Team:Cornell/Project

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=== Design ===
=== Design ===
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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.
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To create a biosensor dependent on Cd(II) concentration, we decided to engineer the existing metal ion homeostasis system in Bacillus subtilis, in which metal ion transport is tightly regulated. The organism requires trace amounts of metals for normal growth, while high levels interfere with cellular processes.  
 +
Cd(2+) enters B. subtilis through the manganese ion influx protein MntH.  Intracellular Cd(2+) concentration is regulated by several factors, one of which is the CadA efflux protein. CadA is a P-type ATPase that pumps out Cd(2+). Transcription of the cadA gene is regulated by protein CzrA (formerly YozA), a ArsR/SmtB family repressor. CzRA binds and represses the cadA regulatory region and is released when bound by cadmium ions [1].
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>Briefly describe the two modules and the differences between them<
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Our first cadmium sensing module utilizes the regulatory region of cadA, which activates transcription in the presence of Cd(2+). By attaching this regulatory region to an appropriate ribosome binding site and the gene for Cyan Fluorescent Protein (BBa_E0020), the production of CFP becomes a function of intracellular Cd(2+) concentration. Measuring the peak emission wavelength of CFP 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.
[[Image:Module 1 Inactive.jpg|600px|center]]
[[Image:Module 1 Inactive.jpg|600px|center]]
[[Image:Module 1 Active.JPG|600px|center]]
[[Image:Module 1 Active.JPG|600px|center]]
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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. As intracellular Cd(2+) concentrations rise, we expect to see a decrease in fluorescence at the peak emission wavelength for YFP at 527 nm.
-
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.
+
[[Image:Module 2 Inactive.JPG‎|600px|center]]
[[Image:Module 2 Inactive.JPG‎|600px|center]]
[[Image:Module 2 Active.jpg|600px|center]]
[[Image:Module 2 Active.jpg|600px|center]]
-
 
By using two modules we can enhance our signal to noise ratio and cancel out stochastic error in our readings. The regulatory proteins for both are modules are not completely specific to the Cd(2+) ion. In order to correct for false positive readings we will compare our fluorescence measurements to baseline values in cells induced without Cd(2+).
By using two modules we can enhance our signal to noise ratio and cancel out stochastic error in our readings. The regulatory proteins for both are modules are not completely specific to the Cd(2+) ion. In order to correct for false positive readings we will compare our fluorescence measurements to baseline values in cells induced without Cd(2+).

Revision as of 20:32, 17 October 2009

Contents

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 which metal ion transport is tightly regulated. The organism requires trace amounts of metals for normal growth, while high levels interfere with cellular processes.

Cd(2+) enters B. subtilis through the manganese ion influx protein MntH. Intracellular Cd(2+) concentration is regulated by several factors, one of which is the CadA efflux protein. CadA is a P-type ATPase that pumps out Cd(2+). Transcription of the cadA gene is regulated by protein CzrA (formerly YozA), a ArsR/SmtB family repressor. CzRA binds and represses the cadA regulatory region and is released when bound by cadmium ions [1].

>Briefly describe the two modules and the differences between them<

Our first cadmium sensing module utilizes the regulatory region of cadA, which activates transcription in the presence of Cd(2+). By attaching this regulatory region to an appropriate ribosome binding site and the gene for Cyan Fluorescent Protein (BBa_E0020), the production of CFP becomes a function of intracellular Cd(2+) concentration. Measuring the peak emission wavelength of CFP 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.

Module 1 Inactive.jpg
Module 1 Active.JPG

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. As intracellular Cd(2+) concentrations rise, we expect to see a decrease in fluorescence at the peak emission wavelength for YFP at 527 nm.

Module 2 Inactive.JPG
Module 2 Active.jpg

By using two modules we can enhance our signal to noise ratio and cancel out stochastic error in our readings. The regulatory proteins for both are modules are not completely specific to the Cd(2+) ion. In order to correct for false positive readings we will compare our fluorescence measurements to baseline values in cells induced without Cd(2+).

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

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