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

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(<b>''lac'' promoters</b>)
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=='''Intro'''==
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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.
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.
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=='''What is an "all-or-none" promoter?'''==
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==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.
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.
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====References====
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===References===
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#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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<biblio>
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#Novick-PNAS-1957 pmid=16590055
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</biblio>
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==<b>''lac'' promoters</b>==
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==''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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*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 (see figure below). <cite>Jensen-EurJBiochem-1993</cite>
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====References====
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[[Image:DelLacYInduction.PNG|thumb|LacY+/- induction|left]]
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#A. Khlebnikov and J. D. Keasling. Effect of lacY expression on homogeneity of induction from the P<sub>tac</sub> and P<sub>trc</sub> promoters by natural and synthetic inducers. Biotechnol Prog, 18:672–4, 2002.
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=='''pBAD promoters'''==
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<br style="clear:both" />
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*Lactose metabolism is likely a problem in these cells (see P<sub>BAD</sub> section below), but this can be avoided by using IPTG, which is not metabolized.
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===References===
 +
<biblio>
 +
#Khlebnikov-BiotechnolProg-2002 pmid=12052093
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#Jensen-EurJBiochem-1993 pmid=8425528
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#Oehler2006 pmid=16432263
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#Kuhlman2007 pmid=17376875
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</biblio>
 +
 
 +
==pBAD promoters==
*Import of arabinose into cells is mediated by the ''araE'' gene. Induction of the arabinose transporter encoded by ''araE'' can be uncoupled from the endogenous P<sub>BAD</sub> promoter by deleting the chromosomal ''araE'' gene and replacing it with a plasmid-borne copy of ''araE'' under control of a constitutive promoter (1). However, this does not seem to be enough to allow for homogenous expression from P<sub>BAD</sub> promoters in a population of cells (2).  
*Import of arabinose into cells is mediated by the ''araE'' gene. Induction of the arabinose transporter encoded by ''araE'' can be uncoupled from the endogenous P<sub>BAD</sub> promoter by deleting the chromosomal ''araE'' gene and replacing it with a plasmid-borne copy of ''araE'' under control of a constitutive promoter (1). However, this does not seem to be enough to allow for homogenous expression from P<sub>BAD</sub> promoters in a population of cells (2).  
*At low concentrations of arabinose, degradation of the sugar within cells also effects the homogeneity of expression from P<sub>BAD</sub> promoters (2). Arabinose degradation is mediated by the ''araBAD'' genes. Strains lacking functional  ''araE'', ''araFGH'' (another transporter), and ''araBAD'' can be made to be responsive to arabinose for P<sub>BAD</sub> promoter induction (2). This is achieved by introduction of a mutant ''lacY'' gene. LacY A177C allows for downhill transport of arabinose, as well as maltose, palatinose, sucrose, and cellobiose (3), but does not actively transport these sugars (4). Lactose import is not affected in this mutant. So, P<sub>BAD</sub> promoters in cells lacking endogeneous arabinose importers and containing LacY A177C are linearly responsible to arabinose at the individual cell level.
*At low concentrations of arabinose, degradation of the sugar within cells also effects the homogeneity of expression from P<sub>BAD</sub> promoters (2). Arabinose degradation is mediated by the ''araBAD'' genes. Strains lacking functional  ''araE'', ''araFGH'' (another transporter), and ''araBAD'' can be made to be responsive to arabinose for P<sub>BAD</sub> promoter induction (2). This is achieved by introduction of a mutant ''lacY'' gene. LacY A177C allows for downhill transport of arabinose, as well as maltose, palatinose, sucrose, and cellobiose (3), but does not actively transport these sugars (4). Lactose import is not affected in this mutant. So, P<sub>BAD</sub> promoters in cells lacking endogeneous arabinose importers and containing LacY A177C are linearly responsible to arabinose at the individual cell level.
*By the way, AraC is the repressor of the P<sub>BAD</sub> promoter. It is encoded on the pBAD vector series and is still present in the above-described strains.
*By the way, AraC is the repressor of the P<sub>BAD</sub> promoter. It is encoded on the pBAD vector series and is still present in the above-described strains.
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====References====
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===References===
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#A. Khlebnikov, K. A. Datsenko, T. Skaug, B. L. Wanner, and J. D. Keasling. Homogeneous expression of the P<sub>BAD</sub> promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiology, 147(Pt 12):3241–7, 2001.
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<biblio>
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#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.
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#Khlebnikov-Microbiology-2001 pmid=11739756
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#B. Rotman. On the rate limiting step in downhill transport via the LacY permease of ''Escherichia coli''. J Supramol Struct. 7(1):29-35, 1977.
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#Morgan-Kiss-PNAS-2002 pmid=12032290
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#S.C. King and T.H. Wilson. Identification of valine 177 as a mutation altering specificity for transport of sugars by the ''Escherichia coli'' lactose carrier. J. Biol. Chem., 265 (17): 9638–9644, 1990.
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#Rotman-JSupramolStruct-1977 pmid=415184
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#King-JBS-1990 pmid=2190983
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#Siegele-PNAS-1997 pmid=9223333
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#Zhang96 pmid=8613984
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#Guzman95 pmid=7608087
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#Khlebnikov2002 pmid=12080425
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</biblio>
==Constructing a strain that allows homogenous, titratable induction of both ''lac'' and P<sub>BAD</sub> promoters==
==Constructing a strain that allows homogenous, titratable induction of both ''lac'' and P<sub>BAD</sub> promoters==
*From what I can tell, this is impossible to do with the current technology. To get homogenous, titratable induction of P<sub>BAD</sub> promoters, LacY A177C is necessary. To get homogenous, titratable induction of ''lac''-type promoters, LacY cannot be present. How do we get around this?
*From what I can tell, this is impossible to do with the current technology. To get homogenous, titratable induction of P<sub>BAD</sub> promoters, LacY A177C is necessary. To get homogenous, titratable induction of ''lac''-type promoters, LacY cannot be present. How do we get around this?
 +
**What about a strain that has ''araBAD'', ''araE'' and ''araFGH'' deleted and ''lacY A177C'' under the control of a constitutive promoter?  My understanding was that ''lacY'' is problematic because the upstream promoter is inducible via IPTG and thus cells which happen to have a few more copies of LacY initially will import more IPTG and thus make more LacY than cells with fewer copies of LacY.  By eliminating the positive feedback, you should still be able to achieve titrable control.
*It seems like the best that can be done at this point is use of ''araE'' under the control of a constitutive promoter in a ''lacY''<sup>—</sup> strain. Does anyone have any other suggestions?
*It seems like the best that can be done at this point is use of ''araE'' under the control of a constitutive promoter in a ''lacY''<sup>—</sup> strain. Does anyone have any other suggestions?
 +
**'''[[User:Jasonk|Jasonk]] 07:30, 14 June 2006 (EDT):'''You could consider using the pPRO [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16269719&query_hl=2&itool=pubmed_docsum Propionate-inducible] induction system instead of pBAD.
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 +
==Related work==
 +
<biblio>
 +
#Lee-ApplEnvironMicrobiol-2007 pmid=17644634
 +
</biblio>
 +
[[Category:Escherichia coli]]

Current revision

Contents

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.

References

  1. Novick A and Weiner M. . pmid:16590055. PubMed HubMed [Novick-PNAS-1957]

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 (see figure below). [2]
LacY+/- induction
LacY+/- induction


  • Lactose metabolism is likely a problem in these cells (see PBAD section below), but this can be avoided by using IPTG, which is not metabolized.

References

  1. Jensen PR, Westerhoff HV, and Michelsen O. . pmid:8425528. PubMed HubMed [Jensen-EurJBiochem-1993]
  2. Khlebnikov A and Keasling JD. . pmid:12052093. PubMed HubMed [Khlebnikov-BiotechnolProg-2002]
  3. Oehler S, Alberti S, and Müller-Hill B. . pmid:16432263. PubMed HubMed [Oehler2006]
  4. Kuhlman T, Zhang Z, Saier MH Jr, and Hwa T. . pmid:17376875. PubMed HubMed [Kuhlman2007]
All Medline abstracts: PubMed HubMed

pBAD promoters

  • Import of arabinose into cells is mediated by the araE gene. Induction of the arabinose transporter encoded by araE can be uncoupled from the endogenous PBAD promoter by deleting the chromosomal araE gene and replacing it with a plasmid-borne copy of araE under control of a constitutive promoter (1). However, this does not seem to be enough to allow for homogenous expression from PBAD promoters in a population of cells (2).
  • At low concentrations of arabinose, degradation of the sugar within cells also effects the homogeneity of expression from PBAD promoters (2). Arabinose degradation is mediated by the araBAD genes. Strains lacking functional araE, araFGH (another transporter), and araBAD can be made to be responsive to arabinose for PBAD promoter induction (2). This is achieved by introduction of a mutant lacY gene. LacY A177C allows for downhill transport of arabinose, as well as maltose, palatinose, sucrose, and cellobiose (3), but does not actively transport these sugars (4). Lactose import is not affected in this mutant. So, PBAD promoters in cells lacking endogeneous arabinose importers and containing LacY A177C are linearly responsible to arabinose at the individual cell level.
  • By the way, AraC is the repressor of the PBAD promoter. It is encoded on the pBAD vector series and is still present in the above-described strains.

References

  1. Khlebnikov A, Datsenko KA, Skaug T, Wanner BL, and Keasling JD. . pmid:11739756. PubMed HubMed [Khlebnikov-Microbiology-2001]
  2. Morgan-Kiss RM, Wadler C, and Cronan JE Jr. . pmid:12032290. PubMed HubMed [Morgan-Kiss-PNAS-2002]
  3. Rotman B. . pmid:415184. PubMed HubMed [Rotman-JSupramolStruct-1977]
  4. King SC and Wilson TH. . pmid:2190983. PubMed HubMed [King-JBS-1990]
  5. Siegele DA and Hu JC. . pmid:9223333. PubMed HubMed [Siegele-PNAS-1997]
  6. Zhang X, Reeder T, and Schleif R. . pmid:8613984. PubMed HubMed [Zhang96]
  7. Guzman LM, Belin D, Carson MJ, and Beckwith J. . pmid:7608087. PubMed HubMed [Guzman95]
  8. Khlebnikov A, Skaug T, and Keasling JD. . pmid:12080425. PubMed HubMed [Khlebnikov2002]
All Medline abstracts: PubMed HubMed

Constructing a strain that allows homogenous, titratable induction of both lac and PBAD promoters

  • From what I can tell, this is impossible to do with the current technology. To get homogenous, titratable induction of PBAD promoters, LacY A177C is necessary. To get homogenous, titratable induction of lac-type promoters, LacY cannot be present. How do we get around this?
    • What about a strain that has araBAD, araE and araFGH deleted and lacY A177C under the control of a constitutive promoter? My understanding was that lacY is problematic because the upstream promoter is inducible via IPTG and thus cells which happen to have a few more copies of LacY initially will import more IPTG and thus make more LacY than cells with fewer copies of LacY. By eliminating the positive feedback, you should still be able to achieve titrable control.
  • It seems like the best that can be done at this point is use of araE under the control of a constitutive promoter in a lacY strain. Does anyone have any other suggestions?

Related work

  1. Lee SK, Chou HH, Pfleger BF, Newman JD, Yoshikuni Y, and Keasling JD. . pmid:17644634. PubMed HubMed [Lee-ApplEnvironMicrobiol-2007]
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