10.3 Molecular Carriers Or Vectors

For the synthesis of recombinant DNA (rDNA), a molecular vector is needed to transfer recombinant DNA into a host cell. Vectors act as a vehicle for carrying foreign DNA (gene of interest) into a host cell for multiplication and its expression for the production of specific protein e.g., insulin. One common type of vector is a plasmid. Plasmids are small circular DNA molecules naturally found in bacterial cells.

Definition

Vectors: Vectors act as a vehicle for carrying foreign DNA (gene of interest) into a host cell for multiplication and its expression for the production of specific protein e.g., insulin.

Plasmids: Plasmids are small circular DNA molecules naturally found in bacterial cells. They are separate from the chromosomal DNA so called extra chromosomal and can replicate independently.

Main Idea

• Vectors are needed to transfer recombinant DNA into a host cell.
• Vectors act as a vehicle for carrying foreign DNA.
• The purpose is multiplication and expression of the gene of interest.
• One common type of vector is a plasmid.
• Plasmids are small circular DNA molecules.
• They are naturally found in bacterial cells.
• Examples of vectors other than plasmids are:
• Lambda phage DNA
• Cosmid
• Yeast artificial chromosomes (YACs)
• Bacterial artificial chromosomes (BACs)
• Plasmids were discovered by investigators studying the sex life of the intestinal bacterium Escherichia coli.
• Plasmids are primarily found in bacterial cells.
• They can also be found in some eukaryotes e.g., yeast and some higher plants.
• They are separate from chromosomal DNA; called extra chromosomal.
• They can replicate independently.
• Plasmids carry genes for antibiotic resistance and fertility etc.
• One of the common plasmids discovered earlier pSC-101 has antibiotic resistance gene for tetracycline.
• pBR-322 has antibiotic resistance genes for both tetracycline and ampicillin.

Core Concept

• Vectors = vehicle for carrying foreign DNA
• Purpose = multiplication and expression of gene of interest
• Plasmids = small circular DNA molecules
• Plasmids = naturally found in bacterial cells
• Plasmids = extra chromosomal; replicate independently
• Plasmids = carry genes for antibiotic resistance and fertility
• Other vectors = lambda phage DNA, cosmid, YACs, BACs
• **pSC-101** = tetracycline resistance
• pBR-322 = tetracycline and ampicillin resistance

Detailed Explanation

Molecular Carriers or Vectors

• For the synthesis of recombinant DNA (rDNA), a molecular vector is needed to transfer recombinant DNA into a host cell.
• Vectors act as a vehicle for carrying foreign DNA (gene of interest) into a host cell for multiplication and its expression for the production of specific protein e.g., insulin.
• One common type of vector is a plasmid.
• Plasmids are small circular DNA molecules naturally found in bacterial cells.
• Example of vectors other than plasmids are: lambda phage DNA, Cosmid (combination of plasmid and phage DNA), Yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs) etc.

Plasmids

• Plasmids were discovered by investigators studying the sex life of the intestinal bacterium Escherichia coli.
• Plasmids are primarily found in bacterial cells but can also be found in some eukaryotes e.g. Yeast and some higher plants.
• They are separate from the chromosomal DNA so called extra chromosomal and can replicate independently.
• Plasmids carry genes for antibiotic resistance and fertility etc.
• One of the common plasmids discovered earlier pSC-101 has antibiotic resistance gene for tetracycline, whereas pBR-322 has antibiotic resistance genes for both tetracycline and ampicillin.

Characteristics of Plasmids as Vectors

Plasmids are commonly used as vectors in genetic engineering due to many characteristics essentially needed as vector:

• Plasmids contain an origin of replication (Ori) site which is required for self-regulated replication of the plasmid in the host cell.
• Plasmids are ideal vector for producing multiple copies of a gene inserted in it.

• Markers help in the selecting of cells (bacteria) that have successfully taken up the recombinant plasmids.
• The markers used for selection are the antibiotic resistance genes.
• Plasmids usually contain antibiotic resistance genes (e.g. genes for ampicillin or tetracycline resistance).
• When these genes are present in a bacteria it will be resistant to the specific antibiotic.

• A vector should have a restriction site for the commonly used endonucleases.
• The plasmid which has at least one restriction site is required for the insertion of foreign DNA fragment.
• Presence of unique restriction sites provide flexibility in the choice of endonucleases for the easy insertion of foreign DNA.

iv. Promoter regions for gene expression: - Plasmids may contain promoter sequences that enable the transcription of inserted genes in the host cell, thus facilitating the expression of the gene of interest.

v. Small Size: - Plasmids are much smaller than chromosomal DNA, so they are easily purified as intact molecular vector and easily inserted into host cells.

vi. High Copy Number: - Some plasmids are found in high copy numbers inside the host cell, resulting in large amounts of the cloned DNA.

Summary Table of Vectors

Summary of Plasmid Characteristics

Summary of Common Plasmids

Intermediate Level

Molecular Carriers or Vectors

Characteristics of Plasmids as Vectors

Common Plasmids

Formula / Mathematical Form

Not applicable to this topic.

Solved Example

Not applicable to this topic (descriptive topic).

Important Points

• 1. Vectors are needed to transfer recombinant DNA into a host cell.
• 2. Vectors act as a vehicle for carrying foreign DNA.
• 3. Purpose is multiplication and expression of gene of interest.
• 4. Plasmids are small circular DNA molecules.
• 5. Plasmids are naturally found in bacterial cells.
• 6. Plasmids are separate from chromosomal DNA.
• 7. Plasmids can replicate independently.
• 8. Plasmids carry genes for antibiotic resistance and fertility.
• 9. Other vectors include lambda phage DNA, cosmid, YACs, BACs.
• 10. Plasmids were discovered in E. coli.
• 11. Plasmids are found in bacteria and some eukaryotes.
• 12. pSC-101 has tetracycline resistance.
• 13. pBR-322 has tetracycline and ampicillin resistance.
• 14. Plasmids have origin of replication.
• 15. Plasmids have antibiotic resistance genes.
• 16. Plasmids have restriction sites.
• 17. Plasmids have promoter regions.
• 18. Plasmids are small in size.
• 19. Plasmids can have high copy number.
• 20. Plasmids are commonly used as vectors.

Remember

• - Vectors = vehicle for carrying foreign DNA
• - Purpose = multiplication and expression of gene of interest
• - Plasmids = small circular DNA molecules
• - Plasmids = naturally found in bacterial cells
• - Plasmids = extra chromosomal; replicate independently
• - Plasmids = carry genes for antibiotic resistance and fertility
• - Other vectors = lambda phage DNA, cosmid, YACs, BACs
• - **pSC-101** = tetracycline resistance
• - pBR-322 = tetracycline and ampicillin resistance

Common Mistakes

• 1. Confusing vectors and plasmids — Plasmids are a type of vector.
• 2. Forgetting the purpose of vectors — Multiplication and expression of gene of interest.
• 3. Mixing up the types of vectors — Plasmids, lambda phage DNA, cosmid, YACs, BACs.
• 4. Forgetting the characteristics of plasmids — Origin of replication, antibiotic resistance genes, restriction sites, promoter regions, small size, high copy number.
• 5. Mixing up the plasmids — pSC-101 (tetracycline); pBR-322 (tetracycline and ampicillin).
• 6. Believing plasmids are only found in bacteria — They are also found in some eukaryotes.
• 7. Mixing up the functions — Carrier of foreign DNA; multiplication; expression.
• 8. Forgetting the role of origin of replication — Self-regulated replication.
• 9. Mixing up the antibiotic resistance genes — Selectable markers.
• 10. Thinking plasmids are not important — They are commonly used as vectors.

Quick Revision

For the synthesis of recombinant DNA (rDNA), a molecular vector is needed to transfer recombinant DNA into a host cell. Vectors act as a vehicle for carrying foreign DNA (gene of interest) into a host cell for multiplication and its expression for the production of specific protein e.g., insulin. One common type of vector is a plasmid. Plasmids are small circular DNA molecules naturally found in bacterial cells. Example of vectors other than plasmids are: lambda phage DNA, Cosmid (combination of plasmid and phage DNA), Yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs) etc. Plasmids were discovered by investigators studying the sex life of the intestinal bacterium Escherichia coli. Plasmids are primarily found in bacterial cells but can also be found in some eukaryotes e.g. Yeast and some higher plants. They are separate from the chromosomal DNA so called extra chromosomal and can replicate independently. Plasmids carry genes for antibiotic resistance and fertility etc. One of the common plasmids discovered earlier pSC-101 has antibiotic resistance gene for tetracycline, whereas pBR-322 has antibiotic resistance genes for both tetracycline and ampicillin. Plasmids are commonly used as vectors in genetic engineering due to many characteristics essentially needed as vector: i. Origin of Replication site: Plasmids contain an origin of replication (Ori) site which is required for self-regulated replication of the plasmid in the host cell. Plasmids are ideal vector for producing multiple copies of a gene inserted in it. ii. Antibiotics resistant genes as selectable Marker: Markers help in the selecting of cells (bacteria) that have successfully taken up the recombinant plasmids. The markers used for selection are the antibiotic resistance genes. Plasmids usually contain antibiotic resistance genes (e.g. genes for ampicillin or tetracycline resistance). When these genes are present in a bacteria it will be resistant to the specific antibiotic. iii. Restriction sites of different enzymes: A vector should have a restriction site for the commonly used endonucleases. The plasmid which has at least one restriction site is required for the insertion of foreign DNA fragment. Presence of unique restriction sites provide flexibility in the choice of endonucleases for the easy insertion of foreign DNA. iv. Promoter regions for gene expression: Plasmids may contain promoter sequences that enable the transcription of inserted genes in the host cell, thus facilitating the expression of the gene of interest. v. Small Size: Plasmids are much smaller than chromosomal DNA, so they are easily purified as intact molecular vector and easily inserted into host cells. vi. High Copy Number: Some plasmids are found in high copy numbers inside the host cell, resulting in large amounts of the cloned DNA.

60-Second Revision

• Vectors = vehicle for carrying foreign DNA
• Purpose = multiplication and expression of gene of interest
• Plasmids = small circular DNA molecules
• Plasmids = naturally found in bacterial cells
• Plasmids = extra chromosomal; replicate independently
• Plasmids = carry genes for antibiotic resistance and fertility
• Other vectors = lambda phage DNA, cosmid, YACs, BACs
• pSC-101 = tetracycline resistance
• pBR-322 = tetracycline and ampicillin resistance
• Warning: Plasmids are a type of vector; they are not the only type of vector

NEXT TOPIC

• Chapter:
• 10. Biotechnology

• Topic:
• 10.3.1 pBR322 AS AN EXAMPLE OF PLASMIDS

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