Transcript Jet-swirl nozzle design for producing nanoscale polymer

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:
• gene therapies
• virus resistant crops/animals
• diagnostics for detecting genetic diseases • genetic diversity for conservation
• recombinant vaccine such as for malaria • microorganisms to clean up toxic
waste (oil spills)
Ancient Biotechnology
10,000 years ago
Farmers collected the seed of wild plants with the
most desirable traits and set them aside for planting
the following year. This activity, called artificial
selection, eventually produced new varieties.
2000 B.C.
The domestication of dogs, cattle and sheep
1800 B.C.
6000 B.C.
4000 B.C.
Fermented food and beverage.
[Fermentation is a microbial process which
enzymatically controlled transformations of organic
compounds occur]
Juice to wine
Bread
Beer
Yogurt
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?
Gene =100
• Human chromosomes consist of to 1000 bases
linear DNA molecules
• Genes are specific base
sequences on the DNA molecule
Sugar-phosphate
• Genes are the encoded
backbone
instructions for manufacturing
proteins
Nucleoside base
(A,T,G or C)
[Image from McKee & McKee, Biochemistry an Introduction]
What is a DNA?
Deoxyribonucleic acid is a long polymer
chain consisting of repeating units called
[Image from SR Barnum Biotechnology an Introduction]
deoxyribonucleotides
3 Basic Components
– Deoxyribose or sugar
– Phosphate group
– Nitrogen containing base
4 Nitrogen Bases
–
–
–
–
Adenine [A]
} double ring - purine
Guanine [G]
Tymine [T]
Cytosine [C] } single ring-pyrimide
[Image from SR Barnum Biotechnology an Introduction]
How’s it get it’s structure?
• Bases project inwards from sugarphosphate 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
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
H
H3N
O
 C  C  O
R
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
[Image from McKee & McKee, Biochemistry an Introduction]
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
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
-organic solvents
-high salt concentrations
-temperature changes
-reducing agents
-detergents
-heavy metals
-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
–
1.
2.
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
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]
[Image from McKee & McKee, Biochemistry an Introduction]
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!
Non-viral vectors
Supercoiled
Open Circle
• 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
Linear
Non-viral vectors
(2) Exterior shell that activates receptor
molecules or promotes transport
• Encapsulation of DNA within lipid sphere
• Chemical linking of DNA
Lipid bilayer
Aqueous DNA solution
[Image from http://web.bham.ac.uk/can4psd4/nonviral/polymer.html]
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
Supercoiled
Open Circle
• 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
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
[Image from SR Barnum Biotechnology an Introduction]
• Use aerobic microorganisms
• Need oxygen, consistent pH and
temperature, nutrients and antifoaming 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
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!
250.00
Rate Constant
200.00
20 cm (ID = 0.508 mm)
20 cm (ID = 0.254 mm)
25 cm (ID = 0.254 mm)
30 cm (ID = 0.254 mm)
15 cm (0.254 mm)
10 cm (0.508 mm)
15 cm (0.508 mm)
25 cm (0.508 mm)
30 cm (0.508 mm)
30 cm G (0.25 mm)
30 cm G (0.53 mm)
50 cm G (0.25 mm)
50 cm G (0.53 mm)
150.00
100.00
50.00
0.00
0.0E+00 1.0E+05 2.0E+05 3.0E+05 4.0E+05 5.0E+05 6.0E+05
Average G [1/s]
Notice increasing plasmid size increases degradation rate!
% Supercoil Structure
100%
5.0 kB
9.8 kB
80%
Linear (5.0 kB)
Linear (9.8 kB )
60%
y = -0.0512x + 0.7428
2
R = 0.9929
40%
20%
y = -0.2399x + 0.8431
R2 = 0.9912
0%
0
3
6
9
Time (min)
12
15
DU Bioengineering Group
[Circumventing shear-induced DNA degradation]