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."
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
editEarly 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
editThis section is missing information about annotation: what basepairs are what. (March 2019) |
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
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See also
editReferences
edit- ^ 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.
- ^ 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.
- ^ a b "pBR322 Nucleotide Sequences, NCBI Sequence Viewer v2.0". 30 September 2008.
- ^ a b R.W. Old & S.B. Primrose. Principles of Gene Manipulation (5th ed.). pp. 53–61.
- ^ 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.
- ^ 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.
- ^ 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.
- ^ 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.
- ^ 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.
- ^ 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.
- ^ Paulina Balbás; Argelia Lorence, eds. (April 2004). Recombinant Gene Expression: Reviews and Protocols (2nd ed.). Humana Press Inc. pp. 77–85. ISBN 978-1592597741.