10 replies to this topic

[muchmoa]

Dear all,
Recently I tried to transform two plasmids derived from pGEX-2TK in E. coli BL21.
There were many colonies on the agar plate so I picked 8 for further analysis.
After induction by IPTG, SDS-PAGE showed half of them expressed one recombinant protein and
the other 4 expressed another recombinant protein.
It seems that the cell can only accept one plasmid.
The recombinant proteins are 59 and 70 KDa (including GST tag) and the recombinant plasmids are
about 6.1 Kbp.
Do you have any idea what happened to my experiment?
Due to the copy number of plasmid?
Many thanks for any suggestions.

[HomeBrew]

Two plasmids belonging to the same incompatibility group cannot stably coexist in the same cell. Two identical vectors are by definition members of the same incompatibility group, and thus will segregate into separate daughter cell populations, just as you've seen. If two plasmids share the same replication control and/or the same partitioning functions, they belong to the same incompatibility group, and compete with each other for stable maintenance.

[swanny]

Further to Homebrew,
Either use two different plasmids (different ori's and antibiotic resistance), or go for a bicistronic vector like the pETDuet family (that way you only transform one vector).

[muchmoa]

Alright, thanks a lot.
I think the compatibility is case by case.
I ever tried to transform a plasmid into cells carrying another pGEX-2TK plasmid and there were only
3 colonies in three plates.

[HomeBrew]

muchmoa, on May 7 2009, 04:17 AM, said:

I think the compatibility is case by case.

I'm not sure what you mean by this. If you mean that simultaneously and stably maintaining two different plasmids created using the same vector but containing different inserts can "sometimes work", I disagree -- unless one of the plasmids has acquired a mutation in its replication or partitioning pathways...

[swanny]

basically, don't try to co-transform if the plasmids have the same ori. There are probably some other subtle rules, but if you follow this one, you'll find virtually all of your problems will disappear.

[muchmoa]

HomeBrew, on May 8 2009, 02:26 AM, said:

muchmoa, on May 7 2009, 04:17 AM, said:

I think the compatibility is case by case.

I'm not sure what you mean by this. If you mean that simultaneously and stably maintaining two different plasmids created using the same vector but containing different inserts can "sometimes work", I disagree -- unless one of the plasmids has acquired a mutation in its replication or partitioning pathways...

Oh, I know for plasmids with the same ori, they can not stay together stably.
What I mean was for plasmids with different ori.

[swanny]

I thought you said you wanted to put pGEX-2tk into cells already containing pGEX-2TK... sounds like the same plasmid to me.

[Ah-Do]

HomeBrew, on May 7 2009, 10:26 AM, said:

muchmoa, on May 7 2009, 04:17 AM, said:

I think the compatibility is case by case.

I'm not sure what you mean by this. If you mean that simultaneously and stably maintaining two different plasmids created using the same vector but containing different inserts can "sometimes work", I disagree -- unless one of the plasmids has acquired a mutation in its replication or partitioning pathways...

i have a question. is this "co-transform" rule also apply to "co-transfection"??
say, if i already have a stable transgenic cell, can i transfect another plasmid containg the same promoter but different gene to this transgenic cell?

[marklee]

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[laurequillo]

i have a question. is this "co-transform" rule also apply to "co-transfection"??
say, if i already have a stable transgenic cell, can i transfect another plasmid containg the same promoter but different gene to this transgenic cell?
[/quote]


In transfection I never had such a problem. I introduced 2 or 3 plasmids in the same cell without any problem (I never checked the plasmids, but I´m pretty sure some of them are pcDNA3)