10.3.1 pBR322 AS AN EXAMPLE OF PLASMIDS

pBR322 is the first and most common, purpose-built plasmid vector, artificially developed in the laboratory by Bolivar and Rodriguez in 1977. pBR322 is modified from natural bacterial plasmid. This vector has the origin of replication (ori), restriction site and also antibiotic-resistant genes.

Definition

pBR322: It is the first and most common, purpose built plasmid vector, artificially developed in the laboratory by Bolivar and Rodriguez in 1977. pBR322 is modified from natural bacterial plasmid.

Main Idea

• pBR322 is the first and most common purpose-built plasmid vector.
• It was artificially developed in the laboratory by Bolivar and Rodriguez in 1977.
• It is modified from natural bacterial plasmid.
• It has:
• Origin of replication (ori)
• Restriction sites
• Antibiotic-resistant genes
• Key features of pBR322:

Core Concept

• pBR322 = first purpose-built plasmid vector
• Developed by = Bolivar and Rodriguez
• **Year** = 1977
• Modified from = natural bacterial plasmid
• Features = origin of replication; restriction sites; antibiotic-resistant genes
• Origin of replication = fragment of pMB1
• **Size** = 4,361 bps
• Copy number = ~15 copies per cell
• Cloning sites = unique restriction sites

Detailed Explanation

pBR322 as an Example of Plasmids

• It is the first and most common, purpose built plasmid vector, artificially developed in the laboratory by Bolivar and Rodriguez in 1977.
• pBR322 is modified from natural bacterial plasmid.
• This vector has the origin of replication (ori), restriction site and also antibiotic-resistant genes.
• Here are the key features of the pBR322 plasmid:

i. Origin of Replication

- It carries a fragment of the plasmid pMB1 that acts as an origin for DNA replication to ensure the multiplication of the vector.

ii. Size

• It is relatively small plasmid having 4,361 bps.
• Small size is important, as transformation efficiency is inversely proportional to size of plasmid.
• Transformation efficiency become very low if plasmid size is above 10 kbp, thus DNA segment up to 6 kbp can possibly be inserted in pBR322 plasmid to form it recombinant.

iii. Copy Number

- It has relatively high copy number (~15 copies per cell).

iv. Cloning Sites

• It carries a number of unique restriction sites.
• Some of these are located in one of the antibiotic resistance genes (e.g., sites for PstI, PvuI, and SacI are found in Ampicillin and BamHI and HindIII in Tetracycline).

Science Titbits

Types of Plasmids as Vectors:

• These are used for the cloning of DNA fragments.
• They contain essential elements like Ori, selectable markers, and cloning sites.

• These are designed for the expression of a gene in a host organism.
• They include promoters, ribosome binding sites, and termination sequences.

• Plasmids that can replicate in multiple host species, such as in both prokaryotic and eukaryotic cells, are known as shuttle vectors.
• These are useful for gene transfer between different biological systems.

Summary Table of pBR322 Features

Summary of Plasmid Types

Intermediate Level

pBR322 Features

Types of Plasmids as Vectors

Key Points of pBR322

Formula / Mathematical Form

Not applicable to this topic.

Solved Example

Not applicable to this topic (descriptive topic).

Important Points

• 1. pBR322 is the first purpose-built plasmid vector.
• 2. It was developed by Bolivar and Rodriguez in 1977.
• 3. It is modified from natural bacterial plasmid.
• 4. It has origin of replication.
• 5. It has restriction sites.
• 6. It has antibiotic-resistant genes.
• 7. Origin of replication is from pMB1.
• 8. Size is 4,361 bps.
• 9. Copy number is ~15 copies per cell.
• 10. It carries unique restriction sites.
• 11. PstI, PvuI, SacI are in ampicillin resistance gene.
• 12. BamHI, HindIII are in tetracycline resistance gene.
• 13. Transformation efficiency is inversely proportional to size.
• 14. Transformation efficiency is low if plasmid size is above 10 kbp.
• 15. DNA segment up to 6 kbp can be inserted.
• 16. Cloning vectors are for cloning DNA fragments.
• 17. Expression vectors are for gene expression.
• 18. Shuttle vectors replicate in multiple hosts.
• 19. pBR322 is widely used in genetic engineering.
• 20. pBR322 is a model plasmid vector.

Remember

• - pBR322 = first purpose-built plasmid vector
• - Developed by = Bolivar and Rodriguez
• - **Year** = 1977
• - Modified from = natural bacterial plasmid
• - Features = origin of replication; restriction sites; antibiotic-resistant genes
• - Origin of replication = fragment of pMB1
• - **Size** = 4,361 bps
• - Copy number = ~15 copies per cell
• - Cloning sites = unique restriction sites
• - Ampicillin resistance = PstI, PvuI, SacI sites
• - Tetracycline resistance = BamHI, HindIII sites

Common Mistakes

• 1. Confusing pBR322 with other plasmids — pBR322 is a specific plasmid vector.
• 2. Forgetting the developers — Bolivar and Rodriguez.
• 3. Mixing up the year — 1977.
• 4. Forgetting the origin of replication — from pMB1.
• 5. Mixing up the size — 4,361 bps.
• 6. Forgetting the copy number — ~15 copies per cell.
• 7. Mixing up the restriction sites — PstI, PvuI, SacI in ampicillin; BamHI, HindIII in tetracycline.
• 8. Believing transformation efficiency is not affected by size — It is inversely proportional to size.
• 9. Mixing up the types of plasmids — Cloning, expression, shuttle.
• 10. Thinking pBR322 is not important — It is a model plasmid vector.

Quick Revision

It is the first and most common, purpose built plasmid vector, artificially developed in the laboratory by Bolivar and Rodriguez in 1977. pBR322 is modified from natural bacterial plasmid. This vector has the origin of replication (ori), restriction site and also antibiotic-resistant genes. Here are the key features of the pBR322 plasmid: i. Origin of replication: It carries a fragment of the plasmid pMB1 that acts as an origin for DNA replication to ensure the multiplication of the vector. ii. Size: It is relatively small plasmid having 4,361 bps. Small size is important, as transformation efficiency is inversely proportional to size of plasmid. Transformation efficiency become very low if plasmid size is above 10 kbp, thus DNA segment up to 6 kbp can possibly be inserted in pBR322 plasmid to form it recombinant. iii. Copy number: It has relatively high copy number (~15 copies per cell). iv. Cloning sites: It carries a number of unique restriction sites. Some of these are located in one of the antibiotic resistance genes (e.g., sites for PstI, PvuI, and SacI are found in Ampicillin and BamHI and HindII in Tetracycline). Types of Plasmids as Vectors: 1. Cloning Vectors: These are used for the cloning of DNA fragments. They contain essential elements like Ori, selectable markers, and cloning sites. 2. Expression Vectors: These are designed for the expression of a gene in a host organism. They include promoters, ribosome binding sites, and termination sequences. 3. Shuttle Vectors: Plasmids that can replicate in multiple host species, such as in both prokaryotic and eukaryotic cells, are known as shuttle vectors. These are useful for gene transfer between different biological systems.

60-Second Revision

• pBR322 = first purpose-built plasmid vector
• Developed by = Bolivar and Rodriguez
• Year = 1977
• Modified from = natural bacterial plasmid
• Features = origin of replication; restriction sites; antibiotic-resistant genes
• Origin of replication = fragment of pMB1
• Size = 4,361 bps
• Copy number = ~15 copies per cell
• Cloning sites = unique restriction sites
• Ampicillin resistance = PstI, PvuI, SacI sites
• Tetracycline resistance = BamHI, HindIII sites
• Warning: Transformation efficiency is inversely proportional to plasmid size

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• Chapter:
• 10. Biotechnology

• Topic:
• 10.3.2 MECHANISM OF FORMATION OF RECOMBINANT PLASMID IN RECOMBINANT DNA TECHNOLOGY

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