biotech and its applications

8/4/2019 Biotech and Its Applications

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Biotechnology , a s you would have l ea rn t f rom the

previous cha pter, essent ial ly deals with ind u strial scale

produ ction of biopha rm aceu ticals an d b iologicals u sing

genetically modified microbes, fu ngi, plan ts an d an ima ls.

The applications of biotechnology include therapeutics,diagnostics, genetically modified crops for agriculture,

processed food, bioremediation, waste treatment, and

energy produc t ion . Three c r i t i ca l r e sea rch a reas o f

biotechn ology are:

(i) Providing the best ca talyst in th e form of imp roved

organ ism u su ally a microbe or pure en zyme.

(ii) Creating optima l conditions throu gh engineering for

a cata lyst to act , and

(iii) Downs tream processing techn ologies to pur ify th e

protein/ organ ic compou nd.

Le t us now lea rn how human be ings have usedbiotechnology to improve the quali ty of human l ife,

esp ecially in t h e field of food prod u ction an d h ealth.

1 2 . 1 B IOTECHNOLOGICAL APPLICATIONS INAGRICULTURE

Let us take a look at the th ree options th at can be thou ght

for increa sin g food prod u ction

(i) agro-chemical based agr icul ture ;

CHAPTER 12

BIOTECHNOLOGY AND ITS

APPLICATIONS

12.1 Biotechnological

Applications in

Agriculture

12.2 Biotechnological

Appl icat ions in

Medicine

12.3 Transgenic Animals

12.4 Ethical Is s ues


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(ii) organ ic agr icul ture ; an d

(iii) genetically engineered crop-bas ed agricu ltu re.

Th e Gree n Revo lution su cceeded in tripling th e food su pply but yet

it was n ot enou gh to feed the growing h u ma n popu lation. Increas ed yieldsha ve part ly been du e to the u se of improved crop variet ies, bu t m ainly

du e to the us e of better ma na gemen t practices a nd u se of agrochem icals

(fertiliser s a n d pes ticides ). However, for far m ers in th e developin g world,

agrochemicals are often too expen sive, an d fu rth er increas es in yield with

existin g varieties ar e not poss ible us ing con ventiona l breed ing. Is th ere

an y alternative path th at our u nd erstan ding of genetics can sh ow so tha t

farm ers m ay obtain m aximu m yield from th eir fields? Is th ere a way to

minimise th e u se of fertilisers a nd chem icals so th at th eir h arm fu l effects

on th e environ m ent a re redu ced? Use of genetically modified crops is a

poss ible solu tion .

Plan ts, bacteria, fu ngi an d an imals whose genes h ave been a ltered byma nipu lation a re called Gene tically Modified Organism s (GMO). GM

plants ha ve been u sefu l in ma ny ways. Genetic modification ha s:

(i) ma de crops m ore tolerant to abiot ic s t resses (cold, drought , sa lt ,

heat).

(ii) redu ced relian ce on chem ical pesticides (pest-resistant crops).

(iii) helped to redu ce post h arvest losses.

(iv) increas ed efficiency of mineral u sage by plan ts (this prevents early

exh au st ion of fert ility of soil).

(v) enh an ced n u trition al valu e of food, e.g., Vitamin A enr ich ed rice.

In addit ion to these uses, GM has been used to create tai lor-made

plants to su pply alterna tive resour ces to ind u stries, in th e form of sta rches ,

fu els an d pha rma ceuticals.

Some of the a pplications of biotechn ology in agricultu re th at you will

stu dy in d etail are th e produ ction of pest res ista nt plants , which could

decrease the amount of pest ic ide used. Bt toxin i s produced by a

bacterium called Bacillus t huringiensis (Bt for sh ort). Bt toxin gene h as

been cloned from th e bacteria a nd been express ed in p lants to provide

resista n ce to in sects with out t h e need for ins ecticides ; in effect created a

bio-pesticide. Exam ples are Bt cotton, Bt corn, r ice, tomato, potato a nd

soyabean etc.

Bt Cotton : Some st ra ins of Bacillus thu ringiens is produce proteins thatkill certain ins ects su ch a s lepidopteran s (tobacco bu dworm, arm yworm),

coleopteran s (beetles) an d d ipteran s (flies, m osqu itoes). B. thu ringiens is

forms protein crystals du ring a pa rt icu lar p ha se of their growth. Thes e

crystals conta in a toxic insec ticidal protein . Wh y does th is toxin n ot kill

th e Bacillus? Actu ally, the Bt toxin protein exist a s inactiveprotoxins bu t

once a n insect ingest t he inactive toxin, it is converted int o an active form

of toxin du e to th e alkaline p H of the gut wh ich solubilise th e crystals.

The activated toxin binds to the surface of midgut epithelial cells and


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BIOTECHNOLOGY AND ITS APPLICATIONS

create pores th at cau se cell swelling and lysis a nd eventu ally cau se death

of th e in sect.

Sp ecific Bt toxin gen es were isolat ed from Bacillus thu ringiens is a n d

incorporated into the s everal crop plan ts s u ch a s cotton (Figu re 12.1).

The choice of genes depends upon the crop and the targeted pest , as

most Bt toxins are insect-group specific. The toxin is coded by a gene

named cry . Th ere are a n u mb er of th em, for exam ple, th e protein s en coded

by the genes cryIAc a n d cryIIAb control the cotton b ollworm s, th at of

cryIAb controls corn b orer.

Figure 12 .1 Cotton boll: (a) destroyed by bollworms; (b) a fully mature

cotton boll

(b)

(a )

Pest Resistant Plants :Several nem atodes par as it ise a wide variety of

plan ts an d an ima ls inclu ding hu ma n b eings. A nema tode Meloidegyne

incognitiainfects th e roots of tobacco plan ts a n d cau ses a great redu ction

in yield. A n ovel str at egy was a dopt ed to prevent t h is in festa tion which

was based on the process of RNA inte rferenc e (RNAi). RNAi ta kes pla ce

in all eu ka ryotic organ isms as a m eth od of cellular defens e. Th is m eth od

involves silen cin g of a sp ecific mRNA du e to a comp lem ent ary d sRNA

molecule tha t bind s to a n d pr events tra n slation of th e mRNA (silencing).Th e sou rce of th is comp lem ent ar y RNA could be from a n infection by

viruses having RNA genomes or mobile genetic elements (transposons)

th at r eplicate via a n RNA in term ediate.

U s i n g Agrobacter ium v e c t o r s , n e m a t o d e - sp e c i f i c g e n e s w e r e

introduced into the host plant (Figure 12.2). The introduction of DNA

was su ch th at i t produ ced both s ense an d an t i-sense RNA in th e host

cells. Th ese two RNAs b eing complem ent ar y to each oth er form ed a d ou ble

st ra n ded (dsRNA) th at in itiated RNAi an d th u s, s ilen ced th e specific m RNA


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of the n ema tode. The cons equen ce was tha t th e para site could not su rvive

in a tra n sgen ic h ost expres sin g specific interferin g RNA. Th e tran sgen ic

plan t th erefore got itself protected from th e pa ra site (Figur e 12.2 ).

Figure 12 .2 Host plant -generated dsRNA tr iggers protect ion against nematode infestat ion:

(a) Roots of a typical contr ol plan ts; (b) tran sgenic plan t roots 5 d ays after delibera te

infect ion of nematode but protected through novel mechanism.

(a ) (b )

1 2 . 2 BIOTECHNOLOGICAL APPLICATIONS IN MEDICINE

The r ecomb ina nt DNA techn ological processes h ave mad e imm ens e imp act

in th e area of hea lthca re by ena bling ma ss produ ction of sa fe and moreeffective therap eutic dru gs. Furth er, the recombinan t th erapeu tics do n ot

indu ce un wanted immu nological respons es as is comm on in case of

s imilar products isola ted from n on-hu ma n sources. At presen t , about

30 recombinan t therapeu t ics have been a pproved for hu ma n-u se the

world over. In Ind ia, 12 of th ese ar e pres ent ly being ma rket ed.

12 .2.1 Gene t ical ly Engineered Insul in

Management of adult-onset diabetes is possible by taking insulin at

regular t ime intervals . Wh at w ould a d iabe tic patient d o if enough

hum an-ins ulin w as not available? If you discus s t his, you would soon

realise th at one would ha ve to isolate and u se insu lin from other an ima ls.

Would the insulin isolated from other animals be just as effective as

that secreted by the hu ma n body itsel f and w ould i t not elicit an imm une

res ponse in the hum an body ? Now, im agine if ba cterium were availab le

tha t could m ake h u ma n ins u lin . Su ddenly the whole process becomes

so sim ple. You ca n ea sily grow a large qua n tity of th e bacteria a n d m ak e

as m u ch ins u lin as you need.

Think about w heth er ins ulin can b e orally ad m inistered to diabetic

people or not. W hy ?


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Ins u lin u sed for diabetes was earl ier extracted from

pan creas of slau ghtered catt le an d pigs. Ins u lin from an

an ima l sou rce, th ough cau sed som e patient s to develop

a l l e rgy or o the r types o f reac t ions to the fo re ignp r o t e in . In s u l in c o n s i s t s o f t w o sh o r t p o l yp e p t i d e

ch a ins : chain A an d cha in B, that a re link ed together by

disu lph ide bridges (Figure 12 .3). In m am ma ls, in clu ding

hu ma ns , ins u lin is synth esised as a pro-horm one (like a

pro-enzyme, the pr o-hormon e also needs to be process ed

before it becomes a fu lly matu re an d fun ctional h ormone)

which contains an extra s tretch called the C peptide . This

C pept ide is n ot present in the m atu re ins u lin an d is

r e m o v e d d u r i n g m a t u r a t i o n i n t o i n su l i n . T h e m a i n

cha llen ge for pr odu ction of ins u lin u sing rDNA techn iqu es

was gett ing insulin assembled into a mature form. In1983, Eli Lilly an American company prepared two DNA sequences

corresponding to A an d B, chains of hu ma n insu lin an d introduced th em

in plasmids ofE. coli to produce insulin chains. Chains A and B were

produ ced sepa rately, extracted a nd combined by creating disu lfide bond s

to form hu man ins u lin .

12 .2.2 Gene Therapy

If a pers on is born with a h ereditary disea se, can a corrective thera py

be taken for such a disease? Gene therapy is an a t tempt to do this .

Gene therapy is a collection of methods that al lows correction of a

gene defect tha t h as been diagnosed in a ch ild/ embryo. Here genes

are inser ted into a p ersons ce lls a nd t iss u es to t rea t a disease .

Correction of a genetic defect involves delivery of a normal gene into

th e ind ividu al or emb ryo to take over the fu n ction of an d compen sa te

for the non-functional gene.

Th e first clin ical gene th erap y was given in 19 90 to a 4 -year old girl

with ad enos ine d eam inas e (ADA) deficien cy. Th is en zym e is cr u cial for

the imm u ne s ystem to fu nction. The disorder is cau sed du e to the deletion

of th e gene for ad enos ine dea m inas e. In som e children ADA deficiency

can be cu red by bone m arrow trans plantation; in oth ers it can b e treated

by enzyme replacem ent th erap y, in which fun ction al ADA is given to th e

patient by injection. But the pr oblem with both of these appr oaches th atthey are not com pletely cura tive. As a first step toward s gene t hera py,

lymph ocytes from the blood of the patient a re grown in a cu ltu re outs ide

the body. A functional ADA cDNA (using a retroviral vector) is then

introdu ced into these lymph ocytes, which are su bsequ ently return ed to

the p atient. However, as th ese cells are n ot imm ortal, the patient r equires

periodic in fu sion of su ch gen etically engineered lym ph ocytes. However, if

th e gene isolate from m ar row cells prod u cing ADA is introd u ced into cells

at early embr yonic stages, it could be a perma nen t cure.

Figure 12 .3 Maturat ion of

pro- insul in into insul in

(simplified)


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12 .2.3 Molecular Diagnosis

You k n ow tha t for effective treat men t of a d iseas e, early diagnos is an d

u nd ersta nd ing its pa th ophysiology is very imp ortan t. Using conventional

meth ods of diagnosis (seru m an d u rine an alysis, etc.) early detection is

n ot poss ible. Recomb inan t DNA techn ology, Polymera se Ch ain Reaction

(PCR) an d En zyme Lin ked Imm u n o-sorben t Ass ay (ELISA) ar e som e of

the techn iques tha t serve the pu rpose of early diagnosis.

Presen ce of a p ath ogen (bacteria, virus es, etc.) is n orma lly su sp ected

only when the pa thogen ha s p roduced a d isease s ymptom. By this t ime

th e concent ra tion of path ogen is a lready very high in t h e body. However,

very low concen tra tion of a bacteria or viru s (at a time when th e symp toms

of th e diseas e are n ot yet visible) can be det ected b y am plification of th eir

nucleic acid by PCR. Can y ou explain how PCR can de tect very low

am ounts of DNA? PCR is now rout inely u sed to detect HIV in su sp ected

AIDS patients. I t is b eing u sed to detect m u tations in genes in su spected

can cer pat ients too. It is a powerfu l techqn iqu e to identify ma ny oth er

genetic disorders .

A single stranded DNA or RNA, tagged with a radioactive molecule

(prob e) is allowed to h ybridise to its com plemen tar y DNA in a clon e of

cells followed by detection u sing au tora diogra ph y. Th e clone h aving the

mu tated gene will hen ce not app ear on th e photograph ic film, becau se

the p robe will not ha ve complimen tari ty with the m u tated gene.

ELISA is based on the principle of antigen-antibody interaction.

Infection by pathogen can be detected by the presence of antigens

(proteins , glycoproteins , etc.) or by detecting the an tibodies synt h esisedagains t th e path ogen.

1 2 .3 TRANSGENIC ANIMALS

Animals tha t h ave had th eir DNA ma nipulated to possess a nd express an

extra (foreign ) gene a re kn own a s transgenic animals . Tran sgenic rats ,

rab bits, pigs, sheep, cows a nd fish ha ve been p rodu ced, althou gh over

95 per cent of al l exist ing tran sgenic anima ls are m ice. Why are these

an im als being produced ? How can m an b ene fit from s uch m odifications ?

Let us try and explore some of the comm on reas ons :

(i) No r m a l p h ys i ol o gy a n d d eve l o p m en t : Trans genic an ima ls can

be specifically designed to al low the study of how genes areregulated, an d h ow th ey affect the n orma l fu nctions of the body

an d its d evelopm en t, e.g., stu dy of comp lex factors involved in growth

su ch a s ins u lin-like growth factor. By introdu cing genes from oth er

species that al ter the formation of this factor and studying the

biological effects th at resu lt , informa tion is obtained abou t th e

biological role of th e fact or in th e body.

(ii) Study of d isease:Many transgenic animals are designed to increase

our understanding of how genes contribute to the development of


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disease. Thes e are sp ecially made to s erve as m odels for h u ma n

diseas es so tha t investigation of n ew treatm ents for disea ses is ma de

possible. Today tran sgenic models exist for m an y hu ma n diseases

su ch a s can cer, cystic fibrosis, rh eu ma toid a rth ritis a n d Alzheimers.

(iii) Bio log i ca l p rod uct s : Medicin es required to treat certain hu ma n

diseases can conta in biological produ cts , but su ch produ cts are

often expens ive to ma ke. Tran sgenic an ima ls th at pr odu ce useful

biological produ cts can be created b y the in trodu ction of th e portion

of DNA (or genes) which codes for a particular product such as

hu man prote in (-1-an titrypsin) u sed to treat emph ysema . Similar

attem pts are being ma de for treatm ent of phenylketonu ria (PKU)

an d cystic fibrosis. In 1 997 , th e first tran sgenic cow, Rosie, produ ced

human prote in-enr iched milk (2.4 grams per l i t re) . The mi lk

conta ined th e hu man a lpha- lac ta lbu min an d was n u tr it ional ly a

more balanced product for hu man babies th an na tura l cow-milk.

(iv) Vaccine sa fe t y :Transgenic mice are being developed for use in

test ing the safety of vaccines before they are used on humans.

Tran sgenic mice are being u sed to test th e sa fety of th e polio vaccine.

If su ccessful an d foun d to be reliab le, they could replace the u se of

mon keys to test th e safety of ba tch es of th e vaccine.

(v) Chemical safety tes t ing:Th is is kn own as toxicity/ sa fety testin g.

Th e procedur e is th e sam e as th at u sed for testing toxicity of dru gs.

Transgenic animals are mad e that carry genes which m ake them more

sens itive to toxic su bsta nces th an non -trans genic animals. They are

then exposed to the toxic su bs tan ces an d th e effects stu died. Toxicity

testing in s u ch a nima ls will allow u s to obtain res u lts in less time.

1 2 . 4 ETHICAL ISSUES

The m an ipulation of living organism s by the h u ma n ra ce cann ot go on

an y fu rth er, withou t regulation. Some ethical stan dar ds a re required to

evalu ate th e mora lity of all h u m an activities th at m igh t h elp or h arm living

organisms.

Goin g beyon d th e mora lity of su ch issu es, th e biological sign ifican ce

of su ch th ings is also importan t . Genetic modification of organ ism s can

ha ve un predicatable resul ts when s u ch organ ism s a re int rodu ced intothe ecosystem .

Therefore, the Indian Governm ent h as se t u p organisa t ions su ch a s

GEAC (Genetic Engineering Approval Committee), which will make

decisions regarding the val idi ty of GM research and the safe ty of

introdu cin g GM-organism s for pu blic services.

Th e m odification/ u sa ge of livin g organism s for pu blic s ervices (as food

an d m edicin e sour ces, for exam ple) ha s also created problems with p aten ts

granted for the sam e.


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There is growing public anger that certain companies are being

granted patents for products and technologies that make use of the

genetic mat erials, plants an d oth er biological resources t ha t h ave long

been identified, developed a nd u sed b y farm ers a nd ind igenou s p eopleof a sp ecific region/ coun try.

Rice is an important food grain, the presence of which goes back

thou san ds of years in Asias agricultur al history. There a re an estimated

20 0,00 0 varieties of rice in In dia a lon e. Th e divers ity of rice in Ind ia is

one of the richest in the world. Basm ati rice is dist inct for i ts u nique

arom a an d flavour a nd 27 docu men ted variet ies of Basm ati are grown

in India. There is reference to Basmati in ancient texts, folklore and

poetry, as i t has been grown for centuries. In 1997, an American

compan y got paten t rights on Bas ma ti rice throu gh the US Patent an d

Tra dem ar k Office. This a llowed th e comp an y to sell a n ew variety of

Basm ati, in the US an d a broad. This new variety of Basm ati h ad actu ally

been d erived from Ind ian farm ers varieties. In dian Bas m ati was cross ed

with sem i-dwarf varieties a n d claimed as an inven tion or a n ovelty. Th e

patent extends to functional equivalents, implying that other people

selling Bas ma ti rice cou ld be restricted by th e paten t. Several attem pts

ha ve a lso been m ade to patent us es, produ cts and processes bas ed on

Ind ian trad it iona l herba l medicines, e.g. , tu rmeric neem . If we ar e n ot

vigilant a nd we do not immediately cou nter thes e paten t ap plications ,

other cou ntries/ individu als ma y encas h on our rich legacy and we may

not be able to do anything about i t .

Biopiracy is the term used to refer to the use of bio-resources by

mul t inat ional companies and other organisa t ions wi thout proper

au thor i sa t ion f rom the count r i e s and people concerned wi thout

compens atory payment .

Most of the indu strial ised na tions are rich fina ncially bu t p oor in

biodiversity and traditional knowledge. In contrast the developing and

the underdeveloped world i s r ich in biodiversi ty and t radi t ional

kn owledge related to bio-resou rces . Trad itiona l kn owledge related to

bio-resou rces can be exploited to develop modern ap plications a n d can

also be used to save t ime, effor t and expendi ture dur ing thei r

commercialisation.

There has been growing realisation of the injustice, inadequate

compensation and benefit sharing between developed and developing

coun tries. Th erefore, some n ations a re developing laws to p revent s u ch

unauthor i sed explo i t a t ion of the i r b io- resources and t rad i t iona l

knowledge.

The Indian Parl iam ent h as recently cleared the second am endm ent

of the Indian Patents Bil l , that takes such issues into consideration,

inc lud ing pa ten t t e rms emergency prov i s ions and resea rch and

development initiative.


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EXERCISES

1. Crystals of Bt toxin produ ced by some bacter ia do not ki ll the bacter iathemselves because

(a) bacteria a r e r es is t an t to the toxin

(b ) t oxin i s im m a t u r e;

(c ) t ox in i s inac t ive ;

(d) bacteria encloses toxin in a s pec ia l sac .

2 . What a r e t r ansgenic bac ter ia? Illus t r a t e us ing any one example.

3 . Compare and cont r as t t he advantages and d isadvantages of product ion

of genetically modified crops.

SUMMARY

Biotechnology has given to humans several useful products by usingmicrobes, plant , animals and thei r metabol ic machinery. Recombinant

DNA technology has made i t possible to engineer microbes, plants

and an i m a l s such t ha t t hey have nove l capab i l i t i e s . G ene t i ca l l y

Modif ied Organisms have been created by using methods other than

natural methods to t ransfer one or more genes f rom one organism to

a n o t h e r , g e n e r a l l y u s i n g t e c h n i q u e s s u c h a s r e c o m b i n a n t D N A

technology.

GM plants have been useful in increasing crop yields, reduce post-

harvest losses and make crops more tolerant of stresses. There are

several GM crop plants with improved nutritional value of foods and

reduced the reliance on chemical pesticides (pest-resistant crops).

Recombinant DNA technological processes have made immense

impact in the area of heal thcare by enabl ing mass product ion of safe

and more ef fect ive therapeut ics. Since the recombinant therapeut ics

a r e i d e n t i c a l t o h u m a n p r o t e i n s , t h e y d o n o t i n d u c e u n w a n t e d

immunological responses and are free from risk of infection as was

observed in case of similar p roducts isolated f rom n on-hu ma n sources.

Human insul in i s made in bac ter i a ye t i t s s t ruc ture i s absolu te ly

identical to that of the natural molecule.

T r a n s g e n i c a n i m a l s a r e a l s o u s e d t o u n d e r s t a n d h o w g e n e s

contribute to the development of a disease by serving as models for

human diseases, such as cancer , cyst ic f ibrosis , rheumatoid ar thr i t i s

and Alzheimers.

Gene th erap y is t he insertion of genes into an individua ls cells

and t i ssues to t reat diseases especial ly heredi tary diseases. I t does

so by replacing a defective mutant allele with a functional one or

gene targeting which involves gene amplification. Viruses that attackthei r hosts and int roduce thei r genet ic mater ial into the host cel l as

part of their replication cycle are used as vectors to transfer healthy

genes or more recently portions of genes.

The current interest in the manipulat ion of microbes, plants, and

animal s has r a i sed ser ious e th ica l ques t ions .


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4 . Wh at are Cry proteins? Name an organism that produce i t . How has

man exploited this protein to his benefit?

5 . Wh a t is g e n e t h e r a p y? Illu s t r a t e u s in g t h e e x a m p le o f a d e n o s in e

deaminase (ADA) deficiency.

6 . D ig r a m m a t ic a lly r e p r e s e n t t h e e xp e r im e n t a l s t e p s in c lo n in g a n d

expressing an human gene (say the gene for growth hormone) into a

bacterium like E. coli?

7 . Can you su gges t a m ethod to remove o il (hydrocarbon) from seeds based

on your understanding of rDNA technology and chemistry of oil?

8 . F ind out from in terne t what i s golden r ice .

9 . D oes ou r b lood have p r ot ea se s and nu c lea ses?

1 0 . C o n s u lt in t e r n e t a n d fin d o u t h o w t o m a k e o r a lly a c t i ve p r o t ei n

pharmaceut ical . What i s the major problem to be encountered?

biotech and its applications

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