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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
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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 #$uare
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F1 cross HhRr x HhRr
!ossible combinationof genes in gametes HR Hr hR hr
HR Hr hR hr
HR
Hr
hR
hr
HHRR HHRr HhRR HhRr
HHRr HHrr HhRr Hhrr
HhRR HhRr hhRR hhRr
HhRr Hhrr hhRr hhrr
The F1 does not breed true. Of the 16 possible combinations
of genes ! do not have the combined beneficial genes
F1 cross&
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rbreeding transfers the complete genome of one variety tother(
means that many new and unpredictable gene combinatiy be formed in addition to those intended
s method of genetic recombination can take place only betieties of the same or closely related species
etic engineering makes it possible to transfer single gen
e genes can also be transferred from one species to a totallerent species
*enetic engineering
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There are several ways in which genes from one organism can beinserted into a di)erent organism
They can be coated on to microscopic gold particles and ,-red.into the cells
They can be delivered by viruses
They can be transmitted by using structures/ called plasmids, present in bacteria
For e"ample/ the human gene for making insulin can be transferredto bacteria/ which are then allowed to reproduce in a culture mediumfrom which the insulin can be e"tracted
!lasmids
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!lasmid
Chromosome: ost bacteria have one circular 2NA chromosome ranging in
si3e from 1/ to +/ kilobase pairs(
Plasmid: 5"trachromosomal genetic element also made of a circular 2NAmolecule(
Bacterial Genome6 The collection of all of the genes present on thebacteria.s chromosome or its e"trachromosomal genetic elements(
2NA is the genetic material of mostorganisms 7from bacteria to humans8
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in addition to a loop of "#$% %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
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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
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The recombinant plastids are
inserted into a bacterium (
the insulin gene ma)es the
bacterium produce insulin
9ecombinantplastids
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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
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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
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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
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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
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* :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
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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
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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
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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
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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<
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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
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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’