Presentation on theme: "Introduction to Bioengineering Lecture #1: Biotechnology"— Presentation transcript:
1Introduction 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/animalsdiagnostics for detecting genetic diseasesrecombinant vaccine such as for malariagene therapiesgenetic diversity for conservationmicroorganisms to clean up toxic waste (oil spills)
3Modern Biotechnology & Therapeutics Modern biotechnology is directed a therapeutic effectOur ability to manipulate living organisms precisely requires knowledge of :(1) Cell structure/behavior(2) Biochemical reactions(3) Genetic codeA result of 300 years of knowledge
4The 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]
5The Cell Plasma membrane - composed of lipid and protein molecules. Lipids provide the structureproteins act as receptors (binding to specific molecules) changing cell activityperform transport mechanismsNucleus – composed largely of DNAContains hereditary informationRegulates cell function[Image from McKee & McKee, Biochemistry an Introduction]
6What is a gene? Human chromosomes consist of linear DNA molecules to 1000 basesHuman chromosomes consist of linear DNA moleculesGenes are specific base sequences on the DNA moleculeGenes are the encoded instructions for manufacturing proteinsSugar-phosphatebackboneNucleoside base(A,T,G or C)[Image from McKee & McKee, Biochemistry an Introduction]
7What is a DNA?Deoxyribonucleic acid is a long polymer chain consisting of repeating units called deoxyribonucleotides3 Basic ComponentsDeoxyribose or sugarPhosphate groupNitrogen containing base4 Nitrogen BasesAdenine [A]Guanine [G]Tymine [T]Cytosine [C][Image from SR Barnum Biotechnology an Introduction]} double ring - purine} single ring-pyrimide
8How’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 backboneHydrogen bonds between opposite bases hold 2-strands togetherLinks between the repeating unit at the number 5 to 3 carbons give helical structurePurines always link to pyrimidesDeoxyribonucleotided every 3.4ÅEach helical turn is 34 ÅDouble helix is 20 Å in diameter
9How 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 copyingCopies 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
10Building ProteinsEach amino acid forming a protein is specified by a triplet of bases on the RNA[Image from SR Barnum Biotechnology an Introduction]
11ProteinsProtein molecules perform most of life’s functions and make up the majority of cellular structureProteins are large organic compounds-enzymes (catylize reactions) hormones (regulate activities)-antibodies (immune response) movement proteins-structural proteins (determine shape of cell)-transcription or transport proteinsProteins are composed of amino acids joined by covalent bonds called “peptide bonds”20 standard amino acidsMassive variety in function result from the numerous amino acid combinations possible, length, and 3-D conformationPeptides = less than 50 amino acids in lengthProteins or polypetides = larger than 50 amino acids in length
12Amino AcidsEach amino acid has the same basic backbone with an unique side group (R) to determine characteristics4 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
13Problem -- SolutionAltered genes manufacture faulty proteins that are unable to carry out normal function (this is called a genetic disorder)Initial binding to the wrong locationfragmented DNA/RNA strandsmutation in the codon sequenceExample:THE BIG RED DOG WAS SADHEB IGR EDD OGW ASS ADRemember, 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
14Protein TherapyThe 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 deliveryTo understand the magnitude of this problem we must discuss the structure of proteins
15Protein Structure Primary structure: Secondary structure: [Image from McKee & McKee, Biochemistry an Introduction]Protein StructurePrimary structure:amino acid sequencedetermined by DNASecondary structure:Stabilized by hydrogen bonds between backbone and R-groups(a) a-helixrigid rod formed by polypeptide chain twist3.6 amino acids/turn, pitch = 54 nmR-groups face outwards(b) b-sheetTwo or more polypeptide chain segments line up side by side.Fully extended sheetTertiary structure:3-D conformation, consequence of side chain interactionhydrophobic, electrostatic, hydrogen bonding, covalent bonding
16Protein StructureThe 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 bioactivityDenaturing agents include-strong acids or bases reducing agents-organic solvents detergents-high salt concentrations heavy metals-temperature changes mechanical stressLigand = molecules that bind to specific sites on large molecules[Image from McKee & McKee, Biochemistry an Introduction]
17Protein EngineeringProtein engineering, the process of changing a protein in a predictable precise manner to bring about a change in function, is closely linked to genetic engineeringMost 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 antibodiesEnzymesCatalyze reactionsWork has focused on isolating the genes that produce useful enzymesWork has also focused on modification of existing enzymes to make them more stableAntibodiesBind 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
18Therapeutic RNA Antisense technology Antisense technology involves the inhibition of gene expression by blocking translation to mRNA into proteinThis is achieved by antisense RNA binding to mRNAAntisense RNA are exactly complementary in sequence and opposite in polarity to the normal mRNASuch complementary binding generates a double-stranded RNA molecule that cannot be translated into a protein, and are quickly degraded in the cell cytoplasm
19What is gene therapy?Gene therapy is the technique(s) for correcting defective genes responsible for diseaseApproaches included:Inserting a normal gene into a nonspecific location (most common)Swapping the abnormal gene for a normal geneRepairing the abnormal geneTurning off or on specific gene
20How does gene therapy work? [Inserting a normal gene] Delivers the therapeutic gene to the target cellThe gene must then translocate into the cell nucleus[Video from
21Gene 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 transferES are isolated and cultured in vitro with a specific gene. Transformed ES cell are microinjected back into the embryoGene targetingIs the insertion of DNA into a specific chromosomal location. This is achieved using viral and non viral vectorsViral vectorsNon viral vectors[Image from SR Barnum Biotechnology an Introduction]
22Viral vectorsViruses 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
23Viral vectors Candidate viruses [Image from McKee & McKee, Biochemistry an Introduction]Viral vectorsCandidate virusesRetroviruses [e.g., HIV]RNA virus that infect humansAbility to target genesDividing cells onlyRisk of mutagenesis8kbAdenoviruses [e.g., virus that causes common cold]Not highly pathogenicDo not integrate into the genomeCan be aerosolizedTransient gene expression8-10kbAdeno-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!
24Non-viral vectors Supercoiled Open Circle Non-viral vectors will provide unlimited access to the human cell, but efficient delivery is the critical issueOptimizing delivery is being achieved in two ways or a combination of both:(1)Smaller molecule size decreases resistance to nuclear transportChemical linking of DNA decreases sizeSupercoiled structure is smallest sizeAides in activating receptor moleculesLinear
25Non-viral vectors(2) Exterior shell that activates receptor molecules or promotes transportEncapsulation of DNA within lipid sphereChemical linking of DNALipid bilayerAqueous DNA solution[Image from
26Current status of gene therapy? Gene therapy is still considered experimental as the FDA has not approved any for commercial saleThe first clinical trials started in 1990 and little progress has been madeMajor set backs include:The death of Jesse Gelsinger in 1999 from multiple organ failure caused by a sever immune response to the adenovirus carrier moleculeThe 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.
27What 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 benefitsTherefore patients must undergo multiple rounds of gene therapyImmune 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 exposureGene delivery vehicles:Beyond toxicity, immune and inflammatory response there is some concern viral vectors may recover its ability to cause diseaseNon-viral alternatives have not yet become as efficient in gene delivery
28What factors keep gene therapy from becoming a reality? Multigene disordersHeart disease, high blood pressure, Alzheimer’s, arthritis and diabetes are all cause by the combined effects of variations in many genesLarge scale manufacturing:The growth, separation, purification and encapsulation in a delivery vehicle is a complicated and expensive processSome manufacturing steps degrade DNA(1) considerable quantity of therapeutic is lost(2) degraded DNA is an difficult impurity to separateSupercoiledOpen CircleLinear[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.
31FermentationThe development of the fermentation process, provides the scientific foundation for many industrial processes and the development of modern biotechnologyExample: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 dehydroxylationShifting the direction of a cells metabolism can produce large amounts of a specific amino acid or metaboliteFermentation provides the cell growth required to amplify a specific plasmid
32Fermentation System Use aerobic microorganisms [Image from SR Barnum Biotechnology an Introduction]Use aerobic microorganismsNeed oxygen, consistent pH and temperature, nutrients and anti-foaming agentsOxygen supplied by bubbling or agitationCells and liquids are separated by sedimentation and filtration after harvestingmetabolites/enzymes collected from liquid phaseproteins and other cell product are purified after cells have been lysed
33DU Bioengineering Group [Circumventing shear-induced DNA degradation] We are investigating the degradation rate of plasmid DNA by shear stress in pipe flowWe vary flow rate, pipe diameter, pipe surface roughness, residence time, fluid viscosity, and plasmid size
34Notice as strain rate increases so does degradation rate!