Vectors
This page contains various information relating to vectors used in OpenWetWare labs.
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)
Online Vector Databases
You can often find vector information at NCBI, either directly or in their list of vectors screened for contamination of new sequence at Vecscreen.
VectorDB contains information about many common vectors, including yeast vectors.
The EMBL Hamburg outstation maintains a large database of vectors.
I.M.A.G.E. Consortium Vectors contains information about plasmids commonly used in EST collections such as those sold by OpenBioSystems and Invitrogen.
For eukaryotic vectors (Fish, Xenopus) see Minnesota.
The Forsburg Lab maintains a list of Fisson Yeast vectors.
Promega maintains a list of their vectors.
NEB maintains a list of common vectors.
Epicentre also maintains its own list.
Lucigen provides transcription-free vectors for cloning AT-rich and other difficult DNAs.
Addgene's Vector DB contains most of the information from Stanford's VectorDB, plus more vector information they have curated from commercial websites and added through our plasmid curation efforts. (However, it seems to be rather sparse when it comes to Escherichia coli vectors.) Note that Addgene is a a non-profit plasmid repository where scientists can archive and share their plasmids. They encourage and invite labs to deposit plasmids at Addgene. They help you with data submission and all tech transfer issues. Plasmids can be requested from Addgene for a fee to cover expenses.
Bioinfoman also has a 5000+ long list of vector sequences.
PlasmID is an online database and DNA repository maintained for plasmids, including empty vectors useful for a variety of techniques, and open reading frame (ORF) clones relevant to human, yeast, and several bacterial pathogens. All clones can be requested individually, and ORF clones can also be viewed and requested as grouped collections. PlasmID is maintained by the DF/HCC DNA Resource Core at the Harvard Institute of Proteomics, Harvard Medical School.
Annotation
PlasMapper: "automatically generates and annotates plasmid maps using only the plasmid DNA sequence as input. Plasmid sequences up to 20,000 bp may be annotated and displayed. Plasmid figures may be rendered in PNG, JPG, SVG or SVGZ format." It can also output GenBank format. Reference: Xiaoli Dong, Paul Stothard, Ian J. Forsythe, and David S. Wishart "PlasMapper: a web server for drawing and auto-annotating plasmid maps" Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W660-4.
- One drawback to this tool is that although it finds ORFs, it doesn't necessarily identify them. -- RS
- A very nice aspect of PlasMapper is that it identifies features of a sequence and puts them into GenBank format - very handy to prepare sequences for submission to GenBank. --User:Rgodiska
Also check out APe, A Plasmid Editor.
- Has some built-in features for annotation (a few genes, many restriction enzymes). Allows you to make custom "feature libraries" for annotating sequences
- Can edit features found, unlike Plasmapper --mel 18:50, 12 June 2007 (EDT)
Nomenclature
- 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 |
Escherichia coli
Information
Common vectors
| Plasmid | Replicon | Copy number |
| pBR322 and its derivatives | pMB1 | 15-20 |
| pUC vectors | pMB1 | 500-700 |
| pACYC and its derivatives | p15A | 10-12 |
| pSC101 and its derivatives | pSC101 | 5 |
| ColE1 | ColE1 | 15-20 |
Replicon Compatibility
| Incompatibility group | Negative control element | Comment |
| colE1, 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, p15A | 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)
Reference
- ISBN:0-87969-577-3
- 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)
- 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 sharing DNA, their transformation and replication.
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.
Other references
- Novagen pET vector table
- table with links to properties/sequences of pET vectors
- note: slow to load
