Difference between revisions of "IGEM:MIT/2007/Heavy Metals"

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==Heavy Metal Binding Proteins/Peptides==
 
==Heavy Metal Binding Proteins/Peptides==
 +
 +
 +
There are several classes of metal binding proteins to consider.  (for review, see Cobbett02, below)
 +
 +
First, those that bind to many metal types (Cd, Hg, Pb, Cu, Zn, etc).  These are nice in that they are multifunctional, but the disadvantage is that they will also bind non-toxic ions like Zn, thus decreasing their efficiency.
 +
 +
**Metallothioneins (MT's) -- small animal proteins which contain many cysteine motifs (usually CxxC)
 +
**Phytochelatins (PC's) -- plant peptides of the form (EC)n-G where n=# repeats (usually 2-10, but can be as many as 20) (see the Bae papers)
 +
 +
Also, certain artificially selected sequences bind selectively to certain metals. 
 +
**The MT-like motif CCAA was found to highly selective for Hg, while CAAC had broad specificity (see DeSilva02)
 +
**The motif (CGCCG)3 was found to bind both cadmium and mercury (see Pazirandeh98)
 +
 +
Seems that Mejare01 does a nice job of comparing the different Cd-binding methods which have been tried.  According to them, the methods which gave the biggest increase (>60-fold) in binding capacity were those that expressed entire MT proteins (or domains of them).  '''However, they did not consider the papers where PCs were used, which seem to have much higher binding capacities.'''
 +
 +
The Bae papers seem to indicate that the EC20 motif can bind both Cd and Hg (higher affinity for Hg), and the absolute accumulation is higher than for the MT-derived peptides.  Also, EC20 binding still works in the presence of up to 200mM salt and even EDTA.  Might be a good idea to ask them for their Lpp-OmpA-EC20 expression construct! 
 +
 +
 +
'''Binding Sequences To Try'''
 +
*The full Pazirandeh98 Hg/Cd-specific binding sequence (CGCCG) is:  CGCCGKGHCGCCGKGHCGCCG
 +
*For good non-specific binding, best bet seems to be the PC-derived EC20 sequence: (EC)20-Gly
 +
 +
*From DeSilva02, the MerP-derived Hg-specific binding sequence (GMTCCAA) could be repeated 3x:  GMTCCAAGMTCCAAGMTCCAA
 +
*For Cd-specific binding, could later use the CadA-derived sequence (GFTCANC)
 +
 +
 +
 +
 
<biblio>
 
<biblio>
 
#Samuelson00 pmid=10698802
 
#Samuelson00 pmid=10698802
Line 23: Line 51:
 
#Lu01 pmid=11710062
 
#Lu01 pmid=11710062
 
#Bae00 pmid=11042548
 
#Bae00 pmid=11042548
 +
#Bae01 pmid=11679366
 +
#Bae02 pmid=11803043
 +
#Kotrba99b pmid=10071794
 +
#Desilva02 pmid=12115136
 +
#Opella02 pmid=12039007
 +
#Sousa98 pmid=9573175
 +
#Mauro00 pmid=10736021
 +
#Cobbett02 pmid=12221971
 +
#Pazirandeh98 pmid=9758845
 +
#Chen98 pmid=9758654
 
</biblio>
 
</biblio>
  
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===Contamination Levels/Limits===
 
===Contamination Levels/Limits===
*MCL(Maximum Containment Level): 5 ppb
+
*MCL(Maximum Containment Level): 5 ppb (equal to about 45nM)
 
*MCLG(Maximum Containment Level Goal): 5 ppb
 
*MCLG(Maximum Containment Level Goal): 5 ppb
 
<biblio>
 
<biblio>
Line 62: Line 100:
 
==Mercury==
 
==Mercury==
 
===Contamination Levels/Limits===
 
===Contamination Levels/Limits===
*MCL: 2 ppb
+
*MCL: 2 ppb (equal to about 10 nM)
 
*MCLG: 2 ppb
 
*MCLG: 2 ppb
 
*20 ppb makes the fish mercury buildup significant
 
*20 ppb makes the fish mercury buildup significant
  
 
===Polluted sites===
 
===Polluted sites===
**Onondaga lake [http://www.aslf.org/OnondagaLake/] [http://www.aslf.org/ONONDAGALAKE/gallery1.html] [http://www.upstatefreshwater.org/html/onondaga_lake.html]
+
*Onondaga lake [http://www.aslf.org/OnondagaLake/] [http://www.aslf.org/ONONDAGALAKE/gallery1.html] [http://www.upstatefreshwater.org/html/onondaga_lake.html]
**Salt Plains National Wildlife Refuge [http://ojas.ucok.edu/03/paper/daniels.htm]
+
*Salt Plains National Wildlife Refuge [http://ojas.ucok.edu/03/paper/daniels.htm]
 
 
  
 
===Detection (to assess our system)===  
 
===Detection (to assess our system)===  

Latest revision as of 20:04, 13 July 2007

Reviews

  • Excellent Summary of Bacterial Metal Resistance (a must read)
  1. Silver S and Phung le T. A bacterial view of the periodic table: genes and proteins for toxic inorganic ions. J Ind Microbiol Biotechnol. 2005 Dec;32(11-12):587-605. DOI:10.1007/s10295-005-0019-6 | PubMed ID:16133099 | HubMed [Silver05]
  • A Good Overview on Pollution (though slightly back-dated): Quantitative assessment of worldwide contamination of air, water and soil by trace metals [1]
  • A summary of Pollutants from the EPA [2]
  • Review Papers on Bioremediation
  1. Paul D, Pandey G, Pandey J, and Jain RK. Accessing microbial diversity for bioremediation and environmental restoration. Trends Biotechnol. 2005 Mar;23(3):135-42. DOI:10.1016/j.tibtech.2005.01.001 | PubMed ID:15734556 | HubMed [Paul05]
  2. Valls M and de Lorenzo V. Exploiting the genetic and biochemical capacities of bacteria for the remediation of heavy metal pollution. FEMS Microbiol Rev. 2002 Nov;26(4):327-38. PubMed ID:12413663 | HubMed [Valls02]
  3. Bruins MR, Kapil S, and Oehme FW. Microbial resistance to metals in the environment. Ecotoxicol Environ Saf. 2000 Mar;45(3):198-207. DOI:10.1006/eesa.1999.1860 | PubMed ID:10702338 | HubMed [Bruins00]
All Medline abstracts: PubMed | HubMed
  • Paul - discusses advantages/disadvantages/problems of bioremediation
  • Bruins, Valls - discusses various kinds of bioremediation systems

Heavy Metal Binding Proteins/Peptides

There are several classes of metal binding proteins to consider. (for review, see Cobbett02, below)

First, those that bind to many metal types (Cd, Hg, Pb, Cu, Zn, etc). These are nice in that they are multifunctional, but the disadvantage is that they will also bind non-toxic ions like Zn, thus decreasing their efficiency.

    • Metallothioneins (MT's) -- small animal proteins which contain many cysteine motifs (usually CxxC)
    • Phytochelatins (PC's) -- plant peptides of the form (EC)n-G where n=# repeats (usually 2-10, but can be as many as 20) (see the Bae papers)

Also, certain artificially selected sequences bind selectively to certain metals.

    • The MT-like motif CCAA was found to highly selective for Hg, while CAAC had broad specificity (see DeSilva02)
    • The motif (CGCCG)3 was found to bind both cadmium and mercury (see Pazirandeh98)

Seems that Mejare01 does a nice job of comparing the different Cd-binding methods which have been tried. According to them, the methods which gave the biggest increase (>60-fold) in binding capacity were those that expressed entire MT proteins (or domains of them). However, they did not consider the papers where PCs were used, which seem to have much higher binding capacities.

The Bae papers seem to indicate that the EC20 motif can bind both Cd and Hg (higher affinity for Hg), and the absolute accumulation is higher than for the MT-derived peptides. Also, EC20 binding still works in the presence of up to 200mM salt and even EDTA. Might be a good idea to ask them for their Lpp-OmpA-EC20 expression construct!


Binding Sequences To Try

  • The full Pazirandeh98 Hg/Cd-specific binding sequence (CGCCG) is: CGCCGKGHCGCCGKGHCGCCG
  • For good non-specific binding, best bet seems to be the PC-derived EC20 sequence: (EC)20-Gly
  • From DeSilva02, the MerP-derived Hg-specific binding sequence (GMTCCAA) could be repeated 3x: GMTCCAAGMTCCAAGMTCCAA
  • For Cd-specific binding, could later use the CadA-derived sequence (GFTCANC)



  1. Samuelson P, Wernérus H, Svedberg M, and Ståhl S. Staphylococcal surface display of metal-binding polyhistidyl peptides. Appl Environ Microbiol. 2000 Mar;66(3):1243-8. PubMed ID:10698802 | HubMed [Samuelson00]
  2. Pazirandeh M, Wells BM, and Ryan RL. Development of bacterium-based heavy metal biosorbents: enhanced uptake of cadmium and mercury by Escherichia coli expressing a metal binding motif. Appl Environ Microbiol. 1998 Oct;64(10):4068-72. PubMed ID:9758845 | HubMed [Biosorbants]
  3. Kotrba P, Dolecková L, de Lorenzo V, and Ruml T. Enhanced bioaccumulation of heavy metal ions by bacterial cells due to surface display of short metal binding peptides. Appl Environ Microbiol. 1999 Mar;65(3):1092-8. PubMed ID:10049868 | HubMed [Kotrba99]
  4. Mejáre M and Bülow L. Metal-binding proteins and peptides in bioremediation and phytoremediation of heavy metals. Trends Biotechnol. 2001 Feb;19(2):67-73. PubMed ID:11164556 | HubMed [Mejare01]
  5. Lu Y, Berry SM, and Pfister TD. Engineering novel metalloproteins: design of metal-binding sites into native protein scaffolds. Chem Rev. 2001 Oct;101(10):3047-80. PubMed ID:11710062 | HubMed [Lu01]
  6. Bae W, Chen W, Mulchandani A, and Mehra RK. Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins. Biotechnol Bioeng. 2000 Dec 5;70(5):518-24. PubMed ID:11042548 | HubMed [Bae00]
  7. Bae W, Mehra RK, Mulchandani A, and Chen W. Genetic engineering of Escherichia coli for enhanced uptake and bioaccumulation of mercury. Appl Environ Microbiol. 2001 Nov;67(11):5335-8. DOI:10.1128/AEM.67.11.5335-5338.2001 | PubMed ID:11679366 | HubMed [Bae01]
  8. Bae W, Mulchandani A, and Chen W. Cell surface display of synthetic phytochelatins using ice nucleation protein for enhanced heavy metal bioaccumulation. J Inorg Biochem. 2002 Jan 15;88(2):223-7. PubMed ID:11803043 | HubMed [Bae02]
  9. Kotrba P, Pospisil P, de Lorenzo V, and Ruml T. Enhanced metallosorption of Escherichia coli cells due to surface display of beta- and alpha-domains of mammalian metallothionein as a fusion to LamB protein. J Recept Signal Transduct Res. 1999 Jan-Jul;19(1-4):703-15. DOI:10.3109/10799899909036681 | PubMed ID:10071794 | HubMed [Kotrba99b]
  10. DeSilva TM, Veglia G, Porcelli F, Prantner AM, and Opella SJ. Selectivity in heavy metal- binding to peptides and proteins. Biopolymers. 2002 Aug 5;64(4):189-97. DOI:10.1002/bip.10149 | PubMed ID:12115136 | HubMed [Desilva02]
  11. Opella SJ, DeSilva TM, and Veglia G. Structural biology of metal-binding sequences. Curr Opin Chem Biol. 2002 Apr;6(2):217-23. PubMed ID:12039007 | HubMed [Opella02]
  12. Sousa C, Kotrba P, Ruml T, Cebolla A, and De Lorenzo V. Metalloadsorption by Escherichia coli cells displaying yeast and mammalian metallothioneins anchored to the outer membrane protein LamB. J Bacteriol. 1998 May;180(9):2280-4. PubMed ID:9573175 | HubMed [Sousa98]
  13. Mauro JM and Pazirandeh M. Construction and expression of functional multi-domain polypeptides in Escherichia coli: expression of the Neurospora crassa metallothionein gene. Lett Appl Microbiol. 2000 Feb;30(2):161-6. PubMed ID:10736021 | HubMed [Mauro00]
  14. Cobbett C and Goldsbrough P. Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annu Rev Plant Biol. 2002;53:159-82. DOI:10.1146/annurev.arplant.53.100301.135154 | PubMed ID:12221971 | HubMed [Cobbett02]
  15. Pazirandeh M, Wells BM, and Ryan RL. Development of bacterium-based heavy metal biosorbents: enhanced uptake of cadmium and mercury by Escherichia coli expressing a metal binding motif. Appl Environ Microbiol. 1998 Oct;64(10):4068-72. PubMed ID:9758845 | HubMed [Pazirandeh98]
  16. Chen S, Kim E, Shuler ML, and Wilson DB. Hg2+ removal by genetically engineered Escherichia coli in a hollow fiber bioreactor. Biotechnol Prog. 1998 Sep-Oct;14(5):667-71. DOI:10.1021/bp980072i | PubMed ID:9758654 | HubMed [Chen98]
All Medline abstracts: PubMed | HubMed

Cadmium

Summary

  • Cd causes damage to cells primarily by the generation of reactive oxygen species (ROS), which causes single-strand DNA damage and disrupts the synthesis of nucleic acids and proteins. Cd is also an inhibitor of the DNA mismatch repair system…Results of this study confirmed that Cd toxicity caused profound changes in gene expression in which several stress response systems were induced simultaneously.
  • known carcinogen
  • Most recent(June06) paper on e.coli bioaccumulation of Cd
  1. Deng X, Yi XE, and Liu G. Cadmium removal from aqueous solution by gene-modified Escherichia coli JM109. J Hazard Mater. 2007 Jan 10;139(2):340-4. DOI:10.1016/j.jhazmat.2006.06.043 | PubMed ID:16890348 | HubMed [Deng06]

Contamination Levels/Limits

  • MCL(Maximum Containment Level): 5 ppb (equal to about 45nM)
  • MCLG(Maximum Containment Level Goal): 5 ppb
  1. Staessen JA, Lauwerys RR, Ide G, Roels HA, Vyncke G, and Amery A. Renal function and historical environmental cadmium pollution from zinc smelters. Lancet. 1994 Jun 18;343(8912):1523-7. PubMed ID:7911869 | HubMed [Staessen94]
  2. Satarug S, Baker JR, Urbenjapol S, Haswell-Elkins M, Reilly PE, Williams DJ, and Moore MR. A global perspective on cadmium pollution and toxicity in non-occupationally exposed population. Toxicol Lett. 2003 Jan 31;137(1-2):65-83. PubMed ID:12505433 | HubMed [Satarug03]
All Medline abstracts: PubMed | HubMed


Cadmium Contamination Sites

  1. Ishihara T, Kobayashi E, Okubo Y, Suwazono Y, Kido T, Nishijyo M, Nakagawa H, and Nogawa K. Association between cadmium concentration in rice and mortality in the Jinzu River basin, Japan. Toxicology. 2001 May 28;163(1):23-8. PubMed ID:11376862 | HubMed [Ishihara01]
Plachimada, India: [3]
    • statistics on Pb, Cd, Cr levels in well water surrounding a Coca-Cola plant (2006)
    • pictures too :)


Promoters

Pubmed: Plasmid pI258 (from S.aureus) cadmium resistance (cadA) gene, complete cds [4]

  1. Brocklehurst KR, Megit SJ, and Morby AP. Characterisation of CadR from Pseudomonas aeruginosa: a Cd(II)-responsive MerR homologue. Biochem Biophys Res Commun. 2003 Aug 22;308(2):234-9. PubMed ID:12901859 | HubMed [Brocklehurst03]
  • Brocklehurst - contains sequence for ZntR/CadR and PzntA/PcadA
    • Semmie's article on CadC linked with GFP [5]

Mercury

Contamination Levels/Limits

  • MCL: 2 ppb (equal to about 10 nM)
  • MCLG: 2 ppb
  • 20 ppb makes the fish mercury buildup significant

Polluted sites

  • Onondaga lake [6] [7] [8]
  • Salt Plains National Wildlife Refuge [9]

Detection (to assess our system)


Promoter

  1. Hamlett NV, Landale EC, Davis BH, and Summers AO. Roles of the Tn21 merT, merP, and merC gene products in mercury resistance and mercury binding. J Bacteriol. 1992 Oct;174(20):6377-85. PubMed ID:1328156 | HubMed [Hamlett92]
  2. Park SJ, Wireman J, and Summers AO. Genetic analysis of the Tn21 mer operator-promoter. J Bacteriol. 1992 Apr;174(7):2160-71. PubMed ID:1312997 | HubMed [Park92]
  3. Condee CW and Summers AO. A mer-lux transcriptional fusion for real-time examination of in vivo gene expression kinetics and promoter response to altered superhelicity. J Bacteriol. 1992 Dec;174(24):8094-101. PubMed ID:1334070 | HubMed [Condee92]
  4. Gambill BD and Summers AO. Versatile mercury-resistant cloning and expression vectors. Gene. 1985;39(2-3):293-7. PubMed ID:4092936 | HubMed [Gambill85]
  5. Hansen LH and Sørensen SJ. Versatile biosensor vectors for detection and quantification of mercury. FEMS Microbiol Lett. 2000 Dec 1;193(1):123-7. PubMed ID:11094290 | HubMed [Hansen00]
All Medline abstracts: PubMed | HubMed
  • Condee - Contains sequence for merO/P region
  • Hansen - Plasmid with Pmer, MerR, and reporter (lux, lac, or gfp) tested in e.coli

Sponge

  • Accumulation of Hg ions in cell membrane [10]

Current Industry Filtration Techniques

Mercury: approved by EPA - Coagulation/Filtration; Granular Activated Carbon; Lime softening; Reverse osmosis.