Introduction to Biotechnology!

Defining Biotechnology

Biotechnology- the study and manipulation of living things or their

component molecules, cells, tissues, or organs.

The beginning of biotechnology

• Humans have been manipulating living things for thousands of years

• Selective breeding- manipulating living things with desired characteristics

• Why?

• Examples?

Beginning of Biotechnology

• Over 100 breeds of dogs created through selective breeding

Beginning of Biotechnology

• Different varieties of apples commonly found in grocery stores

Beginning of Biotechnology

• Range of size, color, and fragrance for a variety of roses

Beginning of Biotechnology

• Cows, goats, sheep, and chickens for milk, meat, and egg production

Beginning of Biotechnology

• Fermentation of foods and beverages

The 1970’s

• Scientists began experimenting with molecules, cells, tissues, and organs (moving away from entire organisms). WHY?

• New technologies are applied to the research and development of products from plant and animal tissues

• The term “Biotechnology” was coined

Biotechnology today

• Focuses on DNA, not RNA or proteins– Manipulate at the earliest

source possible (very difficult to control outcome later)

Uses The Central Dogma of Biology. Moving genes into cells to produce new proteins is the basic principle in genetic engineering.

The Central Dogma of Biology.

The Increasing Variety of Biotechnology Products

“In the past 100 years, scientists have increased the pace of searching for products that improve the quality of life.”

As the methods of manipulating livings have become more sophisticated, the number and variety of biological products have increased at an incredible rate

Recent uses of biotechnology

• Insulin- made in bacteria cells to treat diabetes

• Originally insulin was harvested from the pancreas of a slaughtered animal for treatment

Recent uses of biotechnology

• Proteases- proteins that break down other proteins

• Commonly used in stain removal products

Recent uses of biotechnology

• Antibiotics- proteins developed by the immune system that recognize a specific molecule (antigen)

• Used to fight diseases

Recent uses of biotechnology

• Indiage- protein (enzyme) that causes denim to fade to produce “stonewashed” appearance

Recent uses of Biotechnology

• Mouse cells “tricked” into growing outer portion of a human ear which can be surgically transferred to a human patient

Recent Advances in Biotechnology come from Manipulation of DNA!

What do you think of when you hear this?

Types of Genetic Manipulation

• 1.Recombinant DNA (rDNA) technology- cutting and recombining DNA molecules

E. Coli transformation

What Makes rDNA Possible?

• Polymerase Chain Reaction or PCR: copying short pieces of DNA (genes)

• Restriction enzymes – cuts nucleotide sequence at specific sites on DNA molecule

• Gel Electrophoresis: Separate DNA fragments by size

• DNA ligase – enzyme that pastes DNA molecules together allowing for new combinations

Gel Electrophoresis

Gel Electrophoresis• Electrophoresis allows separation of

molecules in an electrical field on the basis of size/molecular/weight and shape.

• A molecule with a negative charge (anion) will migrate toward the positive electrode (anode), and a molecule with a positive charge (cation) will migrate toward the negative electrode (cathode)

• The migration and separation of molecules are carried out using a solid matrix (i.e. agarose, polyacrylamide).

• The matrix retards the movement of molecules by a seiving effect – small molecule navigate the matrix more quickly than larger ones.

Gel Electrophoresis

• Electrophoresis is used chiefly for analysis and purification of large molecules such as nucleic acids, but can be a applied to any charged molecules.

• The relative mobility of the fragments– how fast they travel though the matrix relative to each other

- will depend on several parameters.

Gel Parameters

• Migration through the gel can be effected by:• Size• Shape• % Agarose• Voltage

Gel Parameters

• Size– Smaller fragments of DNA will travel further than the larger

ones.

• Shape– DNA can roll into a ball, making it migrate faster. In

addition, if damaged, it will migrate slower.

• % Agarose– Fragments larger than the matrix pore size cannot enter

the gel and are not resolved. At the other extreme, fragments smaller than the pore size are not retarded at all.

Gel Parameters

VoltageIn addition to Ohm’s law (V=IR), a

fundamental equation in electrophoresis is the power equation

P=VIThe higher the voltage, the

greater the power – heat!• gel melts• Non-uniform heat

distribution results in smiling bands (heat is more rapidly dispersed at edges of gel)

5 – 8 cm /V

Gel Electrophoresis

• Loading dye – Glycerol (gives weight to the DNA sample so it will not float

out well of the gel into the buffer)– Dye (so we can see the DNA enter the gel and migrate)

• Visualization– Ethidium Bromide is a dye that intercalates between the

base of nucleic acids. When exposed to UV light, it will fluoresce, making the DNA visible.

– We will then find the size of the DNA fragments, and determine the quality and quantity of DNA we have

Markers

A DNA marker, a sample of DNA fragments of known sizes and mass, is used as a reference to estimate the size of unknown DNA molecules.

You run it on the gel with the DNA samples and compare the sizes of your fragments to the sizes of these known fragments

Quality and Quantity Assessment

Quality: Is the band a distinct band or a smear? A smear indicates broken or degraded DNA.

Quantity: How bright is the band. The brighter the band=the more DNA

Steps of rDNA?

• 1. Use Restriction Enzymes to remove the gene of interest from an organism that produces it naturally.

• 2. Use Gel Electrophoresis to resolve fragments.

• 3. Copy the gene of interest millions of times with PCR.

• 4. Use Restriction enzymes to cut the DNA of the organism that will receive the gene of interest. Again, use gel electrophoresis to resolve fragments.

• 5. Use DNA ligase to seal the new gene into the receiving organism. Use gel electrophoresis to confirm size of DNA.

Steps of rDNA

• Production of rDNA can be done directly or with the use of plasmids

• Plasmids – tiny circular pieces of DNA usually from bacteria that is used to insert recombinant DNA into an organism

rDNA to GMO

• Make Recombinant DNA – DNA created from two or more sources

• Genetically modified organism (GMO) – organism that contains DNA from another organism and produces new proteins encoded on the acquired DNA

Restriction enzymes, Gel Electrophoresis, DNA ligase, and plasmids:

leads to

What is Genetic Engineering?

• Genetic engineering refers to the modification of genetic material to achieve specific goals

• We can modify organisms to express genes they never had and make proteins they never have before= A GENEATICALLY MODIFIED ORGANISM

What is the Difference Between rDNA and a GMO

• rDNA: Just the DNA has been genetically modified– DNA of 2 organisms spliced together

• GMO: the genome of an organism has been genetically modified– Organisms is expressing genes that did not occur

naturally– A new gene was inserted into the genome of an

organism

Gene Engineered Plant. Scientists have learned how to insert genes that code for certain traits and transfer them from one species to another. The organism that gets the new genes will then have the potential to express the new traits coded in the newly acquired genes.

Examples of GMOs

Examples of GMOs

Research Animals

MedicinesHumans make only a small amount of human tissue plasminogen activator (t-PA) naturally. By genetically modifying Chinese hamster ovary (CHO) cells, scientists can make large amounts of t-PA for therapeutic purposes, such as to clear blood vessels in the event of a heart attack or stroke.

Examples of

GMOs

• Recombinant DNA and genetic engineering produces 100’s of products

Examples of GMOs

Examples of GMO’s

• Almost all produce in grown in the United States has been genetically modified

Why do you think we have GMO foods?

• Growing human population

• Loss of farmable land

• Remediation of soil

• Enrich nutrient content

•What are some desirable traits for a GMO to have?

Desirable Traits

• Pest Resistance

• Herbicide Tolerance

• Viral Resistance

• Drought Resistance

• Increased Nutritional Value

• Improved Fruit

• Altered Ripening

What are some Arguments against

GMO’s?

Opponents argue

• Creation of super pests

• Creation of super weeds

• Loss of biodiversity

• Biotechnology companies control agriculture

• Health concerns

Types of Genetic Manipulation

• 2. Cloning- producing identical organisms

Steps of Cloning

• Eukaryotes:• 1. Copy the genome of the organism to be cloned• 2. Impregnate a female with this genome• 3. Allow the clone to be born

• Prokaryotes:• Occurs naturally• Can use plasmids to create the organism you

want, then allow it to replicate

Who is practicing Biotechnology?

The Biotechnology Workplace Biotechnology Companies - goal is to produce

and sell commercial “for-profit” productsStaff- scientists, researchers, lab technicians, manufacturing, marketing, sales

Universities - conduct “pure science” research, nonprofitReport results in scientific journals or meetings for the “public good”

• Government Agencies

• CDC Centers for Disease Control and Prevention-national research center for developing and applying disease prevention and control, environmental health, and health promotion and education activities to improve public health

• NIH National Institutes of Health- the federal agency that funds and conducts biomedical research

The Biotechnology Workplace

The Biotechnology Workplace• DNA fingerprinting used by:

– Forensic scientists within police departments to identify suspects

– Ecologists to determine parentage of endangered species for breeding purposes

– Wildlife biologists/customs agents to identify illegally poached or transported animals (bear gall bladders, rhinoceros horns, ivory from tusks, exotic birds)

– Evolutionary biologists to determine relatedness of organisms (red panda is more closely related to the raccoon than it is to the black and white panda bear)

Federal agencies governing product development

Food and Drug Administration (FDA)- regulates the use and production of food, feed, food additives, veterinary drugs, human drugs, and medical devices

Environmental Protection Agency (EPA)- enforces environmental laws including the use and production of microorganisms, herbicides, pesticides, and genetically modified microorganisms

United States Department of Agriculture (USDA)- regulates the use and production of plants, plant products, plant tests, veterinary supplies and medications, and genetically modified plants and animals

The Human Genome Project

Determined the human DNA sequence Scientists now work on:

Identifying all of the genes Determining their functions Understanding how and when genes are

turned on and off

What can this project do to advance biotechnology?