Difference between revisions of "Part:BBa K3470017"

 
 
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<partinfo>BBa_K3470017 short</partinfo>
  
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==Circuit==
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'''Constitutive Promoter – RBS – MerR - PmerT promoter - (RBS – MerT – RBS – MerP – RBS – MerE – RBS - MerC)*- RBS – MerA – RBS – GFP - Double Terminator (Deletion of MerB)'''
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==Usage and Biology==
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MerA encodes the mercury reductase enzyme. It reduces Hg (II) to relatively inert and volatile Hg (0) in an NADPH dependent reaction. (Parks et al., 2009) .MerB encodes the organomercurial lyase enzyme and is usually found immediately downstream to MerA. It catalyzes breaking the bond between carbon and mercury through the protonolysis of compounds such as methylmercury. This produces the less mobile Hg (II) which is then reduced to Hg (0) by MerA. (Miki et al., 2008).
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==Proposed experimentation==
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Methylmercury concentrations in the presence and absence of MerA and MerB must be checked with 3 circuits.
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The first with presence of both MerA and MerB, the second and third with deletion of MerA and MerB respectively and the control with absence of both MerA and MerB. The team tested to see the increase in the Mer spectrum with the introduction of MerB and MerA to conclude that the addition of the two genes confer to a better resistance to methylmercury.
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MTT assay must be performed to map the resistance provided by each gene MerA and MerB.
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The principle of the MTT assay is that for most viable cells mitochondrial activity is constant and thereby an increase or decrease in the number of viable cells is linearly related to mitochondrial activity. Thus, any increase or decrease in viable cell number can be detected by measuring formazan concentration reflected in optical density (OD) using a plate reader at 540 and 720 nm. For drug sensitivity measurements, the OD values of wells with cells incubated with drugs are compared to the OD of wells with cells not exposed to drugs. (Van Meerloo, Kaspers and Cloos, 2011)
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The resistance provided by each gene must be quantitatively mapped using the graphs. The introduction of MerB and MerA is expected to increase the Mer spectrum. The resistance provided is expected to be in the order Control < Circuit 3 < Circuit 2 < Circuit 1. Hence it is hypothesized that the addition of the two genes confers better resistance to methylmercury.
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==Sequence and features==
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<partinfo>BBa_K3470017 SequenceAndFeatures</partinfo>
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==References==
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Parks, J. M., Guo, H., Momany, C., Liang, L., Miller, S. M., Summers, A. O., & Smith, J. C. (2009). Mechanism of Hg-C protonolysis in the organomercurial lyase MerB. Journal of the American Chemical Society, 131(37), 13278–13285. https://doi.org/10.1021/ja9016123
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van Meerloo, J., Kaspers, G. J., & Cloos, J. (2011). Cell sensitivity assays: the MTT assay. Methods in molecular biology (Clifton, N.J.), 731, 237–245. https://doi.org/10.1007/978-1-61779-080-5_20

Latest revision as of 14:06, 23 October 2020

Methylmercury breakdown (Deletion of MerB)

Circuit

Constitutive Promoter – RBS – MerR - PmerT promoter - (RBS – MerT – RBS – MerP – RBS – MerE – RBS - MerC)*- RBS – MerA – RBS – GFP - Double Terminator (Deletion of MerB)

Usage and Biology

MerA encodes the mercury reductase enzyme. It reduces Hg (II) to relatively inert and volatile Hg (0) in an NADPH dependent reaction. (Parks et al., 2009) .MerB encodes the organomercurial lyase enzyme and is usually found immediately downstream to MerA. It catalyzes breaking the bond between carbon and mercury through the protonolysis of compounds such as methylmercury. This produces the less mobile Hg (II) which is then reduced to Hg (0) by MerA. (Miki et al., 2008).

Proposed experimentation

Methylmercury concentrations in the presence and absence of MerA and MerB must be checked with 3 circuits.

The first with presence of both MerA and MerB, the second and third with deletion of MerA and MerB respectively and the control with absence of both MerA and MerB. The team tested to see the increase in the Mer spectrum with the introduction of MerB and MerA to conclude that the addition of the two genes confer to a better resistance to methylmercury.

MTT assay must be performed to map the resistance provided by each gene MerA and MerB.

The principle of the MTT assay is that for most viable cells mitochondrial activity is constant and thereby an increase or decrease in the number of viable cells is linearly related to mitochondrial activity. Thus, any increase or decrease in viable cell number can be detected by measuring formazan concentration reflected in optical density (OD) using a plate reader at 540 and 720 nm. For drug sensitivity measurements, the OD values of wells with cells incubated with drugs are compared to the OD of wells with cells not exposed to drugs. (Van Meerloo, Kaspers and Cloos, 2011)

The resistance provided by each gene must be quantitatively mapped using the graphs. The introduction of MerB and MerA is expected to increase the Mer spectrum. The resistance provided is expected to be in the order Control < Circuit 3 < Circuit 2 < Circuit 1. Hence it is hypothesized that the addition of the two genes confers better resistance to methylmercury.


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
    Illegal NheI site found at 652
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1878
    Illegal NgoMIV site found at 3162
    Illegal NgoMIV site found at 3210
    Illegal NgoMIV site found at 3272
    Illegal NgoMIV site found at 3483
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 4317
    Illegal SapI site found at 637

References

Parks, J. M., Guo, H., Momany, C., Liang, L., Miller, S. M., Summers, A. O., & Smith, J. C. (2009). Mechanism of Hg-C protonolysis in the organomercurial lyase MerB. Journal of the American Chemical Society, 131(37), 13278–13285. https://doi.org/10.1021/ja9016123

van Meerloo, J., Kaspers, G. J., & Cloos, J. (2011). Cell sensitivity assays: the MTT assay. Methods in molecular biology (Clifton, N.J.), 731, 237–245. https://doi.org/10.1007/978-1-61779-080-5_20