Difference between revisions of "Part:BBa K1420000"

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<partinfo>BBa_K1420000 short</partinfo>
 
<partinfo>BBa_K1420000 short</partinfo>
  
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== Function and Components of the ''mer'' operon ==
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== Overview and Molecular Function ==
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<p>The ''mer'' operon is a set of genes that function in synchrony to convey mercury resistance to bacterial cells. For our purposes the operon can be used to bioremediate methylmercury by converting it to its less toxic ionic form, Hg(II), and then into volatile Hg(0). While variations in the operon exist, with not all genes being present in all organisms and some having extra genes, the operon we have focused on contains five genes: ''merR, merT, merP, merA'' and ''merB.''</p>
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<p>The ''mer'' operon is a set of genes that function in synchrony to convey mercury resistance to bacterial cells. For our purposes the operon can be used to bioremediate methylmercury by converting it to its less toxic ionic form, Hg(II), and then into volatile Hg(0). While variations in the operon exist, with not all genes being present in all organisms and some having extra genes, the operon we have focused on contains five genes: ''merR, merT, merP, merA'' and ''merB.''(For more information on the each individual gene of the ''mer'' operon, see the following parts pages: BBa_K1420001 for ''merA'', BBa_K1420002 for ''merB'', BBa_K1420003 for ''merP'', BBa_K1420004 for ''merR'', and BBa_K1420005 for ''merT''.)</p>
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<p>''merR'', located upstream of the rest of the mer operon mercury resistance genes, serves to regulate the mer operon by activating transcription in the presence of Hg(II) and acting as a weak repressor in the absence of Hg(II).  The effector binding region of MerR family proteins can vary allowing for great diversity in MerR-like promoters that can respond to a variety of heavy metals as well as antibiotics and oxidative stress. This variable nature of MerR family proteins makes them a valuable tool for various heavy metal detection and bioremediation. Once transcription is activated, the ''mer T'' and ''merP'' genes encode for proteins that work collectively to transport Hg(II) species into the cell. ''merP'' encodes the periplasmic transport protein, MerP, which binds a single Hg(II) ion at two conserved cysteine residues that define its metal binding motif. The MerP cysteine residues take up a Hg(II) ion and remove any attached ligands before passing the ion to the MerT transmembrane protein. ''merT'' on the other hand encodes a transmembrane mercuric binding enzyme, MerT, which transports Hg(II) species from the periplasm through the membrane. Hg(II) is transferred from the periplasmic cysteine pair on the first transmembrane helix to the cytoplasmic loop cysteine, where it is finally transferred to a cysteine pair at the N-terminus of the protein. Once the Hg(II) species are in the cytoplasm, the ''merB'' gene, which is often found immediately downstream of ''merA'', is essential for the detoxification and bioremediation of organic toxic mercury compounds in congruence with ''merA''. The MerB enzyme is a lyase that catalyzes the breaking of carbon-mercury bonds through protonolysis of toxic mercury compounds, such as methylmercury. This produces the less toxic and less mobile Hg<sup>2+</sup> which is then completely volatilized to Hg<sup>0</sup>.  Lastly, enzyme MerA catalyzes the reduction of the mercuric ion, Hg<sup>0</sup>, to the relative inert, volatile monoatomic mercury in a NADPH dependent reaction. (For more information on the each individual gene of the ''mer'' operon, see the following parts pages: BBa_K1420001 for ''merA'', BBa_K1420002 for ''merB'', BBa_K1420003 for ''merP'', BBa_K1420004 for ''merR'', and BBa_K1420005 for ''merT''.)</p>
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<p>''merR'', located upstream of the rest of the mer operon mercury resistance genes, serves to regulate the mer operon by activating transcription in the presence of Hg(II) and acting as a weak repressor in the absence of Hg(II).  The effector binding region of MerR family proteins can vary allowing for great diversity in MerR-like promoters that can respond to a variety of heavy metals as well as antibiotics and oxidative stress. This variable nature of MerR family proteins makes them a valuable tool for various heavy metal detection and bioremediation. Once transcription is activated, the ''mer T'' and ''merP'' genes encode for proteins that work collectively to transport Hg(II) species into the cell. ''merP'' encodes the periplasmic transport protein, MerP, which binds a single Hg(II) ion at two conserved cysteine residues that define its metal binding motif. The MerP cysteine residues take up a Hg(II) ion and remove any attached ligands before passing the ion to the MerT transmembrane protein. ''merT'' on the other hand encodes a transmembrane mercuric binding enzyme, MerT, which transports Hg(II) species from the periplasm through the membrane. Hg(II) is transferred from the periplasmic cysteine pair on the first transmembrane helix to the cytoplasmic loop cysteine, where it is finally transferred to a cysteine pair at the N-terminus of the protein. Once the Hg(II) species are in the cytoplasm, the ''merB'' gene, which is often found immediately downstream of ''merA'', is essential for the detoxification and bioremediation of organic toxic mercury compounds in congruence with ''merA''. The MerB enzyme is a lyase that catalyzes the breaking of carbon-mercury bonds through protonolysis of toxic mercury compounds, such as methylmercury. This produces the less toxic and less mobile Hg<sup>2+</sup> which is then completely volatilized to Hg<sup>0</sup>.  Lastly, enzyme MerA catalyzes the reduction of the mercuric ion, Hg<sup>0</sup>, to the relative inert, volatile monoatomic mercury in a NADPH dependent reaction. </p>
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== Construction of the ''mer'' operon ==
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<p>The construct used for our iGEM project was assembled from the ''mer'' operon in ''Serratia marscecens'' in the plasmid pDU1358, which was designed to contain an upstream regulatory gene merR, two transport proteins merP (periplasmic) and merT (transmembrane), a gene encoding mercuric reductase MerA, and finally a gene encoding organomercurial lyase MerB. A diagram of how the construct was inserted into a BioBrick compatible vector is shown in ''Figure 1''. This system is regulated by a bidirectional promoter so that merR on one side of the operon is constitutively expressed and allows for the repression of the mer operon in the absence of mercury ions, and the downstream activation and transcription of merT, P, A, B when mercury ions are in close proximity. MerT and MerP were selected as transporters for their high turnover rates to bring in mercury ions, which are subsequently bound by MerA to catalyze their conversion into volatile mercury eventually captured within a carbon filter in our device and disposed of sustainably. The organic and more toxic form, methylmercury, can diffuse into the cytosol of the bacteria where MerB catalyzes its conversion into mercury ions, which are then bound to MerA and converted into less toxic, volatile elemental mercury in an NADP dependent reaction. The system is very tightly regulated and allows for continuous turnover within our bacterial chassis as the mercury ions are volatalized and then captured externally rather than sequestered within our bacteria which would eventually lead to cell death and the requirement to replace the cells.</p>
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== Mechanism ==
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<p></p>
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[[File:Ecolimer.JPG|880px|]]
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== Construction of the pBBRBB::''mer'' plasmid ==
 
<p></p>
 
<p></p>
 
[[File:mer_operon_assembly.JPG|880px|]]
 
[[File:mer_operon_assembly.JPG|880px|]]
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<p>''Figure 1.'' BioBrick assembly of the ''mer'' operon. </p>
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<p>''Figure 1.''</p>
 
<p></p>
 
<p></p>
  

Revision as of 21:43, 17 October 2014

Mer operon, biological system found to detoxify organic and inorganic forms of mercury

Overview and Molecular Function

The mer operon is a set of genes that function in synchrony to convey mercury resistance to bacterial cells. For our purposes the operon can be used to bioremediate methylmercury by converting it to its less toxic ionic form, Hg(II), and then into volatile Hg(0). While variations in the operon exist, with not all genes being present in all organisms and some having extra genes, the operon we have focused on contains five genes: merR, merT, merP, merA and merB.(For more information on the each individual gene of the mer operon, see the following parts pages: BBa_K1420001 for merA, BBa_K1420002 for merB, BBa_K1420003 for merP, BBa_K1420004 for merR, and BBa_K1420005 for merT.)

merR, located upstream of the rest of the mer operon mercury resistance genes, serves to regulate the mer operon by activating transcription in the presence of Hg(II) and acting as a weak repressor in the absence of Hg(II). The effector binding region of MerR family proteins can vary allowing for great diversity in MerR-like promoters that can respond to a variety of heavy metals as well as antibiotics and oxidative stress. This variable nature of MerR family proteins makes them a valuable tool for various heavy metal detection and bioremediation. Once transcription is activated, the mer T and merP genes encode for proteins that work collectively to transport Hg(II) species into the cell. merP encodes the periplasmic transport protein, MerP, which binds a single Hg(II) ion at two conserved cysteine residues that define its metal binding motif. The MerP cysteine residues take up a Hg(II) ion and remove any attached ligands before passing the ion to the MerT transmembrane protein. merT on the other hand encodes a transmembrane mercuric binding enzyme, MerT, which transports Hg(II) species from the periplasm through the membrane. Hg(II) is transferred from the periplasmic cysteine pair on the first transmembrane helix to the cytoplasmic loop cysteine, where it is finally transferred to a cysteine pair at the N-terminus of the protein. Once the Hg(II) species are in the cytoplasm, the merB gene, which is often found immediately downstream of merA, is essential for the detoxification and bioremediation of organic toxic mercury compounds in congruence with merA. The MerB enzyme is a lyase that catalyzes the breaking of carbon-mercury bonds through protonolysis of toxic mercury compounds, such as methylmercury. This produces the less toxic and less mobile Hg2+ which is then completely volatilized to Hg0. Lastly, enzyme MerA catalyzes the reduction of the mercuric ion, Hg0, to the relative inert, volatile monoatomic mercury in a NADPH dependent reaction.

Mechanism

Ecolimer.JPG

Construction of the pBBRBB::mer plasmid

Mer operon assembly.JPG

Figure 1.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 551
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2420
    Illegal NgoMIV site found at 2468
    Illegal NgoMIV site found at 2530
    Illegal NgoMIV site found at 2741
    Illegal NgoMIV site found at 3334
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 536