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Biotechnology and Genetic

Engineering

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Biotechnology

The use of living cells to makeproducts such aspharmaceuticals, foods, andbeverages

The use of organisms such asbacteria to protect theenvironment

The use of DNA science for theproduction of products,diagnostics, and research

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Recombinant DNA

The manipulation andcombination of DNA from twosources

Bacterial DNA + human genefor insulin

Plant DNA + bacterial DNA -Agrobacterium tumefaciens

Mouse DNA + human DNA =transgenic

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Recombination Insert a foreign gene into a host

Plasmid ( for example, exogenous DNA)into the bacterial cell transformationor transfection-organism referred to as

transgenic ( eukaryote ) orrecombinant( prokaryote)

Goal To produce many copies ( clones)of a particular gene

Reporter gene tags gene of interest to identify the presence of a gene

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Vectors

Plasmids

Viruses

Particles ( DNA coatedbullets)

Exogenous DNA

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Characteristics of a Vector

Can replicate independently inthe host cell contains an Ori

Has restriction sites in the

vector- Polylinker cloning region Has a reporter gene that will

announce its presence in thehost cell

Is a small size in comparison tothe host chromosome for easeof isolation

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Restriction Enzymes and Vectors

Cut Plasmid with restrictionenzyme

Cut gene of interest withrestriction enzyme

Splice together gene ofinterest and vector

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Tools for Recombination

Restriction enzymes

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Recombinant DNA

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Pharmaceuticals insulinfor diabetics factor VIIIfor males suffering from hemophilia A factor IXfor hemophilia B human growth hormone(GH) erythropoietin(EPO) for treating anemia three types of interferons

several interleukins granulocyte-macrophage colony-stimulating factor(GM-CSF) for stimulating

the bone marrow after a bone marrow transplant tissue plasminogen activator(TPA) for dissolving blood clots adenosine deaminase(ADA) for treating some forms of severe combined

immunodeficiency(SCID) angiostatinand endostatinfor trials as anti-cancer drugs

parathyroid hormone leptin hepatitis B surface antigen(HBsAg) to vaccinate against the hepatitis B virus

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Golden Rice- Agrobiotech

Golden rice is the resultof an effort to developrice varieties thatproduce provitamin-A(beta-carotene) as a

means of alleviatingvitamin A (retinol)deficiencies in the dietsof poor anddisadvantaged people indeveloping countries.

Because traditional ricevarieties do not produceprovitamin-A, transgenictechnologies wererequired.

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Transgenic Mice Manipulation ofembryo( blastocyst)

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Growth hormone and therapy

Growth hormone, also knownas somatotropin, is aprotein hormone of about190 amino acids that issynthesized and secreted

by cells called somatotrophsin the anterior pituitary.Itis a major participant incontrol of several complexphysiologic processes,including growth and

metabolism. Growth hormoneis also of considerableinterest as a drug used inboth humans and animals.

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Transgenic mice

Two baby mice - sameage

Human Growth hormoneinserted into theembryo of the mouse onthe left. Causes rapidgrowth in the newborn

The mouse on the right

is a normal sized mouse

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Insect Resistance

B. thuringiensis(commonlyknown as 'Bt') is aninsecticidal bacterium,marketed worldwide forcontrol of many importantplant pests - mainly

caterpillars of theLepidoptera (butterflies andmoths) but also mosquitolarvae, and simuliid blackfliesthat vector river blindness inAfrica. Bt products representabout 1% of the totalagrochemical market(fungicides, herbicides andinsecticides)

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Agrobacterium tumefaciens

Agrobacteriumtumefacienscausescrown gall disease byfirst transferring part

of its DNA into anopening in the plant. TheDNA then integratesitself into the plant's

genome and causes theformation of the gall.

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Crown Gall Plant tumor

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Natures Genetic Engineering

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Agrobacterium tumefaciens

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Vaccines

Bananas have potential tobecome the world's firstedible vaccine due toAgrobacterium. An ediblevaccine doesn't need sterilesyringes, costly

refrigeration, or multipleinjections. According to theWorld Health Organization(WHO), more than 2 millionchildren die worldwide each

year from diarrhea that canbe prevented easily withvaccines. Thus, researcherslead by Dr. Charles Arntzenare looking into making thefood vaccines to preventdiarrhea caused byEscherichia coliand Vibrio

cholarabacteria.

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pGlo GfpGreen fluorescent protein

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Fluorescent

In the laboratory,fluorescence is easilyachieved by exposingthe protein to longrange UV light or black" light.

The fluorophoreabsorbs light in theUV-B region (395 nm..plus a smallerabsorbance peak at

470 nm) It emits light

(fluoresces) at 509nm, which is in thegreen part of the

visible spectrum

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Gfp and Land Mines

Neal Stewart at theUniversity of North Carolina isdeveloping plants that candetect land mines

Plants could be idealbiosensors for land mines asseeds would be spread widelyand evenly in a suspect field

The gene that can announcethe presence of land mines isgfp

The gene will be expressed in

the presence of a land mine

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Green Fluorescent Protein and Plants

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GFP and mice

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Glo fish

Fluorescent zebrafish were speciallybred to help detectenvironmental

pollutants. By adding anatural fluorescencegene to the fish,scientists are able toquickly and easilydetermine when ourwaterways are

contaminated

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pGlo

Transformation of E. coliwith the pGlo plasmid

Ori Gene for Gfp The plasmid contains the

genes for the Arabinosepromoter

The plasmid contains thegenes for ampicillinresistance

If the bacterium uptakes theplasmid it should glow inresponse to long range uvlight

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pGlo

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Heat Shock

Leave cells intransformationsolution on ice forten minutes

Transfer to waterbath at 42oC for 90seconds

Return cells to ice

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Lac Operon

Operons Organization of genes formetabolic pathways in bacteria Lac Operon - All genes for the

metabolism of lactose connected

with one Open reading frame orORF Promoter for binding RNA

polymerase for transcription of thegene

Repressor molecule turns offtranscription by binding to anoperator next to the start 3 TAC