Introduction to Bioengineering

lowlytoolboxBiotechnology

Oct 22, 2013 (3 years and 11 months ago)

100 views

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 &
Therapeutics



Modern biotechnology is directed a therapeutic
effect



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:

(a)
prokaryotic cells

-

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

(b)
eukaryotic cells

-

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

Both contain a chromosome

(a)
prokaryotic cells

-

the chromosome is a circular DNA
molecule called a plasmid

(b)
eukaryotic cells

-

the chromosome is a long linear DNA
strand

[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
activity


perform transport mechanisms

Nucleus


composed largely of
DNA


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
proteins

Nucleoside base

(A,T,G or C)

Gene =100

to 1000 bases

[Image from McKee & McKee,
Biochemistry an Introduction
]

Sugar
-
phosphate


backbone

What is a DNA?

D
eoxyribo
n
ucleic
a
cid is a long polymer
chain consisting of repeating units called
deoxyribonucleotides


3 Basic Components


Deoxyribose or sugar


Phosphate group


Nitrogen containing base


4 Nitrogen Bases


Adenine [A]


Guanine [G]


Tymine [T]


Cytosine [C]


}
double ring
-

purine

}
single ring
-
pyrimide

[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
RNA


[Image from SR Barnum
Biotechnology an Introduction
]

Proteins


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

(1)
Non
-
polar/neutral
--

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

(2)
Polar/neutral

--

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

(3)
Acidic

[(
-
) charge/polar]

(4
) Basic

[(+) charge/polar]
--

form ionic bonds and play a catalytic activity

Problem
--

Solution


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


Example:

THE BIG RED DOG WAS SAD

HEB IGR EDD OGW ASS AD



Remember,
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
-
groups

(a)
a
-
helix


rigid rod formed by polypeptide chain twist


3.6 amino acids/turn, pitch = 54 nm


R
-
groups face outwards

(b)
b
-
sheet


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


Fully extended sheet



Tertiary structure:


3
-
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
changes.



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



Denaturing agents include


-
strong acids or bases
-
reducing agents


-
organic solvents
-
detergents


-
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


Enzymes


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


Antibodies


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
-
stranded
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
nucleus



[Video from www.biosciednet.org/portal]

Gene Transfer Modes

Microinjection


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

1.
Viral vectors

2.
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
manner.



Scientist are attempting to take
advantage of natures delivery
system.



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



[Video from www.biosciednet.org/portal]

Viral vectors

Candidate viruses


Retroviruses [e.g., HIV]


RNA virus that infect humans


Ability to target genes


Dividing cells only


Risk of mutagenesis


8kb


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


Not highly pathogenic


Do not integrate into the genome


Can be aerosolized


Transient gene expression


8
-
10kb


Adeno
-
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
]

Non
-
viral vectors


Non
-
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
size


Aides in activating receptor
molecules


Supercoiled


Open Circle

Linear

Non
-
viral vectors


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


Encapsulation of DNA within lipid sphere


Chemical linking of DNA


[Image from http://web.bham.ac.uk/can4psd4/nonviral/polymer.html]

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
molecule


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
scrambled.


What factors keep gene therapy from
becoming a reality?


Short
-
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


Non
-
viral alternatives have not yet become as efficient in gene
delivery

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
separate


Supercoiled


Open Circle

Linear

[Images from McKee & McKee,
Biochemistry an Introduction
]

[Circumventing shear
-
induced DNA degradation]

Motivation:

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
reality.


Background:

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


Objective:

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
-
dimensional
parameter accounts for molecular size and flexibility
effects as well as fluid properties.

Bioprocessing

Fermentation

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


Example:


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
amounts

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
agitation


Cells and liquids are separated by
sedimentation and filtration after
harvesting


metabolites
/
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]