Origin of replication - more, better ? (Jan/17/2007 )

hi all,

Why most of the vector contain 2 Origin of replication (ori) ?? why not 1 or 3 ??

can I just delete one of it, say, f1 ori from pRsetA and so that I can reduce the copy number of the the vector ?

(indeed, I just want a small vector like pRsetA, about 3k in size but with low copy number.........
is there any other choice other than constructing on my own ? )

thx

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hi,

the presence of multiple ori's in a plasmid is because they all serve a different purpose. The frequently used ColE1 is used to replicate your plasmid in bacteria, the 2µ ori is required for replication in yeast and the f1 ori is the origin of replication used by phage f1 (this ori is sometimes used to produce ssDNA). I've never needed the f1 ori, so I believe that it's possible to remove it from your plasmid. However, I don't think this will influence the copy number of your plasmid if it still contains a ColE1 ori. Instead, if you want a vector with low copy number, you should choose a different ori for instance the ori found in pBR322 or pACYC and derivatives (pMB1* or p15A ori; low copy; 10-20/cell) or pSC101 and derivatives (very low copy ~5/cell).

I hope this helps.

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Thank you very much, dpo, it help a lot

and I have to construct a vector on my own.....but the problem is I don't have those vector......

how about pLys ? this should be a low copy number vector ?



I modified some vector previously, and I have to PCR the whole vector (6k in size) with using BioRad iProof DNA polymerase and do further cloning procedure.
It work, but is there any other suggestion ? as the it is not easily to PCR and I just afraid some mutation occur.... (maybe pfu ultra from stratagene is better?)

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The pBR322 origin is a colE1 origin. The lower copy number is due to the presence of genes which control copy number, which have knocked out in plasmids such as pUC19. What properties do you want in your plasmid. We may have one or more which would be useful to you. Check this page: http://parts.mit.edu/r/parts/partsdb/pgrou...?pgroup=Plasmid

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thank you very much phage434, you construct lot of plasmid....

I think I should clarify something......Is that higher the copy number of the vector, more they can enter into E coli during transformation ? or during transformation, only one vector can enter one cell but duplicate again and again inside the cell ?

Actually, I would like to built a library, and I don't want lot of vectors that contain different mutants present in one E coli or colony.....that would mix up those mutants.......

in addition, if the copy number is too larger, I cannot see the difference between individual mutant....therefore, lower expression as well as lower expression level is more favor to me....

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well each time only one plasmid enter the coli cell.

Then during growing of the cell and divisions, the plasmid is replicated +/- efficientely.
If you have a high copy origin, you may have up to 500copies of the SAME plasmid in one cell.
For low copy numbers, may have 10-30 copies per cell.

pMB1 origin is present at 10 - 30 copies/cell. And i do believe that pUC is at a much higher copy number (>300 copies/cell).

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1. More than one plasmid can easily enter the cell during transformation. In addition, the outside of the cell is coated with additional ligated products as well as the surplus inserts. (Remember that 3:1 insert:vector ratio. The unused inserts are still in the ligation mix.)

2. By the time you grow a colony and harvest it for growth in 3 mL of media, the cell goes through approximately 30+ generations. This allows time for segregation to occur. If two or more incompatible plasmids entered the cell, only one is left after 30 generations, even if the difference is only a few base pairs. This also explains why you can pick a colony from the agar plate and then it fails to grow in liquid or there is no plasmid when you harvest the liquid culture for plasmid prep. If the plasmid is toxic or if the antibiotics are inactive, then segregation will allow cells that lack the plasmid to compete and take over the culture. High copy number plsmids that generate beta lactamase can inactivate the ampicillin fairly rapidly. Methicillin is more resistant to beta lactamase (and more expensive) and can be used to maintain high copy number plasmids.

3. Plasmid maps in catalogs frequently indicate that the origin of a pUC-derived plasmid is ColE 1. While closely related, these are more accurately described as ColE 1-type origins, as the RNA primer contains sequence and secondary structural variations. pUC-type plasmids contain a single mutation in the pBR322 (pMB1) RNA primer sequence that renders them high copy number in the absence of rop.
pBR322 = 15 - 25 copies/cell
pBR322 minus rom/rop = 45 - 60 copies/cell
pUC = 300 - 700 copies/cell
pUC plus rom/rop in cis or in trans = 15 - 25 copies/cell
ColE 1 = 10 - 20 copies/cell

4. Plasmids that are compatible with the plasmids in part 3 include pACYC (10 - 12 copies/cell), pSC101 (5 - 6 copies/cell), and R plasmids (variable but low copy number). This means that a single cell can contain 1 plasmid from part 3 and one each of the 3 plasmid types listed in part 4.

5. Actual copy number varies. Because plasmids grow in bacteria, plasmid copy number is affected by the strain of E. coli used and the presence of advantageous mutations such as endA-, the formulation, osmolarity, and the buffering capacity of the media, the aeration available in the growth chamber, the temperature, the shaking speed, the growth phase at harvest, the age and condition of the inoculum (and whether it is fresh or frozen) and the rate at which concentrations of cells or reagents are changed, nutritional deficiencies, the effects of growth inhibitors (antibiotics) will vary according to how fast the cells are growing, the presence of “poison” genes that inhibit the growth of the bacteria, the presence of strong promoters pointed in the same direction as the plasmid replication transcript, the addition of inhibitors for regulated promoters, the origin of replication, and the skill of the lab personnel in harvesting plasmid DNA. Commercial kits are not designed to harvest 100% of the plasmid DNA in the cell. Some high copy number plasmids cause bacterial division to fail under certain conditions, resulting in long snake-like bacteria.
Use of an endA- strain of E. coli is preferable when maximum yields are desired.

6. pRsetA is derived from pBluescript and contains the pUC origin.

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