PBR322

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A schematic representation of the pBR322 vector with restriction sites indicated in blue. PBR322.svg
A schematic representation of the pBR322 vector with restriction sites indicated in blue.

pBR322 is a plasmid and was one of the first widely used E. coli cloning vectors. Created in 1977 in the laboratory of Herbert Boyer at the University of California, San Francisco, it was named after Francisco Bolivar Zapata, the postdoctoral researcher and Raymond L. Rodriguez. The p stands for "plasmid," and BR for "Bolivar" and "Rodriguez."

Contents

pBR322 is 4361 base pairs in length [1] and 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 more than forty restriction enzymes. Eleven of these forty 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 are from pSC101 for Tetracycline and RSF2124 for Ampicillin. [2]

The circular sequence is numbered such that 0 is the middle of the unique EcoRI site and the count increases through the TetR gene. If we have to remove ampicillin for instance, we must use restriction endonuclease or molecular scissors against PstI and then pBR322 will become anti-resistant to ampicillin. The same process of Insertional Inactivation can be applied to Tetracycline. The AmpR gene is penicillin beta-lactamase. Promoters P1 and P3 are for the beta-lactamase gene. P3 is the natural promoter, and P1 is artificially created by the ligation of two different DNA fragments to create pBR322. P2 is in the same region as P1, but it is on the opposite strand and initiates transcription in the direction of the tetracycline resistance gene. [3]

Background

Early cloning experiments may be conducted using natural plasmids such the ColE1 and pSC101. Each of these plasmids may have its advantages and disadvantages. For example, the ColE1 plasmid and its derivatives have the advantage of higher copy number and allow for chloramphenicol amplification of plasmid to produce a high yield of plasmid, however screening for immunity to colicin E1 is not technically simple. [4] The plasmid pSC101, a natural plasmid from Salmonella panama, [5] confers tetracycline resistance which allows for simpler screening process with antibiotic selection, but it is a low copy number plasmid which does not give a high yield of plasmid. Another plasmid, RSF 2124, which is a derivative of ColE1, confers ampicillin resistance but is larger.

Many other plasmids were artificially constructed to create one that would be ideal for cloning purpose, and pBR322 was found to be most versatile by many and was therefore the one most popularly used. [4] It has two antibiotic resistance genes, as selectable markers, and a number of convenient unique restriction sites that made it suitable as a cloning vector. The plasmid was constructed with genetic material from 3 main sources – the tetracycline resistance gene of pSC101, the ampicillin resistance gene of RSF 2124, and the replication elements of pMB1, a close relative of the ColE1 plasmid. [6] [7]

A large number of other plasmids based on pBR322 have since been constructed specifically designed for a wide variety of purposes. [8] [9] Examples include the pUC series of plasmids. [10] 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 chromosome. [11]

DNA sequence

The sequence in pBR322 is [3]

pBR322 
       1 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa       61 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg      121 caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt      181 gcgggatatc gtccattccg acagcatcgc cagtcactat ggcgtgctgc tagcgctata      241 tgcgttgatg caatttctat gcgcacccgt tctcggagca ctgtccgacc gctttggccg      301 ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga tcatggcgac      361 cacacccgtc ctgtggatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac      421 aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca      481 cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg      541 actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct      601 caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgaccgat      661 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt      721 cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct      781 ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct      841 tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa      901 acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccgacgcgc tgggctacgt      961 cttgctggcg ttcgcgacgc gaggctggat ggccttcccc attatgattc ttctcgcttc     1021 cggcggcatc gggatgcccg cgttgcaggc catgctgtcc aggcaggtag atgacgacca     1081 tcagggacag cttcaaggat cgctcgcggc tcttaccagc ctaacttcga tcactggacc     1141 gctgatcgtc acggcgattt atgccgcctc ggcgagcaca tggaacgggt tggcatggat     1201 tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg     1261 ggccacctcg acctgaatgg aagccggcgg cacctcgcta acggattcac cactccaaga     1321 attggagcca atcaattctt gcggagaact gtgaatgcgc aaaccaaccc ttggcagaac     1381 atatccatcg cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg     1441 tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg     1501 ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc     1561 tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg     1621 taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca     1681 ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga     1741 ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa     1801 cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt     1861 ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc     1921 aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt     1981 ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac     2041 gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac     2101 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc     2161 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc     2221 cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg     2281 tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc     2341 gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc     2401 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata     2461 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg     2521 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct     2581 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa     2641 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc     2701 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt     2761 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg     2821 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg     2881 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct     2941 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc     3001 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg     3061 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc     3121 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt     3181 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa     3241 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat     3301 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct     3361 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg     3421 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag     3481 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta     3541 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg     3601 ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg     3661 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct     3721 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta     3781 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg     3841 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc     3901 cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg     3961 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga     4021 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg     4081 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat     4141 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc     4201 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca     4261 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct     4321 ataaaaatag gcgtatcacg aggccctttc gtcttcaaga a

See also

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References

  1. ↑ Watson, N. (1988). "A new revision of the sequence of plasmid pBR322". Gene. 70 (2): 399–403. doi:10.1016/0378-1119(88)90212-0. PMID   3063608.
  2. ↑ 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. doi:10.1016/0378-1119(86)90307-0. PMID   3034735.
  3. 1 2 "pBR322 Nucleotide Sequences, NCBI Sequence Viewer v2.0". 30 September 2008.
  4. 1 2 R.W. Old & S.B. Primrose. Principles of Gene Manipulation (5th ed.). pp. 53–61.
  5. ↑ Manen D, Caro L (February 1991). "The replication of plasmid pSC101". Mol. Microbiol. 5 (2): 233–7. doi:10.1111/j.1365-2958.1991.tb02103.x. PMID   2041467. S2CID   37314534.
  6. ↑ Bolivar F, Rodriguez RL, Betlach MC, Boyer HW (1977). "Construction and characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the plasmid pMB9". Gene. 2 (2): 75–93. doi:10.1016/0378-1119(77)90074-9. PMID   344136.
  7. ↑ Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa JH, Falkow S (1977). "Construction and characterization of new cloning vehicles. II. A multipurpose cloning system". Gene. 2 (2): 95–113. doi:10.1016/0378-1119(77)90000-2. PMID   344137.
  8. ↑ S.B. Primrose & R.M Twyman (17 January 2006). Principles of Gene Manipulation and Genomics (PDF) (7th ed.). Wiley-Blackwell. pp. 64–65. ISBN   978-1405135443.
  9. ↑ 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. doi:10.1016/0378-1119(86)90307-0. PMID   3034735.
  10. ↑ 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.
  11. ↑ Paulina Balbás; Argelia Lorence, eds. (April 2004). Recombinant Gene Expression: Reviews and Protocols (2nd ed.). Humana Press Inc. pp.  77–85. ISBN   978-1592597741.
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