8/4/2019 Biotech Principles and Processes
1/16
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
8/4/2019 Biotech Principles and Processes
2/16
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 )
8/4/2019 Biotech Principles and Processes
3/16
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
8/4/2019 Biotech Principles and Processes
4/16
194
BIOLOGY
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
8/4/2019 Biotech Principles and Processes
5/16
BIOTECHNOLOGY : PRINCIPLES AND PROCESSES
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
8/4/2019 Biotech Principles and Processes
6/16
196
BIOLOGY
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
8/4/2019 Biotech Principles and Processes
7/16
BIOTECHNOLOGY : PRINCIPLES AND PROCESSES
197
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
8/4/2019 Biotech Principles and Processes
8/16
198
BIOLOGY
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)
8/4/2019 Biotech Principles and Processes
9/16
BIOTECHNOLOGY : PRINCIPLES AND PROCESSES
199
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.
8/4/2019 Biotech Principles and Processes
10/16
200
BIOLOGY
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
8/4/2019 Biotech Principles and Processes
11/16
BIOTECHNOLOGY : PRINCIPLES AND PROCESSES
201
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
8/4/2019 Biotech Principles and Processes
12/16
202
BIOLOGY
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
202
8/4/2019 Biotech Principles and Processes
13/16
BIOTECHNOLOGY : PRINCIPLES AND PROCESSES
203
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
8/4/2019 Biotech Principles and Processes
14/16
204
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 )
8/4/2019 Biotech Principles and Processes
15/16
BIOTECHNOLOGY : PRINCIPLES AND PROCESSES
205
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.
8/4/2019 Biotech Principles and Processes
16/16
206
BIOLOGY
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