Hi Everyone,
I have recently began working with the TetR inducible gene expression system in S. cerevisiae. I am currently using the rtTA reverse trans-activator (rTetR-VP16) under control of the myo2 yeast promoter.
I would like to reduce basal activity as much as possible while maintaining the ability to tune gene expression levels by varying the concentration of inducer.
I see a number of possible solutions that have been published and I am unsure of which to move forward with.
I understand that the dual repressor/activator system can reduce basal levels to near zero, however I cannot seem to find any information on this dual system's dose-response beyond "no inducer" and "maximum induction". I read something about the dimerization of the Tet protein possibly leading to some interaction between rTetR-VP16 and the TetR-repressor complex at intermediate concentrations of inducer. I am wondering whether this dual system will maintain a smooth graded response in gene expression when the inducer concentration is varied between 0 and 12ug/L.
I also have read about the rtTA-S2 mutant, which reduces the basal activity and maintains similar levels of maximum induction. However, I could find little followup work using this mutant in S. cerevisiae.
Are there any other variants of this system I should consider? If anyone here has any experience with this problem and has tried multiple solutions, what was the best solution in your opinion?
Thanks
Sofie, for expression in procaryontes, there is a multitude of vector systems. I like the pET15 series (http://www.addgene.org/vector-database/2543/) that one comes with aHIs(6x)-Tag for purification) But there are many different systems. Another popular one is the pGEx-SYstem , wich uses a GST-tag. As I said recently perople like SUMO-fusions (In Bacteria WITH the GG of SUMO!) It also depends what you intend to do with your protein (Enzymatic assays? Immunization?) That will affect your choice of vectors Here a little overview: http://en.wikipedia.org/wiki/Expression_vector#Bacterial
I apologize for neglecting to answer my own question sooner, but In case anyone stumbles upon this question and is hoping for an answer:
The rtTA-S2 mutant worked very well for reducing basal expression to nearly zero in S. cerevisiae. I highly recommend it if anyone is looking to try and control gene expression with an inducer.
Because this mutant was not available on Addgene, we had to synthesize and do some basic clonework and GFP characterization. In hopes of aiding those who might wish to use the rtTA-S2 mutant, we will soon submit a few plasmids to addgene so that other researchers may use this system more easily.
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