Difference between revisions of "Part:BBa K1509000:Design"

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===Design Notes===
 
===Design Notes===
−
According to the mechanisms of smt locus represented above, gene smtB should function with the accompany of smt operator-promoter. Previous cases selected smtA whose product functions by encoding MT-like to sequester metal ions, while our case intended to take advantage of the trigger lies in this locus. By imitating former researches, in which a promoter less lacZ gene was fused upstream of smtA, we respectively splice two selected reporter genes and sequence from smt operator-promoter to smtB, equivalent to replacing smtA with reporter genes, to visualize the effects of metal ions on the locus.
+
According to the mechanisms of smt locus represented above, gene smtB should function with smt operator-promoter together. Previous cases selected smtA whose product functions by encoding MT-like to sequester metal ions, while our case intended to take advantage of the trigger lies in this locus. By imitating former researches, in which a promoter less lacZ gene was fused upstream of smtA, we respectively splice two selected reporter genes and sequence from smt operator-promoter to smtB, equivalent to replacing smtA with reporter genes, to visualize the effects of metal ions on the locus.
  
 
===Source===
 
===Source===
 
Metallothioneins(MTs), discovered in horse kidney in 1957 by Margoshes and Vallee are identified as low-molecular weight and sulphydryl rich proteins which bind metal ions in metal-thiolate clusters and whose synthesis increases in response to elevated concentrations of certain metals.
 
Metallothioneins(MTs), discovered in horse kidney in 1957 by Margoshes and Vallee are identified as low-molecular weight and sulphydryl rich proteins which bind metal ions in metal-thiolate clusters and whose synthesis increases in response to elevated concentrations of certain metals.
−
In 1988, Olafson and co-workers reported the amino acid sequence of a metal-associated protein isolated from cyanobacterium Synechococcus sp.. With the function of binding metal ions remaining, it only has less than 20% homology in amino acid sequence with known MTs in eukaryotes and thus we call it class II MT or on some occasions MT-like.  
+
In 1988, Olafson and co-workers reported the amino acid sequence of a metal-associated protein isolated from cyanobacterium ''Synechococcus sp.''. With the function of binding metal ions remaining, it only has less than 20% homology in amino acid sequence with known MTs in eukaryotes and thus we call it class II MT or on some occasions MT-like.  
−
Owing to the works of Robinson and Huckle, the corresponding locus of Synechococcus PCC7942, smt locus was isolated and analyzed. It includes smtA which encodes the class II MT and a divergently transcribed gene smtB which encodes a trans-acting, autoregulatory repressor that exerts metal ion-inducible negative control over smtA transcription. Between them lies a 100-bp operator-promoter region(smt O-P, BBa_K1509001), which is responsible for the divergent orientation of smtA and smtB.
+
Owing to the works of Robinson and Huckle, the corresponding locus of ''Synechococcus elongates'' PCC7942, smt locus was isolated and analyzed. It includes smtA which encodes the class II MT and a divergently transcribed gene smtB which encodes a trans-acting, autoregulatory repressor that exerts metal ion-inducible negative control over smtA transcription. Between them lies a 100-bp operator-promoter region(smt O-P, BBa_K1509001), which is responsible for the divergent orientation of smtA and smtB.
  
 
===References===
 
===References===
 +
 +
1.Busenlehner,  L.S., M.A. Pennella, and D.P. Giedroc, <em>The  SmtB/ArsR family of metalloregulatory transcriptional repressors: Structural  insights into prokaryotic metal resistance.</em> FEMS Microbiol Rev, 2003. <strong>27</strong>(2-3): p. 131-43.
 +
 +
2.Robinson,  N.J., S.K. Whitehall, and J.S. Cavet, <em>Microbial  metallothioneins.</em> Adv Microb Physiol, 2001. <strong>44</strong>: p. 183-213.
 +
 +
3.Erbe,  J.L., K.B. Taylor, and L.M. Hall, <em>Metalloregulation  of the cyanobacterial smt locus: identification of SmtB binding sites and  direct interaction with metals.</em> Nucleic Acids Res, 1995. <strong>23</strong>(13): p. 2472-8.
 +
 +
4.VanZile,  M.L., X. Chen, and D.P. Giedroc, <em>Allosteric  negative regulation of smt O/P binding of the zinc sensor, SmtB, by metal ions:  a coupled equilibrium analysis.</em> Biochemistry, 2002. <strong>41</strong>(31): p. 9776-86.
 +
 +
5.Morby,  A.P., et al., <em>SmtB is a metal-dependent  repressor of the cyanobacterial metallothionein gene smtA: identification of a  Zn inhibited DNA-protein complex.</em> Nucleic Acids Res, 1993. <strong>21</strong>(4): p. 921-5.
 +
 +
6.Huckle,  J.W., et al., <em>Isolation of a prokaryotic  metallothionein locus and analysis of transcriptional control by trace metal  ions.</em> Mol Microbiol, 1993. <strong>7</strong>(2):  p. 177-87.
 +
 +
7.VanZile,  M.L., X. Chen, and D.P. Giedroc, <em>Structural  characterization of distinct alpha3N and alpha5 metal sites in the  cyanobacterial zinc sensor SmtB.</em> Biochemistry, 2002. <strong>41</strong>(31): p. 9765-75.
 +
 +
8.Kar,  S.R., et al., <em>The cyanobacterial  repressor SmtB is predominantly a dimer and binds two Zn<sup>2+</sup> ions per subunit.</em> Biochemistry, 1997. <strong>36</strong>(49): p.  15343-8.
 +
 +
9.Cook,  W.J., et al., <em>Crystal structure of the  cyanobacterial metallothionein repressor SmtB: a model for metalloregulatory  proteins.</em> J Mol Biol, 1998. <strong>275</strong>(2):  p. 337-46.
 +
 +
10.Busenlehner, L.S., et al., <em>Spectroscopic properties of the  metalloregulatory Cd(II) and Pb(II) sites of S. aureus pI258 CadC.</em> Biochemistry, 2001. <strong>40</strong>(14): p.  4426-36.

Latest revision as of 01:36, 18 October 2014


Coding for the trans-acting regulator SmtB


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 349
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 260
    Illegal PstI site found at 349
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 349
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 349
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

According to the mechanisms of smt locus represented above, gene smtB should function with smt operator-promoter together. Previous cases selected smtA whose product functions by encoding MT-like to sequester metal ions, while our case intended to take advantage of the trigger lies in this locus. By imitating former researches, in which a promoter less lacZ gene was fused upstream of smtA, we respectively splice two selected reporter genes and sequence from smt operator-promoter to smtB, equivalent to replacing smtA with reporter genes, to visualize the effects of metal ions on the locus.

Source

Metallothioneins(MTs), discovered in horse kidney in 1957 by Margoshes and Vallee are identified as low-molecular weight and sulphydryl rich proteins which bind metal ions in metal-thiolate clusters and whose synthesis increases in response to elevated concentrations of certain metals. In 1988, Olafson and co-workers reported the amino acid sequence of a metal-associated protein isolated from cyanobacterium Synechococcus sp.. With the function of binding metal ions remaining, it only has less than 20% homology in amino acid sequence with known MTs in eukaryotes and thus we call it class II MT or on some occasions MT-like. Owing to the works of Robinson and Huckle, the corresponding locus of Synechococcus elongates PCC7942, smt locus was isolated and analyzed. It includes smtA which encodes the class II MT and a divergently transcribed gene smtB which encodes a trans-acting, autoregulatory repressor that exerts metal ion-inducible negative control over smtA transcription. Between them lies a 100-bp operator-promoter region(smt O-P, BBa_K1509001), which is responsible for the divergent orientation of smtA and smtB.

References

1.Busenlehner, L.S., M.A. Pennella, and D.P. Giedroc, The SmtB/ArsR family of metalloregulatory transcriptional repressors: Structural insights into prokaryotic metal resistance. FEMS Microbiol Rev, 2003. 27(2-3): p. 131-43.

2.Robinson, N.J., S.K. Whitehall, and J.S. Cavet, Microbial metallothioneins. Adv Microb Physiol, 2001. 44: p. 183-213.

3.Erbe, J.L., K.B. Taylor, and L.M. Hall, Metalloregulation of the cyanobacterial smt locus: identification of SmtB binding sites and direct interaction with metals. Nucleic Acids Res, 1995. 23(13): p. 2472-8.

4.VanZile, M.L., X. Chen, and D.P. Giedroc, Allosteric negative regulation of smt O/P binding of the zinc sensor, SmtB, by metal ions: a coupled equilibrium analysis. Biochemistry, 2002. 41(31): p. 9776-86.

5.Morby, A.P., et al., SmtB is a metal-dependent repressor of the cyanobacterial metallothionein gene smtA: identification of a Zn inhibited DNA-protein complex. Nucleic Acids Res, 1993. 21(4): p. 921-5.

6.Huckle, J.W., et al., Isolation of a prokaryotic metallothionein locus and analysis of transcriptional control by trace metal ions. Mol Microbiol, 1993. 7(2): p. 177-87.

7.VanZile, M.L., X. Chen, and D.P. Giedroc, Structural characterization of distinct alpha3N and alpha5 metal sites in the cyanobacterial zinc sensor SmtB. Biochemistry, 2002. 41(31): p. 9765-75.

8.Kar, S.R., et al., The cyanobacterial repressor SmtB is predominantly a dimer and binds two Zn2+ ions per subunit. Biochemistry, 1997. 36(49): p. 15343-8.

9.Cook, W.J., et al., Crystal structure of the cyanobacterial metallothionein repressor SmtB: a model for metalloregulatory proteins. J Mol Biol, 1998. 275(2): p. 337-46.

10.Busenlehner, L.S., et al., Spectroscopic properties of the metalloregulatory Cd(II) and Pb(II) sites of S. aureus pI258 CadC. Biochemistry, 2001. 40(14): p. 4426-36.