Ms. GaynorMs. GaynorHonors GeneticsHonors Genetics

Biotechnology and the Biotechnology and the Use of BacteriaUse of Bacteria

What is Biotechnology?What is Biotechnology?The use of The use of

microorganisms microorganisms to create cellular to create cellular products (ie: products (ie: insulin) for insulin) for human usehuman use

Also known as Also known as genetic genetic engineeringengineering

Often uses Often uses bacteria as the bacteria as the microorganismmicroorganism

What is Bacteria?What is Bacteria?Single celled Single celled

prokaryoteprokaryoteNo nucleus (nucleoid No nucleus (nucleoid

region instead) region instead) Very few (“no”) Very few (“no”)

organellesorganellesHas a cell wall, cell Has a cell wall, cell

membrane, membrane, ribosomes, ribosomes, cytoplasmcytoplasm

Has circular Has circular chromosome and chromosome and plasmid(s)plasmid(s)

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2 Types (domains) of 2 Types (domains) of BacteriaBacteriaEubacteria (can be harmful) Eubacteria (can be harmful) ***used in ***used in

biotechnology! biotechnology! ““regular” bacteriaregular” bacteriaThey live in most environmentsThey live in most environmentstheir cell-wall DOES contain their cell-wall DOES contain

peptidoglycanpeptidoglycanArchaea Bacteria (not harmful)Archaea Bacteria (not harmful)

““older” bacteria; live in older” bacteria; live in EXTREMEEXTREME habitatshabitats

more similar to eukaryotes than to more similar to eukaryotes than to bacteria in several ways: bacteria in several ways: their cell-wall does NOT contain their cell-wall does NOT contain

peptidoglycanpeptidoglycan

Plasmids are used in Plasmids are used in BiotechnologyBiotechnology

• In addition to the chromosome, some In addition to the chromosome, some bacteria have bacteria have plasmidsplasmids – smaller circular DNA moleculessmaller circular DNA molecules

Extra chromosomal DNAExtra chromosomal DNA

How Does Bacteria Get Plasmid DNA?How Does Bacteria Get Plasmid DNA?

TransformationTransformationBacteria takes in naked, foreign Bacteria takes in naked, foreign

DNA ( a plasmid) from the DNA ( a plasmid) from the environment under certain environment under certain conditionsconditions

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DNA TechnologyDNA Technology

DNA technology has launched DNA technology has launched a revolution in the area of a revolution in the area of biotechnologybiotechnologyThe manipulation of The manipulation of

organisms or their genetic organisms or their genetic components to make useful components to make useful productsproductsExample: gene cloningExample: gene cloning

What is Gene Cloning?What is Gene Cloning?

DNA cloning permits production of multiple DNA cloning permits production of multiple copies of a specific gene or other DNA copies of a specific gene or other DNA segmentsegment

To work directly with specific genesTo work directly with specific genesScientists developed methods for making Scientists developed methods for making

pieces of DNA in multiple identical pieces of DNA in multiple identical copies, a process called copies, a process called gene cloninggene cloningUsually uses bacterial plasmidsUsually uses bacterial plasmids

Using Restriction Using Restriction Enzymes to Make Enzymes to Make Recombinant DNARecombinant DNA

Bacterial Bacterial restriction enzymesrestriction enzymesCalled Called restriction endonucleasesrestriction endonucleasesCut DNA molecules at a limited Cut DNA molecules at a limited

number of specific DNA sequences, number of specific DNA sequences, called called restriction sitesrestriction sites

Restriction enzymesRestriction enzymesWhere are restriction enzymes found?Where are restriction enzymes found?

In bacterial cells, to help fight viruses/ foreign DNAIn bacterial cells, to help fight viruses/ foreign DNA

What is a restriction site?What is a restriction site? The area on DNA that the restriction enzyme The area on DNA that the restriction enzyme

recognizesrecognizes Every time this DNA sequence occurs in bacterium’s Every time this DNA sequence occurs in bacterium’s

own DNA, methyl groups (-CH3) are added own DNA, methyl groups (-CH3) are added prevents restriction enzyme from working. prevents restriction enzyme from working.

When foreign DNA (ex: a bacterial virus) enters When foreign DNA (ex: a bacterial virus) enters bacterium, bacterium’s restriction enzyme cut bacterium, bacterium’s restriction enzyme cut phage’s DNA into pieces. phage’s DNA into pieces.

NOT all bacteria have restriction enzymes, many NOT all bacteria have restriction enzymes, many do!do! But there is a wide variety of restriction enzymes that have But there is a wide variety of restriction enzymes that have

been discovered and continue to be discoveredbeen discovered and continue to be discovered

Restriction SitesRestriction SitesWhat is a restriction site?What is a restriction site?

The area on DNA that the restriction enzyme The area on DNA that the restriction enzyme recognizesrecognizes

Many different kinds (EcoRI, BamHI, HindIII, Many different kinds (EcoRI, BamHI, HindIII, etc) etc)

Like palindromeLike palindromeAA palindromepalindrome is a word or phrase which is a word or phrase which

reads the same in both directions. reads the same in both directions.

These enzymes make “blunt” and “sticky” ends. These enzymes make “blunt” and “sticky” ends. What is the difference?What is the difference?

Restriction EnzymesRestriction EnzymesEnzymes usually make many cuts in a DNA Enzymes usually make many cuts in a DNA

moleculemoleculeYields a set of restriction fragmentsYields a set of restriction fragments

Most useful restriction enzymes cut DNA Most useful restriction enzymes cut DNA in a in a staggeredstaggered way way Producing fragments with “sticky ends” Producing fragments with “sticky ends”

that can bond with complementary that can bond with complementary “sticky ends” of other fragments“sticky ends” of other fragments

DNA ligase DNA ligase is an enzyme that seals the is an enzyme that seals the bonds between restriction fragmentsbonds between restriction fragments

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Animation #1 Animation #1

Different Different restrictiorestrictio

n n enzymes enzymes that can that can be usedbe used

Recombinant

Plasmid

Restriction Enzymes: Proteins that cut the DNA in a specific place

Gene of interest Cloning

Vector

Gene Cloning Using a Gene Cloning Using a PlasmidPlasmid

The original plasmid is called theThe original plasmid is called the cloning vectorcloning vectorDNA molecule that can carry foreign DNA molecule that can carry foreign

DNA into a cell and replicate thereDNA into a cell and replicate there

After insertion of the gene of interest After insertion of the gene of interest into the plasmid the plasmid is into the plasmid the plasmid is called called recombinant DNArecombinant DNA

Once the plasmid is inside the Once the plasmid is inside the bacterium (host) cell, the cell is called bacterium (host) cell, the cell is called a a transformed celltransformed cell

REVIEW…REVIEW…

How do you clone How do you clone a gene using a a gene using a

plasmid? plasmid?

Steps in DNA recombinationSteps in DNA recombination

1. Isolate “needed: DNA gene & plasmid2. Cut (“digest”) BOTH DNA samples with the

SAME restriction enzyme3. Mix DNA’s together- they join at sticky ends

via DNA base pairing4. Add ligase to seals up sticky ends5.Product is the recombinant DNA 6. Add CaCl2 & bacteria

• CaCl2 makes bacteria “compentent” 7. Collect products (proteins) of cloned gene

(Ex: insulin, Human Growth Hormone)

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Plasmid CloningPlasmid Cloning

3 main reasons for creating recombinant DNA

1.to create a protein product2.to create multiple copies of genes3.to insert foreign genes into other organisms to give those organisms a new trait

•Recombinant DNA is used widely today to create large amounts of protein for treating illnesses• Ex: In 1982, insulin became the first

recombinant DNA drug to hit the market

Why make DNA recombinants?

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Plasmid CloningPlasmid Cloning