BiotechnologyBiotechnology The use of living cells to make The use of living cells to make

products such as products such as pharmaceuticals, foods, and pharmaceuticals, foods, and beveragesbeverages

The use of organisms such as The use of organisms such as bacteria to protect the bacteria to protect the environmentenvironment

The use of DNA science for the The use of DNA science for the production of products, production of products, diagnostics, and researchdiagnostics, and research

Recombinant DNARecombinant DNA The manipulation and The manipulation and

combination of DNA from two combination of DNA from two sources sources

Bacterial DNA + human gene Bacterial DNA + human gene for insulinfor insulin

Plant DNA + bacterial DNA - Plant DNA + bacterial DNA - Agrobacterium tumefaciensAgrobacterium tumefaciens

Mouse DNA + human DNA = Mouse DNA + human DNA = transgenictransgenic

RecombinationRecombination Insert a foreign gene into a hostInsert a foreign gene into a host Plasmid ( for example, exogenous DNA) Plasmid ( for example, exogenous DNA)

into the bacterial cell – transformation into the bacterial cell – transformation or transfection-organism referred to as or transfection-organism referred to as transgenic ( eukaryote ) or recombinant( transgenic ( eukaryote ) or recombinant( prokaryote)prokaryote)

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

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

VectorsVectors PlasmidsPlasmids VirusesViruses Particles ( DNA coated Particles ( DNA coated

bullets)bullets) Exogenous DNAExogenous DNA

Characteristics of a VectorCharacteristics of a Vector

Can replicate independently in Can replicate independently in the host cell – contains an Orithe host cell – contains an Ori

Has restriction sites in the Has restriction sites in the vector- Polylinker cloning regionvector- Polylinker cloning region

Has a reporter gene that will Has a reporter gene that will announce its presence in the announce its presence in the host cellhost cell

Is a small size in comparison to Is a small size in comparison to the host chromosome for ease the host chromosome for ease of isolationof isolation

Restriction Enzymes and VectorsRestriction Enzymes and Vectors Cut Plasmid with restriction Cut Plasmid with restriction

enzymeenzyme Cut gene of interest with Cut gene of interest with

restriction enzymerestriction enzyme Splice together gene of Splice together gene of

interest and vectorinterest and vector

Tools for RecombinationTools for Recombination Restriction enzymesRestriction enzymes

Recombinant DNARecombinant DNA

PharmaceuticalsPharmaceuticals insulininsulin for diabetics for diabetics factor VIIIfactor VIII for males suffering from hemophilia A for males suffering from hemophilia A factor IXfactor IX for hemophilia B for hemophilia B human growth hormonehuman growth hormone ( (GHGH) ) erythropoietinerythropoietin ( (EPOEPO) for treating anemia ) for treating anemia three types of three types of interferonsinterferons several several interleukinsinterleukins granulocyte-macrophage colony-stimulating factorgranulocyte-macrophage colony-stimulating factor ( (GM-CSFGM-CSF) for stimulating ) for stimulating

the bone marrow after a bone marrow transplant the bone marrow after a bone marrow transplant tissue tissue plasminogenplasminogen activator activator ( (TPATPA) for dissolving blood clots ) for dissolving blood clots adenosine deaminaseadenosine deaminase ( (ADAADA) for treating some forms of ) for treating some forms of

severe combined immunodeficiencysevere combined immunodeficiency ( (SCIDSCID) ) angiostatinangiostatin and and endostatinendostatin for trials as anti-cancer drugs for trials as anti-cancer drugs parathyroid hormone parathyroid hormone leptin leptin hepatitis B surface antigen (hepatitis B surface antigen (HBsAgHBsAg) to vaccinate against the hepatitis B virus ) to vaccinate against the hepatitis B virus

Golden Rice- AgrobiotechGolden Rice- Agrobiotech

Golden rice is the result Golden rice is the result of an effort to develop of an effort to develop rice varieties that rice varieties that produce provitamin-A produce provitamin-A (beta-carotene) as a (beta-carotene) as a means of alleviating means of alleviating vitamin A (retinol) vitamin A (retinol) deficiencies in the diets deficiencies in the diets of poor and of poor and disadvantaged people in disadvantaged people in developing countries. developing countries. Because traditional rice Because traditional rice varieties do not produce varieties do not produce provitamin-A, transgenic provitamin-A, transgenic technologies were technologies were required. required.

Transgenic Mice – Manipulation of embryo( blastocyst)

Growth hormone and therapyGrowth hormone and therapy Growth hormone, also known Growth hormone, also known

as as somatotropinsomatotropin, is a , is a protein hormone of about protein hormone of about 190 amino acids that is 190 amino acids that is synthesized and secreted synthesized and secreted by cells called by cells called somatotrophssomatotrophs in the anterior pituitary.in the anterior pituitary. It It is a major participant in is a major participant in control of several complex control of several complex physiologic processes, physiologic processes, including growth and including growth and metabolism. Growth hormone metabolism. Growth hormone is also of considerable is also of considerable interest as a drug used in interest as a drug used in both humans and animals. both humans and animals.

Transgenic miceTransgenic mice Two baby mice - same Two baby mice - same

ageage Human Growth hormone Human Growth hormone

inserted into the inserted into the embryo of the mouse on embryo of the mouse on the left. Causes rapid the left. Causes rapid growth in the newborngrowth in the newborn

The mouse on the right The mouse on the right is a normal sized mouseis a normal sized mouse

Insect ResistanceInsect Resistance B. thuringiensisB. thuringiensis

(commonly known as (commonly known as 'Bt') is an insecticidal 'Bt') is an insecticidal bacterium, marketed bacterium, marketed worldwide for control of worldwide for control of many important plant many important plant pests - mainly pests - mainly caterpillars of the caterpillars of the Lepidoptera (butterflies Lepidoptera (butterflies and moths) but also and moths) but also mosquito larvae, and mosquito larvae, and simuliid blackflies that simuliid blackflies that vector river blindness in vector river blindness in Africa. Bt products Africa. Bt products represent about 1% of represent about 1% of the total ‘agrochemical’ the total ‘agrochemical’ market (fungicides, market (fungicides, herbicides and herbicides and insecticides) insecticides)

Agrobacterium tumefaciensAgrobacterium tumefaciens AgrobacteriumAgrobacterium

tumefacienstumefaciens causes causes crown gall disease by crown gall disease by first transferring part first transferring part of its DNA into an of its DNA into an opening in the plant. The opening in the plant. The DNA then integrates DNA then integrates itself into the plant's itself into the plant's genome and causes the genome and causes the formation of the gall.formation of the gall.

Crown Gall – Plant tumorCrown Gall – Plant tumor

Nature’s Genetic Engineering

Agrobacterium tumefaciensAgrobacterium tumefaciens

VaccinesVaccines Bananas have potential to Bananas have potential to

become the world's first edible become the world's first edible vaccine due to vaccine due to AgrobacteriumAgrobacterium. . An edible vaccine doesn't need An edible vaccine doesn't need sterile syringes, costly sterile syringes, costly refrigeration, or multiple refrigeration, or multiple injections. According to the injections. According to the World Health Organization World Health Organization (WHO), more than 2 million (WHO), more than 2 million children die worldwide each year children die worldwide each year from diarrhea that can be from diarrhea that can be prevented easily with vaccines. prevented easily with vaccines. Thus, researchers lead by Dr. Thus, researchers lead by Dr. Charles Arntzen are looking into Charles Arntzen are looking into making the food vaccines to making the food vaccines to prevent diarrhea caused by prevent diarrhea caused by Escherichia coliEscherichia coli and and Vibrio Vibrio cholaracholara bacteria. bacteria.

pGlo – GfppGlo – GfpGreen fluorescent proteinGreen fluorescent protein

FluorescentFluorescent In the laboratory, In the laboratory,

fluorescence is easily fluorescence is easily achieved by exposing achieved by exposing the protein to long the protein to long range UV light or “ range UV light or “ black" light. black" light.

The fluorophore The fluorophore absorbs light in the absorbs light in the UV-B region (395 nm.. UV-B region (395 nm.. plus a smaller plus a smaller absorbance peak at absorbance peak at 470 nm) 470 nm)

It emits light It emits light (fluoresces) at 509 (fluoresces) at 509 nm, which is in the nm, which is in the green part of the green part of the visible spectrumvisible spectrum

Gfp and Land MinesGfp and Land Mines Neal Stewart at the Neal Stewart at the

University of North University of North Carolina is developing Carolina is developing plants that can detect plants that can detect land minesland mines

Plants could be ideal Plants could be ideal biosensors for land biosensors for land mines as seeds would be mines as seeds would be spread widely and evenly spread widely and evenly in a suspect fieldin a suspect field

The gene that can The gene that can announce the presence announce the presence of land mines is gfpof land mines is gfp

The gene will be The gene will be expressed in the expressed in the presence of a land minepresence of a land mine

Green Fluorescent Protein and Plants

GFP and miceGFP and mice

Glo fishGlo fish Fluorescent zebra fish Fluorescent zebra fish

were specially bred to were specially bred to help detect environmental help detect environmental pollutants. By adding a pollutants. By adding a natural fluorescence gene natural fluorescence gene to the fish, scientists are to the fish, scientists are able to quickly and easily able to quickly and easily determine when our determine when our waterways are waterways are contaminated contaminated

pGlopGlo Transformation of Transformation of E. E.

colicoli with the pGlo with the pGlo plasmidplasmid

OriOri Gene for GfpGene for Gfp The plasmid contains The plasmid contains

the genes for the the genes for the Arabinose promoterArabinose promoter

The plasmid contains The plasmid contains the genes for ampicillin the genes for ampicillin resistanceresistance

If the bacterium If the bacterium uptakes the plasmid it uptakes the plasmid it should glow in response should glow in response to long range uv lightto long range uv light

pGlopGlo

Arabinose operonArabinose operon araO1araO1 is an operator site. is an operator site. AraCAraC binds to binds to

this site and represses its own this site and represses its own transcription from the transcription from the PCPC promoter. In promoter. In the presence of arabinose, however, the presence of arabinose, however, AraCAraC bound at this site helps to activate bound at this site helps to activate expression of the expression of the PBAD PBAD promoter.promoter.

araO2araO2 is also an operator site. is also an operator site. AraCAraC bound at this site can simultaneously bind bound at this site can simultaneously bind to the to the araIaraI site to repress transcription site to repress transcription from the from the PBADPBAD promoter promoter

araI araI is also the inducer site. is also the inducer site. AraCAraC bound bound at this site can simultaneously bind to the at this site can simultaneously bind to the araO2araO2 site to repress transcription from site to repress transcription from the the PBADPBAD promoter. In the presence of promoter. In the presence of arabinose, however, arabinose, however, AraCAraC bound at this bound at this site helps to activate expression of the site helps to activate expression of the PBADPBAD promoter. promoter.

Trasnformation movieTrasnformation movie Transformation movieTransformation movie

P Glo transformationP Glo transformation

Pick one colony from the starter plate.Pick one colony from the starter plate. Use the sterile loopsUse the sterile loops Swirl the loop in ice cold CaCl2 ( experimental)Swirl the loop in ice cold CaCl2 ( experimental) Place in ice for 10 minutes ( Your tubes will be incubating when Place in ice for 10 minutes ( Your tubes will be incubating when

you enter the room). I have found that a longer incubation you enter the room). I have found that a longer incubation period here increases the yield of transformantsperiod here increases the yield of transformants

While the tubes are incubating label your platesWhile the tubes are incubating label your plates LB AMP these plates eliminate bacteria that do not have gene for LB AMP these plates eliminate bacteria that do not have gene for

antibiotic resistance to ampicillinantibiotic resistance to ampicillin LB/AMP? Ara- These plates contain Arabinose and AmpicillinLB/AMP? Ara- These plates contain Arabinose and Ampicillin These are called the selection plates. The Arabinose will induce These are called the selection plates. The Arabinose will induce

the gene to be turned onthe gene to be turned on LB- Luria Broth Agar – all bacteria should grow on this agarLB- Luria Broth Agar – all bacteria should grow on this agar

Heat ShockHeat Shock Leave cells in Leave cells in

transformation transformation solution on ice for solution on ice for ten minutesten minutes

Transfer to water Transfer to water bath at 42oC for bath at 42oC for 90 seconds90 seconds

Return cells to iceReturn cells to ice

Lac OperonLac Operon Operons –Organization of genes for Operons –Organization of genes for

metabolic pathways in bacteriametabolic pathways in bacteria Lac Operon - All genes for the Lac Operon - All genes for the

metabolism of lactose connected metabolism of lactose connected with one Open reading frame or with one Open reading frame or ORFORF

Promoter for binding RNA Promoter for binding RNA polymerase for transcription of the polymerase for transcription of the genegene

Repressor molecule turns off Repressor molecule turns off transcription by binding to an transcription by binding to an operator next to the start 3’ TACoperator next to the start 3’ TAC

Recovery and PlatingRecovery and Plating Incubate bacteria in Luria Incubate bacteria in Luria

Broth for 10 minutes before Broth for 10 minutes before plating in Petri Dishplating in Petri Dish

Plate your bacteriaPlate your bacteria+ pGlo – LB AMP and + pGlo – LB AMP and

LB/Amp/AraLB/Amp/Ara- pGlo – LB and LB/AMP- pGlo – LB and LB/AMP

Gene Expression and Genetic EngineeringGene Expression and Genetic Engineering

LinksLinks OperonOperon movie in Quick Time movie in Quick Time Lac OperonLac Operon Trp Operon MovieTrp Operon Movie E. coli gene regulation E. coli gene regulation Gene RegulationGene Regulation