Escherichia coli/Vectors
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===Stringent vs. relaxed replication=== | ===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 | + | 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 absence 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 absence 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.--[[User:Bcanton|BC]] 19:05, 3 Sep 2005 (EDT) |
===Nomenclature=== | ===Nomenclature=== | ||
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*[[Vectors]] | *[[Vectors]] | ||
| - | [[Category:Escherichia coli]] [[Category:DNA]] | + | [[Category:Escherichia coli]] [[Category:DNA]] [[Category:Vectors]] |
Current revision
Contents |
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 absence 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 absence 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
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
- pACYC184
References
- Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa JH, and Falkow S. . pmid:344137.
vector pBR322 - Chang AC and Cohen SN. . pmid:149110.
p15A origin - Cohen SN and Chang AC. . pmid:334752.
pSC101 origin - Bazaral M and Helinski DR. . pmid:4939624.
original discovery of ColE1 plasmid - Hershfield V, Boyer HW, Yanofsky C, Lovett MA, and Helinski DR. . pmid:4610576.
use of ColEI as a vector - Joseph Sambrook, David W. Russell. Molecular cloning. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory, 2001. isbn:0-87969-577-3.
- Anderson ES. . pmid:4879515.
Discusses transferable drug resistance in Escherichia coli (i.e. plasmids with resistance markers) - Cohen SN and Chang AC. . pmid:4576014.
Discusses generation of plasmids by shearing DNA, their transformation and replication. - Cohen SN, Chang AC, Boyer HW, and Helling RB. . pmid:4594039.
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
- Plasmids at EcoliWiki.org
- EcoliWiki.org has entered many plasmids that are available for annotation. Included are plasmid maps, features and sequences.
