biotech principles and processes

8/4/2019 Biotech Principles and Processes

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Ever since the days of Rene Descartes, the Frenc h p hilosop her,

ma thema tican and biologist of seventeenth ce ntury, all human

knowledge especially natura l sc ienc es were d irec ted to develop

technologies which add to the creature comforts of human

lives, as a lso va lue to huma n life. The w hole a pp roa c h to

understand ing natural phenomena bec am e anthrop oc entric .

Physics and chem istry gave rise to eng ineering, technolog ies

and industries which all worked for huma n c omfort and we lfare.

The ma jor utility of the b iolog ica l world is as a source of food .

Biotechnology, the twentieeth century off-shoot of modern

biolog y, c ha nge d our da ily life as its prod uc ts b roug htqua litative improvement in hea lth and food produc tion. The

basic principles underlying biotechnological proc esses and som e

ap p lica tions a re highlighted and d iscussed in this unit.

Chap ter 11

Biotec hno logy : Princip les and

Proc esses

Chap ter 12

Biotec hnology a nd Its

Applications


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Herbert Boyer wa s bo rn in 1936 and b roug ht up in a c orner of we stern

Pennsylvania w here railroads and mines were the destiny of most young

men. He completed graduate work at the University of Pittsburgh, in

1963, followed by three years of post-graduate studies at Yale.

In 1966, Boyer took over assistant professorship at the University of

Ca lifornia a t San Franc isc o. By 1969, he p erformed stud ies on a c oup le

of restriction enzymes of the E. c oliba c terium with espec ially usefulp rop erties. Boyer ob served tha t these e nzymes have the c apab ility of

c utting DNA strands in a particular fashion, which left wha t has bec ame

known as stic ky ends on the strands. These c lipped end s ma de pasting

tog ethe r piece s of DNA a p rec ise e xercise.

This d isc overy, in turn, led to a ric h and rew ard ing c onversa tion in

Hawa ii with a Stanford sc ientist na me d Stanley Cohe n. Cohen ha d

been studying small ringlets of DNA called plasmids and which float

ab out freely in the c ytoplasm o f ce rtain bac terial ce lls and rep lic ate

indepe nde ntly from the c od ing strand of DNA. Cohen had de veloped

a me thod of removing these p lasmids from the c ell and then reinserting

them in other cells. Combining this process with that of DNA splicingenabled Boyer and Cohen to recombine segments of DNA in desired

configurations and insert the DNA in bacterial cells, which could then

ac t a s ma nufac turing p lants for spec ific p rote ins. This b rea kthroug h w as

the b asis upo n which the d esc ipline o f biotec hnolog y wa s founded.

HERBERT BOYER(1936 )


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B i o t e c h n o l o g y deals wi th techniques of using l ive

organ ism s or enzymes from organisms to produce produ cts

and processes us eful to hu man s. In this s ense , making

curd, bread or wine, which are al l microbe-mediated

p r o c e s s e s , c o u l d a l s o b e t h o u g h t a s a f o r m o f

biotechn ology. However, it is u sed in a restr icted s ens e

today, to refer to such of those processes which use

genetically modified organ ism s to ach ieve the s am e on a

larger scale. Furth er, ma ny other processes/ techn iques ar e

also includ ed u n der biotechnology. For exam ple, in v itro

fertilisation lead ing to a test -tu be ba by, syn th esising a

gene a n d u sin g it, developing a DNA vaccin e or correcting

a d efective gene, a re a ll pa rt of biotechn ology.

The Eu ropean Federation of Biotechn ology (EFB) h as

given a defin ition of biotechn ology tha t en compa ss es both

traditional view and modern molecular biotechnology.

The d efinition given by EFB is as follows:

The integration of na tu ral science a nd organ isms ,cells, pa rts thereof, an d m olecular a na logu es for pr oducts

an d s ervices.

1 1 . 1 PRINCIPLESOF BIOTECHNOLOGY

Among m an y, the two core techniques tha t ena bled birth

of m odern biotechn ology ar e :

(i) Genet i c eng ineer ing : Techn iqu es to al ter the

ch em istr y of genet ic ma ter ial (DNA an d RNA),

CHAPTER 11

BIOTECHNOLOGY : PRINC IPLES

AND PROCESSES

11.1 Principles of Biotechnology

11.2 Tools of Recombinant DNA

Technology

11.3 Processes of Recombinant

DNA Te chn ology


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to int rodu ce these into host organ ism s an d thu s chan ge the

ph enotype of th e host organism .

(ii) Mainten an ce of sterile (microbial conta mina tion-free) am bience

in chem ical engin eering processes to ena ble growth of only th edesired m icrobe/ eu ka ryotic cell in large qua nt i t ies for the

manufacture of biotechnological products l ike ant ibiot ics ,

vaccin es, enzymes , etc.

Let us now un derstan d th e conceptu al development of the principles

of genetic en gin eering.

You proba bly app reciate th e ad van tages of sexu al reprodu ction over

as exual reprodu ction. The form er p rovides opportu nit ies for variat ions

an d formu lation of u n iqu e com bina tions of genetic setu p, som e of which

may be beneficial to the organism as well as the population. Asexual

reprodu ction p reserves th e genetic in forma tion , while sexu al reprodu ction

perm its variation. Traditional hybridisa tion procedu res u sed in plant a nd

animal breeding, very often lead to inclusion and multiplication of

u nd esirable genes along with the desired genes . The tech niques of genetic

engineer ing which include creat ion of recombinant DNA, use o f

gene c loning a n d gene transfer, overcome th is limitation an d a llows u s

to isolate and introduce only one or a set of desirable genes without

introdu cing u nd esirable genes into th e target organism .

Do you know the likely fate of a piece of DNA, which is somehow

tra n sferred into a n alien organ ism? Most likely, th is piece of DNA would

n ot be ab le to mu ltiply itself in th e progeny cells of th e organism . But ,

when it gets integrated into th e genome of the recipient , i t ma y mu ltiply

an d be inh erited along with th e host DNA. This is becau se th e alien p iece

of DNA has become part of a chromosome, which has the abil i ty to

replicate. In a ch rom osom e there is a specific DNA sequ en ce called th e

origin of replication, which is responsible for initiating replication.

Therefore, for th e m u ltiplicat ion of an y alien piece of DNA in a n organ ism

it needs to be a par t of a ch romosom e(s) which h as a s pecific sequ ence

kn own a s origin of replication. Th u s, a n alien DNA is lin ked with th e

origin of replication, s o th at, t h is alien piece of DNA can replicat e an d

m u ltiply itself in th e hos t organism . Th is ca n also be called as c loning or

m ak ing mu ltiple iden tical copies of an y temp late DNA.

Let u s n ow focus on th e first inst an ce of th e const ru ction of an art ificialrecombinan t DNA molecule. The constr u ction of th e first recombina nt

DNA emer ged from th e pos sibility of linkin g a gen e en coding an tibiotic

resistance with a native plasmid (autonomously replicating circular

extra -chr omos oma l DNA) ofSa lmon ella typh im urium . Stanley Cohen an d

Herbert Boyer accomplished this in 1972 by isolating the antibiotic

res istan ce gen e by cutt ing out a piece of DNA from a plas m id wh ich was

responsible for conferring antibiotic resistance. The cutting of DNA at

specific locations became possible with the discovery of the so-called


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195

m olecula r sciss ors restr ict ion enzymes . The cu t p iece of DNA was

th en linked with t h e plasm id DNA. Th ese plas mid DNA act a s vectors to

tra n sfer the piece of DNA atta ched to it. You p roba bly kn ow th at m osqu ito

acts as an ins ect vector to tran sfer the ma larial para site into h u ma n b ody.In th e sam e way, a plasm id can b e u sed a s vector to deliver an alien p iece

of DNA into th e h ost organ ism. The lin king of an tibiotic resista n ce gen e

with the plasmid vector became possible with the enzyme DNA ligase,

which acts on cu t DNA molecu les an d joins th eir end s. Th is m akes a n ew

combination of circular autonomously replicating DNA created in vitro

an d is kn own as recombinan t DNA. When th is DNA is tr an sferred into

Escherichia coli, a bacterium closely related to Salmonella, it could

replicate u sing th e new h osts DNA polymera se en zym e an d m ak e mu ltiple

copies. The ability to multiply copies of antibiotic resistance gene in

E. coli was called c loning of an tibiotic resista n ce gen e in E. coli.

You can hen ce infer tha t th ere are th ree bas ic steps in geneticallymodifying a n organ ism

(i) identification of DNA with des irable genes ;

(ii) intr oduction of the identified DNA into th e host;

(iii) ma inten an ce of int rodu ced DNA in th e host an d tran sfer of the DNA

to its p rogeny.

1 1 .2 TOOLS OF RECOMBINANT DNA TECHNOLOGY

Now we kn ow from t h e foregoin g discu ss ion t h at gen etic en gin eering or

recomb inan t DNA tech n ology can be a ccomplish ed on ly if we ha ve th e

key tools, i .e., restr iction enzymes, polymer as e en zymes , ligases , vectors

an d th e host organism. Let us t ry to und erstand some of these in deta i l.

11 .2 .1 Res trict ion Enzym es

In th e year 196 3, th e two enzymes respon sible for restrict ing th e growth

of ba cteriopha ge in Escherichia coli were isolated. One of these added

methyl groups to DNA, while the other cut DNA. The later was called

rest rict ion e ndonuclease .

The f i r s t r e s t r i c t ion endonuc lease Hind II, whose func t ion ing

depended on a specific DNA nucleotide sequence was isolated and

cha ra cterised five years later. It was fou n d th at Hind IIalways cu t DNA

molecules at a part icular point by recognising a specific sequence ofs i x b a s e p a i r s . T h i s s p e c i f i c b a s e s e q u e n c e i s k n o w n a s t h e

recognit ion se quence for Hind II. Besides Hind II, today we know more

tha n 900 restriction en zymes th at h ave been isolated from over 230 strains

of ba cteria each of which r ecogn ise different recogn ition sequ ences .

Th e conven tion for na min g these en zym es is th e firs t letter of th e na me

comes from th e genes a nd the s econd two letters come from th e species of

th e pr okar yotic cell from which th ey were isolated, e.g., EcoRI comes from

Esche rich ia coli RY 13 . In EcoRI, th e letter R is der ived from th e n am e of


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stra in. Roman nu mb ers following th e na mes ind icate the order in which

the enzymes were isolated from tha t s train of bacteria.

Restrict ion enzymes belong to a larger class of enzymes called

nuc leases . Th ese a re of two kinds ; exonuc leases a n d endonuc leases.Exonu cleases remove nu cleotides from t he en ds of th e DNA whereas,

endon u cleases ma ke cu ts at specific posit ions within the DNA.

Each rest riction en don u cleas e fu n ction s by insp ectin g th e length of

a DNA sequence. Once i t f inds i ts specific recognit ion sequence, i t

wil l bind to the DNA and cut each of the two strands of the double

h e l i x a t s p e c i f i c p o i n t s i n t h e i r s u g a r - p h o s p h a t e b a c k b o n e s

(Figure 11.1) . Each rest r ic t ion endonuclease recognises a speci f ic

palindromic n ucleot ide sequenc es in t h e DNA.

Figure 11 .1 St eps i n f o r m a t i on o f r ecom bi nan t D N A by ac t i on o f r e s t r i c t i on endonuc l easeenzyme - EcoRI

Do you k n ow wha t pa l in drom es a re? Thes e a re grou ps of le t t e r s

t h a t fo r m t h e s a m e w or d s w h e n r e a d b o t h fo r wa r d a n d b a c k w a r d ,

e.g. , MALAYALAM. As aga ins t a word -pa lin dr om e wh ere t h e s am e

word i s read in bo th d i rect ions , t h e pa l ind rome in DNA is a sequ ence

o f b a se p a ir s t h a t r e a d s s a m e o n t h e t w o s t r a n d s w h e n o r ie n t a t io n o f


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reading is kept th e sa me. For examp le , the following sequ ences r eads

t h e s a m e o n t h e t w o s t r a n d s i n 5 ' 3 ' direction. This is also tru e if

r ead in the 3 ' 5 'direction.

5 ' GAATTC 3 '3 ' CTTAAG 5 '

Restriction en zym es cu t th e str an d of DNA a little away from th e centre

of the pa lind rome si tes, but between the s am e two bases on th e opposite

strands. This leaves single stranded portions at the ends. There are

overha nging stretches called st icky end s on each s tran d (Figure 11 .1).

These are named so because they form hydrogen bonds wi th thei r

complemen tary cu t coun terpar ts. This s t ickiness of th e ends facilitates

th e action of the en zyme DNA ligas e.

Restrict ion en donu cleases are u sed in genetic engineering to form

recom bin an t m olecu les of DNA, wh ich a re com pos ed of DNA from

different sources/ genomes.When cut b y the sa me res triction en zyme, th e resu ltan t DNA fragmen ts

ha ve the sa me k ind of st icky-ends an d, th ese can be joined togeth er

(en d-t o-en d) u sin g DNA ligas es (Figu re 11 .2).

Figure 11.2 Diagrammatic representation of recombinant DNA technology


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You m ay ha ve realised th at n orm ally, u nless on e cuts the vector an d

th e sou rce DNA with the s am e restriction en zyme, th e recomb ina nt vector

molecule cann ot be created.

Separation and is olation of DNA fragme nt s : The cutting of DNA byrest r ic t ion endonucleases resul ts in the f ragmentes of DNA. These

fragments can be separated by a technique known as gel elec trophoresis .

Since DNA fragments are negatively charged molecules they can be

sepa rated by forcing them to move toward s th e anode u nd er an electric

fie ld th rough a medium/ matr ix. Nowadays the m ost commonly u sed

ma trix is a garose which is a n atu ral polymer extracted from s ea weeds .

The DNA fragments separate (resolve) according to their size through

sieving effect provided by th e agar ose gel. Hence, th e sm aller th e fra gmen t

size, the farther it moves. Look at the Figure 11.3 an d gu es s at w hich

end of the gel the sa mple w as loaded.

The s epara ted DNA fragmen ts ca n be

visua lised only after s taining th e DNA

with a compoun d kn own as ethidiu m

brom ide followed by expos u re to UV

radiation (you can not s ee pu re DNA

fragments in the visible l ight and

with out sta ining). You can see br ight

orange coloured bands of DNA in a

e t h i d i u m b r o m i d e s t a i n e d g e l

exposed t o UV light (Figu re 1 1.3 ). The

sepa rated b an ds of DNA are cu t out

from the agarose gel and extracted

from th e gel piece. Th is step is kn own

a s e l u t i o n . T h e D N A f r a g m e n t s

pur i f i ed in th i s way a re used in

constructing recombinant DNA by

join ing th em with cloning vectors.

1 1.2 .2 Cloning Vec tors

You kn ow that plasm ids an d b acteriopha ges h ave the a bili ty to replicate

with in ba cterial cells in depen den t of th e contr ol of chr omos oma l DNA.

Bacteriopha ges b ecau se of their h igh nu mb er per cell, ha ve very highcopy nu mb ers of their genome within th e ba cterial cells. Some plasm ids

m a y h a ve o n l y on e o r t w o c op i e s p e r c e ll wh e r e a s o t h e r s m a y h a v e

1 5- 100 copies p er cell . Their n u mb ers ca n go even higher. If we are ab le

to lin k a n alien p iece of DNA with ba cterioph age or p lasm id DNA, we can

mul t iply i t s numbers equal to the copy number of the plasmid or

bacterioph age. Vectors u sed at presen t, are engineered in su ch way that

th ey help eas y linkin g of foreign DNA an d s election of recomb ina n ts from

non-recombinants.

Figure 11 .3 A typical agarose gel

elect rophoresis showing

migration of undigested

(lane 1) and digested set of

DNA fragments (lane 2 to 4)


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Th e followin g are th e featu res t h at a re requ ired to facilitate clon ing

into a vector.

(i) Orig in o f rep l i ca t ion (or i ) : This i s a sequence from where

replication sta rts an d an y piece of DNA when lin ked to th is s equen cecan be m ade to replicate with in th e host cells. This s equen ce is a lso

resp ons ible for contr olling th e copy nu mb er of the link ed DNA. So,

if one wan ts t o recover m an y copies of th e tar get DNA it sh ou ld be

cloned in a vector whose origin su pport h igh copy nu mb er.

(ii) S e l ec t a b l e m a r k e r : In ad dition to ori, th e vector requ ires a

selectable mark er, which h elps in iden tifying an d elimin ating n on-

t ransformant s and se lec t ive ly pe rmi t t ing the growth of the

t ransformants . Transformation is a procedu re through which a

piece of DNA is in trodu ced in a h ost b acteriu m (you will st u dy th e

process in subsequent section). Normally, the genes encoding

resistance to antibiotics such as ampicil l in, chloramphenicol,tetracycline or ka na mycin, etc., are considered u sefu l selectable

ma rkers for E. coli. The n ormalE. coli cells d o not car ry resistan ce

agains t an y of th ese an tibiotics.

(iii) Cloning s i t es : In order to l ink the

alien DNA, the vector needs to have

v e r y f e w , p r e f e r a b l y s i n g l e ,

recognit ion s i tes for th e comm only

u sed restrict ion en zymes . Presence of

more th an one recognition s ites within

t h e v e c t o r w i l l g e n e r a t e s e v e r a l

fra gmen ts , which will comp licate th eg e n e c l o n i n g ( F i g u r e 1 1 . 4 ) . T h e

ligation of alien DNA is ca rr ied ou t a t

a res trict ion s ite presen t in one of the

two antibiot ic resist ance genes . For

exam ple, you can ligate a foreign DNA

at the Bam H I si te of tetracycline

resista n ce gene in th e vector pBR322 .

The recombinant plasmids will lose

tetracycline resistan ce du e to in sertion

of foreign DNA bu t ca n st ill be selected

out f rom non-recombinant ones byplating th e tran sforma nts on a mp icillin

containing medium. The transformants growing on ampicil l in

conta ining medium are then t rans ferred on a medium conta ining

tetra cycline. Th e recomb ina n ts will grow in am picillin cont ainin g

m e d i u m b u t n o t o n t h a t c o n t a i n i n g t e t r a c y c l i n e . B u t , n o n -

recombinant s w i l l g row on the medium conta in ing bo th the

an tibiotics. In th is case, one an tibiotic resistan ce gene h elps in

selecting the tra ns forman ts, whereas th e other an tibiotic resistan ce

Figure 11 .4 E. coli cloning vector pBR322

s h o w i n g r e s t r i c t i o n s i t e s

(Hind III, EcoR I, Bam H I, S a l I,

Pv u II, Ps t I, Cla I ) , or i and

a n t i b i o t i c r e s i s t a n c e g e n e s

(amp R and t e t R). Rop codes for

t he p r o t e i n s i nvo l ved i n t he

replication of the plasmid.


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gene gets inact ivat ed d u e to ins ertion of alien DNA, an d h elps in

selection of recombina nt s.

Selection of recomb inan ts d u e to in activation of ant ibiotics is a

cum bersome procedu re because i t requires s imu ltaneous pla t ingon two plates having different antibiotics. Therefore, alternative

se lec tab le markers have been deve loped which d i f fe ren t i a t e

recombinan ts from n on-recombinan ts on th e basis of their ability

to produ ce colou r in th e presen ce of a ch romogenic su bstr ate. In

th is, a recomb ina nt DNA is ins erted within th e coding s equen ce of

an enzyme, -galactosidas e. Th is res u lts into inact ivat ion of th e

enzyme, which is referred to as insert ional inact ivat ion . The

pres ence of a ch romogenic su bst rate gives b lue colour ed colonies if

the plasmid in the bacteria does not have an insert . Presence of

insert r esu lts int o in sertiona l inactivation of th e -galactosidase an d

the colonies do not produce any colour, these are identified asrecombina nt colonies.

(iv) Vectors for c lon ing genes in p lan t s and an im al s : You m ay be

su rprised to kn ow th at we ha ve learn t th e lesson of tran sferring genes

into plan ts an d an ima ls from bacteria and viru ses which ha ve known

th is for ages how to deliver genes t o tran sform eu ka ryotic cells an d

force them to do what the bacteria or viruses want. For example,

Agrobacterioum tumifaciens, a p ath ogen of several dicot plants is

ab le to deliver a piece of DNA kn own a s T-DNA to tra n sform n orm al

plan t cells in to a tumor an d direct these tu mor cells to produ ce the

chem icals requ ired by th e path ogen. Similarly, retroviru ses in a n ima ls

h ave the a bility to tran sform norm al cells into cancerous cells. Abetter u nd ersta n ding of th e art of delivering genes by path ogens in

their eukar yotic hosts ha s generated kn owledge to tran sform th ese

tools of pa th ogen s into u seful vectors for delivering genes of interes t

to humans. The tumor inducing (Ti) plasmid ofAgrobacterium

tumifaciens ha s n ow been m odified into a cloning vector which is no

more path ogenic to the plants bu t is still able to us e the m echan ism s

to deliver genes of our interest into a variety of plants. Similarly,

retroviru ses h ave also been disar med a n d are n ow u sed to deliver

des irable genes in to an im al cells. So, once a gene or a DNA fra gmen t

ha s been ligated into a s u itable vector it is tran sferred into a bacterial,

plan t or an imal hos t (where it mu ltiplies).

11 .2 .3 Compet ent Hos t (For Trans format i on wit h

Rec om binant DNA)

Since DNA is a hydrophi l ic molecule , i t cannot pass through cel l

membranes . Why ? In order to force bacteria to tak e up the p las mid, the

ba cterial cells mu st first b e m ad e comp eten t to tak e u p DNA. Th is is

done by treating th em with a specific concentra tion of a divalent cat ion,

su ch a s ca lcium , wh ich in creas es th e efficiency with which DNA ent ers


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the bacterium thr ough pores in its cell wall. Recombinan t DNA can then

be forced into s u ch cells by incu bating th e cells with recombinan t DNA

on ice, followed by placing them briefly at 42 0C (hea t sh ock), an d th en

put t ing them back on ice . This enables the bacter ia to take up therecombina nt DNA.

Th is is n ot th e only way to intr odu ce alien DNA into h ost cells. In a

method known as micro-injection , recom bina n t DNA is directly in jected

int o the nu cleus of an an ima l cell. In a noth er meth od, su itab le for plan ts,

cells are bom ba rd ed with h igh velocity micro-par ticles of gold or tu n gsten

coated with DNA in a m ethod k nown a s biolistics or gene gun . And th e

las t m ethod u ses disarm ed pa th ogen vectors, wh ich when allowed to

in fect the cell, tran sfer the recomb inan t DNA into the h ost.

Now th at we ha ve learn t abou t th e tools for const ru cting recombina nt

DNA, let u s d iscu ss th e process es facilitat ing recombin an t DNA techn ology.

1 1 . 3 PROCESSES OF RECOMBINANT DNA TECHNOLOGY

Recomb inan t DNA techn ology in volves several s teps in s pecific

sequ ence s u ch as isolat ion of DNA, fra gmen tat ion of DNA by

restr iction end onu cleas es, isolation of a des ired DNA fragm ent ,

ligation of the DNA fragment into a vector, transferring the

recombinan t DNA into the h ost , cultu ring the h ost cells in a

medium at large scale and extraction of th e desired produ ct.

Let u s exam ine each of th ese steps in some deta ils.

1 1 .3 .1 Iso lation o f the Gene tic Material (DNA)Recall tha t n u cleic acid is th e genetic m ater ial of all organ isms

w i t h o u t e x c e p t i o n . I n m a j o r i t y o f o r g a n i s m s t h i s i s

deoxyribonucleic acid or DNA. In order to cut the DNA with

restr iction enzymes, it needs to be in pu re form, free from oth er

macro-molecu les . S ince the DNA i s enc losed wi th in the

mem bra n es, we have to break t h e cell open t o releas e DNA along

w i t h o t h e r m a c r o m o l e c u l e s s u c h a s R N A , p r o t e i n s ,

polysa ccha rides an d also lipids. Th is can b e achieved by treating

the ba cterial cells/ plant or an ima l t issu e with enzymes s u ch a s

lysozyme (bacteria), cellulase (plan t cells), chit inase (fungus).

You kn ow tha t genes ar e located on lon g molecu les of DNA

interwined with proteins su ch a s h iston es. The RNA can be removed by

t rea tment w i th r ibonuc lease whereas p ro te ins can be removed by

treatm ent with p rotease. Other m olecules can b e removed by appropriate

treat men ts a n d pu rified DNA u ltima tely precipitates ou t after th e add ition

of chilled ethanol. This can be seen as collection of fine threads in the

su sp ens ion (Figure 1 1.5).

Figure 11.5 DNA that

separa tes out can beremoved by spooling


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1 1.3 .2 Cutt ing o f DNA at Spec ific Locat ion s

Restriction en zyme d igestion s a re perform ed by incu ba ting pu rified DNA

m olecules with t he res triction en zyme, at th e optima l cond itions for th at

specific enzyme. Agarose gel electrophoresis is employed to check the

progress ion of a r estr iction en zym e digest ion . DNA is a n egatively cha rged

molecu le , hence i t moves towards the pos i t ive e l ec t rode (anode)

(Figur e 11.3 ). Th e process is repea ted with th e vector DNA also.

The joining of DNA involves several processes. After having cut the

sou rce DNA as well as th e vector DNA with a s pecific res triction en zyme,

th e cu t ou t gene of interest from th e sou rce DNA an d t he cu t vector with

sp ace are mixed an d l igase is ad ded. This resu lts in th e prepara tion of

recombina n t DNA.

1 1.3 .3 Am pli ficat ion of Gene of Inte rest us ing PCR

PCR stands for Polym erase Chain React ion .In th is rea ction, m u ltiple

copies of the gene (or DNA) of interes t is syn th esised in vitro u sing two

Figure 11 .6 Polymerase chain react ion (PCR) : Each cycle has three steps: ( i ) Denaturat ion;

(ii) Primer annealing; and (iii) Extension of primers

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sets of primers (small chemically synthesised oligonucleotides that are

complemen tar y to th e regions of DNA) an d th e enzyme DNA polymer as e.

The enzyme extends th e primers u sing the nu cleotides pr ovided in th e

reaction a nd th e genomic DNA as temp late. If the pr ocess of replicationof DNA is repeated many times, the segment of DNA can be amplified

to app roxim ately billion times , i.e., 1 billion copies are m ad e. Su ch

repeated amplification is achieved by the use of a thermostable DNA

polymerase (isolated from a bacterium, Thermus aquaticus ), which

remain act ive dur ing the high temperature induced denatura t ion of

dou ble st ran ded DNA. Th e am plified fra gmen t if des ired can n ow be

used to ligate with a vector for further cloning (Figure11.6).

11 .3 .4 Ins ert i on o f Recombinant DNA i n t o t he Hos t

Cell / Organism

Th ere ar e several meth ods of in trodu cin g the ligated DNA into recipien tcells. Recipient cells after m ak ing th em comp etent to receive, tak e u p

DNA pres en t in its s u rrou n ding. So, if a recom bina n t DNA bea ring gen e

for res istan ce to an an tibiotic (e.g., am picillin) is tra n sferred into E. coli

cells, the h ost cells b ecome tra n sform ed into a mp icillin-resista n t cells. If

we spread th e tran sformed cells on a gar plates conta ining am picillin, on ly

tra n sforma n ts will grow, un tra n sformed recipient cells will die. Sin ce, du e

to am picillin resista n ce gene, one is ab le to select a tra n sformed cell in th e

pres ence of amp icillin . Th e am picillin res istan ce gen e in th is cas e is called

a selec table m arker.

11 .3.5 Obtaining the Foreign Gen e Product

When you in sert a p iece of alien DNA into a cloning vector a n d tr an sfer it

into a ba cterial, plan t or a n imal cell, th e alien DNA gets m u ltiplied. In

almost al l recominant technologies, the ult imate aim is to produce a

desirable protein. Hen ce, there is a need for th e recombinan t DNA to be

expressed. The foreign gene gets expressed u nd er app ropriate cond itions .

The expres sion of foreign genes in hos t cells involve u nd ersta nd ing man y

technical details.

After having cloned the gene of interest and having optimised the

condit ions to induce the expression of the target protein, one has to

consider producing i t on a large scale. Can you think of any reason

w hy there is a need for large-s cale production? If any protein encodinggene is expressed in a heterologous host , is called a recombinant

protein . The cells harbouring cloned genes of interest may be grown

on a small scale in the laboratory. The cultures may be used for

extracting th e desired pr otein an d th en pu rifying i t by u sing different

separation techniques.

The cells can a lso be mu ltiplied in a continu ous cultu re system wh erein

the used medium is drained out from one side while fresh medium is

add ed from the other to m aintain the cells in their ph ysiologically most


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BIOLOGY

A stirred-tan k r eactor is u su ally cylind rical or with a cur ved ba se to

facilitate th e mixin g of th e reactor con ten ts. The s tirrer facilitat es even

m ixing an d oxygen a vailability th rou ghou t th e bioreactor. Altern at ively

air can be bu bbled thr ough the reactor. If you look at th e figure closelyyou will see tha t th e bioreactor h as an agitator s ystem, an oxygen delivery

system and a foam control system, a temperature control system, pH

control system a nd s am pling ports so tha t sm all volu mes of the cu ltu re

can be with dra wn periodically.

11 .3 .6 Downstream Proces s ing

After comp letion of th e biosynt h etic sta ge, th e produ ct ha s to be su bjected

th rough a s eries of processes b efore it is ready for ma rketing as a finish ed

active log/ exponential pha se. This type of cultu ring meth od produ ces a

larger bioma ss lead ing to h igh er yields of des ired protein.

Sm all volu m e cultur es can n ot yield ap preciable qu an tities of produ cts.

To produ ce in large qu an tit ies, th e developmen t ofbioreactors , wherelarge volum es (10 0-10 00 litres ) of cu ltur e can be pr ocessed , was r equ ired.

Th u s, bioreactors can be th ought of as vessels in which ra w materials are

biologically con verted into sp ecific prod u cts, individu al enzymes, etc.,

u sing microbial plant , an imal or hu ma n cells. A bioreactor provides t he

opt imal condi t ions for achieving the desi red product by providing

optimu m growth condit ions (temperatu re, pH, su bstra te, sal ts, vitam ins ,

oxygen).

The m ost comm only u sed b ioreacters ar e of st irring type, which a re

sh own in Figu re 11.7.

Figure 11 .7 (a) Simp le st irred-tan k bioreactor; (b) Spa rged stirred-tan k bioreactor th rou gh which

ster i le ai r bubbles are sparged

(a ) (b )


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SUMMARY

Biotechnology deals wi th large scale product ion and market ing of

product s and processes us ing l ive organi sms, ce l l s or enzymes .

Modern biotechnology using genet ical ly modif ied organisms was

made possible only when man learnt to alter the chemistry of DNA

a n d c o n s t r u c t r e c o m b i n a n t D N A . T h i s k e y p r o c e s s i s c a l l e drecombinant DNA technology or genetic engineering. This process

i n v o l v e s t h e u s e o f r e s t r i c t i o n e n d o n u c l e a s e s , D N A l i g a s e ,

appropriate plasmid or viral vectors to isolate and ferry the foreign

DNA into h ost organ isms , express ion of the foreign gen e, pu rification

of the gene product, i .e. , the functional protein and finally making a

suitable formulation for marketing. Large scale production involves

use of bioreactors.

EXERCISES1 . C a n y ou l is t 1 0 r e co m b in a n t p r ot e in s w h i ch a r e u s e d in m e d ic a lpract ice? Find out where they are u sed a s th erapeu t ics (u se the internet ).

2. Make a char t (with diagram ma tic representa t ion) sh owing a rest r iction

enzyme, the subst rate DNA on which i t acts , the si te at which i t cuts

DNA and the product i t produces.

3 . From wha t you have learn t , can you t e ll whether enzymes are b igger or

DNA is bigger in molecular size? How did you know?

4 . Wha t w ou l d be the m o la r concen t r a t ion o f hu m an D NA in a h um an

cell? Consult your teacher.

5 . Do eukaryot ic ce lls h ave r es t r ic t ion endonu cleases? J us t ify your an swer.

6 . Bes ides be t t er aera t ion and mixing proper t ies , what o ther advantages

do st i r red tank bioreactors have over shake f lasks?

7. Collect 5 examples of palindromic DNA sequences by consu lting your tea ch er.

Better try to create a pa lind romic sequ ence by following b as e-pair rules.

8 . Can you r eca ll meios is an d indica te a t what s t age a r ecombinant DNA

is made?

9 . C an you t h ink an d answ er how a r epo rt er enzym e can be u sed t o m on it or

transformation of host cells by foreign DNA in addition to a selectable

m a r k e r ?

produ ct. The processes include sep ara tion a nd pu rification, which ar e

collectively referred to as downs tream process in g. Th e produ ct ha s to be

formu lated with s u itable preservatives. Su ch formu lation has to un dergo

thorou gh clinical tr ials as in case of dru gs. Strict qu ality contr ol test ing

for each produ ct is also required. The downstrea m pr ocess ing an d qua lity

control test ing vary from p rodu ct to produ ct.


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10. Descr ibe br iefly the followings :

(a) Origin of replication

(b) Bioreactors

(c) Downstream processing1 1 . E xp la in b r ie fly

(a) PCR

(b) Restriction enzymes and DNA

(c) Chitinase

12. Discuss with your t eacher and find out h ow to d is t ingui sh be tween

(a) Plasmid DNA and Chromosomal DNA

(b) RNA and DNA

(c) Exonuclease and Endonuclease

biotech principles and processes

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