Titratable control of pBAD and lac promoters in individual E. coli cells

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(''lac'' promoters)
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==''lac'' promoters==
==''lac'' promoters==
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*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.
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===='''Real-world evidence'''====
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*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''<sup>+</sup> and ''lacY''<sup>—</sup> cells. In general, the cells did not grow much, if at all, in ''lacY''<sup>+</sup> cells. However, growth in ''lacY''<sup>�—</sup> 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''<sup>—</sup> cells. I couldn't assess this information for the ''lacY''<sup>+</sup> 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''<sup>—</sup> 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. (--[[User:Kathmc|Kathleen]])
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**The  ''lacY''<sup>—</sup> strain that I have was a gift from [http://www.lifesci.ucsb.edu/mcdb/faculty/hayes/ 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''.
==pBAD promoters==
==pBAD promoters==

Revision as of 16:04, 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.

lac promoters

  • 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.

Real-world evidence

  • 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.

pBAD promoters

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