recombinational cloning harini chandra affiliations this is a novel site-specific recombination...

Recombinational cloning

Harini ChandraAffiliations

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.

Master Layout (Part 1)

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1 This animation consists of 2 parts:Part 1 – Conventional cloning protocolPart 2 – GATEWAY Cloning system

Restriction endonuclease

Plasmid vector

Gene of interest

Ligation

Transformation

Screening & selection

Restriction digestion & insertion

E. coli cells

Antibiotic resistance genes

Origin of replication

Definitions of the components:Part 1 – Conventional cloning protocol

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11. 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.

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.

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.

Definitions of the components:Part 1 – Conventional cloning protocol

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16. 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.

Part 1, Step 1:

Action Audio Narration

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4Description of the action

Fragmentation & insertion:

Plasmid vector

Gene of interest

LigationRestriction endonuclease

DNA Ligase

As shown in animation.

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.

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.

Ampicillin resistance gene

Tetracycline resistance gene

Part 1, Step 2:


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E. coli cells

Plasmids that have taken up gene insert

Plasmids without gene insert

Transformation


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.

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.

Part 1, Step 3:


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(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.

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.

Screening & selectionTetracycline + ampicillin- growth observed

Only tetracycline– growth observed

Tetracyclin + ampicillin – no growth

Transformed cells without gene insert

Transformed cells with & without gene insert

Untransformed cells

Comparison of plate colonies will reveal only those that have taken up gene insert (replica plating)

Master Layout (Part 2)

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1 This animation consists of 2 parts:Part 1 – Conventional cloning protocolPart 2 – GATEWAY Cloning system

BP reaction

ccdB

B1 B2 P1 P2Expression clone Donor vector

Gene

Gene ccdB

Entry/Master clone

By-product

LR reaction

BP Clonase Enzyme mix

L1 L2 R1 R2

Gene

Entry cloneL1 L2

ccdB

R1 R2

LR Clonase Enzyme mix

Destination vector

B1 B2

Expression clone

Gene ccdB

P1 P2

By-product

Definitions of the components:Part 2 – GATEWAY Cloning system

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11. 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.

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.

Definitions of the components:Part 2 – GATEWAY cloning system

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16. 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).

Part 2, Step 1:


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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.

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.

BP reaction

B1 B2

P1 P2

Expression clone

Donor vector

Gene

ccdB

Entry/Master clone

By-product


L1 L2

R1 R2

Gene

ccdB

Transform in E. coli

Kanamycin resistant coloniesIncubate ~ 60

min at 25oC

Part 2, Step 2:


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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.

LR reaction

Entry cloneL1 L2

R1 R2


B1 B2

Expression clone

Gene

ccdB

P1 P2

Destination vector By-product

Incubate ~ 60 min at 25oC

Gene

ccdB

Transform in E. coli

Ampicillin resistant colonies

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.

Part 2, Step 3:


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First show the central figure ‘master clone’. Then show the remaining surrounding figures appearing one at a time as depicted in the animation.

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.

Applications of master clones

Gene

Master clone

Gene

Inducible His6 fusion clone

Gene

CMV GFP fusion clone

Gene

p10 promoter GST fusion

Gene

Tet inducible clone

Gene

MMTV retroviral clone

Gene

2-hybrid clone

Bacterial protein assays

Protein structural studies

High yield protein synthesis

Mammalian protein expression

Mammalian protein functioning

Protein interaction studies

Interactivity option 1:Step No: 1

Boundary/limitsInteracativity Type Options Results

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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.


P1 P2

ccdB

Donor vector

R1 R2

ccdB

His6 fusion destination vector

B1 B2

Gene

Yeast 2-hybrid expression clone

Master clone

L1 L2

Gene

R1 R2

ccdB

Yeast 2-hybrid destination vector


Reaction vessel

Drag & drop User must drag and drop the correct components into the grey reaction vessel.

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’.

Questionnaire1

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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?

Answers: a) Tetracycline-containing medium b) Tetracyline + penicillin medium c) Penicillin-

containing 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?

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:

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 readingReference 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 vitro-expressed 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.