diaz@aecom.UUCP (06/14/87)
A post-doctoral fellow here at Einstein informed me recently that the
Gemini series of plasmids marketed by Promega Biotech (pGEM1, 2, 3, 4,
and blue) have copy numbers on the order of 400 per cell. This is one of
the highest copy numbers I have heard of. We may try to clone an E. coli
gene into one of these plasmids in order to see whether we can get
increased levels of an enzyme we're studying just on the basis of copy
number.
Does anyone know of any plasmids with even higher copy numbers? If so,
where are they available from, and what is their selection?
--
5'gtacggagc dn/dx = Dan Diaz (philabs!aecom!diaz)
Department of Molecular Biology & Snake Oil Dynamics
Albert Slimestein College of Medicine ctataacagcta 3' pell@boulder.UUCP (06/15/87)
(Dizzy Dan) writes: > >A post-doctoral fellow here at Einstein informed me recently that the >Gemini series of plasmids marketed by Promega Biotech (pGEM1, 2, 3, 4, >and blue) have copy numbers on the order of 400 per cell. This is one of >the highest copy numbers I have heard of. We may try to clone an E. coli >gene into one of these plasmids in order to see whether we can get >increased levels of an enzyme we're studying just on the basis of copy >number. Check me on this one, but I believe the "400/cell" number is the amount after 12 hours or so of amplification with chloramphenicol. I have gotten in excess of 6 mgs of twice-banded plasmid from a liter of cells with this vector, which, in molecular bio terms, is "tons." I know of no plasmids that amplify better. I think the copy number in healthy cells is about an order of magnitude down. But, since the pGems are deleted for all copy-number control regions of ColE1, I can't imagine you could do much better (let me hedge a bit, i don't think the pGems make the regulatory RNA, at least not an intact one. BTW the promoters for RNA1 and RNA2 are close and transcribe in opposite directions. I think the two transcripts are complimentary for a hundred or so bases). As for other replicons altogether, i don't think any are higher in copy number than ColE1. happy cloning tony
bchso@uhnix2.UUCP (Dan Davison) (06/20/87)
In article <1137@aecom.YU.EDU> diaz@aecom.YU.EDU (Dizzy Dan) writes: >Does anyone know of any plasmids with even higher copy numbers? If so, >where are they available from, and what is their selection? Have you considered and rejected chloramphenicol amplification? I've heard it can get 1000s (!) per cell. The plasmid must be descended from ColE1, though. dr. dan davison|Los Alamos National Laboratory|Theoretical Biology|T-10, MS K-710, Los Alamos, NM 87545...rice!academ!uhnix1!uhnix2!bchso "Of all the things I've lost I miss my mind the most"
diaz@aecom.YU.EDU (Dizzy Dan Diaz) (06/26/87)
In article <404@uhnix2.UUCP>, bchso@uhnix2.UUCP (Dan Davison) writes: > In article <1137@aecom.YU.EDU> diaz@aecom.YU.EDU (Dizzy Dan) writes: > >Does anyone know of any plasmids with even higher copy numbers? If so, > >where are they available from, and what is their selection? > > Have you considered and rejected chloramphenicol amplification? I've heard > it can get 1000s (!) per cell. The plasmid must be descended from ColE1, > though. If I were simply interested in making a plasmid preparation chloramphenicol would serve my purposes. What I'm interested in, however, is observing whether I can get increased cloned enzyme gene expression levels based on copy number. Chloramphenicol would increase the copy number, but it would also interfere with protein synthesis. -- .... dn/dx = Dan Diaz (philabs!aecom!diaz or diaz@aecom.yu.edu) ~..|.> Department of Wasting Taxpayers' Money on Useless Research \../ Albert Einstein College of Medicine and Bar & Grill
werner@aecom.YU.EDU (Craig Werner) (06/26/87)
In article <404@uhnix2.UUCP>, bchso@uhnix2.UUCP (Dan Davison) writes: > > Have you considered and rejected chloramphenicol amplification? I've heard > it can get 1000s (!) per cell. The plasmid must be descended from ColE1, > though. > > dr. dan davison|Los Alamos National Laboratory|Theoretical Biology|T-10, Only if it retains the _rom_ gene, which is what is repressed and leads to amplification. Many laboratory derivatives of pBR322, such as all the pUC derivatives, have had this gene deleted. As a result, they cannot be chloramphenicol-amplified. However, their basal copy number does increase from the 20 or so of pBR322 to about 150-250, which makes amplification a bit gratuitous. -- Craig "Baby Doc" Werner (future MD/PhD, 3 years down, 4 to go) werner@aecom.YU.EDU -- Albert Einstein College of Medicine (1935-14E Eastchester Rd., Bronx NY 10461, 212-931-2517) "Disinformation is one thing, but misinformation is unforgiveable."
pell@boulder.UUCP (06/29/87)
(Dan Davison) writes: (Dizzy Dan) writes: >>Does anyone know of any plasmids with even higher copy numbers? If so, >>where are they available from, and what is their selection? > >Have you considered and rejected chloramphenicol amplification? I've heard >it can get 1000s (!) per cell. How is one going to do protein overexpression in the presence of a protein synthesis inhibitor??? >The plasmid must be descended from ColE1,though. Not quite, the plasmid must have a means of replication that does not rely on continued protein synthesis--as the coli chromosome requires new synthesis for each round of initiation. ColE1 replication is one such dnaA-independant replicon. For those of you using pACYC or pBR-types that have CAT on them, spectinomycin works fairly well for amplification, by the same mode of action. tony "don't pull that; you never know what it might be connected to." -BB
pell@boulder.Colorado.EDU (Anthony Pelletier) (06/30/87)
(Dan Davison) writes: >> >> Have you considered and rejected chloramphenicol amplification? I've heard >> it can get 1000s (!) per cell. The plasmid must be descended from ColE1, >> though. >> >> dr. dan davison|Los Alamos National Laboratory|Theoretical Biology|T-10, Craig writes: > Only if it retains the _rom_ gene, which is what is repressed >and leads to amplification. Many laboratory derivatives of pBR322, >such as all the pUC derivatives, have had this gene deleted. As a result, >they cannot be chloramphenicol-amplified. However, their basal copy >number does increase from the 20 or so of pBR322 to about 150-250, which >makes amplification a bit gratuitous. > > ok, i should start with a disclaimer. I have not read a thing about this in a while; the last was a pre-print from Tomizawa's lab that I think was due out in "Cell" in the summer of '84. i got it in Feb. of that year. The product of the rom gene (or is rop the gene and rom the protein?) is involved in mediating the interaction of RNA1 and RNA2. One of these serves as the primer for DNA synthesis at the ColE1 ori and the other is made from a promoter on the opposite strand such that the two overlap. This second RNA inhibits initiation by binding to the primer RNA. This is part of the copy-number control of the plasmid. tomizawa was pushing the RNA as the "catalyst" (it is nothing of a sort) and claiming that the protein was helpful, but not a strict reguirement. At the time it was (and still is) quite in vogue to claim that "your favorite RNA" had catalytic fuction. He tried to fit the kinetics of the reaction to Michaelis-Menton and failed for the obvious reason that the steady-state assumtions cannot be made. The fact that the reaction had a first order rate constant was pointed out to him. now, i suppose i should attempt a point here. I thought that knocking out the rom product was only part of the story where amplification was concerned. If rop and the inhibitory RNA are made, the plasmid will not continue to replicate beyond about 40/cell, to be sure. But, the real gift of amplification is that the cell number remains constant and the plasmid continues to replicate; you get about the same number of plasmid molecules as in a stationary culture of the same volume but have orders-of-magnitude fewer cells. This makes lysis and extraction a great deal easier. The basis for this is that the dnaA gene product, which is essential for initiation of chromosomal replication, must be made new for each round of replication. ColE1 and others replicate in a dnaA-independant fashion and so continue to replicate. I have never done careful studies on the amplification of pGems and the like. However, I do get better yields from amplified than stationary cultures. This could be largely due to better lysis. I'd be willing to bet that there is more to it than we know yet. But, for me, if it works, don't fix it. > Craig "Baby Doc" Werner (future MD/PhD, 3 years down, 4 to go) ^(I like this one--not that i think it is for my approval)^ tony pelletier (planning to make my fortune selling yeast alpha factor to the singles crowd as "mating phermone" produced through the miracle of gene splicing)