BG1131: Molecular Cell Biology for Biomedical Engineers

Genetic Engineering

Recombinant DNA Technology

Protein Production & Purification

Genetic Engineering
From wikipedia:

Genetic engineering, also called genetic modification, is the direct

manipulation of an organism's genome using biotechnology. New
DNA may be inserted in the host genome by first isolating and
copying the genetic material of interest using molecular cloning
methods to generate a DNA sequence, or by synthesizing the DNA,
and then inserting this construct into the host organism.

Genetically modified organism (GMO)

An organism generated through genetic engineering

Recombinant DNA technology

Molecular cloning

To overproduce protein(s) by overexpression of gene(s)

Remember: gene is expressed; protein is produced

The hosts

Bacteria (e.g. Escherichia coli)

Bakers yeast (e.g. Saccharomyces cerevisiae)
Insect cells (e.g. Spodoptera frugiperda)
Mammalian cells (e.g. Chinese hamster ovary (CHO) cells)

The DNA shuttle = the vector

Vector

DNA molecule, usually engineered, used to transfer genetic materials from

one cell to another cell

Two types:

Cloning vector lacks machinery for gene expression (protein production)

Expression vector

Components of a vector (*for expression vector):

Promoter*
Ribosome binding site (RBS)*
Multiple cloning site (MCS) where gene is inserted
Gene (the insert)
Terminator*
Selectable marker (e.g. antibiotic resistance)
Origin of replication (ORI)

Vector map

Generated using a software (e.g. Vector NTI from Invitrogen)

Image source: novagen

Types of vectors
Plasmid

Circular extrachromosomal double-stranded DNA that is capable of self-replicating inside

a host
Insert size 1-10 kb
Naming usually starts with a p (e.g. pBR322)

Bacteriophage vector

Derived from virus

Insert size ~ 20kb

Cosmid

Combination of Bacteriophage vector and plasmid

Contains cohesive (cos) sequence
Insert size 30-50 kb

Artificial chromosome

Bacterial or yeast
Insert size 150-350 kb
Usually low copy number

Plasmids
Naturally occurring plasmids lacks
features that are required for a
high-quality cloning vector (the
engineered vector):

Small size; necessary for increased

efficiency.

Unique (single) restriction

endonuclease recognition sites.
One or more selectable markers.

Transfer of plasmids with >15 kb into E.

coli decreases significantly

For identifying recipient cells that carry

the cloning vector-insert DNA construct.

Copy number

Multiple (8-10) types of plasmid can exist in a cell but

each plasmid type exists at limited numbers inside a cell.

The number of plasmid copies that can exist inside a cell

is called copy number:

Low (1 4 /cell)
High (10 100 /cell)

Population of plasmid in a bacterium is ~0.1-5.0% of the

total DNA.

Typical bacterial system

Hosts:

Cloning: E. coli strain DH5, DH10

Lacks ability to make mutation

Good for storing plasmids

Expression: E. coli strain BL21(DE3), BL21(DE3) Codon Plus

(Stratagene)

Vectors:

Cloning: pBR322, pUC19, pCR (Invitrogen)

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Does not contain translation signals

Expression: pET family (Novagen)

Source
DNA

Target
DNA

Cloning
Vector

Enzymatic
fragmentation

Enzymatically
linearized
Vector DNA

Join target DNA and

cloning vector
DNA construct

Introduce DNA into host cell

Isolate cells with cloned gene
Host cell

Protein encoded
by cloned gene

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Produce protein from clones gene

DNA Cloning
Procedure

Process overview of vector construction

Isolate gene
Polymerase
Chain
Reaction
(PCR)

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Digest / Cleave
Cut
Restriction
Endo/Exo
Nuclease
(RE)

Ligate
Glue
Ligase

Transform

Replication
Copy/clone

Extract
plasmid/vector
Mini/Midi/
MaxiPrep

Polymerase Chain Reaction (PCR)

To amplify DNA from a source

In the tube (50 l reaction; volume may vary):

1 l Forward primer
1 l Reverse primer
1 l DNA template (from miniprep)
0.5-1 l DNA polymerase

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Taq from Thermus aquaticus (fast but makes mistake)

Pfu from Pyrococcus furiosus (slow but more accurate)

4 l dNTP (deoxyribosnucleotide triphosphate)

5 l 10x Buffer
38 l Water

72oC

60oC

16

95oC

Time

elongate

anneal

denature

elongate

anneal

denature

elongate

anneal

PCR basic

Typical PCR program

T (C)

Duration

Description

95

1-5 minutes

Denature

95

30 seconds

Denature

3**

? (50-70)

30 seconds

Annealing

4***

72

? (30 sec 5 min)

Elongation

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5 10 minutes

Finalize strand
synthesis

Indefinite ()

Hold

Step

25-40
Cycles*

* Less numbers of cycle = less possible mutations; for

subcloning, 30 cycles will suffice.
** Tannealing is empirically determined; 52C is a good start.
*** Telongation is the optimum temperature for the DNA polymerase
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PCR is used to amplify a DNA sequence

The newly synthesized fragments will serve

as templates

So, how what would be the mathematical

relationship between the number of
cycles and the amount of product?

#
=2
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Primer design

Overlap with the target gene: ~ 20 bp

Preferably start and end with a G or a C (3 H-bond; stronger than T=A)
Maximum length ~ 50 bp; longer primer is possible but is usually more
expensive
Insert sequences of restriction endonuclease (RE) sites

Insert sequences if required for RE activity

Remember: RE sites should be UNIQUE no overlap with any sequence with target
gene
To check which enzymes cut a DNA sequence of the target gene, insert its
sequence to: http://tools.neb.com/NEBcutter2/index.php

Some RE needs cushion

Check the followings:

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Melting temperature of the primer (<70C)

Formation of loop structure and dimer
Online tool: http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/

Why the 20-basepair overlap?

Hint: probability
What is frequency of the same sequence appears
elsewhere in the chromosome/plasmid?

A T G G A T A C C A
basepair# 1 2 3 4 5 6 7 8 9 .
4x4x4x4x4x4x4x4x4x4
#

22

=4

Primer design (continued)

Forward primer

Binds to the template strand

Sequence is the same as the 5 end of the gene

Reverse primer

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Binds to the coding strand

Sequence is complimentary to the 3 end of the gene
Usually contains a stop codon (e.g. taa)

Primer design exercise

LOCUS
E2WT_CODA
783 bp
DEFINITION E2WT_CODA 780 bp 5-->3 + taa (stop)
ORIGIN
1 ATGCTGTCTG TTCCTGGTCC CGCTGCTGCA GAGGAAAAGG CTGCTCCAGC GGCTGCGAAA
61 CCGGCTACTA CTGAAGGTGA ATTCCCTGAA ACCCGTGAAA AAATGTCTGG TATCCGTCGT
--truncated-721 AAAGCCCTGA ACCACATCAA ACGTCTGCTG TCCGACCCGG AACTGCTGCT GATGGAAGCT
781 taa
//

Expected to add the following RE sites at the 5-end of the primers:

NdeI (CATATG) for forward and BamHI (GGATCC) for reverse

Forward primer (5 3):

Reverse primer (5 3): gga

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cat ATG CTG TCT GTT CCT GGT CC

tcc tta AGC TTC CAT CAG CAG CAG TTC

Measuring DNA concentration & purity

Using absorbance spectroscopy at 260 nm

To ensure the numbers are useful, the A260 reading should be within the
instrument's linear range (generally 0.11.0).

A260 of 1.0 = 50g/ml pure dsDNA

= 260 /320

(260 320 )
=
(280 320 )

DNA is not the only species that absorbs at 260 nm

Good-quality DNA will have an A260/A280 ratio of 1.72.0;

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= 260 320 50/

Ratio <1.7 means that there may be some contaminants

Source: www.promega.com

Gel electrophoresis:
to confirm PCR product (and protein)

The composition is semisolid, open meshwork of linear

strands

For DNA: agarose

For protein: polyacrylamide gel electrophoresis (PAGE)
Usually expressed in x%; Higher % = smaller pores

1.0% agarose (x g/100 ml) gel resolves 600-20,000 bp DNA

fragments
10% (acrylamide/bisacrylamide) polyacrylamide gel resolves 20-200
kDa protein

Voltage is applied to separate DNA mixture in the

sample
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Gel electrophoresis:
DNA molecules can be separated by size

Gel electrophoresis:
Determining molecular mass

Separation is based on size

(-) negative

DNA backbone is negatively charged

For most applications, protein samples are treated with an

anionic detergent, sodium dodecyl sulfate (SDS)

SDS binds and dissociates most multichain proteins

Provides uniform negative charge to the proteins
(+) positive

Progress is monitored using tracking dye

Molecular mass can be estimated by comparing the relative

mobility (Rf) to standard

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The relationship is logarithmic

Image sources: wikipedia & Bio-Rad

PCR can be used to obtain genomic or cDNA

clones

PCR can be used to detect the presence of a

viral genome in a blood sample

PCR is used in forensic science

10_30_2_PCR_forensic.jpg

Digestion

After PCR, both PCR product (the insert) and vector are
digested using restriction enzymes

This procedure produces compatible ends to allow

specific insertion of the insert into the vector

In the tube (30 l reaction):

Plasmid or insert
RE
Water

Incubate at recommended tempearture

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Restriction enzymes = nuclease

Enzyme that cuts specific DNA sequence (the

RE site)
Recognizes palindromic sequence

Two strands of DNA that are identical when

either is read in the same polarity, i.e. 5 to 3

Endonuclease
Exonuclease

Sticky end (has overhang)

5-phosphate extension
3-hydroxyl extension

Blunt end

Vector cleavage is usually followed by

alkaline phosphatase treatment to prevent
self-ligation
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EcoRI
Sticky end

HindII
Blunt end

Restriction nucleases cleave DNA at specific

nucleotide sequences

Examples of restriction endonucleases

Name

Sequence

Type of cut end

Notes

EcoRI

5-phosphate
overhang

One of the first type II

endonuclease characterized.
Isolated from E. coli

BamHI

5-phosphate

has some star activity; tends to

over digest

HindIII

5-phosphate

the first isolated in 1970

NdeI

5-phosphate

Contains ATG
Commonly used at the 5 end

PstI

3-hydroxyl

SmaI

Blunt end

Good sources of overview and enzyme finder:

http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/overview.asp#.UJvqc-QUlAI
http://www.neb.com/nebecomm/EnzymeFinder.asp#.UJu0SeQUlAI
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Ligation

Commonly used enzyme: T4 DNA ligase

Formation of phosphodiester bond between

the target gene (the insert) and the cleaved
vector

In the tube (10 l)

T4 DNA ligase

Phosphodiester bond

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i.e. insert is omitted and the volume is balanced

with water.
In the negative control tube, no ligation should
take place.

Target DNA
RE cleavage

RE cleavage
AP treatment

Linearized vector
Insert*
(Molar ratio between insert and vector = 3:1)
T4 DNA ligase
ATP (sometimes already included in the buffer)
Buffer
Water

*It is important to include a negative

control:

Plasmid DNA

Nick

Nick

Phosphodiester bond
Transformation
Host cells
RE = restriction endonuclease
AP = alkaline phosphatase

Ligation of digested DNA fragments

Cloning = vector replication

Vector replication is tied to cell division

As the cell is doubling its chromosome, the vector is also

doubled
Upon cell division, the vectors are also distributed into the
daughter cells

Introducing vectors into host cells

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Transformation: host is bacteria or yeast

Transduction: when viral vector is used
Transfection: host is mammalian cells

Transformation

Process of introducing vector into bacterial host

cells
Remember: the HOST is transformed
Only circular plasmid will be taken up by the host;
linearized (cut/cleaved) vector or insert will not.
Chemical

Use of chemical (i.e. rubidium chloride) to make the

cell membrane leaky
See supplementary info for complete protocol

Electrotransformation

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Use of electric pulse to make cell membrane leaky

Typical system: Bio-Rad Electroporation

Image source: www.bio-rad.com

Transformation of E. coli

In the tube:

Competent E. coli cells

Plasmid or ligation mixture

Basic protocol

Incubate on ice for 30 minutes

Heat shock 30 @ 42C
Incubate on ice for 2 minutes
Add growth medium containing high salt
Grow for 1 hour
Plate on LB containing chemical for selection

Antibiotic (e.g. ampicillin, tetracyclin, chloramphenicol, kanamycin, etc.)

Amino acid

Once the E. coli is transformed, it will then be plated and selected

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Plating
Negative control plate should not have many
colonies (much less compared to sample plate)
i.e. theoretically since water was used as insert
during the ligation step and the RE sites were
usually different, the vector or insert alone were
incapable in entering/replicated in the cell.
So, if you have colonies on the control plate, what may it
tell you?

Sample plate
Only clones that contain plasmids of interest
(positive clones) will be able to survive the selection

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Replication

Once positive clone is identified, plasmids

are further replicated in liquid culture

A cloned DNA fragment can be replicated inside a bacterial cell

Plasmid isolation is usually performed using commercially
available kit

DNA Sequencing

PCR may make some errors, so to confirm that the

sequence that was amplified (copied) is correct, the
plasmids are sent for sequencing.

The basic is by adding dideoxynucleotide (ddNTP) into

PCR mixture.

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When ddNTP is incorporated during strand elongation step,

the reaction is terminated

The enzymatic or dideoxy method is the most

commonly used technique for sequencing DNA

Differentially terminated fragments allow

nucleotide sequencing

DNA sequencing is now fully automated

Also check out Chromas Lite at http://www.technelysium.com.au/

Recombinant Protein Production

& Purification

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Recombinant protein production

Non-native protein
production is metabolically
costly for the host

There is a need to control

the production of the nonnative protein

Typical procedure

Sometimes the non-native

proteins are produced in
an inclusion body.

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Transformation of expression
vector
Overnight seed culture
Expression culture
Induction @ OD600 = 0.6-0.8
Harvest by low speed
(3000xg) centrifugation
Store cells at -80C

Growth curve
E. coli has doubling time (Td) of 20-30 minutes
2.5

Lag phase

Log/ exponential phase

Stationary/dead phase

2.0

OD600 (AU)

Induction
point

1.5

Host w/ vector
Host

1.0

0.5

0
0

Time (hr)
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Graph by Loh Zheng Yi, Hwa Chong, NRP

Promoter & inducer

Most promoter is repressed provides

control of non-native production

Repressed promoter prevents DNA

transcription
Addition of effector (also known as inducer)
will derepress the promoter, allowing
RNA polymerase to bind and start the
transcription
Some promoters are leaky repression
is not complete protein is still produced
without addition of inducer
Types of promoter: strong and weak

Induction = addition of chemical signal

(inducer) to start gene expression

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Commonly used inducer for lac promoter

derivative is IPTG (isopropyl -D-1thiogalactopyranoside)

Lactose

IPTG

Protein purification

Ammonium sulfate
precipitation

Adding (NH4)2SO4 (up to 4 M) to

expose hydrophobic patches on the
protein surface which induces
precipitation.
Precipitation can be reversed.

Chromatography

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Uses medium/resin (usually packed in

a column) that binds samples at a
particular condition.
The samples can be reversibly
attached and detached from the resin.

Protein chromatography
Protein property

Technique

Biorecognition / ligand specificity

Affinity (AC)

Charge

Ion exchange (IEX)

Hydrophobicity

Hydrophobic interaction (HIC)

Size

Gel filtration (GF); also known as size

exclusion (SEC)

AC is the most specific and can be

performed as a single-step purification.
Capture
Intermediate
Polish
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Note that GF is usually performed last

as it requires relatively pure protein.

General protocol for bench-top chromatography

Step 1:
Equilibrate
column

Step 2:
Bind
sample

At least 5 CV

Limited to the
capacity of the
column

Range from 0.5

to 10 ml;
depending on
the desired [P]

If the column is 1 ml, steps 1 and 3 require at least 5 ml.

Binding:

At least 5 CV

Step 4:
Elute
fractions

CV = column volume

Step 3:
Wash
unbound
sample

IEX usually the highest binding capacity

AC the most specific
SEC does not bind protein

Most of the times, buffers for steps 1-3 are the same while step 4
buffer contains competitive ions (for IEX) or lower [ion].
Higher fraction volume = more dilute sample
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Monitoring the progress of protein

purification

The progress of protein purification is usually monitored

using spectrophotometer at 280 nm.

A280 is due to the presence of aromatic amino acid

residues (Tyr, Trp, Phe) in the protein.
Buffer pump

Spect
Sample pump

61

Fraction
collector

Components of affinity medium

Affinity chromatography (AC)

Native protein does

not usually contain
tags but they can be
produced with tags by
producing it as fusion
protein at either the
N-terminus or Cterminus

GST-tag
His-tag
Strep-tag

Histidine tagging of protein as a

purification strategy

Ion exchange chromatography (IEX)

There are 2 types:

Because proteins have different pI-s, binding of

sample is pH dependent.

Anion exchange (+)charge

Cation exchange (-)charge

Binding
-

pH > pI protein is (-)charge; binds to anion

exchange
pH < pI protein is (+)charge; binds to cation
exchange

Protein is eluted from the resin by competition

with charge opposite to the resin.

The most commonly used salt is NaCl

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For anion exchange, Cl- competes with the bound protein

For cation exchange, Na+ competes with the bound
protein

Elution
ClClClCl-

Hydrophobic interaction chromatography

(HIC)

Based on the presence of hydrophobic

patches on the protein

Usually hydrophobic residues are buried

Adding (NH4)2SO4 provides competition in
water ordering in the solution

Water molecules hydrate the ions and provides

less hydrophilic environment near the protein
surfaces, hence the exposure of hydrophobic
patches

Protein is bound at higher and eluted at

lower [(NH4)2SO4]
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Binding
NH42+
SO42NH42+
SO42-

Elution

Size exclusion chromatography (SEC)

The resin is usually porous

Proteins are selected based on the ability of proteins

to pass through a tortuous path
Bigger protein cannot enter the path within the resin and
elute at the void volume

Void volume is the volume that is not occupied by the resin

inside the column
Vcolumn = Vvoid + Vresin

Smaller protein spend longer time in the resin longer

residence time, larger elution volume
Elution time (volume) for larger protein < smaller protein
Elution volume = flow rate (ml/min) x elution time (min)

Relationship between the elution volume and

molecular mass is logarithmic similar to the Rf in gel
electrophoresis

Molecular mass of the unknown protein is determined

by comparing its elution volume to standard proteins
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A280

Vol or time

Learning Points

Protein is made of amino acids

The interaction between amino acids results in 1, 2, 3,
4 structures
Folding is determined by the sequence until it reaches
thermodynamic equilibrium
The conformation is stabilized by different forces
The structure can be determined using X-ray
crystallography and NMR
Parameters that describe proteins
Allostery effect is the change of protein conformation
due to binding of a certain molecule
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How to Study for The Midterm

70

Last Lecture of the Semester

Lets have some fun now!
How to Write Like a Scientist An assay by Adam Ruben
The Story of an ATPSynthase Poster

71

An Excerpt from How to Write Like a Scientist

By Adam Ruben (published on Science Careers on March 23, 2012)

I didnt know whether to take my Ph.D. advisers remark as a compliment. You dont write like a
scientist, he said, handing me back the progress report for a grant that I had written for him. In
my dream world, tears would have come to his eyes, and he would have squealed, You write like
a poet!
In reality, though, he just frowned. He had meant it as a criticism. I dont write like a scientist, and
apparently thats bad. I asked for an example, and he pointed to a sentence on the first page. See
that word? he said. Right there. That is not science.
The word was lone, as in PvPlm is the lone plasmepsin in the food vacuole of Plasmodium
vivax. It was a filthy word. A non-scientific word. A flowery word, a lyrical word, a word worthy
of -- ugh -- an MFA student.
I hadnt meant the word to be poetic. I had just used the word only five or six times, and I
didnt want to use it again. But in his mind, lone must have conjured images of PvPlm perched
on a cliffs edge, staring into the empty chasm, weeping gently for its aspartic protease
companions. Oh, the good times they shared. Afternoons spent cleaving scissile bonds. Lazy
mornings decomposing foreign proteins into their constituent amino acids at a nice, acidic pH.
Alas, lone plasmepsin, those days are gone.
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