Presentation is loading. Please wait.

Presentation is loading. Please wait.

Introduction to Bioengineering Lecture #1: Biotechnology

Similar presentations

Presentation on theme: "Introduction to Bioengineering Lecture #1: Biotechnology"— Presentation transcript:

1 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)

2 Ancient Biotechnology

3 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

4 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]

5 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]

6 What is a gene? Human chromosomes consist of linear DNA molecules
to 1000 bases Human chromosomes consist of linear DNA molecules Genes are specific base sequences on the DNA molecule Genes are the encoded instructions for manufacturing proteins Sugar-phosphate backbone Nucleoside base (A,T,G or C) [Image from McKee & McKee, Biochemistry an Introduction]

7 What is a DNA? Deoxyribonucleic acid 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] [Image from SR Barnum Biotechnology an Introduction] } double ring - purine } single ring-pyrimide

8 How’s it get it’s structure?
[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

9 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

10 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]

11 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

12 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

13 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

14 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

15 Protein Structure Primary structure: Secondary structure:
[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

16 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]

17 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

18 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

19 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

20 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

21 Gene Transfer Modes Microinjection Embryonic stem cell transfer
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]

22 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

23 Viral vectors Candidate viruses
[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!

24 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

25 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

26 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.

27 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

28 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]

29 [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.

30 Bioprocessing

31 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

32 Fermentation System Use aerobic microorganisms
[Image from SR Barnum Biotechnology an Introduction] 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

33 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

34 Notice as strain rate increases so does degradation rate!

35 Notice increasing plasmid size increases degradation rate!

36 DU Bioengineering Group [Circumventing shear-induced DNA degradation]

Download ppt "Introduction to Bioengineering Lecture #1: Biotechnology"

Similar presentations

Ads by Google