Difference between revisions of "Part:BBa K3470006"
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Constitutive Promoter – RBS – MerR - PmerT promoter – GFP - Double Terminator  
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<partinfo>BBa_K3470006 short</partinfo>
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MerR is a homodimer and the regulatory factor of the mer operon. It acts as an activator in the presence of Hg (II) and a weak repressor in its absence, maintaining its expression at a certain level. It binds to the operator site between –35 and –10 elements of mercury inducible promoter PmerT, independent of the presence of mercury. The Hg- bound MerR dimer causes a structural distortion of PmerT, which allows the RNA polymerase to bind, leading to the transcription of genes downstream to it.(Hobman, J. L., Wilkie, J., & Brown, N. L. 2005)  
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We test the response of MerR to methylmercury using this circuit Vs 2 control circuits which do not have the MerR gene present in them. E. coli cells inoculated with methylmercury chloride are grown for the required amount of time according to the results of the preliminary experiment. We check for GFP intensities expecting an exponential increase in fluorescence with an increase in methylmercury concentration for the circuit with MerR present. For the control with PmerT, transcription is initiated but at a lower level then the circuit to be tested. The plot of fluorescence and methylmercury concentration is taken. Exponential increase in fluorescence with an increase in methylmercury concentration in the case of the given circuit. The plot indicates that MerR contributes significantly in methylmercury response. Without MerR, even in the absence of methylmercury, transcription of downstream genes may occur with the help of PmerT.
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==Circuit==
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References:
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'''Constitutive Promoter – RBS – MerR - PmerT promoter – GFP - Double Terminator'''
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==Usage and Biology==
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MerR is a homodimer and the regulatory factor of the mer operon. It acts as an activator in the presence of Hg (II) and a weak repressor in its absence, maintaining its expression at a certain level. It binds to the operator site between –35 and –10 elements of mercury inducible promoter PmerT, independent of the presence of mercury. The Hg- bound MerR dimer causes a structural distortion of PmerT, which allows the RNA polymerase to bind, leading to the transcription of genes downstream to it (Hobman, J. L., Wilkie, J., & Brown, N. L. 2005).
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==Proposed experimentation==
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The response of MerR to methylmercury using this circuit vs 2 control circuits which do not have the MerR gene present in them must be tested. E. coli cells inoculated with methylmercury chloride must be grown for the required amount of time according to the results of the preliminary experiment. GFP intensities should be mapped, where an exponential increase in fluorescence with an increase in methylmercury concentration for the circuit with MerR present is expected.  
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For the control with PmerT, transcription is expected to be initiated but at a significantly lower level than the circuit to be tested. The plot of fluorescence and methylmercury concentration must be drawn. It is expected that there would be an exponential increase in fluorescence with an increase in methylmercury concentration in the case of the given circuit. The plot is expected to indicate that MerR contributes significantly to methylmercury response. However, without MerR, even in the absence of methylmercury, transcription of downstream genes is expected to occur in significantly low amounts with the help of PmerT.
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==Sequence and features==
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<partinfo>BBa_K3470006 SequenceAndFeatures</partinfo>
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==References==
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Brown, N. L., Stoyanov, J. V., Kidd, S. P., & Hobman, J. L. (2003). The MerR family of transcriptional regulators. FEMS Microbiology Reviews, 27(2–3), 145–163. https://doi.org/10.1016/S0168-6445(03)00051-2  
 
Brown, N. L., Stoyanov, J. V., Kidd, S. P., & Hobman, J. L. (2003). The MerR family of transcriptional regulators. FEMS Microbiology Reviews, 27(2–3), 145–163. https://doi.org/10.1016/S0168-6445(03)00051-2  
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Hobman, J. L., Wilkie, J., & Brown, N. L. (2005). A design for life: Prokaryotic metal-binding MerR family regulators. BioMetals, 18(4), 429–436. https://doi.org/10.1007/s10534-005-3717-7
 
Hobman, J. L., Wilkie, J., & Brown, N. L. (2005). A design for life: Prokaryotic metal-binding MerR family regulators. BioMetals, 18(4), 429–436. https://doi.org/10.1007/s10534-005-3717-7

Latest revision as of 15:54, 24 October 2020

Methylmercury control system

Circuit

Constitutive Promoter – RBS – MerR - PmerT promoter – GFP - Double Terminator

Usage and Biology

MerR is a homodimer and the regulatory factor of the mer operon. It acts as an activator in the presence of Hg (II) and a weak repressor in its absence, maintaining its expression at a certain level. It binds to the operator site between –35 and –10 elements of mercury inducible promoter PmerT, independent of the presence of mercury. The Hg- bound MerR dimer causes a structural distortion of PmerT, which allows the RNA polymerase to bind, leading to the transcription of genes downstream to it (Hobman, J. L., Wilkie, J., & Brown, N. L. 2005).

Proposed experimentation

The response of MerR to methylmercury using this circuit vs 2 control circuits which do not have the MerR gene present in them must be tested. E. coli cells inoculated with methylmercury chloride must be grown for the required amount of time according to the results of the preliminary experiment. GFP intensities should be mapped, where an exponential increase in fluorescence with an increase in methylmercury concentration for the circuit with MerR present is expected.

For the control with PmerT, transcription is expected to be initiated but at a significantly lower level than the circuit to be tested. The plot of fluorescence and methylmercury concentration must be drawn. It is expected that there would be an exponential increase in fluorescence with an increase in methylmercury concentration in the case of the given circuit. The plot is expected to indicate that MerR contributes significantly to methylmercury response. However, without MerR, even in the absence of methylmercury, transcription of downstream genes is expected to occur in significantly low amounts with the help of PmerT.

Sequence and features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1231


References

Brown, N. L., Stoyanov, J. V., Kidd, S. P., & Hobman, J. L. (2003). The MerR family of transcriptional regulators. FEMS Microbiology Reviews, 27(2–3), 145–163. https://doi.org/10.1016/S0168-6445(03)00051-2

Hobman, J. L., Wilkie, J., & Brown, N. L. (2005). A design for life: Prokaryotic metal-binding MerR family regulators. BioMetals, 18(4), 429–436. https://doi.org/10.1007/s10534-005-3717-7