pBR322 Vector -Definition, Structure, Sites, Applications

pBR322 is a commonly used cloning vector in E. coli and has tremendous applications in cloning.

pBR322 full form

p = plasmid

BR = Bolivar, and Rodriguez 

322 = numerical designation

  • It was constructed in 1977 in the lab of Herbert Boyer at The University of California in San Francisco. 
  • It is a synthetic plasmid and was the first artificial plasmid to be constructed and used as a cloning vector
  • pBR322 is one of the most studied plasmids.
  • It is 4362 base pairs long. 
  • It is completely sequenced, which means the whole sequence of pBR322 is known and studied. 
  • Its molecular weight is 2.83 x 106 Daltons.
pBR322 Vector
pBR322 Vector.

Structure of pBR322

  1. Origin of replication 
  2. Restriction enzyme sites
  3. Selectable marker sites

Origin of replication

  • The origin of replication in plasmid pBR322 is known as pMB1.
  • The copy number of this plasmid is 15-20. 

Restriction enzyme sites

  • Around 40 different restriction sites are present on the genome of pBR322.
  • Almost 11 different restriction sites are present in the region of tetracycline resistance region.
  • In the ampicillin resistance region 9 restriction sites of different enzymes are present. 
  • Some of the known restriction enzyme sites are – BamHI, HindIII, EcoRI, SaII, and many more.
Figure: pBR322 map. Image Source: NEB.

Selectable marker sites

Two selectable marker sites or antibiotic resistance genes are present on the genome of this plasmid.

  • Ampicillin resistance site – the ampicillin gene codes for β-lactamase, which can be used for screening microorganisms when a foreign DNA is being inserted in the plasmid.
  • Tetracycline resistance site – this gene degrades the antibiotic tetracycline and can be used for screening microorganisms. 
  • These antibiotic resistance genes are useful in screening organisms after cloning. 

Screening of recombinants containing pBR322

  • Consider that the restriction enzyme BamHI is used to cut the plasmid at that specific position. 
  • BamHI lies in the tetracycline resistance region of plasmid pBR322. 
  • Now, the recombinant molecule of pBR322, which includes the newly inserted DNA molecule, will be sensitive to Tetracycline. 
  • But this recombinant molecule is resistant to Ampicillin, as the ampicillin resistance gene is fully functional. 
  • Hence, when the recombinant cells are plated on the medium containing ampicillin and incubated. 
  • The colonies that appear on plates containing a medium with ampicillin are transformed colonies containing cells with the newly inserted DNA molecule. 
  • The replica plate technique is used to confirm the transformed colonies. 
  • To confirm that the colonies on the ampicillin-containing media are transformed colonies, they are plated on the plates containing a medium inclusive of tetracycline. 
  • The colonies that do not grow on the medium inclusive of tetracycline are transformed colonies as the transformed colonies are sensitive to tetracycline. 

Advantages of pBR322

  • Due to its manageable size, plasmid pBR322 is widely used as a cloning vector.
  • The presence of two antibiotic resistance genes eases the selection process of recombinants. 
  • Multiple restriction enzyme sites make the plasmid compatible in many ways. 
  • It has a high copy number which is highly favorable in genetic engineering.

Disadvantages

  • Detection of recombinant cells having the newly inserted DNA molecule is time-consuming. 

References

  1. 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. Gene50(1-3), 3–40. https://doi.org/10.1016/0378-1119(86)90307-0 
  2. PRINCIPLES OF GENE MANUPULATION AND GENOMICS BY PRIMROSE AND TWYMAN 
  3. http://vidyamandira.ac.in/pdfs/e_learning/ds_microbio/PLASMID%20CLONING%20VECTORS%20%20MCBA%20P7%20T.pdf 

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