10.3.2 Mechanism of Formation of Recombinant Plasmid in Recombinant DNA Technology

Cloning of the desired gene through recombinant DNA technology involves the formation of recombinant DNA (gene of interest + vector DNA), transformation into suitable expression system by the recombinant DNA and the identification of transformed clones. The first step in the construction of a recombinant DNA is the isolation and purification of gene of interest and relevant vectors. The gene of interest is cleaved directly from genome by specific restriction endonucleases with specific sticky ends or it may be synthesized from mRNA by reverse transcription using reverse transcriptase. Then specific vector DNA (plasmid) having same restriction site is also cleaved with same restriction enzyme by which gene of interest is cleaved so that compatible sticky ends can be produced. Next both, vector and gene of interest are incubated together in the presence of DNA ligase which connects them by forming phosphodiester linkage. When gene of interest is ligated to plasmid at the complementary sticky ends the resultant plasmid is now called recombinant plasmid (vector).

Definition

Formation of Recombinant Plasmid: The first step in the construction of a recombinant DNA is the isolation and purification of gene of interest and relevant vectors. When gene of interest is ligated to plasmid at the complementary sticky ends the resultant plasmid is now called recombinant plasmid (vector).

Molecular Glue (DNA Ligase): Molecular glue (DNA Ligase) is responsible for the formation of the phosphodiester linkage between two adjacent nucleotides and thus joins two double-stranded DNA fragments; therefore it is called molecular glue.

Main Idea

• Cloning of the desired gene involves:
• First step: isolation and purification of gene of interest and relevant vectors.
• Gene of interest is cleaved directly from genome by specific restriction endonucleases.
• Or it may be synthesized from mRNA by reverse transcription using reverse transcriptase.
• Vector DNA (plasmid) having same restriction site is also cleaved with same restriction enzyme.
• This produces compatible sticky ends.
• Both vector and gene of interest are incubated together in the presence of DNA ligase.
• DNA ligase connects them by forming phosphodiester linkage.
• The resultant plasmid is called recombinant plasmid (vector).

Expression System

• Expression system may be a suitable cell or organism that can act as host for the recombinant vector for its cloning (multiplication) or expression.
• Selection of suitable expression system always depends upon the type of vector which is being used while making recombinant DNA.
• The most important character of an ideal expression system is its short generation time and simplicity of its cellular and genetic system.
• So bacterial cells can act as an ideal expression system.

Transformation of Expression System

• Here transformation refers to the insertion of recombinant DNA into the expression system.
• This can be performed by putting the expression system (bacterial cells that already contain no plasmids) and recombinant plasmids into the same medium.
• Bacterial cells take up recombinant plasmid, especially, if they are treated with calcium chloride which make them more permeable.
• Thereafter, as the cell reproduces, a bacterial clone forms and each new cell contains at least one plasmid.
• Therefore, each of the bacterial cell contains the gene of interest, which will express itself and make its product.

Identification of Transformed Clone

• The transformed clone can be identified by adding a particular antibiotic (for which resistant gene is found in plasmid) into the medium.
• As the transformed clone has got resistance against the antibiotic, so it remains alive and continues to grow.
• Whereas all the untransformed clones are killed by the antibiotic.
• From this transformed bacterial clone, the cloned gene can be isolated for further analysis or its protein product can be separated and used for various purposes.

Core Concept

• Recombinant plasmid = gene of interest + vector DNA
• Isolation = gene of interest and vector
• Cleavage = restriction endonucleases
• Compatible sticky ends = essential
• DNA ligase = joins DNA fragments
• Phosphodiester linkage = formed by DNA ligase
• Expression system = suitable cell or organism
• Ideal expression system = short generation time; simple cellular and genetic system
• Transformation = insertion of recombinant DNA into expression system
• Calcium chloride = makes bacterial cells more permeable
• Identification = antibiotic resistance
• Transformed clone = survives antibiotic
• Untransformed clone = killed by antibiotic

Detailed Explanation

Formation of Recombinant Plasmid

The first step in the construction of a recombinant DNA, is the isolation and purification of gene of interest and relevant vectors.

• The gene of interest is cleaved directly from genome by specific restriction endonucleases with specific sticky ends or it may be synthesized from mRNA by reverse transcription using reverse transcriptase.
• Then specific vector DNA (plasmid) having same restriction site is also cleaved with same restriction enzyme by which gene of interest is cleaved so that compatible sticky ends can be produced.
• Next both, vector and gene of interest are incubated together in the presence of DNA ligase which connects them by forming phosphodiester linkage.
• When gene of interest is ligated to plasmid at the complementary sticky ends the resultant plasmid is now called recombinant plasmid (vector).

Molecular Glue (DNA Ligase)

• Molecular glue (DNA Ligase) is responsible for the formation of the phosphodiester linkage between two adjacent nucleotides and thus joins two double-stranded DNA fragments; therefore it is called molecular glue.
• In rDNA experiments, DNA ligase is used to join two different DNA fragments (plasmid/vector and the foreign DNA) that are annealed by the sticky ends.
• Products of recombinant DNA technology can be produced by using suitable expression system.
• Plasmids are usually made recombinant for the production of specific protein as a result of expression of gene of interest in natural expression system.

Expression System

• Expression system may be a suitable cell or organism that can act as host for the recombinant vector for its cloning (multiplication) or expression.
• Therefore, the selection of suitable expression system always depends upon the type of vector which is being used while making recombinant DNA.
• The most important character of an ideal expression system is its short generation time and simplicity of its cellular and genetic system.
• So bacterial cells can act as an ideal expression system.

Transformation of Expression System

• Here transformation refers to the insertion of recombinant DNA into the expression system.
• This can be performed by putting the expression system (bacterial cells that already contain no plasmids) and recombinant plasmids into the same medium.
• Bacterial cells take up recombinant plasmid, especially, if they are treated with calcium chloride which make them more permeable.
• Thereafter, as the cell reproduces, a bacterial clone forms and each new cell contains at least one plasmid.
• Therefore, each of the bacterial cell contains the gene of interest, which will express itself and make its product.

Identification of Transformed Clone

• The transformed clone can be identified by adding a particular antibiotic (for which resistant gene is found in plasmid) into the medium.
• As the transformed clone has got resistance against the antibiotic, so it remains alive and continues to grow, whereas all the untransformed clones are killed by the antibiotic.
• From this transformed bacterial clone, the cloned gene can be isolated for further analysis or its protein product can be separated and used for various purposes.

Summary Table of Steps

Summary of Expression System

Intermediate Level

Formation of Recombinant Plasmid

Expression System

Transformation

Identification of Transformed Clone

Formula / Mathematical Form

Not applicable to this topic.

Solved Example

Not applicable to this topic (descriptive topic).

Important Points

• 1. Cloning involves formation of recombinant DNA.
• 2. Transformation into suitable expression system.
• 3. Identification of transformed clones.
• 4. First step: isolation and purification.
• 5. Gene of interest is cleaved by restriction endonucleases.
• 6. Gene of interest can be synthesized from mRNA.
• 7. Vector is cleaved with same restriction enzyme.
• 8. Compatible sticky ends are produced.
• 9. DNA ligase joins vector and gene of interest.
• 10. DNA ligase forms phosphodiester linkage.
• 11. DNA ligase is called molecular glue.
• 12. Recombinant plasmid is formed.
• 13. Expression system is a suitable cell or organism.
• 14. Expression system is for cloning and expression.
• 15. Ideal expression system has short generation time.
• 16. Ideal expression system has simple cellular and genetic system.
• 17. Bacterial cells are ideal expression system.
• 18. Transformation is insertion of recombinant DNA.
• 19. Calcium chloride makes cells more permeable.
• 20. Antibiotic is used to identify transformed clones.

Remember

• - Recombinant plasmid = gene of interest + vector DNA
• - Isolation = gene of interest and vector
• - Cleavage = restriction endonucleases
• - Compatible sticky ends = essential
• - DNA ligase = joins DNA fragments
• - Phosphodiester linkage = formed by DNA ligase
• - Expression system = suitable cell or organism
• - Ideal expression system = short generation time; simple cellular and genetic system
• - Transformation = insertion of recombinant DNA into expression system
• - Calcium chloride = makes bacterial cells more permeable
• - Identification = antibiotic resistance
• - Transformed clone = survives antibiotic
• - Untransformed clone = killed by antibiotic

Common Mistakes

• 1. Confusing recombinant plasmid and vector — Recombinant plasmid contains gene of interest; vector is the carrier.
• 2. Mixing up the steps — Isolation, cleavage, ligation, transformation, identification.
• 3. Forgetting the role of DNA ligase — Joins DNA fragments.
• 4. Mixing up restriction endonucleases and DNA ligase — Restriction endonucleases cut; DNA ligase joins.
• 5. Forgetting the role of calcium chloride — Makes cells more permeable.
• 6. Mixing up the expression system — Suitable cell or organism.
• 7. Believing transformation is the first step — Transformation comes after recombinant plasmid formation.
• 8. Mixing up the identification method — Antibiotic resistance.
• 9. Thinking untransformed clones survive — Untransformed clones are killed by antibiotic.
• 10. Forgetting the role of compatible sticky ends — Essential for ligation.

Quick Revision

Cloning of the desired gene through recombinant DNA technology involves the formation of recombinant DNA (gene of interest + vector DNA), transformation into suitable expression system by the recombinant DNA and the identification of transformed clones. The first step in the construction of a recombinant DNA, is the isolation and purification of gene of interest and relevant vectors. The gene of interest is cleaved directly from genome by specific restriction endonucleases with specific sticky ends or it may be synthesized from mRNA by reverse transcription using reverse transcriptase. Then specific vector DNA (plasmid) having same restriction site is also cleaved with same restriction enzyme by which gene of interest is cleaved so that compatible sticky ends can be produced. Next both, vector and gene of interest are incubated together in the presence of DNA ligase which connects them by forming phosphodiester linkage. When gene of interest is ligated to plasmid at the complementary sticky ends the resultant plasmid is now called recombinant plasmid (vector). Molecular glue (DNA Ligase) is responsible for the formation of the phosphodiester linkage between two adjacent nucleotides and thus joins two double-stranded DNA fragments; therefore it is called molecular glue. In rDNA experiments, DNA ligase is used to join two different DNA fragments (plasmid/vector and the foreign DNA) that are annealed by the sticky ends. Products of recombinant DNA technology can be produced by using suitable expression system. Plasmids are usually made recombinant for the production of specific protein as a result of expression of gene of interest in natural expression system. Expression system may be a suitable cell or organism that can act as host for the recombinant vector for its cloning (multiplication) or expression. Therefore, the selection of suitable expression system always depends upon the type of vector which is being used while making recombinant DNA. The most important character of an ideal expression system is its short generation time and simplicity of its cellular and genetic system. So bacterial cells can act as an ideal expression system. Here transformation refers to the insertion of recombinant DNA into the expression system which can be performed by putting the expression system (bacterial cells that already contain no plasmids) and recombinant plasmids into the same medium. Bacterial cells take up recombinant plasmid, especially, if they are treated with calcium chloride which make them more permeable. Thereafter, as the cell reproduces, a bacterial clone forms and each new cell contains at least one plasmid. Therefore, each of the bacterial cell contains the gene of interest, which will express itself and make its product. The transformed clone can be identified by adding a particular antibiotic (for which resistant gene is found in plasmid) into the medium. As the transformed clone has got resistance against the antibiotic, so it remains alive and continues to grow, whereas all the untransformed clones are killed by the antibiotic. From this transformed bacterial clone, the cloned gene can be isolated for further analysis or its protein product can be separated and used for various purposes.

60-Second Revision

• Recombinant plasmid = gene of interest + vector DNA
• Isolation = gene of interest and vector
• Cleavage = restriction endonucleases
• Compatible sticky ends = essential
• DNA ligase = joins DNA fragments
• Phosphodiester linkage = formed by DNA ligase
• Expression system = suitable cell or organism
• Ideal expression system = short generation time; simple cellular and genetic system
• Transformation = insertion of recombinant DNA into expression system
• Calcium chloride = makes bacterial cells more permeable
• Identification = antibiotic resistance
• Transformed clone = survives antibiotic
• Untransformed clone = killed by antibiotic
• Warning: DNA ligase joins DNA fragments; it does not cut them

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