THE RAW MATERIALS OF BIOTECHNOLOGY Learning Outcomes Identify

THE RAW MATERIALS OF BIOTECHNOLOGY

Learning Outcomes Identify the levels of biological organization and explain their relationships Describe cell structure and its significance in biotechnology research and product development Discuss the types of organisms researched and the types of cells grown and studied in biotechnology facilities plus the product with which they are associated

Learning Outcomes Distinguish between the cellular organization of prokaryotic and eukaryotic cells List the 4 main classes of macromolecules and describe their structure and function Define genetic engineering and identify products created with this technology Explain the Central Dogma of Biology and its importance in genetic engineering

Vocabulary Fluorometer Organism Cell Escherichia coli Multicellular Cytology Anatomy Physiology Respiration Unicellular Chlorophyll Photosynthesis Steroids nucleotide Tissue Organ Protein Eukaryote Protist Organelles Mitochondria Sugar Starch Nucleic acid Pancreas Hormone R group ribonuicleic acid

Vocabulary Chloroplast Cytoplasm Lysosome Ribosome Cell wall Cellulose Plasma membrane Glucose ATP Nucleus Chromosomes Enzyme Cellular respiration Deoxyribose Hydrophobic Triglycerides Ribose Phospholipids Pigments m. RNA Amino acid Polypeptide Chinese hamster ovary cells Vero cells Prokaryote Organic Carbohydrates Cytoskeleton Monomer Polymer Monosaccharide Disaccharide Polysacccharide Fructose Sucrose Lactose Hydrophillic

Organisms & Their Components To manufacture biotechnology products for medical, industrial, or agricultural applications, biotechnicians must work either directly or indirectly with organisms or their components � Entire multicellular organisms � Bacteria � Tissues/cells

Organisms & Their Components Working in any area of biotechnology requires a thorough understanding of the characteristics of life and the structures that compose organisms You must have at least a minimal understanding of � Biochemistry: organic molecules structure, function, & interactions � Cytology: branch of life science, which deals with the study of cells in terms of structure, function and chemistry

The Living Condition Biologists estimate that there are well in excess of 20 million different species, with some estimates being as high as 150 million All living things can be classified into one of the following categories Prokaryotes Eubacteria Archaeabacteria Protists Plants Animals Fungi

The Living Condition Biologists estimate that there are well in excess of 20 million different species, with some estimates being as high as 150 million All living things can be classified into one of the following categories Prokaryotes Eukaryotes (Unicellular) (mostly multicellular) Eubacteria Archaeabacteria Protists Plants__ Animals__ Fungi

Characteristics of Life Growth & Development Reproduction Organized structure composed of 1 or more cells Response to stimuli Conversion of energy � Respiration � Metabolism Catabolism Anabolism

Here We Go Again! Cells & Their Parts The cells of both unicellular and multicellular organisms are tiny microscopic factories that produce thousands of different molecules Biotechnology companies exploit the biologiical manufacturing capabilities of cells and trick them into producing particular molecules in large quantities � These become biotechnology products Organelles: a specialized subunit within a cell that has a specific function, and is usually separately enclosed within its own lipid bilayer � The name organelle comes from the idea that these structures are to cells what an organ is to the body

Eukaryotic Cell Structure

Organelles involved in Energy Production Mitochondria A membrane bound organelle consisting of an outer membrane and a highly folded innermembrane; it is responsible for generating large amounts of ATP through the process of aerobic cellular respiration � � Found in all eukaryotic cells ATP: adenosine triphosphate a nucleotide that serves as the main source of usable energy for ALL living things Chloroplast A specialized organelle in plants and some protists that uses the energy from sun light to convert inorgaic molecules (CO 2 & H 2 O) into energy storing organic molecules (glucose) through the process of photosynthesis

Nucleus: The Organelle that Stores the Hereditary Material Nuclear Envelope a double lipid bilayer that encloses the genetic material in eukaryotic cells. � Nuclear Contents serves as the physical barrier, separating the contents of the nucleus from the cytosol nuclear pores are inserted in the nuclear envelope, which facilitate and regulate the exchange of materials (proteins such as transcription factors, and RNA) between the nucleus and the cytoplasm. The outer membrane is continuous with the rough endoplasmic reticulum The outer and inner nuclear membrane are fused at the site DNA: a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms � � main role is the long-term storage of information Used as a template to make RNA through the priocess of transcription Nucleolus: a non-membrane bound structure composed of proteins and nucleic acids found within the nucleus. � Ribosomal RNA (r. RNA) is transcribed and assembled within the nucleolus, forming

Ribosomes: involved in Protein Synthesis Non-membrane bound organelles composed of r. RNA & protein that provide the appropriate microenvironment to catalyze peptide bond formation between amino acids based on the instructions it reads on an m. RNA molecule through the process of translation � Chemistry Connect: Remember, all organic polymers are formed through condensation reactions, aka dehydration synthesis

Organelles involved in Trafficking Rough Endoplasmic Reticulum A highly folded lipid bilayer whose surface ois studded with protein-manufacturing ribosomes giving it a "rough" appearance Ribosomes bound to the RER at any one time are not a stable part of this organelle's structure as ribosomes are constantly being bound and released from the membrane. � A ribosome only binds to the ER once it begins to synthesize a protein destined for the secretory pathway Golgi Apparatus The Golgi apparatus processes and packages macromolecules, such as proteins and lipids, after their synthesis and before they make their way to their destination; it is particularly important in the processing of proteins for secretion.

Organelles involved in Trafficking Rough Endoplasmic Reticulum A highly folded lipid bilayer whose surface is studded with protein-manufacturing ribosomes giving it a "rough" appearance Ribosomes bound to the RER at any one time are not a stable part of this organelle's structure as ribosomes are constantly being bound and released from the membrane. � A ribosome only binds to the ER once it begins to synthesize a protein destined for the secretory pathway Golgi Apparatus The Golgi apparatus processes and packages macromolecules, such as proteins and lipids, after their synthesis and before they make their way to their destination; it is particularly important in the processing of proteins for secretion.

Organelles involved in Metabolism Smooth Endoplasmic Reticulum Functions in several metabolic processes, including synthesis of lipids and steroids, metabolism of carbohydrates, regulation of calcium concentration, drug detoxification, attachment of receptors on cell membrane proteins, and steroid metabolism. It is connected to the nuclear envelope. Lysosomes: contain acid enzymes to break up waste materials and cellular debris. � � � found in animal cells, while in yeast and plants the same roles are performed by lytic vacuoles. digest excess or worn-out organelles, food particles, and engulfed viruses or bacteria The membrane around a lysosome allows the digestive enzymes to work at the 4. 5 p. H they require.

Metabolism: is the set of chemical reactions that happen in living organisms to maintain life Catabolism is the set of pathways that break down molecules into smaller units These reactions release energy Anabolism the set of metabolic pathways that construct molecules from smaller units. These reactions require energy Critical Thinking: Which organelles are catabolic and which are

The Endomembrane System Is composed of the different membranes that are suspended in the cytoplasm within a eukaryotic cell. These membranes divide the cell into functional and structural compartments, or organelles. In eukaryotes the organelles of the endomembrane system include: the nuclear envelope, the endoplasmic reticulum, the Golgi apparatus, lysosomes, vacuoles (plants), vesicles (all eukaryotes), and the cell

Cellular Membranes Acts as a semi-permeable barrier that regulates what enters or leaves the cell or organelle They consist of phospholipids which spontaneously arrange so that the hydrophobic "tail" regions are shielded from the surrounding polar fluid, causing the more hydrophilic "head" regions to associate with the cytosolic and extracellular faces of the resulting bilayer.

Things in/on Cellular Membrane Proteins: function in cellular communication, catalyzing reactions, cell, signaling, cell movement � 4 main types Receptor proteins bind signal molecules and initiate signal transduction pathways Enzymes: speed up chemical reactions Channel proteins: open or close to let target molecules in or out in response to a signal Marker proteins: specific for each cell type; also, help the immune system distinguish self from non-self Cholesterol: establishes proper membrane permeability & fluidity

Cell Walls All living things except animals and some protists have cell walls Cell walls are semipermeable boundaries that are more rigid than, and surround the plasma membrane provides structural support and protection, and also acts as a filtering mechanism � Keeps cells from bursting in hypotonic environments � Different organisms have cell walls made of different organic polymers � Plants: cellulose Bacteria: peptidoglycan Fungi (some): chitin

Cell Tissue Organism Depending on the type of cell hundreds of different molecules are being manuifactured at any given moment Some molecules are unique to a specific type of cell, others are produced in all cells Example: Pancreas organ of the digestive system that lies in your abdomen, behind your stomach, with 2 main functions: � producing digestive enzymes to break down food; and � producing the hormones insulin and glucagon to control sugar levels in your body.

Organism Case in Point: Pancreas People Organ Pancreas Tissue Islets of langerhans Acinar tissue Secrete hormones Secrete digestive enzymes Cells Liver cells are called hepatocytes α-cells: secrete glucagon β-cells secrete insulin Acinar cells: secrete enzymes that allow your body to digest protein, fat and starch from your food

A closer look at the BIG Picture When you, the organism, eats a meal, your stomach (with the help of digestive enzymes from your pancreas and other organs) break down the complex carbohydrates into glucose Glucose is absorbed through the intestines (small I think) into the blood stream Rising glucose in the blood stream is detected by the pancreas as the glucose binds glucose receptors on pancreatic cells. This stimulates β -cells to release insulin

Insulin: pancreatic homeostasis Insulin works by improving the uptake of glucose from the blood across cell membranes and into the cells of the body by binding to insulin receptors on cells Specifically, when insulin binds the insulin receptor, the receptor protein transports phosphate groups from ATP to other proteins in the cell, initiating signal transduction pathways that open glucose channels allowing passive diffusion of glucose into the cell � Once in the cells, the glucose is used as the energy to fuel the cells doing their different jobs or is stored in the liver or muscle cells as glycogen. � This results in the glucose level of the blood dropping,

Critical Thinking Insulin and glucagon are both hormones that work together to regulate blood glucose levels. Knowing how insulin works, and that glucagon's effect is opposite that of insulin, propose a mechanism by which glucagon may work � Hint: remember that unused glucose is converted to and stored in the form of glycogen in the pancreas

Glycogenolysis Glucagon helps maintain the level of glucose in the blood. Glucose is stored in the liver in the form of glycogen, which is a starch-like polymer chain made up of glucose molecules. Liver cells (hepatocytes) have glucagon receptors. When glucagon binds to the glucagon receptors, the liver cells convert the glycogen polymer into individual glucose molecules, and release them into the bloodstream, in a process known as glycogenolysis.

Why all this matters The purpose of biotechnology is to increase quality and/ or quantity of life Life is dependant on the bodies abilities to � Maintain homeostasis � Transform energy � Reproduce � Grow and develop � Respond to stimuli Internal: Biotechnology focuses on making organisms better able to accomplish one or more of these tasks for example a change in levels of blood sugar External: for example a change in temperature

Central Dogma If the goal of biotechnology is to increase an organisms ability to accomplish one or more of the characteristics of life, then it is important to understand how the organism naturally carries these tasks out! One of the most important aspects of understanding this is to understand the central dogma of biology

Chromosomes & Genes We know chromosomes are in the nucleus We know genes are on chromosomes, and are segments of DNA that code for protein What we need to look at more specifically is HOW the DNA instructs your body to build a protein

Central Dogma: an Overview DNA is made up of 2 strands of nucleotides that coil around each other forming a double helix Each strand is composed of varying arrangements of 4 nucleotides � Adenine Cytosine Thymine Guanine When genes are used to instruct the body to build a protein, they must be transcribed into a complementary sequence of messenger RNA

Central Dogma: an Overview This m. RNA can leave the nucleus and be read by a ribosome which actually constructs the protein through the process of translation In translation, the ribosome links amino acids together in the order specified by the sequence of nucleotides in the m. RNA These amino acids are held together by special covalent bonds called peptide bonds � There are 20 different amino acids � A chain of amino acids are called a polypeptide

Nucleus Ribosome in cytoplasm or on RER

Central Dogma The process of Gene Expression is universally found in all cells � How the DNA code is rewritten into m. RNA and then decoded into a protein It is called “The Central Dogma of Biology” because it helps explain how virtually all molecules are made either � directly protein & nucleic acids � or indirectly carbohydrates and lipids because their synthesis is controlled by the proteins

Tying it together DNA is used to make protein Proteins do most of the work of the cell � The other organic molecules are important too, but because their production/ destruction is controlled by proteins we focus on the protein The cell has specialized organelles, containing different collections of proteins, giving each organelle a unique function based on the proteins (and other organic molecules) in/on it The organelles work together to help the cell carry out all of the characteristics of life

Tying it together Not all cells, even within the same individual, are the same Some cells become specialized to perform a specific function � β-cells: Secrete insulin � Rods: sense light Cells are able to perform these specialized functions BECAUSE of the proteins they produce � Different types of cells produce different types/quantities of proteins at different times

Critical Thinking If all cells within one organism have the same set of DNA (in other words, the DNA in your nerve cells is identical to the DNA in the epithelium of your little toe), how is it that all cells are not the same?

Cells Commonly Used in Biotechnology In biotech applications, some cells are used more than others Chinese Hamster Ovary (CHO)cells Vero Cells: African green monkey kidney epithelial cells Other Words He. La cells: human epithelial In cells You need to know Fungal cells how to keep cells � Aspergillus alive in culture, in � yeast order to do most Prokaryotic cells � E. coli biotechnology � Staphylococcus research

NOTE On page 46 of your text book it claims that “Due to their lack of mitochondria, bacteria conduct ONLY anaerobic respiration” This is WRONG & the author is a MORON There are SOME strictly anaerobic bacteria but MOST need or at least can survive in oxygen environments!

Read section 2. 3 on your own! Things to pay attention to � Carbohydrates � � � lipids why are lipids referred to as hydrocarbons The 3 general groups of lipids and their functions � Glycogen: what is it Structural polysaccharides of cell wall How polysaccharides interefere with purification procedures The text book reads “Cells break the bonds in glucose, releasing energy in a form that cells can use” why do I hate that definition Figure 2. 23 Triglycerides Phospholipids Steroids Structure of a phospholipid

“DNA is the Flash, Proteins are the Cash!” Water makes up 75% of a cells mass Of 25% of dry mass, 75% of it is protein In biotechnology, proteins are often the manufactured product Often, 50 -75% of a companies staff is devoted to protein research (proteomics)

Proteins fall into 9 different categories depending on their function Structural: proteins with the primary purpose of producing the essential structural components of the cell Enzyme: speed up chemical reactions by lowering activation energy Transport: involved in the movement of substances across biological membranes; or carries specific molecules (example oxygen is transported by hemoglobin) Contractile: involved in muscle contraction, also in the functioning of the mitotic spindle Hormone: a chemical released by a cell or a gland in one part of the body that sends out messages to affect cells in

Proteins fall into 9 different categories depending on their function Antibody: used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses Pigment: a material that changes the color of reflected or transmitted light as the result of wavelength-selective absorption Marker/ Recognition: proteins usually found on the extracellular membrane that allows other molecules to identify the cell type Toxins: small molecules, peptides, or proteins that are capable of causing disease on contact with or absorption by body tissues interacting with biological macromolecules such as enzymes or cellular receptors

Proteins Grouping is based on functions, and often proteins within the same group are more similar to each other than to proteins in different groups Note: some proteins can have different functions, even within the same organism, depending on which cell they are produced in A typical cell produces about 2, 000 different proteins Proteins are the work horses of the cell, each able to accomplish its task due to its specific structure and properties

Proteins The structure of a protein is determined by the amino acid sequence Chains of amino acids are called polypeptides Polypeptides are not functional until they fold into their 3 -dimensional shape Folded polypeptide chains are called proteins The way a polypeptide folds into a functional protein is determined by the sequence of the amino acids and their properties The sequence of amino acids is determined by the sequence of m. RNA which is determined by the sequence of DNA

There are 20 different amino acids All amino acids have the same basic structure

R-Groups R- groups are the chemical side groups on an amino acid that varies between different amino acids It is the R-group that is responsible for the unique characteristics of each amino acid The chemical nature of each R-group results in attractions and repulsions between certain amino acids � Example: the negatively charged R-group of glutamic acid is attracted to the positively charged R-group of arginine � The various folding patterns of each protein are a

R-Groups The R-groups of protein chains can interact between proteins as well Many proteins function by attracting or repelling other protein chains Many recognition proteins, antibodies, enzymes, and protein hormones function because the R-group on one of their amino acids is strategically placed (active site) to interact with its target molecule (ligand)

Nucleic Acids Information carrying molecules that direct the synthesis of all cellular molecules, including themselves Ultimately, each protein, carbohydrate, and lipid molecules production can be traced back to genetic information stored in the sequence of DNA, which is packaged into chromatin or chromosomes, depending on what stage of the cell cycle the cell is in

Quick Comparison of Prokaryotic Chromosomes to Eukaryotic Chromosomes Prokaryotes Eukaryotes Chromosome located in the cytoplasm in a region known as the nucleoid Chromosomes found in the nucleus -Genetic 1 chromosome (may have additional plasmids) informatio # of chromosomes varies n with species Chromosome is circular -Contains Chromosomes are linear A, C, T, G No introns, exons only DNA contains both DNA is NOT associated Composed introns & exons with histone proteins of DNA is associated with nucleotide histone proteins s

Overview of Gene Expression DNA is composed of long stretches of nucleotides covalently linked together Genetic information lies in the sequence of nitrogenous bases � There are 5 nitrogenous bases DNA: adenine, cytosine, guanine, thymine RNA: adenine, cytosine, guanine, uracil Cytosine on 1 strand always pairs with guanine on the other strand Adenine pairs with either thymine (if you’re copying DNA) or Uracil, (if your transcribing RNA

DNA inisthethe Occurs _______ on a template to. Occurs build RNA REPLICATIO N T T A C A G G TRANSCRIPTI ON C A G A T ________ in the cytoplasm or attached to the RER TRANSLATION

Genetic Code This is how a molecule with only 4 nitrogenous bases can code for over 25, 000 different proteins! Because RNA and so the gene is “read” in groups of 3 We can use this knowledge to perhaps not just treat but actually fix genetic mistakes and or give organisms new characteristics that make them more fit, substantially improving the quality and quantity of life

Genetic Engineering Companies employ genetic engineers to isolate and alter the DNA codes for a particular protein or group of proteins Sometimes the protein, like insulin, is the product itself The goal, then, is to produce it in sufficient quantities to sell in the marketplace and make a profit

The “New” Biotechnology Organisms & their products have been harvested and improved for centuries The most significant breakthrough in the manipulation of plant and animal cells occurred when scientists learned how to move pieces of DNA within and between organisms The key to this discovery was restriction enzymes and DNA ligase, which allow us to cut and paste DNA to make r. DNA

Recombinant DNA r. DNA usually contains fragments from different organisms They are usually novel molecules not seen in existence anywhere else, and thus are referred to as being “engineered” DNA fragments containing genes of interest can be pasted into vectors and carried back into cells Once in cells they are transcribed and translated into protein molecules that the recipient cell has never produced This organism has been “genetically

The 1 st Genetic Engineering Experiment Occurred in 1973 when 3 scientists � Stanley Cohen (Stanford) � Herb Boyer (University of California) � Paul Berg (Stanford) Excised (cut out) a segment of amphibian DNA from the African Clawed Toad, Xenopus, and pasted it into a small ring of bacterial DNA called a plasmid The new recombinant plasmid (r-plasmid) was placed in an E. coli cell which then transcribed the DNA into ribosomal RNA

Human Insulin The 1 st genitically engineered product to reach the market place was human insulin Scientists used similar methods as before to excise a healthy human insulin gene using restriction enzymes, paste it into a bacterial plasmid using DNA ligase, and then transferred the plasmid to E. coli cells which then transcribed the DNA into RNA, and then translated the m. RNA into the insulin protein The cells were grown in large volumes, and then the insulin was purified out of the cell culture The FDA approved r-human insulin (rhinsulin)in 1982

Genentech In 1976 Robert Swanson & Herb Boyer founded the 1 st biotechnology company called Genentech in San Francisco Their 1 st product was rh. Insulin Genentech has grown into one of the largest pharmaceutical companies in the world It currently markets or is developing � t-PA: treatment of heart attachs � Nutropin: a human growth hormone � Rituxin: an antibody that targets cancer cellsin B-cell non-Hodgkins lymphoma � Pulmozyme: for cystic fibrosis treatment