6. Rekayasa genetika

8/18/2019 6. Rekayasa genetika

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Genetic Engineering

Fatchul Anam Nurlaili


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HHrr

high yield

low resistance

pollen

grainovule

hhRR

low yield

high resistance

The F1

consists of

plants with high yield

and good resistance

zygote

4


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Can you see any disadvantages in this method ofmanipulating genes ?

Try working out what would happen if you tried to breed fromthe F1

ork out the various gene combinations in the gametes

!ut them into a4"4 !unnett #$uare

%


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F1 cross HhRr x HhRr

!ossible combinationof genes in gametes HR Hr hR hr

HR Hr hR hr

HR

Hr

hR

hr

HHRR HHRr HhRR HhRr

HHRr HHrr HhRr Hhrr

HhRR HhRr hhRR hhRr

HhRr Hhrr hhRr hhrr

The F1 does not breed true. Of the 16 possible combinations

of genes ! do not have the combined beneficial genes

F1 cross&


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rbreeding transfers the complete genome of one variety tother(

means that many new and unpredictable gene combinatiy be formed in addition to those intended

s method of genetic recombination can take place only betieties of the same or closely related species

etic engineering makes it possible to transfer single gen

e genes can also be transferred from one species to a totallerent species

*enetic engineering

+


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There are several ways in which genes from one organism can beinserted into a di)erent organism

They can be coated on to microscopic gold particles and ,-red.into the cells

They can be delivered by viruses

They can be transmitted by using structures/ called plasmids, present in bacteria

For e"ample/ the human gene for making insulin can be transferredto bacteria/ which are then allowed to reproduce in a culture mediumfrom which the insulin can be e"tracted

!lasmids

0


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!lasmid

Chromosome: ost bacteria have one circular 2NA chromosome ranging in

si3e from 1/ to +/ kilobase pairs(

Plasmid: 5"trachromosomal genetic element also made of a circular 2NAmolecule(

Bacterial Genome6 The collection of all of the genes present on thebacteria.s chromosome or its e"trachromosomal genetic elements(

2NA is the genetic material of mostorganisms 7from bacteria to humans8


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in addition to a loop of "#$% %bacteria also contain numerous

rings of "#$ called plasmids

cell wall

cytoplasm

cell membrane the plasmids can be

e&tracted and used for

genetic engineering

'.''1mm

A bacterium 1

11


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plasmid

restriction

enzyme cuts

plasmid

the samerestriction

enzyme cuts

the insulin gene

out of the

human "#$

human "#$

strand

insulin

gene

'nserting agene

the insulin geneis inserted into

the plasmid

11


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The recombinant plastids are

inserted into a bacterium (

the insulin gene ma)es the

bacterium produce insulin

9ecombinantplastids

1:


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;nly about 1 in 1/ bacteria take up the recombined plasmids

There are techni$ues for identifying and isolating these bacteria

The bacteria with the insulin gene are then allowed to reproduce in a culture solution from which the insulin can be e"tracted<

=uman growth hormone can be made in a similar way

Factor >'''/ needed by haemophiliacs/ 7blood clotting disorders8can be produced from hamster cells containing plasmids with thefactor >''' genes

Chymosin/ used for clotting milk in cheesemaking/ can beproduced from yeast cells with recombinant plasmid 2NA

Applications

1@


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As well as producing useful substances from genetically alteredcells/ whole organisms can be genetically modi-ed(#ome e"amples are (

A bacterial gene which makes an insecticide can be introduced intocrop plants/ e(g( mai3e and cotton/ to make them resistant to attackby moth caterpillars

A gene which confers resistance to herbicides has been insertedinto crop plants so that spraying kills weeds but not the crop plants

A gene introduced to oilseed rape makes the oil more suitablefor commercial processes/ e(g( detergent production

*enes which control the production of human en3ymes have beeninserted into sheep so that the en3ymes can be recovered fromtheir milk

Applications

14


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*enetic engineering does not always have to involve gene transferbetween unrelated organisms

*enes in a single organism can be modi-ed to improve theircharacteristics or their products

A gene for the production of B carotene 7a precursor of >itamin A8has been introduced to rice to bene-t countries where rice is thestaple diet and >itamin A de-ciencies are common<

The ne"t slide shows tomatoes which have been geneticallymodi-ed to suppress production of an en3yme which causes thefruit to soften as it ripens( This improves the keeping $ualities

Applications

1%


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n organisms reproduce ase"ually/ all the o)spring receive

of genes from the parent(

As a result they are identical to each other andto the parent5"amples are 6

acteria and singlecelled organisms

!lants with vegetative reproduction by bulbs/ corms etc(

Fungi

#ome of the lower invertebrates

population of identical individuals arising from ase"ual

production is called a clone

Cloning1+

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A clone of crocuses

10

* :1


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cells in sheep $*s

mammary gland

one cell

isolated

diploid

nucleus

egg cell +ovum,

from sheep -

nucleus

removedthe two cells

are fused together (

embryo implanted

in uterus of sheep

cloned lamb

born

cell division produces

early embryo

2olly

:1

::


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'f the process becomes cheap and reliable it means that bene-cialgenes will be present in all the o)spring/ thus eliminating thechances of their being lost during conventional breeding

efore the early embryo is implanted in the surrogate mother/ it canbe broken up into its individual cells( 5ach of these can develop intoa new embryo

#heep/ pigs/ horses/ cows and/ by now/ probably many more animalshave been cloned

#o far/ this is being done on an e"perimental basis

=undreds of embryos have to be prepared and implanted to obtainone or two successful births

::

:@


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fertilised frog egg

cell division to form

an embryo

growth and development to

produce tadpole and frog

at the /0cell stage any one of these

cells can develop into a frog

:@

Cl f :4


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/0cell frog embryo

cells separated

each cell can develop into a frog

Clone offrogs

:4

:%


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cells from the +cell embryo are called embryonic stem c

because each one can form all the cells and tissues to

oduce a complete frog

After the 1&cell stage/ the cells lose this abilityand can only

produce specialised cells such as blood/ bone andnerve cellsells capable of dividing to produce specialised cells are

lled stem cells

ecialised cells normally lose the power to divide and may h

imited life span

:%

e tissues produced by specialised cells usually contain somem cells which retain the power of division

:0


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section through a 1'0day

human embryo

'. mm

these cells will contribute

to the placenta

these cells will form

the embryo +stem cells,

stem cells cultured

+cloned,

nutrient medium(

stem cells transferred

to culture dish

=uman 5#Cs

:0

@


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All the cells in the body have a full set of genes

hen the cells become specialised/ they lose their ability to divideand many of the genes are ,switched o).

For e"ample/ the genes for producing hydrochloric acid in a stomachcell would not be functional in a skin cell

5ven though tissues consist mainly of specialised cells/ most of themalso contain their own stem cells

't may become possible to treat stem cells from specialised tissueswith hormones and growth factors that cause them to produce awider range of specialised cells<

@

pp ca ons o s em @1


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pp ca ons o s emcells

t applications of stem cells are in the e"perimental stage/ a

ergoing clinical trials or have been tried on very few patient

!ossibilities are 6

9eplacement of damaged tissues such as heart muscle/ sbone and cartilage

Treatment of disease/ e(g( diabetes by inDecting islet cellsinto the pancreasE or !arkinson.s disease by inDecting ner

stem cells into the brain

e stem cells can be derived from the patient.s own tissue/ction by the immune system is avoided

@1

h t 2 't T


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hat 2oes 't ean6 ToCloneG? Clone: a collection of molecules or cells, all identicalto an original molecule or cell

2 To Hclone a geneH is to make many copies of it for

e"ample/ by replicating it in a culture of bacteria(2 Cloned gene can be a normal copy of a gene 7I

wild typeG8(

2 Cloned gene can be an altered version of a gene 7I

mutantG8(2 9ecombinant 2NA technology makes manipulatinggenes possible(


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9estriction 5n3ymes

2 acteria have learned to HrestrictH the

possibility of attack from foreign 2NA bymeans of Hrestriction en3ymesG(

2 Cut up foreignG 2NA that invades the cell(

2 Type '' and ''' restriction en3ymes cleave 2NA

chains at selected sites(2 5n3ymes may recogni3e 4/ & or more bases in

selecting sites for cleavage(

2 An en3yme that recogni3es a &base se$uence

is called a Hsi"base cutterG(


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asics of type '' 9estriction5n3ymes

2 No AT! re$uirement(

2 9ecognition sites in double stranded 2NA have a:fold a"is of symmetry J a palindromeG(

2 Cleavage can leave staggered or HstickyH ends orcan produce HbluntG ends(


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Type '' restriction en3yme nomenclature

2 Eco9' J Escherichia coli strain 9/ 1st en3yme2 Bam=' J Bacillus amyloliquefaciens strain =/ 1st

en3yme2 Dpn' J Diplococcus pneumoniae/ 1st en3yme2 Hind''' J Haemophilus inuenzae/ strain 2/ @rd

en3yme

2 Bgl'' J Bacillus globigii/ :nd en3yme

2 Pst ' J Proidencia stuartii 1&4/ 1st en3yme2 !au@A' J !taphylococcus aureus strain @A/ 1st

en3yme

2 "pn' J "lebsiella pneumoniae/ 1st en3yme

#hy the funny names$


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2NA Kigase Doins 2NA fragmentstogether

2 5n3ymes that cut with staggered cuts result incomplementary ends that can be ligatedtogether(

2 Hind''' leaves %. overhangs 7stickyG8

5’ --A AGCTT--3’ 5’ --AAGCTT-- 3’

3’ --TTCGA A--5’ 3’ --TTCGAA-- 5’

2 #ticky ends that are complementary 7fromdigests with the same or di)erent en3ymes8 canbe ligated together(

2 #ticky ends that are not complementary cannot


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2NA fragments with blunt ends generated by di)erent

en3ymes can be ligated together 7with lowereLciency8/ but usually cannot be recut by eitheroriginal restriction en3yme(

2 SmaI -CCC GGG-

2 DraI -AAA TTT-

2 Kigations that reconstitute a !ma' or Dra' site 7CCC*** or AAATTT8can be recut by !ma' or Dra'(

2 i"ed ligation products 7CCCTTT M AAA***8 cannot be recut by!ma' or Dra'(

2NA Kigase can also Doin blunt ends

-CCCGGG-

-AAATTT-

-CCCTTT-

-AAAGGG-

asm s ve c es or


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asm s J ve c es orcloning

2 !lasmids are naturally occurringe"trachromosomal 2NA

molecules(

2 !lasmids are circular/ double

stranded 2NA(

2 !lasmids are the means by whichantibiotic resistance is often

transferred from one bacteria to

another(

2!lasmids can be cleaved byrestriction en3ymes/ leaving

sticky or blunt ends(

2 Arti-cial plasmids can be

constructed by linking new 2NA

fragments to the sticky ends of

Tetr

Ampr

;ripBR!!

"#1bp

;ri

p$C1%

Ampr

&C'

(ac)


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Cloning >ectors

2 A cloning vector is a plasmid thatcan be modi-ed to carry new

genes(

2 !lasmids useful as cloning vectors

must have6

3 An origin of replication( 3 A selectable marker 7antibiotic

resistance gene/ such as ampr

and tetr8(

3 ultiple cloning site 7C#8 7site

where insertion of foreign 2NA

will not disrupt replication or

inactivate essential markers8(

3 5asy to purify away from host

2NA(

Tetr

Ampr

;ripBR!!

"#1bp

;rip$C1%

Ampr

&C'

(ac)

;lder cloning vector

Newer cloning vector


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Chimeric !lasmids

2 Named for mythological beast

7chimera8 with body parts from

several creatures(

2 After cleavage of a plasmid with a

restriction en3yme/ a foreign 2NAfragment can be inserted(

2 5nds of the plasmidfragment are

closed to form a Hrecombinant

plasmidG(

2 !lasmid can replicate when placedin a suitable bacterial host( ;ri

p$C1%*hCFTR

Ampr

&C'

(ac)

C F T 9


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2NA cloning re$uiresrestriction endonucleaseand 2NA ligase

Consider a plasmid with a uni$ue Eco9'site6

5' NNNNGAATTC NNNN 3'

3’ NNNNCTTAAGNNNN 5'

An Eco9' restriction fragment of foreign

2NA can be inserted into a plasmid

having an Eco9' cloning site by6

a8 cutting the plasmid at this site with

Eco9'/

b8 annealing the lineari3ed plasmid withthe Eco9' foreign 2NA fragment/ and/

c8 sealing the nicks with 2NA ligase(

5' NNNNGAATTCNNNN 3'

3' NNNNCTTAAGNNNN 5’

Documents

6. Rekayasa genetika