Introduction to Bioengineering


22 Οκτ 2013 (πριν από 3 χρόνια και 5 μήνες)

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Introduction to Bioengineering

Lecture #1: Biotechnology

Biotechnology is any technique that uses living
organisms or substances from those organisms to make
or modify a product, to improve plants or animals, or to
develop microorganisms for specific use.

Contributions include:

virus resistant crops/animals

diagnostics for detecting genetic diseases

recombinant vaccine such as for malaria

gene therapies

genetic diversity for conservation

microorganisms to clean up toxic
waste (oil spills)

Ancient Biotechnology

Modern Biotechnology &

Modern biotechnology is directed a therapeutic

Our ability to manipulate living organisms
precisely requires knowledge of :

(1) Cell structure/behavior

(2) Biochemical reactions

(3) Genetic code

A result of 300 years of knowledge

The Cell

All living things are composed of either:

prokaryotic cells


those lacking a nucleus such as
bacteria where the genetic information is found in
nucleoid matter

eukaryotic cells


complex cells having a nucleus
similar to the animal cell shown here.

Both contain a chromosome

prokaryotic cells


the chromosome is a circular DNA
molecule called a plasmid

eukaryotic cells


the chromosome is a long linear DNA

[Image from McKee & McKee,
Biochemistry an Introduction

The Cell

Plasma membrane


composed of lipid and protein molecules.

Lipids provide the structure

proteins act as receptors (binding to specific molecules) changing cell

perform transport mechanisms


composed largely of

Contains hereditary information

Regulates cell function

[Image from McKee & McKee,
Biochemistry an Introduction

What is a gene?

Human chromosomes consist of
linear DNA molecules

Genes are specific base
sequences on the DNA molecule

Genes are the encoded
instructions for manufacturing

Nucleoside base

(A,T,G or C)

Gene =100

to 1000 bases

[Image from McKee & McKee,
Biochemistry an Introduction



What is a DNA?

cid is a long polymer
chain consisting of repeating units called

3 Basic Components

Deoxyribose or sugar

Phosphate group

Nitrogen containing base

4 Nitrogen Bases

Adenine [A]

Guanine [G]

Tymine [T]

Cytosine [C]

double ring


single ring

[Image from SR Barnum
Biotechnology an Introduction

How’s it get it’s structure?

Bases project inwards from sugar
phosphate backbone

Hydrogen bonds between opposite bases
hold 2
strands together

Links between the repeating unit at the
number 5 to 3 carbons give helical structure

Purines always link to pyrimides

Deoxyribonucleotided every 3.4Å

Each helical turn is 34 Å

Double helix is 20 Å in diameter

[Image from SR Barnum
Biotechnology an Introduction

How is the information transferred to protein?

The enzyme RNA polymerase reads a specific nucleotide sequence

(gene) from the DNA template while proteins called transcription
factors facilitate the copying

Copies are made in the form of Ribonucleic acid (RNA)

RNA resembles DNA except:

base thymine [T] and adenine [A] are replace with uracil [U]

pentose sugars are ribose molecules rather than deoxyribose

single stranded molecule

Building Proteins

Each amino acid forming a protein is
specified by a triplet of bases on the

[Image from SR Barnum
Biotechnology an Introduction


Protein molecules perform most of life’s functions and make up the majority
of cellular structure

Proteins are large organic compounds

enzymes (catylize reactions)
hormones (regulate activities)

antibodies (immune response)
movement proteins

structural proteins (determine shape of cell)

transcription or transport proteins

Proteins are composed of amino acids joined by covalent bonds called
“peptide bonds”

20 standard amino acids

Massive variety in function result from the numerous amino acid
combinations possible, length, and 3
D conformation

Peptides = less than 50 amino acids in length

Proteins or polypetides = larger than 50 amino acids in length

Amino Acids

Each amino acid has the same basic backbone with an unique
side group (R) to determine characteristics

4 Main Classes of Side Groups


Hydrophobic, play a role in 3
D structure, and can
catalyze reactions



Hydrophilic, capable of hydrogen bonding, play role in
structure and stability


) charge/polar]

) Basic

[(+) charge/polar]

form ionic bonds and play a catalytic activity



Altered genes manufacture faulty proteins that are unable to carry out
normal function (this is called a genetic disorder)

Initial binding to the wrong location

fragmented DNA/RNA strands

mutation in the codon sequence




Biotechnology is any technique that uses living organisms or
substances from those organisms to make or modify a product, to improve plants or
animals, or to develop microorganisms for specific use.

Possible therapeutic solutions:

(1) Dose patient with missing proteins

(2) Does patient with specific RNA to synthesis desired proteins

(3) Gene Therapy

Protein Therapy

The major problem with protein therapy is the cost of
large repetitive dosing [i.e., insulin]

Proteins are extremely unstable and therefore lose
therapeutic activity during processing and delivery

To understand the magnitude of this problem we must
discuss the structure of proteins

Protein Structure

Primary structure:

amino acid sequence

determined by DNA

Secondary structure:

Stabilized by hydrogen bonds between backbone and R


rigid rod formed by polypeptide chain twist

3.6 amino acids/turn, pitch = 54 nm

groups face outwards


Two or more polypeptide chain segments line up side by side.

Fully extended sheet

Tertiary structure:

D conformation, consequence of side chain interaction

hydrophobic, electrostatic, hydrogen bonding, covalent bonding

[Image from McKee & McKee,
Biochemistry an Introduction

Protein Structure

The biological activity of proteins is often regulated by small
ligands binding to proteins and inducing specific confirmation

Therefore changes in the interaction between protein subunits
can substantially impact bioactivity

Denaturing agents include

strong acids or bases
reducing agents

organic solvents

high salt concentrations
heavy metals

temperature changes
mechanical stress

Ligand = molecules that bind to specific
sites on large molecules

[Image from McKee & McKee,
Biochemistry an Introduction

Protein Engineering

Protein engineering, the process of changing a protein in a
predictable precise manner to bring about a change in function,
is closely linked to genetic engineering

Most research has been directed to using physical property data
to develop computerized models that predict protein structure
and function in order to modify existing enzymes and antibodies


Catalyze reactions

Work has focused on isolating the genes that produce useful enzymes

Work has also focused on modification of existing enzymes to make them
more stable


Bind to specific chemical structures (antigens)

Work has focused on custom design antibodies to attach to specific types
of cells such as cancer in order to improve drug delivery methods

Therapeutic RNA

Antisense technology

Antisense technology involves the inhibition of gene
expression by blocking translation to mRNA into protein

This is achieved by antisense RNA binding to mRNA

Antisense RNA are exactly complementary in sequence and
opposite in polarity to the normal mRNA

Such complementary binding generates a double
RNA molecule that cannot be translated into a protein, and
are quickly degraded in the cell cytoplasm

What is gene therapy?

Gene therapy is the technique(s) for correcting
defective genes responsible for disease

Approaches included:

Inserting a normal gene into a nonspecific location
(most common)

Swapping the abnormal gene for a normal gene

Repairing the abnormal gene

Turning off or on specific gene

How does gene therapy work?

[Inserting a normal gene]

Delivers the therapeutic
gene to the target cell

The gene must then
translocate into the cell

[Video from]

Gene Transfer Modes


Foreign gene is injected before the first cell
division occurs so all the cells of the
organism harbor the gene (transgenic
animals or plants)

Embryonic stem cell transfer

ES are isolated and cultured in vitro with a
specific gene. Transformed ES cell are
microinjected back into the embryo

Gene targeting

Is the insertion of DNA into a specific
chromosomal location. This is achieved
using viral and non viral vectors

Viral vectors

Non viral vectors

[Image from SR Barnum
Biotechnology an Introduction

Viral vectors

Viruses have evolved a way of
encapsulating and delivering genes
to human cells in a pathogenic

Scientist are attempting to take
advantage of natures delivery

Viruses would be genetically altered
to carry the desired normal gene and
turn off the natural occurring disease
within the virus.

[Video from]

Viral vectors

Candidate viruses

Retroviruses [e.g., HIV]

RNA virus that infect humans

Ability to target genes

Dividing cells only

Risk of mutagenesis


Adenoviruses [e.g., virus that causes common cold]

Not highly pathogenic

Do not integrate into the genome

Can be aerosolized

Transient gene expression


associated virus [inserts only at chromosome 19]

Herpes simplex virus [e.g., virus that causes cold sores]

Viral vectors will only be effective a few times before the
body becomes resistant!

[Image from McKee & McKee,
Biochemistry an Introduction

viral vectors

viral vectors will provide
unlimited access to the human
cell, but efficient delivery is
the critical issue

Optimizing delivery is being
achieved in two ways or a
combination of both:

(1)Smaller molecule size decreases
resistance to nuclear transport

Chemical linking of DNA
decreases size

Supercoiled structure is smallest

Aides in activating receptor


Open Circle


viral vectors

(2) Exterior shell that activates receptor
molecules or promotes transport

Encapsulation of DNA within lipid sphere

Chemical linking of DNA

[Image from]

Aqueous DNA solution

Lipid bilayer

Current status of gene therapy?

Gene therapy is still considered experimental as the FDA
has not approved any for commercial sale

The first clinical trials started in 1990 and little progress
has been made

Major set backs include:

The death of Jesse Gelsinger in 1999 from multiple organ failure
caused by a sever immune response to the adenovirus carrier

The appearance of leukemia
like conditions in two French children
successfully treated by gene therapy for X
linked severe combined
immunodeficiency disease. The retroviral vector employed
originally contained a leukemia gene sequence that had been

What factors keep gene therapy from
becoming a reality?

lived nature:

Problems with integrating therapeutic DNA into the genome and
rapidly dividing nature of cells prevent any long
term benefits

Therefore patients must undergo multiple rounds of gene therapy

Immune response:

The body is designed to attack foreign matter, thus the body itself is
designed to make gene therapy less effective.

Immune system response is enhanced on repeated exposure

Gene delivery vehicles:

Beyond toxicity, immune and inflammatory response there is some
concern viral vectors may recover its ability to cause disease

viral alternatives have not yet become as efficient in gene

What factors keep gene therapy from
becoming a reality?

Multigene disorders

Heart disease, high blood pressure,
Alzheimer’s, arthritis and diabetes are all
cause by the combined effects of variations in
many genes

Large scale manufacturing:

The growth, separation, purification and
encapsulation in a delivery vehicle is a
complicated and expensive process

Some manufacturing steps degrade DNA

(1) considerable quantity of therapeutic is lost

(2) degraded DNA is an difficult impurity to


Open Circle


[Images from McKee & McKee,
Biochemistry an Introduction

[Circumventing shear
induced DNA degradation]


While delivery efficiency is continually being
improved, little attention has been paid to critical
bioprocessing issues that drive production costs and could
prevent this new class of pharmaceuticals from becoming a


Several current processing steps fragment
plasmids that render them biologically in affective and
provide a source of contamination.


To date no one has correlated degradation rate to
shear stress or strain rate in a way that is efficient for
design.Our goal is develop a correlation of degradation rate
to non
dimensional strain rate where the non
parameter accounts for molecular size and flexibility
effects as well as fluid properties.



The development of the fermentation process, provides
the scientific foundation for many industrial processes
and the development of modern biotechnology


Cholesterol can be converted to estrogen through the addition
of an OH group to the cholesterol ring. Microorganisms can
readily carry out the hydroxylation and dehydroxylation

Shifting the direction of a cells metabolism can produce large

of a specific amino acid or metabolite

Fermentation provides the cell growth required to amplify a
specific plasmid

Fermentation System

Use aerobic microorganisms

Need oxygen, consistent pH and
temperature, nutrients and anti
foaming agents

Oxygen supplied by bubbling or

Cells and liquids are separated by
sedimentation and filtration after

enzymes collected from
liquid phase

proteins and other cell product are
purified after cells have been lysed

[Image from SR Barnum
Biotechnology an Introduction

DU Bioengineering Group

[Circumventing shear
induced DNA degradation]

We are investigating the degradation
rate of plasmid DNA by shear stress in
pipe flow

We vary flow rate, pipe diameter, pipe
surface roughness, residence time,
fluid viscosity, and plasmid size

Notice as strain rate increases so does degradation rate!

Notice increasing plasmid size increases degradation rate!

DU Bioengineering Group

[Circumventing shear
induced DNA degradation]