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

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Intro

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.

What is an "all-or-none" promoter?

Inducing gene expression from an "all-or-none" promoter at subsaturating inducer concentrations results in a heterogeneous population of cells in which some are fully induced and others are induced very little, if at all. What is often confusing about this phenomenon in practice is that a population of cells will typically respond in a linear manner to increased concentration of inducer. What is really happening, though, is that more cells in the population are being turned on as inducer concentration is increased, but there are still some cells in the population that are not induced at all. The "on" phenotype is a result of inducer importers being turned on when a cell is exposed to the inducer, resulting in increased uptake of the inducer. At subsaturating inducer concentrations, there is not enough inducer to go around for all of the cells, so those that get their importer turned on first get all of the the inducer.

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.

References

  1. A. Khlebnikov and J. D. Keasling. Effect of lacY expression on homogeneity of induction from the Ptac and Ptrc promoters by natural and synthetic inducers. Biotechnol Prog, 18:672–4, 2002.

pBAD promoters

References

  1. R. M. Morgan-Kiss, C. Wadler, and J. E. J. Cronan. Long-term and homogeneous regulation of the Escherichia coli araBAD promoter by use of a lactose transporter of relaxed specificity. Proc Natl Acad Sci USA, 99(11):7373–7, 2002.
  2. A. Khlebnikov, K. A. Datsenko, T. Skaug, B. L. Wanner, and J. D. Keasling. Homogeneous expression of the PBAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiology, 147(Pt 12):3241–7, 2001.

References

  1. A. Novick and M. Weiner. Enzyme induction as an all-or-none phenomenon. Proc Natl Acad Sci USA, 43(7):553–66, 1957.
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