Biotechnology and Genetic Engineering - Ph.D Positions

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Feb 20, 2013 (4 years and 4 months ago)

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Biotechnology and Genetic
Engineering

Historical Developments


It

should

be

recognized

that

biotechnology

is

not

something

new

but

represent

a

developing

and

expanding

series

of

technologies

dating

back

thousands

of

years,

to

when

humans

first

began

to

use

microbes

to

produce

foods

and

beverages

such

as

bread

and

beer

and

to

modify

plants

and

animals

through

progressive

selection

for

desired

traits
.



Biotechnology

encompasses

many

traditional

processes

such

as

brewing,

baking,

wine

making,

cheese

production,

the

production

of

oriental

foods

such

as

soy

sauce

and

sewage

treatment

where

the

use

of

microorganisms

has

been

developed

somewhat

empirically

over

countless

years
.


Historical Developments


However,

it

was

the

discovery

of

antibiotics

in

1929

and

their

subsequent

large
-
scale

production

in

the

1940
s

that

created

the

greatest

advances

in

fermentation

technology
.

Since

then

we

have

witnessed

a

phenomenal

development

in

the

technology,

not

only

in

the

production

of

antibiotics

but

in

many

other

useful,

simple

or

complex

biochemical

products,

e
.
g
.

organic

acids,

polysaccharides,

enzymes,

vaccines

and

hormones
.



Thus,

biotechnology

is

not

a

sudden

discovery

but

rather

a

coming

of

age

of

a

technology

that

was

initiated

several

decades

ago
.


Historical Developments

Thus,

from

a

historical

perspective,

biotechnology

dates

back

to

the

time

when

yeast

was

first

deliberately

used

to

ferment

beer

and

when

bacteria

were

first

used

to

make

yogurt
.


The

term

"biotechnology"

was

created

in

1917

by

a

Hungarian

engineer,

Karl

Ereky,

to

describe

an

integrated

process

for

the

large
-
scale

production

of

pigs

by

using

sugar

beets

as

the

source

of

food
.

According

to

Ereky,

biotechnology

was

“all

lines

of

work

by

which

products

are

produced

from

raw

materials

with

the

aid

of

living

things”
.

Historical Developments


The

origin

of

biotechnology

can

be

traced

back

to

prehistoric

times,

when

microorganisms

were

already

used

for

processes

like

fermentation,

Although

a

molecular

biologist

may

consider

cloning

of

DNA

to

be

the

most

important

event

in

the

history

of

biotechnology,

the

latter

has

actually

been

rediscovered

in

1970
's

for

the

third

time

during

the

present

cen'tury
.


In

1920
's

Clostridium

acetoblltylicum

was

used

by

Chaim

Weizmann

for

converting

starch

into

butanol

and

acetone
;

the

latter

was

an

essential

component

of

explosives

during

World

War

I
.

This

raised

hopes

for

commercial

production

of

useful

chemicals

through

biological

processes,

and

may

be

considered

as

the

first

rediscovery

of

biotechnology

in

the

present

century
.


Historical Developments



During

World

War

II

(in

1940
's),

the

production

of

penicillin

(as

an

antibiotic

discovered

by

Alexander

Fleming

in

1929
)

on

a

large

scale

from

cultures

of

Pencillium

notatum,

marked

the

second

rediscovery

of

biotechnology
.

This

was

the

beginning

of

an

era

of

antibiotic

research
.


The

third

rediscovery

of

biotechnology

is

its

recent

reincarnation

in

the

form

of

recombinant

-

DNA

technology,

which

led

to

the

development

of

a

variety

of

gene

technologies

and

is

thus

considered

to

be

the

greatest

scientific

revolution

of

this

century
.



Thus,

even

though

biotechnology

had

its

origin

in

the

past,

it

has

assumed

special

significance

only

in

1970
's

and

1980
's
.


Historical Developments


During

1980
s,

biotechnology

became

the

major

growth

area
.

This

change

came

about

through

a

single

development
;

the

ability

to

explain

together

in

vitro

DNA

molecules

derived

from

different

sources
.

These

gene

splicing

ability

is

referred

to

as

'gene

manipulation
.
'

This

is

also

known

as

recombinant

DNA

technology
.



Recombinant

DNA

technology

and

genetic

engineering

techniques

found

several

useful

applications

in

the

area

of

vaccines,

foods,

antibiotics,

alcohols,

hormones

and

monoclonal

antibodies


Historical Developments in Genetic
Engineering and Biotechnology

1917

Karl Ereky coins the term biotechnology

1940

A. Jost coins the term genetic engineering

1943

Penicillin produced on an industrial scale

1961

The journal
Biotechnology and Bioengineering

is established

1970

First restriction endonuclease isolated

1973

Boyer and Cohen establish recombinant DNA technology

1976

First guidelines for the conduct of recombinant DNA research issued

1978

Genentech produces human insulin in
E. coli

1980

U.S. Supreme Court rules in the case of Diamond v. Chakrabarty that
Genetically manipulated microorganisms can be patented

1982

First animal vaccine produced by recombinant DNA methodologies
approved for use in Europe

1983

Engineered T1 plasmids used to transform plants

1988

U.S. patent granted for a genetically engineered mouse susceptible to cancer

1990

Approval granted in the United States for a trial of human somatic cell gene
therapy

1990

Recombinant chymosin used for cheese making in the United States

1996

First recombinant protein, erythropoietin, exceeds $1 billion in annual sales

1996

Commercial planting of genetically modified crops

1997

Nuclear cloning of a mammal
-
a sheep
-
with a differentiated cell nucleus

1998

FDA approves first antisense drug

1999

FDA approves recombinant fusion protein (diphtheria toxin
-
interleukin
-
2) for
cutaneous T
-
cell lymphoma

2000

Monoclonal antibodies exceed $2 billion in annual sales

2000

Development of "golden rice" (provitamin
-
A
-
producing rice) announced

2001

Human genome sequenced

2002

Complete human gene microarrays (gene chips) commercially available

By

2000
,

dozens

of

new

drugs

that

were

produced

by

recombinant

DNA

technology

had

been

certified

for

use

with

humans
;

over

200

were

in

the

process

of

being

tested

in

human

trials,

and

more

than

750

other

recombinant

pharmaceutical

products

are

being

developed
.


Similarly,

many

new

molecular

biotechnology

products

for

enhancing

crop

and

livestock

yields

have

been

created

and

are

being

marketed
.


Biotechnology Defined


Biotechnology,

as

word

indicates,

is

the

product

of

interaction

between

the

science

of

biology

and

technology
.

This

relationship

between

science

and

technology

has

been

observed

to

be

complex,

so

that

not

only

the

science

has

influenced

technology,

but

the

technology

has

also

influenced

science


Biotechnology Defined


Biotechnology

has

been

defined

in

various

ways
.

mostly

unsatisfatorily
.

Biotechnology

has

been

defined

as

the

application

of

biological

organisms,

systems

or

processes

to

manifacturing

and

service

industries

(Coombs,

1984
)
.



It

is

also

defined

as

the

application

of

scientific

and

engineering

principals

to

the

processing

of

materials

by

biological

agents

to

produce

goods

and

services

(Coleman,

1986
)
.

In

this

word

‘agent’

denotes

a

wide

range

of

biological

things

such

as

enzymes,

whole

cell

or

multi
-
cellular

organisms
.

Services

and

goods

mean

such

processes

as

waste

and

water

treatment
.



The

scientific

and

engineering

principles

are

chiefly

microbiology,

biochemistry,

genetics

and

biochemical

and

chemical

engineering
.


Biotechnology Defined


Biotechnology

is

commercial

exploitation

of

live

organisms

or

their

components

(Primrose,

1987
)
.



It

is

also

defined

as

the

industrial

exploitation

of

biological

systems

or

processes

and

it

is

largely

based

upon

the

expertise

of

biological

systems

in

recognition

and

catalysis

(Higgins,

1985
)
.



While

biotechnology

has

been

defined

in

many

forms,

in

essence,

it

implies

the

use

of

microbial,

animal

or

plant

cells

or

enzymes

to

synthesize,

breakdown

or

transform

materials
.


Biotechnology

is

the

integrated

use

of

biochemistry,

microbiology

and

engineering

sciences

in

order

to

achieve

technological

(industrial)


Biotechnology

may

be

defined

as

"the

application

of

scientific

and

Engineering

principles

to

the

processing

of

material

by

biological

agents

to

provide

goods

and

services
.
"


Biotechnology Defined


Biotechnology

is

"the

integrated

use

of

biochemistry,

microbiology,

and

engineering

sciences

in

order

to

achieve

technological

(industrial)

application

of

the

capabilities

of

micro
-
organisms,

cultured

tissue

cells

and

parts

thereof'

(European

Federation

of

Biotechnology)
.



Biotechnology

is

"a

technology

using

biological

phenomena

for

copying

and

manufacturing

various

kinds

of

useful

substances"

(Japenese

Biotechnologists)

.



Biotechnology

is

“the

controlled

use

of

biological

agents,

such

as

micro
-
organisms

or

cellular

components,

for

beneficial

use"

(US

National

Science

Foundation)
.


Genetic Engineering


The

conveyance

of

a

functional

unit

of

inheritance

(gene)

from

one

organism

to

another

was

based

on

the

strategy

devised

by

Stanley

Cohen

and

Herbert

Boyer

in

1973
.



This

became

possible

because

vectors

like

plasmids

and

phages

reproduce

in

a

host

(e
.
g
.

E
.

coli)

in

their

usual

manner

even

after

insertion

of

foreign

DNA,

so

that

the

inserted

DNA

will

also

replicate

faithfully

with

the

parent

DNA
.

This

technique

is

called

gene

cloning
.



With

this

technique,

genes

can

be

isolated,

cloned

and

characterized,

so

that

the

technique

has

led

to

significant

progress

in

all

areas

of

molecular

biology
.

Genetic Engineering


In

recent

years,

techniques

for

manipulating

prokaryotic

as

well

as

eukaryotic

DNA

have

witnessed

a

remarkable

development
.

This

has

allowed

breakage

of

a

DNA

molecule

at

two

desired

places

to

isolate

a

specific

DNA
.

segment

and

then

insert

it

in

another

DNA

molecule

at

a

desired

position
.

The

product

thus

obtained

is

called

recombinant

DNA

and

the

technique

often

called

genetic

engineering
.



Using,

this

technique

we

can

isolate

and

clone

single

copy

of

a

gene

or

a

DNA

segment

into

an

indefinite

number

of

copies,

all

identical
.


Genetic Engineering


Recombinant

DNA

technology,

which,

is

also

called

gene

cloning

or

molecular

cloning,

is

an

umbrella

term

that

encompasses

a

number

of

experimental

protocols

leading

to

the

transfer

of

genetic

information

(DNA)

from

one

organism

to

another
.



The

DNA

(cloned

DNA,

insert

DNA,

target

DNA,

or

foreign

DNA)

from

a

donor

organism

is

extracted,

enzymatically

cleaved

(cut,

or

digested),

and

joined

(ligated)

to

another

DNA

entity

(a

cloning

vector)

to

form

a

new,

recombined

DNA

molecule

(cloning

vector
-
insert

DNA

construct,

or

DNA

construct)
.

Genetic Engineering


This

cloning

vector
-
insert

DNA

construct

is

transferred

into

and

maintained

within

a

host

cell
.

The

introduction

of

DNA

into

a

bacterial

host

cell

is

called

transformation
.



Those

host

cells

that

take

up

the

DNA

construct

(transformed

cells)

are

identified

and

selected

(separated,

or

isolated)

from

those

that

do

not
.


If

required,

a

DNA

construct

can

be

created

so

that

the

protein

product

encoded

by

the

cloned

DNA

sequence

is

produced

in

the

host

cell
.

Tools of Genetic engineering


Enzymes


DNA or Gene fragment


Cloning Vectors


Recipient Cells

Plasmids as vectors


Plasmids

are

termed

as

autonomous

elements,

whose

genomes

exist

in

the

cell

as

extra

chromosomal

units
.

They

are

self

replicating

circular

(only

rarely

linear)

duplex

DNA

molecules,

which

are

maintained

in

a

characteristic

number

of

copies

in

a

bacterial

cell,

yeast

cell

or

even

in

organelles

found

in

eukaryotic

cells
.

These

plasmids

can

be

single

copy

plasmids

that

are

maintained

as

one

plasmid

DNA

per

cell

or

multicopy

plasmids,

which

are

maintained

as

10
-
20

genomes

per

cell
.



There

are

also

plasmids,

which

are

under

relaxed

replication

control

thus

permitting

their

accumulation

in

very

large

numbers

(up

to

1000

copies

per

cell)
.

These

are

the

plasmids

which

are

used

as

cloning

vectors,

due

to

their

increased

yield

potential
.




Circular

plasmid

DNA

which

is

used

as

a

vector,

can

be

cleaved

at

one

site

with

the

help

of

a

restriction

enzyme

to

give

a

linear

DNA

molecule
.

A

foreign

DNA

segment

can

now

be

inserted,

by

joining

the

ends

of

broken

circular

DNA

to

the

two

ends

of

foreign

DNA,

thus

regenerating

a

bigger

circular

DNA

molecule

that

can

now

be

separated

by

gel

electrophoresis

on

the

basis

of

its

size


Plasmids as vectors




Selection

of

chimeric

DNA

is

also

facilitated

by

the

resistance

genes,

which

the

plasmid

may

carry

against

one

or

more

antibiotics
.

If

a

plasmid

has

two

such

genes

conferring

resistance

against

two

antibiotics

and

if

the

foreign

DNA

insertion

site

lies

within

one

of

these

two

genes,

then

the

chimeric

vector

loses

resistance

against

one

antibiotic,

the

gene

for

which

has

foreign

DNA

inserted

within

its

structure
.

In

such

a

situation,

the

parent

vector

in

bacterial

cells

can

be

selected

by

resistance

against

two

antibiotics

and

the

chimeric

DNA

can

be

selected

by

retention

of

resistance

against

only

one

of

the

two

antibiotics
.


Plasmids as vectors

Plasmids as vectors


pBR
322

and

pBR
327

vectors
:

The

naturally

occurring

plasmids

may

not

possess

all

the

above

and

other

essential

properties

of

a

suitable

cloning

vector
.

Therefore,

one

may

have

to

restructure

them

by

inserting

genes

of

relaxed

replication

and

genes

for

antibiotic

resistance
.

This

has

actually

been

done

and

suitable

plasmid

vectors

have

been

obtained
.

One

of

the

standard

cloning

vectors

widely

used

in

gene

cloning

experiments

is

pBR
322

(derived

from

E
.

coli

plasmid

CoIEl),

which

is

4
,
362

bp

DNA

and

was

derived

by

several

alterations

in

earlier

cloning

vectors

(pBR
322

was

named

after

Bolivar

and

Rodriguez,

who

prepared

this

vector)
.

It

has

genes

for

resistance

against

two

antibiotics

(tetracycline

and

ampicillin),

an

origin

of

replication

and

a

variety

of

restriction

sites

for

cloning

of

restriction

fragments

obtained

through

cleavage

with

a

specific

restriction

enzyme
.


Plasmids as vectors


Another

vector

pBR
327

was

derived

from

pBR
322
,

by

deletion

of

nucleotides

between

1
,
427

to

2
,
51
.

These

nucleotides

are

deleted

to

reduce

the

size

of

the

vector

and

to

eliminate

sequences

that

were

known

to

interfere

with

the

expression

of

the

cloned

DNA

in

eukaryotic

cells
.

pBR
327

still

contains

genes

for

resistance

against

two

antibiotics

(tetracycline

and

ampicillin)
.

Both

pBR
322

and

pBR
327

are

very

common

plasmid

vectors
.

Plasmids as vectors

pUC

vectors
:

Another

series

of

plasmids

that

are

used

as

cloning

vectors

belong

to

pUC

series

(after

the

place

of

their

initial

preparation

i
.
e
.

University

of

California)
.

These

plasmids

are

2
,
700
bp

long

and

possess,

(i)

ampicillin

resistance

gene,

(ii)

the

origin

of

replication

derived

from

pBR
322

and

(iii)

the

lac

Z

gene

derived

from

E
.

coli
.

Within

the

lac

region

is

also

found

a

polylinker

sequence

having

unique

restriction

sites

(identical

to

those

found

in

phage

M
13
)
.

When

DNA

fragments

are

cloned

in

this

region

of

pUC,

the

lac

gene

is

inactivated
.


Plasmids as vectors

These

plasmids

when

transformed

into

an

appropriate

E
.

coli

strain,

which

is

lac
-

(
e
.
g
.

JM
103
,

JM
109
),

and

grown

in

the

presence

of

IPTG

(isopropyl

thiogalactoside,

which

behaves

like

lactose,

and

induces

the

synthesis

of

β
-
galactosidase

enzyme)

and

X
-
gal

(substrate

for

the

enzyme),

will

give

rise

to

white

or

clear

colonies
.

Plasmids as vectors


On

the

other

hand,

pUC

having

no

inserts

and

transformed

into

bacteria

will

have

an

active

lac

Z

gene

and

therefore

will

produce

blue

colonies,

thus

permitting

identification

of

colonies

having

pUC

vector

with

cloned

DNA

segments
.


The

cloning

vectors

belonging

to

pUC

family

arc

available

in

pairs

with

reversed

orders

of

restriction

sites

relative

to

lac

Z

promoter
.

pUC
8

and

pUC
9

make

one

such

pair
.

Other

similar

pairs

include

pUC
12

and

pUC
13

or

pUC
18

and

pUC
19
.


Plasmids as vectors


In

pBR
322

and

pBR
327
,

the

DNA

is

inserted

at

a

site

located

in

one

of

the

two

genes

for

resistance

against

antibiotics,

so

that

it

will

inactivate

one

of

the

two

resistance

genes
.

The

insert

bearing

plasmid

can

be

selected

by

their

ability

to'

grow

in

a

medium

containing

only

one

of

the

two

antibiotics

and

by

their

failure

to

grow

in

a

medium

containing

both

the

antibiotics
.



The

plasmids

carrying

no

insert

on

the

other

hand,

will

be

able

to

grow

in

media

containing

one

or

both

the

antibiotics
.

In

this

manner,

the

presence

of

lac

Z

gene

in

pUC

and

resistance

genes

against

ampicillin

and

tetracycline

in

pBR
322

and

pBR
327

allow

selection

of

E
.

coli

colonies

transformed

with

plasmids

carrying

the

desired

foreign

cloned

DNA

segment
.

Molecular biotechnology ought to contribute
unprecedented benefits to humanity

It

should



provide

opportunities

to

accurately

diagnose

and

prevent

or

cure

a

wide

range

of

infectious

and

genetic

diseases
.



Significantly

increase

crop

yields

by

creating

plants

that

are

resistant

to

insect

predation,

fungal

and

viral

diseases,

and

environmental

stresses

such

as

short
-
term

drought

and

excessive

heat
.



Develop

microorganisms

that

will

produce

chemicals,

antibiotics,

polymers,

amino

acids,

enzymes,

and

various

food

additives
.



Develop

livestock

and

other

animals

that

have

enhanced

genetically

determined

attributes



Facilitate

the

removal

of

pollutants

and

waste

materials

from

the

environment

Some microorganisms that have been genetically

engineered to perform biotechnological processes



Acremonium chrysogenum


Bacillus brevis


Bacillus subtilis


Bacillus thuringiensis


Corynebacterium glutamicum


Erwinia herbicola


Escherichia coli


Pseudomonas
spp.


Rhizobium
spp.


Streptomyces
spp.


Trichoderma reesei


Xanthomonas campestris


Zymomonas mobilis