pBR322 Plasmid Vector: Introduction, Mapping, Applications, and Modifications

Introduction

The pBR322 plasmid vector is one of the most widely used and well-characterized plasmids in molecular biology. Developed in 1977 by Francisco Bolivar and Raymond Rodriguez, it has played a crucial role in advancing genetic engineering and recombinant DNA technology. pBR322 is a circular, double-stranded DNA plasmid, 4 361 base pairs (Balbás et al., 1986) that contains important features such as antibiotic resistance genes for ampicillin (AmpR) and tetracycline (TetR), as well as multiple restriction sites ((at least 40, restriction sites (EcoR1, HindIII, Pst1), making it a versatile tool for cloning and gene expression studies. The molecular weight is 2.83 x 10⁶ daltons. 

Due to its simplicity, stability, and ease of manipulation, pBR322 has become a standard vector in molecular cloning experiments. It has paved the way for numerous modifications, allowing for the incorporation of various foreign DNA sequences. Its application extends from gene mapping and sequencing to the production of recombinant proteins. Understanding the construction, features, and functionality of the pBR322 plasmid provides valuable insights into the principles of vector design and the evolution of molecular cloning techniques.

pBR322 Vector Map

pBR322 is 4361 base pairs in length. It has two antibiotic resistance genes – the gene bla encoding the ampicillin resistance (AmpR) protein, and the gene tetA encoding the tetracycline resistance (TetR) protein. It contains the origin of replication of pMB1, and the rop gene, which encodes a restrictor of plasmid copy number. The plasmid has unique restriction sites for at least 40 restriction enzymes including EcoR1, HindIII, and Pst1. Eleven sites lie within the TetR gene. There are two sites for restriction. Enzymes HindIII and ClaI within the promoter of the TetR gene. There are six key restriction sites inside the AmpR gene. The source of these antibiotic resistance genes is from pSC101 for Tetracycline and RSF2124 for Ampicillin.

The Nomenclature of pBR322

The pBR322 was designed and constructed at the University of California, San Francisco in 1977 by Bolivar et al. P: plasmid (indicates the type of vector), BR: “Bolivar” and “Rodriguez” lead researchers who developed it. 322: The plasmid vector model number differencing it from others developed in the same lab, like 327

Construction of pBR322

Three major steps were involved in the construction of pBR322.

1. The AmpR (ampicillin resistance) gene has been incorporated from the natural plasmid RSF 2124 of E. coli.
2. The TetR (tetracycline resistance) gene is derived from pSC101 natural plasmid.
3. The origin of replication (Ori) is derived from pMB1 plasmid
4. It contains a rop gene, which encodes a restrictor of plasmid copy number.
5. The plasmid has more than 40 unique restriction sites for restriction enzymes.
6. Out of these restriction sites, 11 fall within the sequence of the TetR gene, whereas 6 fall within the sequence of the AmpR gene.
7. The total size of the developed plasmid is 4361bp and can carry genetic inserts of size 100bp to 10kb.

Advantages and limitations of pBR322

Advantages

Small size (~ 4.3 kb) facilitates the isolation, purification, and manipulation of pBR322 avoiding problems such as DNA breakdown. it carries two sets of antibiotic-resistance genes. Ampicillin or tetracycline resistance can be used as a selectable marker for easy selection of recombinant plasmids, and each marker gene includes unique restriction sites that can be used in cloning experiments. A moderate number of copies can be generated inside a host cell.

Limitations

1. Chances of loss of vector or mix-population contamination due to high mobility of pBR322 via conjugation between bacteria.
2. Instability in the absence of selection: The pBR322 vector is prone to instability in the absence of selection, which can lead to the loss of the inserted foreign DNA fragment.
3. The pBR322 vector does not have a direct selection scheme for recombinant DNA molecules, which can make it difficult to identify and isolate the desired clones.
4. The pBR322 vector has a limited capacity for large DNA inserts, which can limit its usefulness for cloning large genes or DNA sequences.
5. The pBR322 vector can be incompatible with other plasmids in the host cell, which can lead to the formation of hybrid plasmids and complicate the cloning process

Modification of pBR322

A large number of other plasmids based on pBR322 have since been constructed specifically designed for a wide variety of purposes and advantages. Examples include:

1. pBR327, which was produced by removing a 1089 bp segment from pBR322. This deletion left the ampR and tetR genes intact but changed the replicative and conjugative abilities of the resulting plasmid.
2. pUC series of plasmids.
3. Most expression vectors for extrachromosomal protein expression and shuttle vectors contain the pBR322 origin of replication, and fragments of pBR322 are very popular in the construction of intraspecies shuttle or binary vectors and vectors for targeted integration and excision of DNA from chromosomes.

References

1. https://pubmed.ncbi.nlm.nih.gov/3034735/
2. https://www.sciencedirect.com/science/article/abs/pii/0378111986903070
3. Yanisch-Perron C, Vieira J, Messing J (1985). “Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors”. Gene. 33 (1): 103–19. doi:10.1016/0378-1119(85)90120-9. PMID 2985470
4. pBR322 – Wikipedia
5. pBR322 Vector | NEB
6. Balbas, Paulina, et al. “The Plasmid, PBR322.” Vectors, 1987, pp. 5-41, https://doi.org/10.1016/B978-0-409-90042-2.50007-6. Accessed 29 Sept. 2024.
7. Balbás, P., Soberón, X., Merino, E., Zurita, M., Lomeli, H., Valle, F., Flores, N., & Bolivar, F. (1986). Plasmid vector pBR322 and its special-purpose derivatives — a review. Gene, 50(1–3), 3–40. https://doi.org/10.1016/0378-1119(86)90307-0

*Sequencing data from Watson (confirmed at New England Biolabs, Inc.) has shown pBR322 to be 4,361 base pairs, not 4,363 base pairs as previously reported.