The Raw Materials of Biotechnology

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 mRNA 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

Anatomy: structure of living things and their components

Physiology: the processes and functions of living systems

The Living Condition Biologists estimate that there are well in

excess of 20million different species, with some estimates being as high as 150 million

All living things can be classified into one of the following categories

Prokaryotes EukaryotesEubacteria

Archaeabacteria ProtistsPlants

AnimalsFungi

The Living Condition Biologists estimate that there are well in

excess of 20million different species, with some estimates being as high as 150 million

All living things can be classified into one of the following categories

Prokaryotes(Unicellular)

Eukaryotes(mostly multicellular)

EubacteriaArchaeabacteria

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

A membrane bound organelle consisting of an outer membrane and a highly folded inner-membrane; 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

A specialized organelle in plants and some protists that uses the energy from sun light to convert inorgaic molecules (CO2 & H2O) into energy storing organic molecules (glucose) through the process of photosynthesis

Mitochondria Chloroplast

Nucleus: The Organelle that Stores the Hereditary Material

a double lipid bilayer that encloses the genetic material in eukaryotic cells. 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 of nuclear pore complexes.

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 (rRNA) is

transcribed and assembled within the nucleolus, forming ribosomes

Nuclear Envelope Nuclear Contents

Ribosomes: involved in Protein Synthesis

Non-membrane bound organelles composed of rRNA & protein that provide the appropriate microenvironment to catalyze peptide bond formation between amino acids based on the instructions it reads on an mRNA molecule through the process of translation Chemistry Connect: Remember, all organic

polymers are formed through condensation reactions, aka dehydration synthesis

Organelles involved in Trafficking

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

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.

Rough Endoplasmic Reticulum Golgi Apparatus

Organelles involved in Trafficking

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

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.

Rough Endoplasmic Reticulum Golgi Apparatus

Organelles involved in Metabolism

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 pH they require.

Smooth Endoplasmic Reticulum Lysosome

http://en.wikipedia.org/wiki/Vacuole

Metabolism: is the set of chemical reactions that happen in living organisms to maintain life

is the set of pathways that break down molecules into smaller units

These reactions release energy

the set of metabolic pathways that construct molecules from smaller units.

These reactions require energy

Catabolism Anabolism

Critical Thinking: Which organelles are catabolic and

which are anabolic?

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 membrane

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 Organ 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.

Case in Point: Pancreas

CellsLiver cells are called hepatocytes

α-cells: secrete glucagonβ-cells secrete insulin

Acinar cells: secrete enzymes that allow your body to digest protein, fat and starch from

your food

TissueIslets of langerhans

Secrete hormones

Acinar tissueSecrete digestive

enzymes

Organ

Pancreas

Organism

People

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, which then triggers the pancreas to switch off the release of insulin

http://bcs.whfreeman.com/thelifewire/content/chp50/5002002.html

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: for example a change in levels of blood sugar

External: for example a change in temperature

Biotechnology focuses on making organisms better

able to accomplish one or more of these

tasks

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

The mRNA transcript is made of nucleotides Adenine Cytosine Uracil Guanine

Central Dogma: an Overview This mRNA 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 mRNA

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 mRNA

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 HeLa cells: human epithelial cells Fungal cells

Aspergillus yeast

Prokaryotic cells E. coli Staphylococcus Streptococcus

In Other WordsYou need to know how to keep cells alive in culture, in order to do most

biotechnology 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 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

lipids why are lipids referred to as hydrocarbons The 3 general groups of lipids and their functions

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 other parts of the organism

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 mRNA 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 result of these interactions

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

Chromosome located in the cytoplasm in a region known as the nucleoid

1 chromosome (may have additional plasmids)

Chromosome is circularNo introns, exons only

DNA is NOT associated with histone proteins

Chromosomes found in the nucleus

# of chromosomes varies with species

Chromosomes are linearDNA contains both introns

& exonsDNA is associated with

histone proteins

-Genetic information-Contains A,C,T,G-Composed of nucleotides

Prokaryotes Eukaryotes

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 is the template to build RNA

TTAGGCAGATAC

REPLICATION

TRANSCRIPTION

TRANSLATION

Occurs on a _______________ in the

cytoplasm or attached to the RER

Occurs in the _______

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 rDNA

Recombinant DNA rDNA 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 engineered”

The 1st 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 1st 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 mRNA 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 1st biotechnology company called Genentech in San Francisco

Their 1st product was rhInsulin 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

The Raw Materials of Biotechnology

Documents

Transcript of The Raw Materials of Biotechnology