Sistem Hospes dan Vektor

dalam Kloning Gen

Tri Wibawa
Departemen Mikrobiologi
Fakultas Kedokteran Kesehatan Masyarakat dan
Keperawatan (FKKMK) - UGM
 DNA Technology
DNA Technology is the application of our learned properties of
DNA

I. Amplification of DNA
– Cloning (Amplification / expression)
– PCR
II. Detection of DNA
– Gel electrophoresis
– Sequencing
– Hybridization
 Amplification of DNA
• DNA amplification to generate multiple copies
of a specific gene or DNA segment, a small
fraction of chromosomal DNA, mitochondrial
DNA, or plasmid DNA.
– Cloning: amplification by replication inside cells
– PCR : amplification by DNA polymerase enzymes
outside cells (artificial process)
• The objectives of Recombinant DNA
technology include:
– Identifying genes
– Isolating genes
– Modifying genes
– Re-expressing genes in other hosts or
organisms
Molecular Cloning
MCS
Bacterial
plasmid
vector
Origin of
replication

Multiply
 Gene Cloning
• Isolation and amplification of an individual
gene sequence by insertion of that sequence
into a cells where it can be replicated
• Involves the construction of novel DNA
molecules by joining DNA from different
sources
• Product is Recombinant DNA (rDNA)
 Basic Events in Gene Cloning
 Isolation and amplification of gene of interest
 Incorporate gene into a vector (small replicating
DNA molecule, usually circular)
 Introduce recombinant vector into host cell via
transformation
 Select for the cells that have acquired the
recombinant DNA molecule
 Multiply recombinant vector within host cell to
produce a number of identical copies of the
cloned gene
 Extract the vector to obtain the copy of the gene
 Components of Gene Cloning

• Vectors (cloning vehicles)

• Enzymes for cutting and joining the DNA
fragments
• The DNA fragments (Target DNA)
• Selection process
 Vectors
• Must contain a replicon that enables it to replicate in
host cells (region of DNA that is amplified, i.e.: has
origin of replication)
• Small enough and unlikely to degrade during
purification.
• Several marker genes
• Unique cleavage site(s)
• For expression, must contain control elements, such
as promoters, terminators, ribosome binding sites,
etc…
 Types of Vectors
• Plasmids
• Cosmids
• Fosmids
• Phages
• Yeast Artificial Chromosomes (YACs)
• Transposons
• Bacterial Artificial Chromosomes (BACs)
• Viruses
– retroviruses
– adenoviruses
– adeno-associated viruses
– herpes simplex virus
– rhinoviruses
– Human Immunodeficiency Virus (HIV)
Vectors for Bacterial Cells
 Plasmid Vectors
• The most widely used vectors for bacterial cells.
• These vectors have their origin from extra-
chromosomal circular DNA (plasmid) found in certain
bacterial cells.
• Typically less than 5 kb.
• Large DNA molecules are difficult to handle and often
subject to degradation.
• The efficiency of transformation decreases with
increasing size of the plasmid
 Plasmid Vectors …….. cont
• Double stranded, circular DNA which exist in
bacteria.
• May exist as single copy per cell or multi-copy
per cell (10-20 genomes/cell), or even under
relaxed replication control where up to 1000
copies/cell can be maintained
• Size of rDNA insertions limited to ~10kb
 Plasmid vector’s structural
elements.
• Replication origin
• Cloning sites (multiple cloning sites=MCS)
• Selectable markers: These are usually antibiotic
resistance genes
• Expression vector: contain a promoter upstream of
MCS.
• Optional but popular feature: polyhistidine sequence
(e.g. 5'-CACCACCACCACCACCAC encoding 6
histidines)
Purifica
tion of
his-
tagged
protein
on an
affinity
column
 Characteristics of Plasmids as Cloning Vectors

• Natural vectors
• Useful cloning vectors
– Small
– Easy to isolate and purify
– Independently replicating
– Multiply copy number
– Presence of selectable markers
• Antibiotic resistance genes
 High and Low Copy Plasmids.
• Plasmids can be grouped into:
– high copy (≥100 copies/cell); ex: pUC
– low copy plasmids (1 -25 copies/cell); ex: pBR322
• High copy:
– Good for yield
– Not good if it has adverse effect
• Copy number is depend on:
– Origin or replication
– Size of plasmid and associated insert
 Structure of Plasmid pBR322

Antibiotic
resistance
Restriction
enzyme cut
site
 Advantages of Using pBR322

• Small • Complete sequence

• Relative stable in E. known
coli with 20 – 30 • Single cut restriction
copies/cell sites
• Can be amplified to • Amp and tet
1000 copies/cell resistance as tags
• Up to 10 kbp can be
inserted
Cloning Genes
with pBR322
pUC Plasmids
• The β-lactamase gene (ampicillin
resistance, AmpR)
• The lac operon in pUC contains a
truncated lacZ (β-galactosidase)
gene
• MCS is inserted into the lacZ'
region
• The pUC plasmids are expression
vectors, because the lac operon is
active when isopropyl-P-D-
thiogalactopyranoside (IPTG) is
supplied.
 The ampicillin
resistance
gene is a
selectable
marker.

In precence of IPTG
α-peptide will be produced by
vector Lac Z
to complete β-galactosidase
of the host
Promoters and RNA Polymerases.
• The bacteriophage T3, T7, and
SP6 promoters are also used in
the construction of bacterial
expression vectors
• These promoters are only
recognized specifically by their
respective RNA polymerases,
and not by the E. coli RNA
polymerases.
• T7 RNA polymerase produced by
selected host which induced by
IPTG
 Topoisomerase-based Cloning.
• Topoisomerase is to cleave and rejoin DNA during
replication
• Binding and cleavage occur at a pentameric motif 5'-
(C or T)CCTT in duplex DNA.
• Both sticky end and blunt end ligations can be
achieved
 Bacteriophage Vectors
• Viruses that attack specific bacteria
• Mostly for the construction of cDNA or genomic
libraries
• Must first deactivate lysogenic growth component of
phage (phage DNA inserts into host DNA, creating
prophage)
• Allow lytic growth – cell death after infection and
replication. Cell death revealed as plaques
• Steps:
– Insert rDNA into phage (usu. up to 25kb)
– Infect bacteria with phage
– Infected bacteria form plaques
• Advantage: Transformation, selection very easy
Bacteriophages
 λ genome

λ insertion λ Replacement
vector R vector R R

New DNA
Inserted

R R R R
Bacteriophage λ
Life Cycle.
 Bacteriophage λ Vectors
• Designed to facilitate:
– DNA insertion,
– Screening for recombinants
– Gene expression.
• Contains a lacZ gene and a unique EcoRl restriction site at the
5' end of the gene
• Insertion of a DNA segment or a gene at the unique restriction
site interrupts the lacZ gene sequence.
– Recombinant phages  clear plaques
– Blank phages  blue plaques
• The cloned DNA or gene sequence is expressed as a fusion
protein with β-galactosidase --- It can also be screened by
immunodetection methods
 Bacteriophage λ Vectors ….. cont
• The genes related to integration are deleted, and
thus no induction is required to switch from
lysogenic to the lytic mode
• A region containing the terminator for RNA synthesis
is deleted
• Nonsense mutations are introduced in the genes
required for lytic growth ----- A reversion of this
effect of mutation can be achieved by suppression in
the anticodon of the tRNA carried out by the host
strain (Ex: specific E. coli)
Genetic map of bacteriophage λ and λgt11 vector
Host for Cloning
 Recombinant DNA technology today

Gene source Isolate the gene

Which?
Sequence known? Screen cDNA library
(degenerate primers?)
RT-PCR (degenerate primers?)

Screen expression library

(antibody to YFP)
Synthetic gene

Express Clone
and purify
Choose organism
Choose expression vector
Mutations or other changes?
 “What the general issues to cloning in a heterologous
host?”

 Selection of host and vector

 Can regulate expression by choice of vector
 Genetic modification of host and cloned gene
 Choice of location of product
 Modification of protein produced
 Ease of production and scale
 Can facilitate purification
 Choice of Hosts for
Cloning Vectors
• Depends on
– Final application
– Posttranslational modifications
– Compatibility between vector and host
– Cost
• Desired characteristics
– Grow rapidly on inexpensive media
– Nonpathogenic
– Capable of taking up DNA
– Genetically stable in culture
– Allow replication of vector
Prokaryotic and fungi chosen more often than
eukaryotic hosts
• Cultured in large quantities in short time
• Abundant and constant supply of desired
proteins
• Proteins usually more stable than from
plant/animal
• Genetic manipulation usually easy
• Proteins can be directed to cytoplasm,
membrane, secreted…
 Problems of Production of a protein in a
heterologous system

• Expression (more protein is not always better

protein
• Folding (E. coli can produce inclusion bodies)
• Other properties of the protein produced
Potential Problems in Prokaryotic Expression Systems

• Low expression
• Degradation by bacterial proteases
• Improper folding
• Oxygen limitations
• Biofilm formation
 There are many systems for expression.
Some commonly used ones
• Escherichia coli
• Other bacteria
• Yeast
• Pichia pastoris
• Baculovirus
• Animal cell culture
• Plants
• Animals (sheep, cows, goats)
 Advantages

Disadvantages

Fig. 31.2
 Expression systems
USE EASY DIFFICULT

COST LOW HIGH

Prokaryotic
Yeast Mammalian

Insect

- +
POST-TRANSLATIONAL
MODIFICATIONS