Difference between revisions of "Part:BBa K3470016"
 
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<partinfo>BBa_K3470016 short</partinfo>
  
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Constitutive Promoter – RBS – MerR - PmerT promoter - (RBS – MerT – RBS – MerP – RBS – MerE – RBS - MerC)*- RBS – MerB – RBS – GFP - Double Terminator (Deletion of MerA)
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==Circuit==
  
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'''Constitutive Promoter – RBS – MerR - PmerT promoter - (RBS – MerT – RBS – MerP – RBS – MerE – RBS - MerC)*- RBS – MerB – RBS – GFP - Double Terminator (Deletion of MerA)'''
  
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==Usage and Biology==
  
 
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).  
 
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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The team checked for methylmercury concentrations in the presence and absence of MerA and MerB 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. The team performed the MTT assay to map the resistance provided by each gene MerA and MerB.  
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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)  
 
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 team could quantitatively map the resistance provided by each gene using the graphs. The introduction of MerB and MerA increases the Mer spectrum. The resistance provided should be in the order Control<Circuit 3< Circuit 2< Circuit 1. Hence the addition of the two genes confers better resistance to methylmercury.
 
  
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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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References:
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<partinfo>BBa_K3470016 SequenceAndFeatures</partinfo>
  
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==References==
  
 
Barkay, T., Miller, S. M., & Summers, A. O. (2003). Bacterial mercury resistance from atoms to ecosystems.  
 
Barkay, T., Miller, S. M., & Summers, A. O. (2003). Bacterial mercury resistance from atoms to ecosystems.  
 
FEMS Microbiology Reviews, 27(2–3), 355–384. https://doi.org/10.1016/S0168-6445(03)00046-9  
 
FEMS Microbiology Reviews, 27(2–3), 355–384. https://doi.org/10.1016/S0168-6445(03)00046-9  
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Miki, K., Watanabe, S., Kita, A., & Kobayashi, K. (2008). Crystal structure of the [2Fe-2S] transcriptional activator SoxR bound to DNA. Acta Crystallographica Section A Foundations of Crystallography, 64(a1), C89–C89. https://doi.org/10.1107/s0108767308097122  
 
Miki, K., Watanabe, S., Kita, A., & Kobayashi, K. (2008). Crystal structure of the [2Fe-2S] transcriptional activator SoxR bound to DNA. Acta Crystallographica Section A Foundations of Crystallography, 64(a1), C89–C89. https://doi.org/10.1107/s0108767308097122  
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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-
 
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
 
61779-080-5_20

Latest revision as of 14:05, 23 October 2020

Methylmercury breakdown( Deletion of MerA)

Circuit

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

Usage and Biology

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 2376
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 3270
    Illegal SapI site found at 637

References

Barkay, T., Miller, S. M., & Summers, A. O. (2003). Bacterial mercury resistance from atoms to ecosystems. FEMS Microbiology Reviews, 27(2–3), 355–384. https://doi.org/10.1016/S0168-6445(03)00046-9

Miki, K., Watanabe, S., Kita, A., & Kobayashi, K. (2008). Crystal structure of the [2Fe-2S] transcriptional activator SoxR bound to DNA. Acta Crystallographica Section A Foundations of Crystallography, 64(a1), C89–C89. https://doi.org/10.1107/s0108767308097122

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