic strain level “fingerprints” of bacteria and comprises them
against thousands of other patterns to identify organisms.
 This critical information facilitates probiotic formulations that are safe, highly targeted,
effective and efficient.

Riboprinter

Anaerobic chamber
Application

 It is a bactron workstations that allows efficient free handling and treatment of

samples in an oxygen free environment
 Modular system within the bactron that provide anerobic medium for
completion of procedures, incubation and inoculation in the absence of oxygen.

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 Bactron

Colour Q-Count
Application
 It is an automated colony counter apparatus
 It can count and save data of bacterial colony
 Its range of counting is 20-300 colonies
 It can recognize different colonies of different strains by giving them different
colors.

Colour Q-Count

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 Spectramax 384 Plus Microtiter Plate Reader
Application

 Get absorbance measurements from 100 to 1000 nm quickly for samples in test
tubes, cuvettes, and 96- or 24- or 384-well microplates.
 The SpectraMax® Plus 384 Microplate Reader gives walk-up convenience for
both standard spectrophotometer and microplate reader applications on the
same instrument
 Monochromator wavelength selection saves the time and trouble of ever having
to change out filters.
 Applications
I. DNA and RNA quantification
II. Enzyme kinetic assays
III. ELISA based assays.

Spectramax plate reader

Bioflux

Application
 It helps to study the cellular behaviour under shear stress from fluid flow.
 The high shear BIOFLUX plate provides a well-controlled platform for
studying drug and cell response in high shear condition.
 It offers a wide variety of cellular applications such as biofilm growth,
mutant screening, microbial adhesion, antibacterial or antifungal screening
and host pathogen interaction.

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 Bioflux

Safety cabinet

Application
 It is designed for protection of user environment and samples from hazardous
contamination
 It provides ventilated lab work place for safety working with materials
contaminated with pathogens required a defined biosafety level

Safety cabinet

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 Autoclave
Application

 It is the physical method for disinfection and sterilization under specific

temperature and pressure. This is the effective way to kill microorganism and
spores.
 It is also used to treat the biological waste contain bacteria,viruses and other
medical waste before disposal. Also used to sterilize media and instruments.

Autoclave

Gram staining

Gram staining is used to differentiate and classify the bacterial species into two major
group i.e gram-positive and gram-negative.

Principle
Gram staining differentiate the bacteria by their cell wall peptidoglycan composition. Gram
positive have thick layer of it and that’s why those retain the primary dye after washing with
alcohol whereas gram negative have thin layer and dissolve during washing. After stain with
safranin gram positive remain purple while gram negative show pink colour.
.

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Protocol

 Smear Preparation

 First of all I took a new slide and removed its grease by heating.
 Placed a drop of water on it and bacterial colony was picked from plate and
mix with yellow tip.
 Make a thumb like smear and allowed to dry then fixed it by heating.

 Gram Staining

 Flooded the smear for 1 minute with crystal violet staining reagent.
 Wash the slide for 2 sec in a gentle and indirect stream of water.
 Flooded the slide with mordant Gram’s iodine stain and wait for 1 minute.
 Wash the slide for 2 sec in a indirect stream of water.
 Flooded the slide again with decolorizing agent and wait for 10-15 sec or
added drop by drop to slide until decolorizing agent running from the slide runs
clear.
 Lastly, flooded the slide with counterstain, safranin and wait for 30 sec to 1
minute.
 Wash the slide under indirect stream of water then dry the slide.
 Observed the staining result under BX 63 microscope.

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Stained slides observed under BX 63 microscope

71