Escherichia coli/Vectors

General information

Stringent vs. relaxed replication

Plasmid replication control is usually controlled by balancing the levels of a positive and a negative regulator of replication. For some plasmids (pMB1/colE1 replicons) the positive regulator is an RNA and in others (e.g. pSC101) it is a protein. Plasmids with a protein positive regulator will not replicate in the abscence of protein production - stringent control (although not the same as the stringent response due to a shortage of loaded tRNAs). Plasmids with an RNA positive regulator will continue to replicate in the abscence of protein production. This is termed relaxed control. High yields of plasmid may be obtained by halting protein production (via chloroamphenicol) when the culture reaches a high density and then continuing incubation for a number of hours. This might be of practical relevance when prepping the 1 and 3 series of Synthetic Biology plasmids.--BC 19:05, 3 Sep 2005 (EDT)

Nomenclature

1. Novick RP, Clowes RC, Cohen SN, Curtiss R 3rd, Datta N, and Falkow S. Uniform nomenclature for bacterial plasmids: a proposal. Bacteriol Rev. 1976 Mar;40(1):168-89. DOI:10.1128/br.40.1.168-189.1976 |

Common vectors

Plasmid	Replicon	Copy number
pBR322[2] and its derivatives	pMB1	15-20
pUC vectors	pMB1	500-700
pACYC and its derivatives	p15A[3]	10-12
pSC101 and its derivatives	pSC101[4]	5
ColE1	ColE1[5, 6]	15-20

Replicon compatibility

Incompatibility group	Negative control element	Comment
colE1[5, 6], pMB1	RNAI	controls processing of pre-RNAII into primer. pUC is derived from pBR322 (a single mutation in the pBR322 Primer RNA and deletion of the rop gene) which is derived from a pMB1 replicon, and cannot co-reside with the colE1 incompatibility group.
IncFII, pT181	RNA	controls synthesis of RepA protein
P1, F, R6K, pSC101[4], p15A[3]	iterons	sequesters RepA protein

Replication origins in different incompatibility groups are compatible. Replication origins in the same incompatibility group are not.

Some sets of vectors with compatible origins are available as a part of the Novagen Duet system. (from TK)

Individual vector links

Note: searching for cloning vector <insert vector name> when looking for vector sequences in NCBI Entrez Nucleotide search. It helps to cut down on the number of hits.

• pUC19
• pBeloBAC11
• pBACe3.6
• F plasmid
• pSCANS genbank vector info cookbook
• pSC101 replication origin
• pBR322

References

2. Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa JH, and Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95-113.

   vector pBR322

3. Chang AC and Cohen SN. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141-56. DOI:10.1128/jb.134.3.1141-1156.1978 |

   p15A origin

4. Cohen SN and Chang AC. Revised interpretation of the origin of the pSC101 plasmid. J Bacteriol. 1977 Nov;132(2):734-7. DOI:10.1128/jb.132.2.734-737.1977 |

   pSC101 origin

5. Bazaral M and Helinski DR. Circular DNA forms of colicinogenic factors E1, E2 and E3 from Escherichia coli. J Mol Biol. 1968 Sep 14;36(2):185-94. DOI:10.1016/0022-2836(68)90374-4 |

   original discovery of ColE1 plasmid

6. Hershfield V, Boyer HW, Yanofsky C, Lovett MA, and Helinski DR. Plasmid ColEl as a molecular vehicle for cloning and amplification of DNA. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3455-9. DOI:10.1073/pnas.71.9.3455 |

   use of ColEI as a vector

7. ISBN:0-87969-577-3
8. Anderson ES. The ecology of transferable drug resistance in the enterobacteria. Annu Rev Microbiol. 1968;22:131-80. DOI:10.1146/annurev.mi.22.100168.001023 |

   Discusses transferable drug resistance in Escherichia coli (i.e. plasmids with resistance markers)

9. Cohen SN and Chang AC. Recircularization and autonomous replication of a sheared R-factor DNA segment in Escherichia coli transformants. Proc Natl Acad Sci U S A. 1973 May;70(5):1293-7. DOI:10.1073/pnas.70.5.1293 |

   Discusses generation of plasmids by shearing DNA, their transformation and replication.

10. Cohen SN, Chang AC, Boyer HW, and Helling RB. Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci U S A. 1973 Nov;70(11):3240-4. DOI:10.1073/pnas.70.11.3240 |

    Discusses use of vector to propagate other DNA fragments

External links

• Novagen pET vector table
  • table with links to properties/sequences of pET vectors
  • note: slow to load

See also

• Bacterial artificial chromosomes
• F plasmids
• Synthetic Biology vectors
• Vectors