Biotechnology

BIOTECHNOLOGY

BIOTECHNOLOGY

FOR THE YOUNG MINDSDr. NEERAJA SOOD DYAL SINGH COLLEGE UNIVERSITY OF DELHINEW DELHIBIOTECHNOLOGYThe use of living organisms or their products to enhance our lives and our environment.

A more narrow definition is;The commercial application of living organisms or their products, which involves the deliberate manipulation of their DNA molecules"

WHY BIOTECHNOLOGY?Traditional methods of improvement are limited in scope due to genetic barriersImprovement may not be of high standardsMethods of improvement are time consuming.Markers associated with improvement are limited (diagnostic tests to identify genes for inherited diseases like cystic fibrosis, Huntingtons disease).

TECHNIQUES USED IN BIOTECHNOLOGYR-DNA TECHNOLOGYHYBRIDOMA TECHNOLOGYEMBRYO TRANSFER TECHNOLOGYTRANSGENICSPROTEIN ENGINEERINGDNA ENGINEERINGVACCINESMICROBIAL BIOTECHNOLOGYr-DNA TECHNOLGY

TRANSgenesis

TRANSGENICSA large number of transgenic animals have been createdMice Cows Pigs Sheep Goats Fish Frogs InsectsCurrently, no transgenic animal or animal product is approved by the FDA or USDA for human consumption

TRANSGENIC CATTLEDairy cows carrying extra copies of two types of casein genes produce 13% more milk proteinNot only will this make the milk more nutritious, it would allow for less milk to make more cheeseCurrently the milk from these animals is under FDA reviewThe important difference between this & other transgenics is that the DNA added is not foreign

EnviroPig TMTransgenic pigs express phytase in their salivary glandsPhytic acid in the pig meal is degraded releasing phosphorusThe phosphorus is absorbed by the pigNormally the phytic acid/phosphorus complex is excreted Pig waste causes eutrophication of lakes & streams

Transgenic FishTilapiaSalmon/troutCatfishAll can grow up to 6 times faster than wildtype fish Most have extra copies of growth hormone (GH) gene

TRANSGENIC FISHGloFish, originally developed in Singapore as a way to monitor water pollution The normally black-and-silver zebrafish was turned green or red by inserting various versions of the GFP geneGlofish are on sale throughout the US except in CaliforniaGlofish retail for about $5 per fish. Normal zebra-fish cost around one tenth of the price

Transgenic CropsTransgenic crops resistant to insect pestsTransgenic crops resistant to diseasesDrought tolerant transgenic cropsHerbicide tolerant transgenic cropsTransgenics with nutritive traits like Golden RiceTransgenics with agronomic traits like delayed ripening

Insect Protective GenesBt cry genes from Bacillus thuringiensishighly effective, highly specific, safe to environment and higher animals, amenable to genetic engg.Trypsin Inhibitor genesLectin genes

DNA ENGINEERING

VACCINES (EDIBLE)

IMMOBILISATION OF ENZYMES

LACONESCONSERVATION CLONINGMany endangered or extinct animals are being cloned or considered for cloningGaurBucardo mountain goat MammothQuaggaBanteng

pBR322CLONING

Dolly, First Mammal Cloned From an Adult Cell

Dolly, as an adult

GENE CLONINGIDENTIFICATION OF DESIRABLE GENES (cut DNA by RE -> gel electrophoresis -> elution of band/desirable DNA)INTRODUCTION OF IDENTIFIED DNA INTO THE HOST(use of cloning vectors/RE/ligases/transformation )MAINTAINENCE OF INTRODUCED DNA IN THE HOST AND ITS TRANSFER IN PROGENY(use of selectable marker)OBTAINING THE RECOMBINANT PROTEIN (bioreactor)pBR322

BACTERIOPHAGE LAMDA

CLONING VECTOR

VECTORS

Fig 1.BACFig 2. YAC31RE

32r-Plasmid DNA

COLONY HYBRIDISATION

PCR

HUMAN INSULIN PRODUCTION BY BACTERIA

FERMENTORS

Mammalian Cell Bioreactor

DOWNSTREAM PROCESSING

Bacterial and Animal Biotechnology Products

Biotech chymosinenzyme used to curdle milk products gene from yeast harvested from GE bacteria replaces the calf enzyme

bST (bovine somatotropin)increases milk production gene from cow protein harvested from GE bacteria replaces cow protein originally harvested from pituitary glands of slaughtered cows

SCOPE OF BIOTECHNOLOGY Human Health Diagnostics (e.g., biosensors, immunodiagnostics, geneprobes) Therapeutics (e.g., vaccines, immune stimulants,biopharmaceuticals) Drug Delivery Bioinformatics Genomics and Molecular Modelling (e.g., DNA/RNA/proteinsynthesizing and databases for humans, plants, animals, andmicro-organisms) Gene Therapy (e.g., gene identification, gene constructs, genedelivery)

SOME MORE AREASEnvironment Air, Water and Soil (e.g., bioremediation, diagnostics, phytoremediation,biofiltration)

Natural Resources Energy (e.g., microbiologically enhanced petroleum recovery,industrial bioprocessing, biodesulphurization) Mining (e.g. microbiologically enhanced mineral recovery,industrial bioprocessing, biodesulphurization) Forest Products (e.g., biopulping, biobleaching, biopesticides,tree biotechnology, industrial bioprocessing)

ADVANCES:-In 1977, Genentech reports production of first human protein, Somatostatin, in bacteria

Genentech Inc and the City of Hope National Medical Center announces production of human insulin using recombinant technology

US supreme court allowed patenting of genetically altered life; oil eating microbe patented

GE plants are also patented

USDA approves release of GE tobacco plants under field conditions

Recombinant hepatitis B vaccine approvedCentocars CA125TM test for ovarian cancer approvedGenentech Inc. gets approval to market rt-PA (GE tissue plasminogen activator) to treat heart attacksAnticancer drugs, Interleukin-2 and polyethylene glycol IL-2 licensedKary Mullis introduced PCR in 1988The Human Genome Project, the international effort to map all of the genes in the human body, was launched. Estimated cost: $13 billion. 1990 Formal launch of the international Human Genome Project

Southern Blotting

VNTRsEvery strand of DNA has pieces that contain genetic information which informs an organism's development (exons) and pieces that, apparently, supply no relevant genetic information at all (introns). Although the introns may seem useless, it has been found that they contain repeated sequences of base pairs. These sequences, called Variable Number Tandem Repeats (VNTRs), can contain anywhere from twenty to one hundred base pairs. Every human being has some VNTRs. To determine if a person has a particular VNTR, a Southern Blot is performed, and then the Southern Blot is probed, through a hybridization reaction, with a radioactive version of the VNTR in question. The pattern which results from this process is what is often referred to as a DNA fingerprint. A given person's VNTRs come from the genetic information donated by his or her parents; he or she could have VNTRs inherited from his or her mother or father, or a combination, but never a VNTR either of his or her parents do not have. Shown below are the VNTR patterns for Mrs. Nguyen [blue], Mr. Nguyen [yellow], and their four children: D1 (the Nguyens' biological daughter), D2 (Mr. Nguyen's step-daughter, child of Mrs. Nguyen and her former husband [red]), S1 (the Nguyens' biological son), and S2 (the Nguyens' adopted son, not biologically related [his parents are light and dark green]).

VNTR

DNA FINGERPRINTING

The chemical structure of everyone's DNA is the same. The only difference between people (or any animal) is the order of the base pairs. There are so many millions of base pairs in each person's DNA that every person has a different sequence. Using these sequences, every person could be identified solely by the sequence of their base pairs. However, because there are so many millions of base pairs, the task would be very time-consuming. Instead, scientists are able to use a shorter method, because of repeating patterns in DNA. These patterns do not, however, give an individual "fingerprint," but they are able to determine whether two DNA samples are from the same person, related people, or non-related people. Scientists use a small number of sequences of DNA that are known to vary among individuals a great deal, and analyze those to get a certain probability of a match.5' T-T-G-A-C-T-A-T-C-C-A-G-A-T-C 3'3' A-A-C-T-G-A-T-A-G-G-T-C-T-A-G 5'The chemical structure of DNA is as follows:

DNA FINGERPRINTING

Different individuals carry different alleles.Most alleles useful for DNA fingerprinting differ on the basis of the number of repetitive DNA sequences they contain.HGPHuman Genome Project has been called a Mega Project because of the following factors:1. The human genome has approx. 3.3 billion base-pairs; if the cost of sequencing is US $3 per base-pair, then the approx. cost will be US $10 billion.2. If the sequence obtained were to be stored in a typed form in books and if each page contains 1000 letters and each book contains 1000 pages, then 3300 such books would be needed to store the complete information.However, if expressed in computer storage units (3.3 billion base-pairs) x (2 bits per pair) = 825 megabytes of raw data. Which is about the same size of one music CD. If further compressed, this data can be expected to fit in less than 20 Megabytes.

CENTRAL DOGMA

RNA- INTERFERENCE

NEXT STEP?

PLAYING GOD!Synthetic Mycoides refers to an artificial life form created by Craig Venter at the J Craig Venter Institute in May 2010. A synthetic genome was transferred into an empty cell to form the bacterium.

J. CRAIG VENTOR

THANKS!