Download Bacteria and Recombinant DNA

Recombinant DNA
Aulanni’am
Biochemistry Laboratory
Chemistry Laboratory
Brawijaya University
Aulani "GE" Presentation 8
Definition

To put DNA from one organism into the DNA of bacteria
Methods
Transformation
Transduction
Conjugation
Aulani "GE" Presentation 8
Method: Transformation



The modification of the genotype of a cell (usually
prokaryotic) by introducing DNA from another source
The uptake of DNA from an organism’s environment
The uptake and expression of DNA in a bacterium
Aulani "GE" Presentation 8
Method: Transformation
3
elements required
Suitable host
2. Self-replicating vector
3. Means of selecting of host cell that have taken up
the gene
1.
 Plasmids
Small, circular, extra-chromosomal DNA
molecules found in bacteria which can replicate
on their own outside of a host cell
 Rapid growth rate
 Cloning limit 0.1-10 kb

Aulani "GE" Presentation 8
Method: Transformation

Plasmid (cont.)




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Fertility-(F-)plasmids, which contain only tra-genes. Their
only function is to initiate conjugation.
Resistance-(R-)plasmids, which contain genes that can build
a resistance against antibiotics or poisons.
Col-plasmids, which contain genes that code for (determine
the production of) colicines, proteins that can kill other
bacteria.
Degrative plasmids, which enable the digestion of unusual
substances, e.g., toluole or salicylic acid.
Virulence plasmids, which turn the bacterium into a
pathogen.
Aulani "GE" Presentation 8
Method: Transformation


The modification of the genotype of a cell (usually
prokaryotic) by introducing DNA from another source
The uptake of DNA from an organism’s environment
 Cosmids
Extrachromosomal circular DNA molecules that
combines features of plasmids and phage
 Cloning limit 35-50 kb

Aulani "GE" Presentation 8
Method: Transduction
 Process
of transfection with phage
introduction(equivalent to
transformation)
 Phages

Derivatives of bacteriophage lambda

Virus which infects E. coli
Linear molecules
 Transformation efficiency 100X greater than
plasmid vector
 Cloning limit 8-25 kb

Aulani "GE" Presentation 8
Method: Conjugation
 Bacterial
(BAC)
 Based
Artificial Chromosomes
on bacterial mini-F plasmids
Small pieces of episomal bacterial DNA that
give the bacteria the ability to initiation
conjugation with adjacent bacteria
 Cloning limit 75-300 kb

Aulani "GE" Presentation 8
Definitions for rDNA process
 Restriction
 Natural

endonuclease
function
to protect bacteria by cutting and
inactivating the DNA of infecting viruses
 rDNA

Recognize specific base sequences and cleave
the DNA molecule



EcoR1 cuts at GAATTC
BamHI cuts CCTAGG
HindIII cuts TTCGAA
Aulani "GE" Presentation 8
Definitions for rDNA process
 “Sticky
ends”
 Cloning
Vector
 Single
stranded extensions of DNA
created from a restriction enzyme
A
DNA molecule that carries foreign
DNA into a host cell, where it replicated
Phages
 Plasmid
 Cosmid

Aulani "GE" Presentation 8
Definitions for rDNA process
 Donor
 Cell
cells
supplying the snippet of DNA
 Recipient
cells
 The
cell supplying the cell as well as the
rest of the bacterial chromosome
 DNA
ligase
 Enzyme
used to bind sticky ends
Aulani "GE" Presentation 8
rDNA Steps
1.
2.
3.
4.
5.
(generalized)
Plasmid isolated and cut with enzyme
DNA isolated from donor cell and cut
with same enzyme
Donor and plasmid DNA mixed and
sealed with DNA ligase
rDNA introduced into a bacterium
Replication of plasmids
Aulani "GE" Presentation 8
Results
3
results
 Cells
that lack plasmids (picked up human
DNA)
 Cells that contain plasmid that do not
contain a foreign gene (picked up plasmid
DNA)
 Cells that contain plasmid that have picked
up a foreign gene (picked up rDNA)
Aulani "GE" Presentation 8
Distinguishing rDNA

Antibiotic resistance marker(s) added to
plasmid (ex. Ampicillin and tetracycline
genes)



Human DNA only – sensitive to both
Plasmid DNA only – resistant to both
rDNA –resistant to ampicillin but sensitive to
tetracycline (the human DNA is inserted into
the tetracycline coding sequence, thus
disrupting the gene)
Aulani "GE" Presentation 8
Other info
 Gene
isolation
 Genomic

library
collection of recombinant bacteria that harbor
pieces of a genome
 DNA
probe (gene sequence in question)
A labeled short sequence of DNA that
corresponds to a specific gene.
 Emits a signal when it binds to its complement
in a bacterial cell that contains a recombinant
plasmid

Aulani "GE" Presentation 8
Applications

Epidermal growth factor

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
Insulin

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Accelerates healing of wounds and burns
treats gastric ulcers
Allows cells to take up glucose in treatment of
type I diabetes mellitus
Renin inhibitor

Lowers blood pressure

Treats kidney cancer
Interleukin-2
Aulani "GE" Presentation 8