IGEM:IMPERIAL/2008/Projects/Water filter/Useful genes

Promoters

Cadmium promoter

• pCad has been shown to have a dose response to the concentration of cadmium. The lowest threshold pcad response is 10^-11 and 10^-9 Molar of Cadmium. [1] [2]. Has been shown to work successfully in E.coli but requires the expression of the protein CadC regulatory protein. Note of caution, we need to consider that many bacterium including E.coli have developed mechanisms to actively pump out many of the ions out of the cells. For example, P-type ATPase, ZntA that extrudes Cd(II), Zn(II) and Pb(II) and so we may consider either using KO versions of these or expression ion transport channels.

Mercury Promoter

• The MerR regulatory protein forms a dimer that binds to the single DNA site in the Mer operon. In the absence of mercury the DNA represses the promoter in the absence of mercury and in the presence of mercury it causes activation of the promoter. [3] [4]

Lead Promoter

• Lead response element within E.coli that acts similar to that to the mercuary response element. Like the mercuary and the cadmium these promoters have been used within biosensors. [5] [6]

Metal Binding Proteins

Figure 1: Example of membrane binding protein [7]

We can make a distinction between the mechanisms that organisms use to deal with heavy metal ions. These are intraceullar and extracellular mechanisms.

• Binding proteins such as metallothioneins (MTs) or phytochelatins (PCs) provide potential parts to bind and sequest heavy metal ions such as cadmium. The principle of these proteins is that they contain high levels of cysteine residues that are able to bind heavy metals. There is a huge diversity in the choice of MTs or PCs that we could choose, including several modified proteins designed to give high affinity binding.
• extracellular mechanisms use MTs fused to membrane proteins and have been successfully synthesized in a number of examples. The advantages of these are really two fold, firstly it can make removal and isolation of purified cadmium easier and secondly, there is no interference with intracellular mechanisms of the cell. Figure 1 below shows a design that was used to bind the membrane protein. One downside is that if you trap the cadmium at the surface of the E.coli it will prevent entry into the cell and prevent an input into our devices and stop expression. [7]

Transport Proteins

Cadium

• A manganese transport gene has been previously used previously to transport cadmium in engineeered E.coli. The results of this showed that using this gene cells could increase the transport of cadmium by six times compared to cells without this gene. Wild type E.coli do naturally accumulate cadmium via a zinc transporter, however use of a specific transporter clearly specifically increases uptake significantly.
• The relative rates of uptake have been shown to be 90% maximal at 15 minutes. [8]
• Some genes that have been used before include mntA and cdtB. [9].

References

1. Shetty RS, Deo SK, Shah P, Sun Y, Rosen BP, and Daunert S. Luminescence-based whole-cell-sensing systems for cadmium and lead using genetically engineered bacteria. Anal Bioanal Chem. 2003 May;376(1):11-7. DOI:10.1007/s00216-003-1862-9 |
2. Endo G and Silver S. CadC, the transcriptional regulatory protein of the cadmium resistance system of Staphylococcus aureus plasmid pI258. J Bacteriol. 1995 Aug;177(15):4437-41. DOI:10.1128/jb.177.15.4437-4441.1995 |
3. Summers AO. Untwist and shout: a heavy metal-responsive transcriptional regulator. J Bacteriol. 1992 May;174(10):3097-101. DOI:10.1128/jb.174.10.3097-3101.1992 |

4. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=413224

5. Tauriainen S, Karp M, Chang W, and Virta M. Luminescent bacterial sensor for cadmium and lead. Biosens Bioelectron. 1998 Oct 15;13(9):931-8. DOI:10.1016/s0956-5663(98)00027-x |

6. GFP expressing bacterial biosensor to measure lead contamination in aquatic environment.

   http://www.sciencemag.org/cgi/content/abstract/261/5122/715

7. Rüegg M, Metzger V, and Susi H. Computer analyses of characteristic infrared bands of globular proteins. Biopolymers. 1975 Jul;14(7):1465-71. DOI:10.1002/bip.1975.360140712 |
8. Kim SK, Lee BS, Wilson DB, and Kim EK. Selective cadmium accumulation using recombinant Escherichia coli. J Biosci Bioeng. 2005 Feb;99(2):109-14. DOI:10.1263/jbb.99.109 |
9. 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 |