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    Broad-Host-Range Expression Vectors with Tightly Regulated Promoters and Their Use To Examine the Influence of TraR and TraM Expression on Ti Plasmid Quorum Sensing

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    Author
    Khan, Sharik R.; Gaines, Jennifer M.; Roop, R. Martin II; Farrand, Stephen K.
    Abstract
    Experiments requiring strong repression and precise control of cloned genes can be difficult to conduct because of the relatively high basal level of expression of currently employed promoters. We report the construction of a family of vectors that contain a reengineered lacIq-lac promoter-operator complex in which cloned genes are strongly repressed in the absence of inducer. The vectors, all based on the broad-host-range plasmid pBBR1, are mobilizable and stably replicate at moderate copy number in representatives of the alphaand gammaproteobacteria. Each vector contains a versatile multiple cloning site that includes an NdeI site allowing fusion of the cloned gene to the initiation codon of lacZ . In each tested bacterium, a uidA reporter fused to the promoter was not expressed at a detectable level in the absence of induction but was inducible by 10- to 100-fold, depending on the bacterium. The degree of induction was controllable by varying the concentration of inducer. When the vector was tested in Agrobacterium tumefaciens, a cloned copy of the traR gene, the product of which is needed at only a few copies per cell, did not confer activity under noninducing conditions. We used this attribute of very tight and variably regulatable control to assess the relative amounts of TraR required to activate the Ti plasmid conjugative transfer system. We identified levels of induction that gave wild-type transfer frequencies, as well as levels that induced correspondingly lower frequencies of transfer. We also used this system to show that the antiactivator TraM sets the level of intracellular TraR required for tra gene activation. Originally published Applied and Environmental Microbiology, Vol. 74, No. 16, Aug 2008
    URI
    http://hdl.handle.net/10342/3316
    Subject
     Cloned genes; Controlled induction; Expression vectors 
    Date
    2008-08
    Citation:
    APA:
    Khan, Sharik R., & Gaines, Jennifer M., & Roop, R. Martin II, & Farrand, Stephen K.. (August 2008). Broad-Host-Range Expression Vectors with Tightly Regulated Promoters and Their Use To Examine the Influence of TraR and TraM Expression on Ti Plasmid Quorum Sensing. Applied and Environmental Microbiology, (74:16), p.5053-5062. Retrieved from http://hdl.handle.net/10342/3316

    Display/Hide MLA, Chicago and APA citation formats.

    MLA:
    Khan, Sharik R., and Gaines, Jennifer M., and Roop, R. Martin II, and Farrand, Stephen K.. "Broad-Host-Range Expression Vectors with Tightly Regulated Promoters and Their Use To Examine the Influence of TraR and TraM Expression on Ti Plasmid Quorum Sensing". Applied and Environmental Microbiology. 74:16. (5053-5062.), August 2008. July 03, 2022. http://hdl.handle.net/10342/3316.
    Chicago:
    Khan, Sharik R. and Gaines, Jennifer M. and Roop, R. Martin II and Farrand, Stephen K., "Broad-Host-Range Expression Vectors with Tightly Regulated Promoters and Their Use To Examine the Influence of TraR and TraM Expression on Ti Plasmid Quorum Sensing," Applied and Environmental Microbiology 74, no. 16 (August 2008), http://hdl.handle.net/10342/3316 (accessed July 03, 2022).
    AMA:
    Khan, Sharik R., Gaines, Jennifer M., Roop, R. Martin II, Farrand, Stephen K.. Broad-Host-Range Expression Vectors with Tightly Regulated Promoters and Their Use To Examine the Influence of TraR and TraM Expression on Ti Plasmid Quorum Sensing. Applied and Environmental Microbiology. August 2008; 74(16) 5053-5062. http://hdl.handle.net/10342/3316. Accessed July 03, 2022.
    Collections
    • Microbiology and Immunology
    Publisher
    East Carolina University

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