1. TransformationsTransformations
of Cellsof Cells (and(and
Transfections too)Transfections too)
Bacteria, Fungi, Plants, and AnimalBacteria, Fungi, Plants, and Animal
Cells & Recombinant DNACells & Recombinant DNA
TechnologyTechnology

2. TransformationTransformation
 TransformationTransformation (def):(def): the genetic alteration ofthe genetic alteration of
a cell resulting from the introduction, uptakea cell resulting from the introduction, uptake
and expression of foreign genetic materialand expression of foreign genetic material
(DNA) in molecular biology(DNA) in molecular biology
 This can be done to Bacteria, Fungi, Plants,This can be done to Bacteria, Fungi, Plants,
and Animal cellsand Animal cells

3. Transformation -Transformation -
HistoryHistory
 1928 - Frederick Griffith transforms nonpathogenic1928 - Frederick Griffith transforms nonpathogenic
pneumococcus bacteria into a virulent variety bypneumococcus bacteria into a virulent variety by
mixing them with heat-killed pathogenic bacteria.mixing them with heat-killed pathogenic bacteria.
 Transformation was demonstrated in 1944 byTransformation was demonstrated in 1944 by
Oswald Avery, Colin MacLeod, and MaclynOswald Avery, Colin MacLeod, and Maclyn
McCarty, who showed gene transfer inMcCarty, who showed gene transfer in
Streptococcus pneumoniaeStreptococcus pneumoniae was pure DNA.was pure DNA.
 Avery, Macleod and McCarty call the uptake andAvery, Macleod and McCarty call the uptake and
incorporation of DNA by bacteria transformation.incorporation of DNA by bacteria transformation.

4. Transformation -Transformation -
MechanismsMechanisms BacteriaBacteria
 transformationtransformation refers to a genetic changerefers to a genetic change
brought about by picking up naked strands ofbrought about by picking up naked strands of
DNA and expressing it, andDNA and expressing it, and competencecompetence refersrefers
to the state of being able to take up DNA.to the state of being able to take up DNA.
 Two different forms of competence should beTwo different forms of competence should be
distinguished, natural and artificial.distinguished, natural and artificial.

5. Transformation -Transformation -
MechanismsMechanisms
 Bacteria -Bacteria - Natural competenceNatural competence
 Some bacteria (around 1% of all species) are naturallySome bacteria (around 1% of all species) are naturally
capable of taking up DNA. Such species carry sets ofcapable of taking up DNA. Such species carry sets of
genes specifying machinery for bringing DNA acrossgenes specifying machinery for bringing DNA across
the cell's membrane or membranes.the cell's membrane or membranes.
 The evolutionary function of these genes isThe evolutionary function of these genes is
controversial. Although most textbooks andcontroversial. Although most textbooks and
researchers have assumed that cells take up DNA toresearchers have assumed that cells take up DNA to
acquire new versions of genes, a simpler explanationacquire new versions of genes, a simpler explanation
that fits most of the observations is that cells take upthat fits most of the observations is that cells take up
DNA mainly as a source of nucleotides, which can beDNA mainly as a source of nucleotides, which can be
used directly or broken down and used for otherused directly or broken down and used for other
purposespurposes

6. Transformation -Transformation -
MechanismsMechanisms
 Bacteria -Bacteria - Artificial competenceArtificial competence
 Artificial competence is not encoded in the cell'sArtificial competence is not encoded in the cell's
genes.genes.
 It is induced by laboratory procedures in which cellsIt is induced by laboratory procedures in which cells
are passively made permeable to DNA, usingare passively made permeable to DNA, using
conditions that do not normally occur in nature.conditions that do not normally occur in nature.
 These procedures are comparatively easy andThese procedures are comparatively easy and
simple, and are widely used to genetically engineersimple, and are widely used to genetically engineer
bacteria.bacteria.
 Artificially competent cells of standard bacterialArtificially competent cells of standard bacterial
strains may also be purchased frozen, ready tostrains may also be purchased frozen, ready to
use.Common Strain ofuse.Common Strain of E. coli -E. coli - DH5DH5αα (alpha)(alpha)

7. Transformation -Transformation -
MechanismsMechanisms
 Bacteria -Bacteria - Artificial competence - TemperatureArtificial competence - Temperature
 Chilling cells in the presence of divalent cationsChilling cells in the presence of divalent cations
such as Ca2+ (in CaClsuch as Ca2+ (in CaCl22) prepares the cell walls to) prepares the cell walls to
become permeable to plasmid DNA.become permeable to plasmid DNA.
 Cells are incubated with the DNA and then brieflyCells are incubated with the DNA and then briefly
heat shocked (42heat shocked (42oo
C for 30-120 seconds), whichC for 30-120 seconds), which
causes the DNA to enter the cell.causes the DNA to enter the cell.
 This method works well for circular plasmid DNAsThis method works well for circular plasmid DNAs
but not for linear molecules such as fragments ofbut not for linear molecules such as fragments of
chromosomal DNA.chromosomal DNA.
 An excellent preparation of competent cells will giveAn excellent preparation of competent cells will give
~10~1088
colonies per μg of plasmid. A poor preparationcolonies per μg of plasmid. A poor preparation
will be about 10will be about 1044
/μg or less. Good non-commercial/μg or less. Good non-commercial
preps should give 10preps should give 1055
to 10to 1066
transformants pertransformants per
microgram of plasmid.microgram of plasmid.

8. Transformation -Transformation -
MechanismsMechanismsWhat is a plasmid again?What is a plasmid again?
 A plasmid DNA molecule containsA plasmid DNA molecule contains
sequences allowing it to be replicated in thesequences allowing it to be replicated in the
cell independently of the chromosome.cell independently of the chromosome.
 Plasmids used in experiments will usuallyPlasmids used in experiments will usually
also contain an antibiotic resistance genealso contain an antibiotic resistance gene
which is placed in a bacterial strain that haswhich is placed in a bacterial strain that has
no antibiotic resistance.no antibiotic resistance.
 Therefore, only transformed bacteria willTherefore, only transformed bacteria will
grow on a media containing the antibiotic.grow on a media containing the antibiotic.

9. Transformation -Transformation -
MechanismsMechanisms
 Bacteria -Bacteria - Artificial competence –Artificial competence –
ElectroporationElectroporation
 Electroporation is another way to make holes inElectroporation is another way to make holes in
cells, by briefly shocking them with an electriccells, by briefly shocking them with an electric
field of 100-200V/cm.field of 100-200V/cm.
 Now plasmid DNA can enter the cell throughNow plasmid DNA can enter the cell through
these holes.these holes.
 Natural membrane-repair mechanisms will closeNatural membrane-repair mechanisms will close
these holes afterwards.these holes afterwards.

10. Transformation -Transformation -
MechanismsMechanisms
 Bacteria -Bacteria - Artificial competence – LipofectionArtificial competence – Lipofection
 LipofectionLipofection (or liposome transfection) is a technique(or liposome transfection) is a technique
used to inject genetic material into a cell by means ofused to inject genetic material into a cell by means of
liposomes which are vesicles that can easily merge withliposomes which are vesicles that can easily merge with
the cell membrane since they are both made of athe cell membrane since they are both made of a
phospholipid bilayer.phospholipid bilayer.
 The vescicle fuses with the cell membrane (similar toThe vescicle fuses with the cell membrane (similar to
how two oil spots at the top of a broth will fuse) and thehow two oil spots at the top of a broth will fuse) and the
contents of the vesicle & the cell are combinedcontents of the vesicle & the cell are combined..

11. Transformation vs.Transformation vs.
TransfectionTransfection
 TransfectionTransfection (def): the introduction of(def): the introduction of
foreign material into eukaryotic cells.foreign material into eukaryotic cells.
 This typically involves opening transientThis typically involves opening transient
pores or 'holes' in the cell plasmapores or 'holes' in the cell plasma
membrane, to allow uptake of material.membrane, to allow uptake of material.

12. Transfection MechanismsTransfection Mechanisms
Yeasts and FungiYeasts and Fungi
 These methods (and more) are currently known toThese methods (and more) are currently known to
transform yeasts:transform yeasts:
 Two-hybrid System Protocol:Two-hybrid System Protocol:
 The two-hybrid system involve the use of two different plasmids in aThe two-hybrid system involve the use of two different plasmids in a
single yeast cell.single yeast cell.
 One plasmid contains a cloned gene or DNA sequence of interestOne plasmid contains a cloned gene or DNA sequence of interest
while the other plasmid contains a library of genomic or cDNA.while the other plasmid contains a library of genomic or cDNA.
(later)(later)
 Frozen Yeast Protocol:Frozen Yeast Protocol:
 Frozen yeast cells that are competent for transformation afterFrozen yeast cells that are competent for transformation after
thawing.thawing.
 Gene Gun Transformation:Gene Gun Transformation:
 Gold or tungsten nanoparticles can be shot at fungal cells growingGold or tungsten nanoparticles can be shot at fungal cells growing
on PDA, transforming them.on PDA, transforming them.
 Protoplast Transformation:Protoplast Transformation:
 Fungal spores can be turned into protoplasts which can then beFungal spores can be turned into protoplasts which can then be
soaked in DNA solution and transformed.soaked in DNA solution and transformed.

13. Transfection MechanismsTransfection Mechanisms
 Plants -Plants - A number of mechanisms are available to transfer DNAA number of mechanisms are available to transfer DNA
into an organism, these include:into an organism, these include:
 AgrobacteriumAgrobacterium is ais a genusgenus ofof Gram-negativeGram-negative bacteriabacteria thatthat
uses horizontal gene transfer to cause tumors in plants.uses horizontal gene transfer to cause tumors in plants.
Agrobacterium tumefaciensAgrobacterium tumefaciens is the most commonly studiedis the most commonly studied
species in this genus.species in this genus.
 Horizontal gene transfer (HGT)Horizontal gene transfer (HGT), also, also Lateral geneLateral gene
transfer (LGT)transfer (LGT), is any process in which an organism, is any process in which an organism
incorporates genetic material from another organism withoutincorporates genetic material from another organism without
being the offspring of that organism. By contrast,being the offspring of that organism. By contrast, verticalvertical
transfertransfer occurs when an organism receives genetic materialoccurs when an organism receives genetic material
from its ancestor, e.g. its parent or a species from which itfrom its ancestor, e.g. its parent or a species from which it
evolved. Most thinking in genetics has focused upon verticalevolved. Most thinking in genetics has focused upon vertical
transfer, but there is a growing awareness that horizontaltransfer, but there is a growing awareness that horizontal
gene transfer is a highly significant phenomenon, andgene transfer is a highly significant phenomenon, and
amongst single-celled organisms perhaps the dominantamongst single-celled organisms perhaps the dominant
form of genetic transfer. Artificial horizontal gene transfer isform of genetic transfer. Artificial horizontal gene transfer is
a form of genetic engineering.a form of genetic engineering.

14. Transfection MechanismsTransfection Mechanisms
 Plants -Plants - A number of mechanisms are available to transferA number of mechanisms are available to transfer
DNA into an organism, these include:DNA into an organism, these include:
 AgrobacteriumAgrobacterium mediated transformation is the easiestmediated transformation is the easiest
and most simple plant transformation. Plant tissue (oftenand most simple plant transformation. Plant tissue (often
leaves) are cut in small pieces, eg. 10x10mm, andleaves) are cut in small pieces, eg. 10x10mm, and
soaked for 10 minutes in a fluid containing suspendedsoaked for 10 minutes in a fluid containing suspended
agrobacterium. Some cells along the cut will beagrobacterium. Some cells along the cut will be
transformed by the bacterium, that inserts its DNA intotransformed by the bacterium, that inserts its DNA into
the cell.the cell.
 Placed on selectable rooting and shooting media, thePlaced on selectable rooting and shooting media, the
plants will regrow. Some plants species can beplants will regrow. Some plants species can be
transformed just by dipping the flowers into suspensiontransformed just by dipping the flowers into suspension
of Agrobacteria and then planting the seeds in aof Agrobacteria and then planting the seeds in a
selective medium.selective medium.
 Unfortunately, many plants are not transformable by thisUnfortunately, many plants are not transformable by this
method.method.

15. Transfection MechanismsTransfection Mechanisms
 PlantsPlants
 ElectroporationElectroporation: make holes in cell walls using: make holes in cell walls using
electricity, that allows DNA to enter.electricity, that allows DNA to enter.
 Viral transformationViral transformation: Package your genetic material into: Package your genetic material into
a suitable plant virus and then use the modified virusa suitable plant virus and then use the modified virus
for infection of the plant.for infection of the plant.
 Genomes of most plant viruses consist of singleGenomes of most plant viruses consist of single
stranded RNA which replicates in the cytoplasm ofstranded RNA which replicates in the cytoplasm of
infected cell.infected cell.
 So this method is not a real transformationSo this method is not a real transformation (why?(why?) …) …
since the inserted genes never reach the nucleus of thesince the inserted genes never reach the nucleus of the
cell and do not integrate into the host genome.cell and do not integrate into the host genome.
 The progeny of the infected plants is virus free and alsoThe progeny of the infected plants is virus free and also
free of the inserted genefree of the inserted gene

16. Transfection MechanismsTransfection Mechanisms
 PlantsPlants
 Particle bombardment (gene gun): Coat small goldParticle bombardment (gene gun): Coat small gold
or tungsten particles with DNA and shoot them intoor tungsten particles with DNA and shoot them into
young plant cells or plant embryos. Some geneticyoung plant cells or plant embryos. Some genetic
material will stay in the cells and transform them.material will stay in the cells and transform them.
This method also allows transformation of plantThis method also allows transformation of plant
plastids.plastids.
 The transformation efficiency is lower than in agrobacterialThe transformation efficiency is lower than in agrobacterial
mediated transformation, but most plants can bemediated transformation, but most plants can be
transformed with this method.transformed with this method.

17. Transfection MechanismsTransfection Mechanisms
 More on the “gene gun”More on the “gene gun”
 The target of a gene gun is often aThe target of a gene gun is often a calluscallus of undifferentiatedof undifferentiated
plant cells growing on gel medium in aplant cells growing on gel medium in a petripetri dishdish. After the. After the
gold particles have impacted the dish, the gel and callus aregold particles have impacted the dish, the gel and callus are
largely disrupted. However, some cells were not obliteratedlargely disrupted. However, some cells were not obliterated
in the impact, and have successfully enveloped a DNAin the impact, and have successfully enveloped a DNA
coated tungsten particle, whose DNA eventually migrates tocoated tungsten particle, whose DNA eventually migrates to
and integrates into a plantand integrates into a plant chromosomechromosome..
 Cells from the entire petri dish can be re-collected andCells from the entire petri dish can be re-collected and
selected for successful integration and expression of newselected for successful integration and expression of new
DNA using modern biochemical techniquesDNA using modern biochemical techniques
 Selected single cells from the callus can be treated with aSelected single cells from the callus can be treated with a
series of plant hormones, such asseries of plant hormones, such as auxinsauxins andand gibberellinsgibberellins,,
and each may divide and differentiate into the organized,and each may divide and differentiate into the organized,
specialized, tissue cells of an entire plant. This capability ofspecialized, tissue cells of an entire plant. This capability of
total re-generation is calledtotal re-generation is called totipotencytotipotency. The new plant that. The new plant that
originated from a successfully shot cell may have neworiginated from a successfully shot cell may have new
genetic (heritable) traits.genetic (heritable) traits.

18. Transfection MechanismsTransfection Mechanisms
 Gene gun with Humans and AnimalsGene gun with Humans and Animals
 Gene guns have also been used to deliverGene guns have also been used to deliver
DNA vaccinesDNA vaccines to experimental animals.to experimental animals.
Theoretically, it may be used in humans asTheoretically, it may be used in humans as
well.well.
 The delivery of plasmids into rat neuronsThe delivery of plasmids into rat neurons
through the use of a gene gun is also used as athrough the use of a gene gun is also used as a
pharmacological precursor in studying thepharmacological precursor in studying the
effects of neurodegenerative diseases such aseffects of neurodegenerative diseases such as
Alzheimer's DiseaseAlzheimer's Disease..
 The Gene gun technique is also popularly usedThe Gene gun technique is also popularly used
in Edible vaccine production technique, wherein Edible vaccine production technique, where
the nano gold particles coated with plant genethe nano gold particles coated with plant gene
under the high vacuum pressurized chamber isunder the high vacuum pressurized chamber is
transformed into suitable plant tissues.transformed into suitable plant tissues.

19. Transfection MechanismsTransfection Mechanisms
 AnimalsAnimals
 MicroinjectionMicroinjection: use a thin needle and inject: use a thin needle and inject
the DNA directly in the core of embryonicthe DNA directly in the core of embryonic
cells.cells.
 Viral transformationViral transformation: Package genetic: Package genetic
material into a virus, which delivers thematerial into a virus, which delivers the
genetic material to target host cells.genetic material to target host cells.