Team:Peking/Project/Expansion

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<font size=6><font color=#585858><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;Expansion</font></font></font>
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<font size=6><font color=#585858><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;EXPANSION</font></font></font>
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<a href="https://2010.igem.org/Team:Peking/Project/Expansion"><font size=4><b><font color=#000000>----Introduction----</font></font></b></a>
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<a href="https://2010.igem.org/Team:Peking/Project/Expansion/LeadBiosensor"><font size=3><font color=#000000>*Lead Biosensor</font></font></a>
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<a href="https://2010.igem.org/Team:Peking/Project/Expansion/LeadBioabsorbent"><font size=2><font color=#000000>*Lead Bioabsorbent </font></font></a>
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<a href="https://2010.igem.org/Team:Peking/Project/Expansion/LeadBioabsorbent"><font size=3><font color=#000000>*Lead Bioabsorbent </font></font></a>
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<a href="https://2010.igem.org/Team:Peking/Project/Expansion/KitOperation"><font size=3><font color=#000000>*Kit Operation </font></font></a>
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<font size=6><font color=#585858><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;INTRODUCTION</font></font></font>
<font size=6><font color=#585858><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;INTRODUCTION</font></font></font>
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[[Team:Peking/Project|Project]] > [[Team:Peking/Project/Expansion|Expansion]] <html>
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==PbrR-based lead bioabsorbent==
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=Expansion Introduction=
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After the completion of MerR-based decontamination kit, we decided to take another example to verify the validness of our design. Based on the previous homology study of MerR family proteins, we took a lead-sensing protein, PbrR, as the second research target.
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&nbsp;&nbsp;&nbsp;&nbsp;The MerR family of transcription activators includes those that respond to the presence of essential and toxic metals; and those that respond to the presence of drugs and other chemical species which cause cellular damage(Brown et al., 2003). The bacterial metal sensing MerR regulators directly bind metals and then activate transcription from unusual promoters as a result of metal binding. A recent phylogenetic analysis of the predicted protein sequences of metal responsive MerR regulators (COG0789) classifies them into several groups – a subgroup of which sense Hg2+ (MerR), Cu+ (CueR/SctR), Cd2+ (CadR), Pb2+ (PbrR), or Zn2+ (ZntR) with high specificity (Fig 1) etc. (Permina et al., 2006).
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Lead contamination is a serious threat to human health and the environment. Lead poisoning is still one of the most common environmentally caused diseases in the world today.[1] As the concentration of such toxic ions is generally low, which present a huge challenge for environmental engineers to both detect and to absorb the pollutant with traditional chemical methods. A revolutionary strategy was taken into consideration, which took the advantage of metalloregulatory proteins with capability of sensing and absorbing the Pb (II) ions.
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Nature has evolved numerous such regulating proteins to control the concentrations of beneficial or toxic metal ions with extraordinary sensitivity and selectivity.[1] As is known, the MerR family is a group of transcriptional activators with similar N-terminal helix-turn-helix DNA binding regions and C-terminal effector binding regions that are specific to the effector recognized.[2] The majority of regulators in the family respond to environmental stimuli, such as oxidative stress, heavy metals or antibiotics. A subgroup of the family activates transcription in response to metal ions. This subgroup shows sequence similarity in the C-terminal effector binding region as well as in the N-terminal region. PbrR is a MerR family protein found in Ralstonia metallidurans CH34, a bacterium specifically adapted to survive under toxic heavy metal environment. The PbrR protein is responsible for regulation of lead (II) efflux pumps involved in lead detoxification inside R. metallidurans.
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Due to the high homology of protein MerR and PbrR (Fig 1), we were able to apply the strategy used for MerR construct to the development of lead bioabsorbent. Based on the crystal structure study of MerR, the metal binding domain of PbrR was recognized by sequence alignment with MerR.[3] (Fig 2)Then we designed PbrR metal binding peptide, with consisted of two tandem duplications of α-helix 5 linked by three amino acids, SSG, and followed by a short peptide sequence.(Fig 3) These direct tandem α-helices fold back on each other into an antiparallel, coiled-coil hairpin. Three Cys-residues located at 79,114,123 specifically binds Pb(II) ions by forming disulfide bonds within the engineered dimer.
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<html><img src="https://static.igem.org/mediawiki/2010/4/4d/LeadintroFig1.jpg"></html>
 
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Figure 1 Sequence alignment of MerR and PbrR
 
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<html><img src="https://static.igem.org/mediawiki/2010/3/3a/Intro1.jpg" alt="Selective metal ion recognition by metalloregulatory proteins." id="imggrey"></html>
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Figure 2 Pb binding domain predicted by sequence alignment
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<br>'''Fig 1. Selective metal ion recognition by metalloregulatory proteins. Adapted from Peng Chen and Chuan He, 2008.'''
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<html><img src="https://static.igem.org/mediawiki/2010/f/f7/LeadintroFig3.jpg"></html>
 
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Figure 3 Design of PbrR metal binding peptide and structure prediction
 
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&nbsp;&nbsp;&nbsp;&nbsp;It is notable that the MerR family is a group of transcriptional activators with similar N-terminal helix-turn-helix DNA binding regions and C-terminal effector binding regions that are specific to the effector recognized. Interestingly, the subgroup shows sequence similarity in the C-terminal effector binding region as well as in the N-terminal region, but it is not yet clear how metal discrimination occurs (Fig 2 and Fig 3). This subgroup of MerR family regulators includes MerR itself and may have evolved to generate a variety of specific metal-responsive regulators by fine-tuning the sites of metal recognition.
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<html><a href="https://static.igem.org/mediawiki/2010/1/12/Intro2.jpg"target="_blank"><img src="https://static.igem.org/mediawiki/2010/1/12/Intro2.jpg" width=90% alt="Sequence alignment of MerR and PbrR."id="imggrey"></a></html>
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<br>'''Figure 2 Sequence alignment of MerR and PbrR. MerR family TFs share a highly conserved homology at their metal binding domains (Brown et al., 2003; Hobman, 2007), which implies that our strategies of bioabsorbent engineering might be applicable to other cases of heavy metals.'''
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<html><a href="https://static.igem.org/mediawiki/2010/4/4e/MerR.swf" target="_blank"><embed src="https://static.igem.org/mediawiki/2010/4/4e/MerR.swf" width=49% alt="MerR"id="imggrey"></a>
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<embed src="https://static.igem.org/mediawiki/2010/6/64/PbrR.swf" width=49% alt="PbrR"id="imggrey"target="_blank"></html>
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<br>'''Figure 3 3D structure modeling of 2 archetypes of MerR family regulators, MerR(left) and PbrR(right). Note that they comprise 2 domains, respectively, a metal binding domain at the C terminal and a DNA binding domain at the N-terminal, joined together by a interface domain. '''
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&nbsp;&nbsp;&nbsp;&nbsp;Inspired by the fact that MerR family TFs share a highly conserved homology at their metal binding domains (Brown et al., 2003; Hobman, 2007), we developed to engineer strategies of bioreporter and bioabsorbent which may be applicable to other cases. Therefore, we then expanded our reverse engineering strategy to another common toxic heavy metal, lead (Borremans et al., 2001; Chakraborty et al., 2008; Chen et al., 2005; Julian et al., 2009; Mergeay et al., 2003). We primarily took lead resistance operon apart, followed by analyze of function, structure and operation of PbrR, the lead responsive regulator. Then modeling was conducted to analyze the characteristics of PbrR and topology of its regulation behavior. Information collected confirmed the possibility to design genetic circuit that makes lead sensor and regulator components more efficient and robuster. We also engineered PbrR into single-chain coiled coil (MBD) via the same method as MerR. Exhilaratingly, following expression of PbrR MBD on the surface, periplasm and in cytosol of bacteria and the lead (II) absorption test showed that our bacteria can absorb more than 50% of 10^-6M Pb (II) in 2 hours, which is comparable to mercury MBD expression in E.coli, proving validness of our engineering strategy. <br>
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==reference==
 
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[1] Peng Chen, Bill Greenberg, Safiyh Taghavi, Christine Romano, Daniel van der Lelie, and Chuan He, An exceptionally selective lead (II)-regulatory protein from Ralstonia metallidurans: development of a fluorescent lead (II) probe, Angew. Chem. 117, 2005,  2775 –2779.
 
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[2] Nigel L. Brown, Jivko V. Stoyanov, Stephen P. Kidd, Jon L. Hobman, The MerR family of transcriptional regulators, FEMS Microbiology Reviews, 27, 2003, 145-163.
 
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[3] Lingyun Song, Jonathan Caguiat, Zhongrui Li, Jacob Shokes, Robert A. Scott, Lynda Olliff, and Anne O. Summers, Engineered Single-Chain, Antiparallel, Coiled Coil Mimics the MerR Metal Binding Site, Journal of Bacteriology, 186(6), 2004,  1861–1868.
 
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=Reference=
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Borremans, B., Hobman, J.L., Provoost, A., Brown, N.L., and van Der Lelie, D. (2001). Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34. J Bacteriol 183, 5651-5658.<br>Brown, N.L., Stoyanov, J.V., Kidd, S.P., and Hobman, J.L. (2003). The MerR family of transcriptional regulators. FEMS Microbiol Rev 27, 145-163.<br>Chakraborty, T., Babu, P.G., Alam, A., and Chaudhari, A. (2008). GFP expressing bacterial biosensor to measure lead contamination in aquatic environment. Current Science 94, 800-805.<br>Chen, P., Greenberg, B., Taghavi, S., Romano, C., van der Lelie, D., and He, C. (2005). An exceptionally selective lead(II)-regulatory protein from Ralstonia metallidurans: development of a fluorescent lead(II) probe. Angew Chem Int Ed Engl 44, 2715-2719.<br>Hobman, J.L. (2007). MerR family transcription activators: similar designs, different specificities. Mol Microbiol 63, 1275-1278.<br>Julian, D.J., Kershaw, C.J., Brown, N.L., and Hobman, J.L. (2009). Transcriptional activation of MerR family promoters in Cupriavidus metallidurans CH34. Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology 96, 149-159<br>Mergeay, M., Monchy, S., Vallaeys, T., Auquier, V., Benotmane, A., Bertin, P., Taghavi, S., Dunn, J., van der Lelie, D., and Wattiez, R. (2003). Ralstonia metallidurans, a bacterium specifically adapted to toxic metals: towards a catalogue of metal-responsive genes. Fems Microbiology Reviews 27, 385-410<br>Permina, E.A., Kazakov, A.E., Kalinina, O.V., and Gelfand, M.S. (2006). Comparative genomics of regulation of heavy metal resistance in Eubacteria. BMC Microbiol 6, 49.<br><br>
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Latest revision as of 17:36, 27 October 2010

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   EXPANSION

        
              *Lead Bioabsorbent


   INTRODUCTION


         Project > Expansion

Expansion Introduction

    The MerR family of transcription activators includes those that respond to the presence of essential and toxic metals; and those that respond to the presence of drugs and other chemical species which cause cellular damage(Brown et al., 2003). The bacterial metal sensing MerR regulators directly bind metals and then activate transcription from unusual promoters as a result of metal binding. A recent phylogenetic analysis of the predicted protein sequences of metal responsive MerR regulators (COG0789) classifies them into several groups – a subgroup of which sense Hg2+ (MerR), Cu+ (CueR/SctR), Cd2+ (CadR), Pb2+ (PbrR), or Zn2+ (ZntR) with high specificity (Fig 1) etc. (Permina et al., 2006).


Selective metal ion recognition by metalloregulatory proteins.
Fig 1. Selective metal ion recognition by metalloregulatory proteins. Adapted from Peng Chen and Chuan He, 2008.


    It is notable that the MerR family is a group of transcriptional activators with similar N-terminal helix-turn-helix DNA binding regions and C-terminal effector binding regions that are specific to the effector recognized. Interestingly, the subgroup shows sequence similarity in the C-terminal effector binding region as well as in the N-terminal region, but it is not yet clear how metal discrimination occurs (Fig 2 and Fig 3). This subgroup of MerR family regulators includes MerR itself and may have evolved to generate a variety of specific metal-responsive regulators by fine-tuning the sites of metal recognition.


Sequence alignment of MerR and PbrR.
Figure 2 Sequence alignment of MerR and PbrR. MerR family TFs share a highly conserved homology at their metal binding domains (Brown et al., 2003; Hobman, 2007), which implies that our strategies of bioabsorbent engineering might be applicable to other cases of heavy metals.



Figure 3 3D structure modeling of 2 archetypes of MerR family regulators, MerR(left) and PbrR(right). Note that they comprise 2 domains, respectively, a metal binding domain at the C terminal and a DNA binding domain at the N-terminal, joined together by a interface domain.


    Inspired by the fact that MerR family TFs share a highly conserved homology at their metal binding domains (Brown et al., 2003; Hobman, 2007), we developed to engineer strategies of bioreporter and bioabsorbent which may be applicable to other cases. Therefore, we then expanded our reverse engineering strategy to another common toxic heavy metal, lead (Borremans et al., 2001; Chakraborty et al., 2008; Chen et al., 2005; Julian et al., 2009; Mergeay et al., 2003). We primarily took lead resistance operon apart, followed by analyze of function, structure and operation of PbrR, the lead responsive regulator. Then modeling was conducted to analyze the characteristics of PbrR and topology of its regulation behavior. Information collected confirmed the possibility to design genetic circuit that makes lead sensor and regulator components more efficient and robuster. We also engineered PbrR into single-chain coiled coil (MBD) via the same method as MerR. Exhilaratingly, following expression of PbrR MBD on the surface, periplasm and in cytosol of bacteria and the lead (II) absorption test showed that our bacteria can absorb more than 50% of 10^-6M Pb (II) in 2 hours, which is comparable to mercury MBD expression in E.coli, proving validness of our engineering strategy.




Reference

Borremans, B., Hobman, J.L., Provoost, A., Brown, N.L., and van Der Lelie, D. (2001). Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34. J Bacteriol 183, 5651-5658.
Brown, N.L., Stoyanov, J.V., Kidd, S.P., and Hobman, J.L. (2003). The MerR family of transcriptional regulators. FEMS Microbiol Rev 27, 145-163.
Chakraborty, T., Babu, P.G., Alam, A., and Chaudhari, A. (2008). GFP expressing bacterial biosensor to measure lead contamination in aquatic environment. Current Science 94, 800-805.
Chen, P., Greenberg, B., Taghavi, S., Romano, C., van der Lelie, D., and He, C. (2005). An exceptionally selective lead(II)-regulatory protein from Ralstonia metallidurans: development of a fluorescent lead(II) probe. Angew Chem Int Ed Engl 44, 2715-2719.
Hobman, J.L. (2007). MerR family transcription activators: similar designs, different specificities. Mol Microbiol 63, 1275-1278.
Julian, D.J., Kershaw, C.J., Brown, N.L., and Hobman, J.L. (2009). Transcriptional activation of MerR family promoters in Cupriavidus metallidurans CH34. Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology 96, 149-159
Mergeay, M., Monchy, S., Vallaeys, T., Auquier, V., Benotmane, A., Bertin, P., Taghavi, S., Dunn, J., van der Lelie, D., and Wattiez, R. (2003). Ralstonia metallidurans, a bacterium specifically adapted to toxic metals: towards a catalogue of metal-responsive genes. Fems Microbiology Reviews 27, 385-410
Permina, E.A., Kazakov, A.E., Kalinina, O.V., and Gelfand, M.S. (2006). Comparative genomics of regulation of heavy metal resistance in Eubacteria. BMC Microbiol 6, 49.

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