IGEM:MIT/2007/Heavy Metals

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Contents 1 Reviews 2 Heavy Metal Binding Proteins/Peptides 3 Cadmium 3.1 Summary 3.2 Contamination Levels/Limits 3.3 Cadmium Contamination Sites 3.4 Promoters 4 Mercury 4.1 Contamination Levels/Limits 4.2 Polluted sites 4.3 Detection (to assess our system) 4.4 Promoter 4.5 Sponge 5 Current Industry Filtration Techniques

Reviews

• Excellent Summary of Bacterial Metal Resistance (a must read)

1. Silver S and Phung le T. . pmid:16133099. PubMed 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

2. Paul D, Pandey G, Pandey J, and Jain RK. . pmid:15734556. PubMed HubMed [Paul05]
3. Valls M and de Lorenzo V. . pmid:12413663. PubMed HubMed [Valls02]
4. Bruins MR, Kapil S, and Oehme FW. . pmid:10702338. PubMed 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)

5. Samuelson P, Wernérus H, Svedberg M, and Ståhl S. . pmid:10698802. PubMed HubMed [Samuelson00]
6. Pazirandeh M, Wells BM, and Ryan RL. . pmid:9758845. PubMed HubMed [Biosorbants]
7. Kotrba P, Dolecková L, de Lorenzo V, and Ruml T. . pmid:10049868. PubMed HubMed [Kotrba99]
8. Mejáre M and Bülow L. . pmid:11164556. PubMed HubMed [Mejare01]
9. Lu Y, Berry SM, and Pfister TD. . pmid:11710062. PubMed HubMed [Lu01]
10. Bae W, Chen W, Mulchandani A, and Mehra RK. . pmid:11042548. PubMed HubMed [Bae00]
11. Bae W, Mehra RK, Mulchandani A, and Chen W. . pmid:11679366. PubMed HubMed [Bae01]
12. Bae W, Mulchandani A, and Chen W. . pmid:11803043. PubMed HubMed [Bae02]
13. Kotrba P, Pospisil P, de Lorenzo V, and Ruml T. . pmid:10071794. PubMed HubMed [Kotrba99b]
14. DeSilva TM, Veglia G, Porcelli F, Prantner AM, and Opella SJ. . pmid:12115136. PubMed HubMed [Desilva02]
15. Opella SJ, DeSilva TM, and Veglia G. . pmid:12039007. PubMed HubMed [Opella02]
16. Sousa C, Kotrba P, Ruml T, Cebolla A, and De Lorenzo V. . pmid:9573175. PubMed HubMed [Sousa98]
17. Mauro JM and Pazirandeh M. . pmid:10736021. PubMed HubMed [Mauro00]
18. Cobbett C and Goldsbrough P. . pmid:12221971. PubMed HubMed [Cobbett02]
19. Pazirandeh M, Wells BM, and Ryan RL. . pmid:9758845. PubMed HubMed [Pazirandeh98]
20. Chen S, Kim E, Shuler ML, and Wilson DB. . pmid:9758654. PubMed 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

21. Deng X, Yi XE, and Liu G. . pmid:16890348. PubMed HubMed [Deng06]

Contamination Levels/Limits

• MCL(Maximum Containment Level): 5 ppb (equal to about 45nM)
• MCLG(Maximum Containment Level Goal): 5 ppb

22. Staessen JA, Lauwerys RR, Ide G, Roels HA, Vyncke G, and Amery A. . pmid:7911869. PubMed HubMed [Staessen94]
23. Satarug S, Baker JR, Urbenjapol S, Haswell-Elkins M, Reilly PE, Williams DJ, and Moore MR. . pmid:12505433. PubMed HubMed [Satarug03]

All Medline abstracts: PubMed HubMed

Cadmium Contamination Sites

24. Ishihara T, Kobayashi E, Okubo Y, Suwazono Y, Kido T, Nishijyo M, Nakagawa H, and Nogawa K. . pmid:11376862. PubMed 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]

25. Brocklehurst KR, Megit SJ, and Morby AP. . pmid:12901859. PubMed 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)

• Gold Nanoparticles http://www.cah.ucf.edu/news/2005-Mercurynose.php

Promoter

26. Hamlett NV, Landale EC, Davis BH, and Summers AO. . pmid:1328156. PubMed HubMed [Hamlett92]
27. Park SJ, Wireman J, and Summers AO. . pmid:1312997. PubMed HubMed [Park92]
28. Condee CW and Summers AO. . pmid:1334070. PubMed HubMed [Condee92]
29. Gambill BD and Summers AO. . pmid:4092936. PubMed HubMed [Gambill85]
30. Hansen LH and Sørensen SJ. . pmid:11094290. PubMed 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.