TRANSFER MATERI

GENETIK
Sri Agung Fitri Kusuma

MEKANISME TRANSFER MATERI GENETIK

Transformasi
Transduksi
Konjugasi

population's genetic diversity

Bacterial transformation
Transformation is the act of putting
foreign DNA into a bacterial cell
Occurs in nature, but VERY RARELY!
Weve perfected an artificial method to
force bacteria to take up DNA

TRANSFORMASI
Proses perpindahan DNA bebas dari lingkungan ke sel resipien yang kompeten
Competence: alterations in the bacterial cell wall that make it permeable to large
DNA molecules
Sel kompeten mengikat DNA bebas 1000 kali lebih tinggi dibandingkan non
kompeten
Naturally transformable : dapat mengambil DNA bebas dari lingkungannya tanpa
perlakuan khusus -Natural competence : Acinetobacter, azotobcate, bacillus,
Streptococcus, Haemophilus, Neisseria dan Thermus.
Transformasi yang perlu perlakuan khusus supaya mudah tertransformasi, pada E.
coli dan beberapa bakteri Gram (-); lingkungan dengan konsentrasi kalsium yang
tinggi dan didinginkan beberpa menit, atu menggunakan electroporation.
Sel kompeten mengikat DNA bebas 1000 kali lebih tinggi dibandingkan non
kompeten.
Efisiensi DNA uptake
Spesifity of DNA uptake, H. influenzae (5 AAGTGCGGTCA 3), N. gonorrhoeae
(5
GCCGTCTCAA 3), only from their own species and others can take up DNA from any
sources.
DNA uptake during growth cycle, but most naturally transformable bacteria usually
just before they reach stationary phase

Transformation

GRIFFITH'S EXPERIMENT

Transformation procedure
probable mechanism
Cells in mid-logarithmic phase are suspended
in cold calcium chloride which makes them
permeable to DNA
They are mixed with the DNA to be inserted
They are heat shocked which drives the
DNA into the cell
They are allowed to recover at 37oC
They are spread on a plate containing a
selection drug(s)

Transformation procedure
E. coli

CaCl2
cold!

E. coli

pAmp/Kan

42oC
Recover
at 37oC
LB Amp+Kan

Incubate at
37oC overnight
LB Amp+Kan

E. coli

Transformation procedure
E. coli

CaCl2
cold!

E. coli

pAmp/Kan

Making cells competent

42oC
Recover
at 37oC
LB Amp+Kan

Incubate at
37oC overnight
LB Amp+Kan

E. coli

Heat shock

elektroforasi

Transformation

Important considerations
Cells must be in log phase when
making competent!
Cells must be kept cold!
Cells must recover after heat shock!

Electroporation

Gene gun method

DNA
pellets

inserted gene
plant
chromosome

Transformed
plant cell

Source: Monsanto

Conjugation
Definition: Gene transfer from
a donor to a recipient by
direct physical contact
between cells
Mating types in bacteria

Donor

Donor
F factor (Fertility factor)

Recipient
F (sex) pilus

Lacks an F factor
Recipient

Plasmids
Definition:

Extrachromosomal genetic
elements that are capable of
autonomous replication (replicon)
Episome - a plasmid that can integrate
into the chromosome

Classification of Plasmids
Transfer

properties

Conjugative
Nonconjugative

Phenotypic

effects

Fertility
Bacteriocinogenic plasmid
Resistance plasmid (R factors)

Conjugation and F+ factor

F+ factor (fertility factor)- a
sophisticated plasmid
OriT, Origin of

transfer

insertion sequences,

genes for own

replication,

genes for own transfer

Mechanism of F x F- Crosses
Pair formation
Conjugation
bridge
DNA

transfer

of
transfer
Rolling circle
replication

F-

F-

Origin

Conjugation cycle

Conjugation converts an F- to F+, and original F+ remains F+

Note: no host genes (in blue) transferred from F+ to F-

Hayes expt:
Take strain A, which is
streptomycin-resistant, and
auxotrophic for biotin and
methionine.
Take strain B, which is
streptomycin-sensitive, and
auxotrophic for threonine and
leucine.
Mix the two on a minimalmedium plate containing
streptomycin.
Wait and see.

J. Lederberg, E. Tatum (1946)

Novel genotypes in mixed cultures of biochemical
mutants of bacteria. Cold Spring Harbor Symp.
Quant. Biol. 11: 113-114.

15.11

Cours de Physiologie et de gntique bactriennes, 1957

Illustration humoristique des cours par une srie de dessins de F. Lavall
Lgendes de Georges Cohen
http://www.pasteur.fr/infosci/archives/mon/im_laval.html

Interruption de la pntration de l'ADN mle dans le cytoplasme de la bactrie

femelle l'aide d'un warring blender (simple mixer mnager).
MCB 140 09-19-07

26

TRANSDUKSI
Proses transfer DNA asing (bukan berasal dari DNA virus itu sendiri)
ke dalam bakteri melalui bakteriofaga yang mengandung DNA asing
tersebut
Generalized and specialized transduction
Generalized transduction, setiap segmen dari DNA bakteri donor
dapat ditransfer
Specialized transduction, hanya gen tertentu yang dekat dengan
situs perlekatan lysoghenic phage saja yang dapat ditransfer.
Transducing phage, phages capable of transduction
Donor and recipient strain
Transductant, cells that have received DNA from another bacterium
by transduction

Transduction
Types of transduction

Generalized - Transduction in which

potentially any dornor bacterial gene can
be transferred.

Specialized - Transduction in which only

certain donor genes can be transferred

Generalized Transduction
Infection of Donor
Phage replication and degradation of host DNA
Assembly of phages particles
Release of phage
Infection of recipient
Legitimate recombination

A second form of gene transfer- transduction

SPESIALIZED TRANSDUCTION

Three mechanisms of gene transfer in bacteria