A study guide for

AS Biotechnology

EOY Exam

The Cells
1. Using bacteria as typical prokaryotic cells, state
functions of their cellular structures: surface
structures (flagella, pili, fimbriae), glycocalyx, cell wall,
cell membrane, ribosomes, cytoplasm, plasmids,
nucleoid and endospore
2. Using animal and higher plant cells as typical
eukaryotic cells, state functions of: cell membrane,
cell wall, cytoplasm, cell vacuoles, nucleus, smooth
and rough endoplasmic reticulum, mitochondria,
chloroplasts, Golgi body and ribosomes
3. compare the structures of typical prokaryotic
(bacteria) and eukaryotic cells (plant and animal)
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Plasmid

necloid

Flagella
M

Cell membrane

ribosome
G
E

Capsule
Text

fimbriae

cell wall

Pili

Microbial Biotechnology (3 topics)

Culture of
bacteria

1. Cell shape and arrangement

Characterization
of a bacterium

4. Nutrient
& energy
requirement

2. Cell wall
Gram stain
KOH test
Action of
penicillin

3. Environmental
factors
pH
temperature
O2 requirement

5. Biochemical reaction

Aseptic technique
16 streak
Serial dilution
Colony count
Growth curve
Culture media

Industrial
microbiology
Enzymes
Agriculture
Food

Culture of bacteria
Important techniques
Aseptic techniques, 16 streaks, serial dilution,
spread plate, colony counting
What is the importance of pure culture?
You are given a mixed culture. Describe
stepwise how you can:
Separate them into pure cultures
Enumerate the number of bacteria
in the culture (cfu/ml)
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9ml fresh
medium

Tube Y

You intend to find out how many E. coli cells are there in
Tube Y. You performed serial dilution, and spread 100 l of
10-6 diluted culture into an agar plate. After overnight
incubation, you found 253 colonies on that agar plate.
Calculate what is the bacterial concentration in Tube Y.

Culture of bacteria
Describe the stages of the bacterial growth
curve in culture
Explain the difference between defined
medium v.s. complex medium
If you were to prepare 35% salt solution, how
much salt do you need to add into 70ml of
water?
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Characterization of bacteria
1. Cell shapes and arrangements
State the 4 basic shapes of bacteria
How are bacteria named according to their shapes
and arrangements?

Characterization of bacteria
2. Cell wall
Compare and contrast Gram +ve and Gram ve
bacterial cell walls
List the steps to perform Gram staininig.
Explain the principle of KOH test
Describe how penicillin exert its effect on bacteria
Which test can be used to determine if penicillin is
effective against a strain of bacteria? Describe
how to perform this test.
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Characterization of bacteria
3. Environmental factors
Define the following:
Neutrophiles, acidophiles, alkaliphiles
Thermophiles, mesophiles, psychrophiles
Obligate aerobe, facultative anaerobe,
strict anaerobe, microaerophile, aerotolerant
anaerobe

Describe how to culture a strict anaerobe in

lab.
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Characterization of bacteria
4. Carbon and energy requirement
Define the following:
Photoautotroph, Photoheterotroph
Chemoautotroph, Chemoheterotroph

5. Biochemical tests
Describe stepwise how to perform the following tests:
Oxidase test, Catalase test, starch hydrolysis test

What do each of these tests tells you about the

character of the bacterium?
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Industrial Microbiology
Microbial enzymes used in industry (MCQ only)
Explain how nitrogen fixation activities of microbes is
essential for the growth of plants
Microbes have been used in the production of the
following food: Yogurt, Cheese, wine/beer, vinegar
Give the scientific name of any one bacterium used in the
production of each type of food
Describe how these food are produced with the help of
the bacteria.

Define the term probiotics. List any 3 benefits of

probiotics.

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Check List 1
The following learning outcomes are listed at SEAB website. Have
you master them?
1. Introduction to Biotechnology
1.1 The Cell
Learning Outcomes:
Upon completion of this unit the student will be able to
(a) identify bacteria as typical prokaryotic cells and state functions of their
cellular structures: surface structures (flagella, pili, fimbriae), glycocalyx,
cell wall, cell membrane, ribosomes, cytoplasm, plasmids, nucleoid and
endospore
(b) identify cellular structures (including organelles) of typical eukaryotic
cells and state their functions: cell membrane, cell wall, cytoplasm, cell
vacuoles, nucleus, smooth and rough endoplasmic reticulum,
mitochondria, chloroplasts, Golgi body and ribosomes
(c) compare the structures of typical prokaryotic (bacteria) and eukaryotic
cells (plant and animal)

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3. Microbial Biotechnology
3.1 Bacteria
Learning Outcomes:
Upon completion of this unit the student will be able to
(a) state the effects of cell wall inhibitors (antibiotics e.g. penicillin) on bacteria
(b) describe the stages of the bacterial growth curve in culture
(c) classify bacteria based on
i. morphology
ii. nutrient requirements and nutritional types
(d) state the different environmental conditions (e.g. temperature, pH, oxygen) that
bacteria can grow
(e) outline the tests used to characterise bacteria (e.g. Gram stain and biochemical tests)
(f) state and perform basic microbiological procedures
i. preparation of agar and broth cultures
ii. serial dilution and determination of bacteria density
iii. determination of culture purity
(g) discuss the applications of bacteria in
i. food (fermentation, probiotics)
ii. industry (production of enzymes)
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iii. agriculture (nitrogen fixation)

DNA

RNA

Protein

What are the relationships between DNA,

genes, chromosomes and genome?
Describe the process of DNA replication.
Keywords: helicase, DNA separation, DNA
polymerase, elongation, 5 3, semi-conservative.

Compare DNA and RNA in terms of

composition, structure, location in cell and
function.
How are RNAs translated into amino acids?
Keywords: start/stop codon, codon redundancy
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Gene Cloning and DNA/protein Analysis

1. Isolate the gene of interest
2. Paste the gene into cloning
vector

5a. OD260 to quantitate the amount of

DNA extracted
5b. OD260/OD280 to determine purity
of DNA extracted
5c. Restriction digestion to confirm the
confirm the gene of interest by size

3. Transform recombinant
plasmid into host cells

4. Select for colonies that

contain the gene of interest
5. Confirm the presence of
the gene of interest in the cell
6. Express the gene of interest
into protein

5d. Agarose gel electrophoresis to

analyse the size of a DNA fragment
5e. PCR to confirm the gene of interest
by size
6a. SDS PAGE to determine the
presence, size and purity of proteins
extracted
6b. Size exclusion chromatography to
purify the proteins of interest
6c. OD280 and Bradford assay to
quantitate the amount of proteins

Gene to be cloned (in bold):

Example:
Figure 1

pUC19 to be used as vector

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Gene Cloning
Step 1: Get that gene out!
How to break open the cells?

What you use to cut out the gene of

interest?
What are the benefits of double digest when
compared to single digest?
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Gene Cloning
Step 2: Paste the gene onto a cloning
vector
What is a cloning vector?
What is the purpose of the following parts /
genes of a cloning vector?
Ori, antibiotic resistance gene, lacZ gene, MCS

How ligase work?

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Gene Cloning
Step 3: Transform recombinant plasmid into
host cells
What are competent cells?
How to perform heat-shock?
Name other alternatives to deliver foreign genes
into host cells (MCQ only)
What are the 3 possible types of transformed cells
you get after this step?

21

Gene Cloning
Step 4: select for the colonies that contain the
gene of interest
How does antibiotic-resistance gene and lacZ gene
help us select the type of transformed cells?
What is the principle of blue-white selection?
What is the role of X-gal?

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Gene cloning
Step 4a: Why the need to spread different
volumes of transformed cells onto agar
plates?

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Wow, full of white

colonies too
good to be true???

What could be the possibilities?

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Gosh! Not even a

single transformed
cell???

What could be the possibilities?

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Step 4b: Why the need to set up controls?

Control 1: host cells + unligated plasmid, spread on agar with
antibiotic
This control checks for the following:
i. Whether transformation steps are successful
ii. Whether the cells are competent
iii. Whether the competent cells are healthy / dead
iv. Whether the medium/agar is well prepared (suitable
pH, nutrient content, X-gal, etc)
Control 2: host cells + buffer, spread on agar with antibiotic
This control checks for the following:
i. Whether the antibiotic in the agar is functional
ii. Whether any contamination occurs
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Gene cloning
Step 4c: What is the ratio of cells transformed
with recombinant plasmid over total
transformed cells?
Example: You have
107 blue colonies
32 white colonies

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After Gene Cloning

Step 5: how to confirm the selected
colonies contain the gene of interest?
5a. OD260 to quantitate the amount of DNA extracted

5b. OD260/OD280 to determine purity of DNA extracted

5c. Restriction digestion to confirm the confirm the gene
of interest by size
5d. Agarose gel electrophoresis to analyse the size of a
DNA fragment

5e. PCR to confirm the gene of interest by size

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5a/5b: Spectrophotometry (OD260 & OD280)

How to determine DNA concentration using
spectrophotometer?
E.g. You perform plasmid extraction and collected 1ml of
plasmid in tube X. You pipette 50 l of plasmid into 950 l of
distilled water slot the cuvette into a spectrophotometer.
The reading at OD260 is 1.45. Calculate the original
concentration of plasmid in tube X.
50 l
Tube X
containing
undiluted
plasmid.
Concentration
unknown

Cuvette
containing 950 l
of distilled water

OD260 = 1.45

5a/5b: Spectrophotometry (OD260 & OD280)

Based on the undiluted pABC concentration in tube
X, determine M and N.
(50 g required)

M l

N l

OD280 of tube X is 1.02. Comment on purity of

plasmid in tube X.
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5c: Restriction digestion to confirm the

confirm the gene of interest by size
After cloning, list out the possible combination(s) of
restriction enzymes that could be used to confirm if
the gene of interest has been successfully ligated in
the plasmid (see slide 2)

If the cloning is successful, what is the approximate

length of the DNA fragment you would expect after
digestion with the restriction enzymes above?
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5d. Agarose gel electrophoresis to

analyse the size of a DNA fragment
Explain the principle of agarose gel electrophoresis in
analysing DNA fragments.

List out step-wise how to prepare an agarose gel.

Explain how the shape and size of a DNA can affect

its migration speed.
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5d. Agarose gel electrophoresis to

analyse the size of a DNA fragment
Measure the distances (mm) travelled
by any five DNA marker bands. Fill in
the table below, and then plot a graph
to determine the size of the DNA band
(pointed by arrow).
Molecular weight
(bp)
DNA Marker 1
DNA Marker 2
DNA Marker 3
DNA Marker 4
DNA Marker 5

LogMW

Distance travelled
(mm)

5e. PCR
List the reagents required for a PCR reaction
and describe their functions.

List the steps of PCR. Describe what occurs in

the test tube during those steps.

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This is a
typical
PCR cycle:

45

30 cycles

Assuming the rest of the settings are correct, what would

happen if
You set 65 C in step 2.
You set 85 C in step 3.
You set 50 C in step 4.
You set 10 cycles for steps 2 to 4.
You set 10 seconds in step 4.
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5e. PCR
Design a pair of primers that can amplify the gene
(page 2). Your primers should be 10bp in length and
have to start from and end at the nucleotides
marked with . You are required to list out the
primer sequences in 5 to 3 orientation.

What do you expect to see on the agarose gel if the

cloning is successful (or unsuccessful)?
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Gene to be cloned (in bold):

pUC19 to be used as vector

37

After correct clone is identified

Step 6: The cloned gene is expressed into
proteins. How to analyze the proteins?
6a. SDS PAGE to determine the presence, size
and purity of proteins extracted

6b. Size exclusion chromatography to purify the

proteins of interest
6c. Bradford assay to quantitate the amount of
proteins extracted
38

6a. SDS PAGE

Outline the principle of SDS PAGE.

Why the proteins need to be denatured?

Describe the purpose of different reagents /

chemicals used in SDS PAGE

39

6a. SDS PAGE

Determine the size (kDa) of
the protein indicated by

Figure 2

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6b. Size Exclusion Chromatography

Explain the principle of SEC.

Which of the following column is more suitable for

purifying the protein sample indicated by in
Figure 2? Describe how you would collect the protein.
Column A: exclusion limit 60 kDa
Column B: exclusion limit 40 kDa

41

6c. OD280 and Bradford assay to

quantitate the amount of proteins
OD280 protein measurement
A protein solution has an absorbance of 1.75 at
when measured with 280nm wavelength, what is
the concentration of protein inside?

42

6c. OD280 and Bradford assay to

quantitate the amount of proteins
Bradford Assay
What is this assay for ?
What is the principle of this assay?
The table below is generated using OD595 readings of
the protein standard solutions. Use this table to
determine the concentration of an unknown protein with
an absorbance of 0.29.
Protein concentration (mg/ml)
0
50
200
400
600
900

OD595 absorbance
0
0.03
0.12
0.22
0.37
0.53

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Bioinformatics
List any 3 applications of Bioinformatics.

Databases (MCQ)
Name the popular DNA databases
Name the popular protein databases
How to search for info using these tools?
BLAST, Align, Entrez

Bioinformatics
How to interpret sequence comparison results?
For example:

Human Genome Project

What are the purposes of Human Genome Project?

List any 3 social ethical issues comes along with the

advancement in genetic sequencing technology.

Take note: Hierarchical sequencing and shotgun sequencing

approaches will NOT be tested. The rest only in MCQ.

Check list 2
The following learning outcomes are listed at SEAB website.
Have you master them?

Culturing Animal Cells in Lab

1. Types of Animal
Cell Culture
Purpose of cell
culture
Primary cell
culture vs cell
line
Adherent vs
suspension
cells

2.
Equipment
3. Aseptic
techniques

5. From Tissue
to Cell Line
Subculturing
Cell counting
Seeding
density

4. Culture
medium

1. Types of Animal Cell Culture

List any 3 purposes of doing animal cell culture in lab.
Give an example for each of them.

Explain the difference between primary cell culture

and cell line.

Describe the appearances of adherent cells. How do

you differentiate them from suspension cells?

2. Equipment
State the purposes of the following equipment
used in a cell culture lab:
Biological Safety Cabinet (BSC)
Cell culture incubator
Inverted microscope
Haemocytometer
Liquid nitrogen storage devices and facilities
Vessels for cell culture (Petri dish, flask,
bioreactor)

4. Cell Culture Medium

What is a cell culture medium?
State the purposes of adding the following
components in a cell culture medium:
Water , Amino acids, Carbohydrates, Serum,
Antibiotics, pH indicator (e.g. phenol red)

How does sodium bicarbonate work to control pH in

the culture medium?

4. Cell Culture Medium

How to prepare the following solutions?
60ml of 2.4% agarose gel solution (w/v)
35ml of 15% HCl solution (v/v)
700ml of 3M CaCl2 solution (molecular mass of
Ca=40, Cl=35.5)
Your stock glucose solution is 85g/ml. How do you
prepare 0.2 liter of glucose solution that is 25g/ml?

Reminder: In your answer, give a statement indicating the

amount of solute and solvent required to prepare the solution.

5. From Tissue to Cell Line

Describe stepwise how do you prepare a cell
line from an animal tissue
What is the function of trypsin?
What are the purposes of subculturing a cell
culture?
List the steps in subculturing an adherent cell
line.

5. From Tissue to Cell Line

5. From Tissue to Cell Line

Determine cell seeding density
You have a cell suspension with 8 X 106
cells/ml. You transfer 1.5ml of cell suspension
into a 75cm2 flask.
Determine the cell density expressed in
no. of cells/cm2
You then top up the medium to 25ml.
Determine the cell density expressed in
no. of cells/ml

Animal Models

Why mouse is an important animal model?

Which type of studies are performed using mice?
Why zebrafish is an important animal model?
Which type of studies are performed using zebrafish?

These animals are commonly used in which types of

experiments / research? (MCQ only)
Rabbit
Pig
Sheep

Check list 3
The following learning outcomes are listed at SEAB website.
Have you master them?

58

What is NOT tested in Sec 3 exam?

Biotech overview, mutations, Forensic Science, HGP
sequencing methodologies, HGP statistics, Electron
transport chain, resistance to antibiotics,
biochemical tests flow charts used in lab diagnosis

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What to bring?
Standard stationeries
Long ruler, calculator
What can you do when you
have doubt during your
revision?
Email me: njy2@np.edu.sg
Consult Miss Teo or Ms Ng
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