Difference between revisions of "Titratable control of pBAD and lac promoters in individual E. coli cells"
|Line 2:||Line 2:|
Revision as of 13:04, 25 October 2005
I started looking into this because I wanted to generate a strain in which I could control the level of induction from both a pTrc and a pBAD promoter in individual cells. After talking to a few people, I was able to sort out that these promoters both exhibit all-or-none activity in "wild-type" E. coli strains. I also discovered that it seems that not everyone knows about this (or at least the details of the process and how to get around it). Below is a summary of the information I was able to assemble on the topic. Hopefully, you know more than I do and can add more information.
- Import of lactose (and IPTG) into E. coli is controlled by the lacY gene. If you knock this gene out, lac-type promoter induction is titratable at non-saturating lactose or IPTG concentrations in individual cells.
- I am expressing a repressor protein that is toxic to cells if it is overexpressed. The theory is that, since it is a DNA binding protein, when the repressor is at high levels it binds to all of the DNA in the cell, wrecking havoc on the critter. I was looking at growth of critters containing the plasmid-borne repressor protein under the control of a pTrc promoter on LB plates containing different amounts of IPTG. I examined this in lacY+ and lacY— cells. In general, the cells did not grow much, if at all, in lacY+ cells. However, growth in lacY�— cells was dependent on the amount of IPTG on the plate; too much IPTG and the critters died. Expression of a protein under the control of the repressor protein was also dependent on the amount of IPTG I had on the plate in lacY— cells. I couldn't assess this information for the lacY+ cells, because any cell that expressed the repressor expressed too much of it and killed the cell. Thus, I appear to have titratable control of the pTrc promoter in lacY— cells. I'm sure there is a much better and more elegant published example of this, I just don't have the reference right now. Please add references here if you know of any. (--Kathleen)
- The lacY— strain that I have was a gift from Chris Hayes at UCSB. In the wild-type strain, lacY was contained on the F plasmid. The strain Chris generated lacks the F plasmid, so it is missing genes in addition to lacY.