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Team:Cornell/Project
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Project Details
Design
To create a biosensor dependent on Cd(II) concentration, we decided to engineer the existing metal ion homeostasis system in Bacillus subtilis. In B. subtilis metal ion transport is tightly regulated. Though the organism requires trace amounts of metals, high levels interfere with cellular processes. Cd(2+) enters B. subtilis through the manganese ion influx protein MntH. Intracellular Cd(2+) concentration is under the regulation of several pathways, one of which is the CadA efflux protein. CadA is a P-type ATPase that effluxes Cd(2+). Transcription of the cadA gene is regulated by CzrA (formerly YozA) a ArsR/SmtB family repressor that binds the cadA regulatory region and is released when bound by cadmium ions.[1] Our first cadmium sensing module is based on the regulatory region of cadA. By attaching this regulatory region to an appropriate ribosome binding site and the gene for Cyan Fluorescent Protein (BBa_E0020), the production of CFP and by extension the fluorescence at the peak emission wavelength of CFP becomes a function of intracellular Cd(2+) concentration. Measuring the emission at 476 nm will allows us to indirectly measure the intracellular Cd(2+) concentration. As intracellular Cd(2+) concentrations rise we expect to see an increase in fluorescence at the peak emmission wavelength for CFP.
Our second cadmium sensing module is based on the transcription of the Mn(2+) and Cd(2+) influx protein MntH. mntH is part of MntR regulon which is downregulated in the presence of Cd(2+).[1] By using regulatory region of mntH and attaching to an appropriate ribosome binding site and Yellow Fluorescent Protein(BBa_E0030), we can use this module as another measure of the intracellular Cd(2+) concentration. Therefore as intracellular Cd(2+) concentrations rise, we expect to see a decrease in fluorescence at the peak emission wavelength for YFP at 527 nm.
In order to accomplish this, we need to delete the mntR gene which is responsible for regulating Mn(II) and Cd(II) import through the mntH transporter. mntH is a member of the NRAMP (natural resistance associated macrophage protein) family of proton-coupled, metal ion transporters, which is specific to Mn(II) and Cd(II). At high Mn(II) or Cd(II) concentrations in B. subtilis, mntR functions as a Mn(II) or Cd(II)-dependent repressor of mntH transcription. This process thereby down-regulates transport of Mn(II) and Cd(II) into the cell. Previous experiments have demonstrated that mntR mutant strains show greater sensitivity to both Mn(II) and Cd(II).5 This indicates that in mntR mutant strains, Mn(II) and Cd(II) influx is deregulated. With Cd(II) influx deregulated, Cd(II) influx becomes directly correlated to extracellular Cd(II) concentration.
To detect Cd(II) influx we propose to use the natural resistance mechanism to Cd(II) in B. subtilis. Cadmium resistance is provided by an operon that consists of the gene cadA which encodes for a P-type ATPase that effluxes Cd(II). In vitro studies have suggested that CzrA (formerly YozA) is an ArsR/SmtB family repressor that binds the cadA regulatory region and is released when bound by cadmium ions.[1] Because these studies have not yet been confirmed in vivo, we plan to demonstrate that a high intracellular concentration of Cd(II) will lead to increased transcription of cadA. The transcription levels of cadA are then an indicator of intracellular Cd(II) concentration and, therefore, extracellular Cd(II) concentration.
By expressing a cadA-reporter gene fusion, we can detect intracellular Cd(II) concentration. We will test two reporter systems and compare the results. We will create a cadA-GFP fusion and detect GFP levels through standard flourimetry techniques. We also plan on testing a cadA-lacZ fusion to determine if a β-galactosidase assay will output similar results. Since the β-galactosidase assay is cheaper to implement, it may serve as a desirable alternative to GFP flourimetry in creating a cost effective system.
The Experiments
Part 3
References
[1] Helmann, John D., Charles M. Moore, Ahmed Gaballa, Monica Hui, Rick W. Ye (2005), Genetic and physiological responses of Bacillus subtilis to metal ion stress. Molecular Microbiology 57(1), p.27-40
