Transcript Welcome

Recombinational cloning
This is a novel site-specific recombination technique
for transferring DNA sequences, which allows one
universal strategy to move DNA sequence to any
vector. The recombination cloning is a promising
approach for high throughput genomics and
proteomics applications.
Harini Chandra
Affiliations
Master Layout (Part 1)
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This animation consists of 2 parts:
Part 1 – Conventional cloning protocol
Part 2 – GATEWAY Cloning system
Restriction endonuclease
2
Antibiotic resistance
genes
Gene of interest
Restriction
digestion &
insertion
Plasmid vector
3
Origin of
replication
Transformation
E. coli cells
Ligation
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5
Screening & selection
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Definitions of the components:
Part 1 – Conventional cloning protocol
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1. Plasmid vector: An expression vector or construct is used to introduce a
particular gene of interest into a target cell for expression. Plasmids are the
most commonly used vectors for gene expression. An efficient expression
vector system must be capable of producing large quantities of the protein
product.
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2. Restriction endonuclease: These are enzymes that cleave double or
single stranded DNA at specific recognition sequences known as restriction
sites. The restriction sites are often found to be palindromic sequences such
as GAATTC. Cleavage can result in formation of either blunt ends or sticky
ends.
3. Gene of interest: The DNA fragment that codes for the desired target
protein that needs to be expressed by means of the expression vector.
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4. Restriction digestion & insertion: The same restriction enzyme is used
to cleave the plasmid vector as well as the DNA sequence containing the
gene of interest at their specific restriction sites. The gene of interest is then
inserted into the plasmid which can re-anneal with the gene sequence due to
the specific sequence that it possesses.
5. Ligation: The process of sealing any gaps that are present in genetic
material. Once the gene has been inserted into the plasmid vector, the ends
are ligated by means of a specific ligase enzyme.
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3
4
5
Definitions of the components:
Part 1 – Conventional cloning protocol
6. Transformation: Once ligation is complete, the vectors containing the gene
insert are introduced into suitable host cells such as E. coli. This is done by
various techniques such as electroporation, chemical sensitization etc.
7. E. coli cells: These bacteria act as suitable host cells for introduction of
expression vectors containing the desired gene insert. They have simple
nutritional requirements for growth and multiply quickly, thereby allowing
protein expression to take place soon.
8. Antibiotic resistance genes: These are genes in the plasmid vector that
confer resistance against various antibiotics like ampicillin, tetracycline etc.
Bacteria that take up these plasmids can be grown even in the presence of
these antibiotics. Insertion of a gene fragment within any of these genes leads
to its inactivation. These are extremely useful for selection of transformed
bacteria.
9. Screening & selection: Once the cells have been grown on a suitable
medium, they are screened to identify those cells that have taken up the
plasmid containing the gene of interest. This is done with the help of selectable
markers that are present in the cloning vectors such as antibiotic resistance
markers or colour selection markers. In order to confirm that the cells also
contain the desired gene insert, further analysis is carried out by PCR or
restriction fragment analysis.
1 Part 1, Step 1:
Fragmentation & insertion:
Ampicillin resistance
gene
DNA Ligase
2
Plasmid vector
Restriction
endonuclease
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4
5
Tetracycline resistance
gene
Ligation
Gene of interest
Action
As
shown
in
animat
ion.
Description of the action
First show the circle with colored fragments and the grey
and blue line below. Next show the pink pie shaped
object which must attach at the places shown and must
be shown to create breaks at these points to give rise to
the figures in the next panel where there are gaps
between the fragments. The blue fragment must then
bend and be inserted in between the green fragment in
the circle. The green oval must then appear at the
breaks and seal these gaps to give the final structure on
the right.
Audio Narration
The conventional cloning protocol makes use of a restriction
enzyme that fragments the selected plasmid vector as well the
DNA sequence containing the gene of interest at the same
recognition sites. The complementary sequence overhangs
that are produced in the plasmid and gene insert during
restriction digestion are useful for proper orientation of the
fragment during insertion. The insert is then ligated by means
of the DNA ligase enzyme. Insertion of the fragment within an
antibiotic resistance gene leads to inactivation of this gene.
1 Part 1, Step 2:
E. coli cells
2
Plasmids that have
taken up gene insert
Transformation
3
Plasmids without
gene insert
4
5
Action
As
shown
in
animat
ion.
Description of the action
First show the circles on top with the label below followed
by the circles below with label. Then show the rectangles
on the right which must have wavy lines that must not be
solid. The circles must then be shown to enter these
rectangles. However, the entry must not be direct. The
circles must be shown to take a maze-like path through the
dotted outlines of the rectangles and must finally be shown
to enter them. This is to indicate difficulty in penetration.
Audio Narration
Once the insert has been introduced into the
plasmid vector in the desired orientation, they
are transformed into suitable bacterial host
cells. This can be done by techniques such as
electroporation, chemical sensitization etc.
which make the cell membrane relatively
permeable thereby allowing the plasmids to
enter.
1, Step 3:
1 Part
Screening & selection
Only tetracycline
– growth observed
Tetracycline + ampicillin
- growth observed
2
Comparison of
plate colonies
will reveal only
those that have
taken up gene
insert (replica
plating)
3
4
5
Tetracyclin + ampicillin –
no growth
Transformed cells
without gene insert
Transformed cells with
& without gene insert
Action Description of the action
As
shown
in
animat
ion.
(Please redraw all figures.)
First show the three grey circles on top
with their labels. The first two must show
small dots as clusters indicating growth
while the last one should not show
anything. Each of these circles must be
zoomed into to depict the figures below.
After zooming out, the double headed
arrow must appear with the text shown
below.
Untransformed cells
Audio Narration
The cells are grown on a suitable medium which contain specific antibiotics that
allow only certain bacterial cells to grow. Cells that have been transformed with the
plasmid but do not contain the gene of interest grow on medium containing both
tetracycline and ampicillin. Those cells that have taken up the plasmid and contain
the gene insert will grow on a tetracycline containing medium but will not grow in
presence of ampicillin. Those cells that do not grow in presence of antibiotics have
not taken up any plasmid and therefore do not have resistance to the antibiotics.
Comparison of colonies grown in the presence of both antibiotics and in presence of
only tetracycline will reveal those that have taken up the gene insert, a technique
known as replica plating.
Master Layout (Part 2)
1
This animation consists of 2 parts:
Part 1 – Conventional cloning protocol
Part 2 – GATEWAY Cloning system
LR reaction
BP reaction
2
Expression clone
B2
B1
3
L2
Gene
Entry/Master
clone
R1
R1
R2
ccdB
BP Clonase
Enzyme mix
L1
5
L2
Gene
ccdB
Gene
4
L1
P2
P1
Destination vector
Entry clone
Donor vector
LR Clonase
Enzyme mix
R2
B2
B1
P2
P1
ccdB
Gene
ccdB
By-product
Expression clone
By-product
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2
3
Definitions of the components:
Part 2 – GATEWAY Cloning system
1. GATEWAY Cloning: This is a powerful new recombinational cloning
technology that facilitates protein expression and cloning of PCR products by
using site-specific recombination enzymes rather than restriction
endonucleases and ligases. This technique makes use of a master clone
having a particular gene that can be rapidly transferred to desired destination
vectors and thereby provides significant benefit over conventional cloning.
2. Expression clone: The clone containing the gene sequence of interest
flanked by attB sites. Orientation of the gene is maintained throughout the
cloning process due to specific interactions between the att sites, which are
DNA segments of a certain defined length. These expression clones can be
produced by means of the LR reaction.
3. Donor vector: The donor vector consists of a counter-selectable gene
flanked by attP sites that recombines with the gene of interest flanked by attB
sites in the BP reaction to produce a master or entry clone.
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4. Entry/Master clone: The vector containing the gene of interest flanked by
attL sites. Entry clones are formed by the BP reaction and used in the LR
reaction for production of expression clones of interest.
5. Destination vector: Vector containing a counter-selectable gene flanked
by attR sites which interacts with the gene from the entry clone to produce an
expression vector in the LR reaction.
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3
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Definitions of the components:
Part 2 – GATEWAY cloning system
6. BP reaction: A site specific recombination reaction between the attB and
attP sites leading to generation of the master or entry clones which can further
be used at any time for specific purposes.
7. LR reaction: The main reaction pathway of the GATEWAY system
consisting of a recombination reaction between a master clone and a
destination vector used for generation of the expression clones. These
expression clones can be used for a variety of applications.
8. BP & LR Clonase enzyme mix: These are specific recombination protein
mixes that are used to carry out the BP and LR reactions respectively.
9. Site specific recombination: A genetic recombination technique where
DNA strand exchange takes place between regions possessing reasonable
degree of sequence homology. Specific recombinase enzymes cleave the
DNA backbone and carry out interchange of DNA helices between specific
sites on two different molecules. The common site specific recombination
technologies currently in use are the GATEWAY Technology (Invitrogen) and
the Creator Technology (BD Clontech).
1 Part 2, Step 1:
BP reaction
Expression clone
L1
L2
B2
B1
Transform in
E. coli
Gene
Gene
2
3
BP Clonase
Enzyme mix
Entry/Master
clone
Incubate ~ 60
min at 25oC
Donor vector
P2
P1
R1
Kanamycin
resistant
colonies
R2
ccdB
ccdB
By-product
4
5
Action
As
shown
in
animat
ion.
Description of the action
First show the figures on the left top and bottom.
Next show the arrows with enzyme name and
conditions of reactions mentioned. The rectangular
portions along with the L shaped objects that are
closest to it must then exchange between the top
and bottom figures as depicted in animation
resulting in the figures on the right. The figure on top
with the violet rectangle must then be shown to
enter grey rectangular cells as shown in slide #6.
Once this happens, this plate must appear with its
label.
Audio Narration
The BP reaction of GATEWAY cloning is a site-specific recombination
reaction between the attB site of an expression clone or a PCR
product and attP site of a donor vector in the presence of BP Clonase
enzyme master mix. The reaction is incubated for just an hour at 25oC
to obtain the entry or master clones containing the gene of interest.
Once this master clone, flanked by attL sites is produced, it can then
be transferred into any destination vector to produce expression
clones for a specific desired application. The reaction yields more
than 90% correct clones.
1 Part 2, Step 2:
LR reaction
B2
B1
Entry clone
L1
L2
Gene
2
3
LR Clonase
Enzyme mix
5
Expression clone
Ampicillin
resistant
colonies
Incubate ~ 60
min at 25oC
R1
R2
Action
As
shown
in
animat
ion.
P2
P1
ccdB
ccdB
Destination vector
4
Transform in
E. coli
Gene
Description of the action
First show the figures on the left top and bottom. Next
show the arrows with enzyme name and conditions of
reactions mentioned. The rectangular portions along
with the L shaped objects that are closest to it must
then exchange between the top and bottom figures
as depicted in animation resulting in the figures on
the right. The figure on top with the violet rectangle
must then be shown to enter grey rectangular cells as
shown in slide #6. Once this happens, this plate must
appear with its label.
By-product
Audio Narration
The LR reaction is essentially the reverse of the BP reactions
where the master clone, flanked by attL sites, recombines with a
destination vector with attR sites. This reaction which takes
place in the presence of the LR Clonase enzyme mix results in
transfer of the gene from the master clone to the destination
vector to produce an expression clone for a specific purpose.
This reaction enables generation of several expression clones
for various applications in very short time, thereby providing
significant advantage over conventional cloning techniques.
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Part 2, Step 3:
Applications of master clones
Gene
2
Mammalian
protein
functioning
MMTV
retroviral
clone
Gene
Inducible
His6 fusion
clone
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5
First show the central figure ‘master
clone’. Then show the remaining
surrounding figures appearing one
at a time as depicted in the
animation.
High yield
protein
synthesis
Gene
Action Description of the action
As
shown
in
animat
ion.
Gene
p10 promoter
GST fusion
Tet
inducible
clone
4
Protein
structural
studies
Gene
Gene
2-hybrid
clone
Gene
CMV GFP
fusion
clone
Master
clone
Protein
interaction
studies
Bacterial
protein
assays
Mammalian
protein
expression
Audio Narration
The gene in the master clone can be transferred to various destination
vectors by means of the LR reaction to produce expression clones for
several applications. Proteins can be efficiently expressed in bacterial, yeast
and mammalian systems and used for a variety of applications such as
structural & functional studies, protein interaction studies, protein assays,
producing high yields of proteins for experimentation etc. The rapid
recombination between clones that is possible with the GATEWAY system
cannot be done with conventional cloning techniques due to which the
GATEWAY protocol is now being extensively adopted.
1 Interactivity option 1:Step No: 1
It is desired to obtain an expression clone from a master clone containing the suitable gene
that can be used for studying protein interactions. From the components given below, drag
and drop the correct components into the reaction vessel that will allow this expression clone
to be generated.
2
L1
L2
Gene
LR Clonase
Enzyme mix
Master clone
BP Clonase
Enzyme mix
B2
B1
Gene
R1
3
P2
P1
R2
Yeast 2-hybrid
expression clone
ccdB
ccdB
Donor vector
Yeast 2-hybrid
destination vector
R1
R2
ccdB
His6 fusion
destination vector
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5
Reaction vessel
Interacativity Type
Options
Drag & drop
User must drag
and drop the
correct
components
into the grey
reaction vessel.
Boundary/limits
Results
User must drag and drop the correct components
into the grey reaction vessel. Only when the
correct components are selected, the output
shown o the right will appear. Otherwise a cross
sign should appear indicating that the reaction
will not take place. The correct components are
‘master clone’, ‘yeast 2-hybrid destination vector’
and orange ‘LR clonase enzyme mix’.
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Questionnaire
1. If a gene fragment is inserted within a tetracyline resistance gene on a plasmid containing
another gene for resistance against penicillin, then on what medium will bacteria transformed
with these plasmids grow?
2
Answers: a) Tetracycline-containing medium b) Tetracyline + penicillin medium c) Penicillincontaining medium d) They will not grow on any medium
2. The BP reaction of the GATEWAY system gives rise to which of the following clones?
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Answers: a) Expression clone b) Master clone c) Donor vector d) Both expression and donor
clones
3. Match the following recombination sites with their corresponding clones or vectors:
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5
a) Master clone
i) attP
b) Expression clone
ii) attB
c) Donor vector
iii) attR
d) Destination vector
iv) attL
Answers: a) a-ii, b-i, c-iii, d-iv b) a-iii, b-ii, c-i, d-iv c) a-iv, b-ii, c-iii, d-i d) a-iv, b-ii, c-i, d-iii
Links for further reading
Reference websites:
www.invitrogen.com
Research papers:
 Park, J. & LaBaer, J. Recombinational Cloning. Current protocols in molecular biology,
2006, 3.20.1 – 3.20.22.
 Hartley, J.L., Temple, G.F. & Brasch, M.A. DNA cloning using in vitro site-specific
recombination. Genome Res. 10, 1788–1795 (2000).
 Goshima, N. et al. Human protein factory for converting the transcriptome into an in vitroexpressed proteome. Nat methods 2008, 5(12), 1011-1017.
 Aguiar, J. C. et al. High-throughput generation of P. falciparum functional molecules by
recombinational cloning. Genome Res. 2004, 14(10B), 2076-82.