Team:St Andrews/metal-binding

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Metal binding protein

Precious and toxic metals frequently find their way into the environment. As their names suggest, such leaks are wasteful and damaging respectively. St Andrews iGEM '12 plans to take the first steps toward solving this problem using synthetic biology.

Project Description

The human race moved from the stone of the Neolithic period and into the metallurgy of the Chalcolithic period with the widespread use of metal tools in their every day work regime (Gale 1991). Little changed over the next 6,000 years. Metals are still a massive part of our everyday lives, but in quantities that dwarf previous usage. These high levels of metal requirements have left the landscape scarred.

Our project was inspired by the work done identifying the platinum and palladium particles present on roads, primarily emitted from catalytic converters (Deplanche. K., et al. 2011). In 2010, 50% of the world's platinum and palladium production was used for catalytic converters, with the largest use in Europe (Jollie 2010). Platinum and palladium appear on road surfaces in small concentrations and in minute particles, < 3 μm (Prichard, Fisher 2012). These precious elements are a finite resource! However, their eventual exhaustion can be postponed by collecting and recycling them.

We have focused on the collection process. We have worked in a similar way to Chung et al, in that we have produced a protein with a metal binding peptide on the N-terminus of an easily expressible protein (Chan Chung, Cao et al. 2008). The difference is that we used glutathione S-transferase (GST tag) rather than ubiquitin, and instead of adding the binding peptide chemically to the protein we expressed both the protein and the peptide in E. coli BL21 (DE3) cells. These cells are particularly suited to large scale protein production. The peptides sequences were taken from various sources (Seker, Demir 2011, Bae, Chen et al. 2000, Song, Caguiat et al. 2004, White, Liljestrand et al. 2007) and codon optimised for E. coli., there nucleotide sequence was modified so they could be produced by E. coli in a highly efficient manner, using an online program Protein to DNA.

We decided to go down two different routes. The first was making an array of proteins that bind specific metals, and the second was to make a protein with multiple metal binding sites for multiple metals.

With the first of the two ideas we thought that it would be possible use a column to imobilise the protein by its GST tag. A solution containing multiple metal ions would then be passed through the column and bind each metal ion specifically. Initially, palladium, platinum and nickel (Seker, Demir 2011). The reason for choosing these metals was twofold. Firstly, because platinum and palladium are particularly precious metals providing an economic argument, and secondly, because nickel columns were readily available to test this idea.

The second route would produce a protein that would act as a general metal scavenging protein. gBlocks (≤ 500 bps) were specifically designed and inserted into a pGEX-6P-1. One of them was designed to bind toxic metals; cadmium, mercury and cobalt (Seker, Demir 2011, Bae, Chen et al. 2000, Song, Caguiat et al. 2004, White, Liljestrand et al. 2007). This was done by inserting flexible linkers between the metal binding sites (Hu, Wang et al. 2007). The linkers were used because they were quite flexible. This prevented hairpins in the structure. The second gBlock designed was for precious metals, not including platinum and palladium. The corresponding base pairs for gold, silver, aluminum and titanium peptides (Seker, Demir 2011) were used. The design of this differed from the other gBlock as myoglobin was hijacked, the loops removed and replaced with our metal binding peptides. .


Synthesizing metal-binding peptides

For detail on our laboratory procedures, please refer to our Protocols.

Sequences were found for short metal binding peptides. To insert these sequences into a plasmid vector in the form of double stranded, functioning piece of DNA, we designed short complementary pairs of primers that would anneal.


 

Primers

All primers are notated 5' to 3'.

Ni1 forward
Ni1 reverse
Ni2 forward
Ni2 reverse
.

Pd forward
Pd reverse
Pt forward
Pt reverse

For primer annealing in the PCR, the primer sequences were combined in the following way:

  • GST Forward and Ni1 Reverse
  • GST Forward and Ni2 Reverse
  • GST Forward and Pd Reverse
  • GST Forward and Pt Reverse

Vector


 

  • Our vector of choice was pGEX-6p-1, as it contains the genetic information needed to produce GST (258, 992) and is ampicillin-resistant.

    pGEX was digested with EcoR1(955) and Xho1(970).


  •  

    Short metal binding peptides

    The primers designed to create our short Ni1, Ni2, Pd and Pt binding inserts were annealed.

    The primer dimers were ligated into the cut pGEX vector on the N terminus of GST.

    The ligated vector was transformed into DH5-α E. coli cells.

    These cells were grown up and a miniprep was carried out.

    The primer dimer insert was too small to detect using a diagnostic digest.

    The miniprep product was used to transform the engineered pGEX vector into BL21 gold E. coli cells.

    These cells were induced and GST+Ni1, Ni2, Pd or Pt was overexpressed.

    GST+ Ni1 and Ni2 were used to prove functionality in our engineered protein. They bound to nickel beads successfully, and further characterisation was carried out by the University Geology department using ICP-MS.

    The Pd and Pt constructs were not fully characterised .


     
    TM

    Two gBlocksTM were designed and ordered from IDT.

    Precious metal scavenging Toxic metal scavenging

    The gBlocks alone were ligated into the iGEM pSB1C3 vector for submission.

    The ligation was successful but the BioBricks remain uncharacterised.


    Biobricks


    References

    BAE, W., CHEN, W., MULCHANDANI, A. and MEHRA, R.K., 2000. Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins. Biotechnology and bioengineering, 70(5), pp. 518-524.

    CHAN CHUNG, K.C., CAO, L., DIAS, A.V., PICKERING, I.J., GEORGE, G.N. and ZAMBLE, D.B., 2008. A high-affinity metal-binding peptide from Escherichia coli HypB. Journal of the American Chemical Society, 130(43), pp. 14056-14057.

    DEPLANCHE, K., MURRAY, A., MENNAN, C., TAYLOR, S. and MACASKIE, L., Biorecycling of Precious Metals and Rare Earth Elements.

    GALE, N.H., 1991. Metals and metallurgy in the Chalcolithic period. Bulletin of the American Schools of Oriental Research, , pp. 37-61.

    HU, X., WANG, H., KE, H. and KUHLMAN, B., 2007. High-resolution design of a protein loop. Proceedings of the National Academy of Sciences, 104(45), pp. 17668.

    JOLLIE, D., 2010. Platinum 2010. Johnson Matthey.

    PRICHARD, H.M. and FISHER, P.C., 2012. Identification of platinum and palladium particles emitted from vehicles and dispersed into the surface environment. Environmental science & technology, .

    SEKER, U.O.S. and DEMIR, H.V., 2011. Material binding peptides for nanotechnology. Molecules, 16(2), pp. 1426-1451.

    SONG, L., CAGUIAT, J., LI, Z., SHOKES, J., SCOTT, R.A., OLLIFF, L. and SUMMERS, A.O., 2004. Engineered single-chain, antiparallel, coiled coil mimics the MerR metal binding site. Journal of Bacteriology, 186(6), pp. 1861-1868.

    WHITE, B.R., LILJESTRAND, H.M. and HOLCOMBE, J.A., 2007. A ‘turn-on’FRET peptide sensor based on the mercury binding protein MerP. Analyst, 133(1), pp. 65-70.

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    University of St Andrews, 2012.

    Contact us: igem2012@st-andrews.ac.uk, Twitter, Facebook

    This iGEM team has been funded by the MSD Scottish Life Sciences Fund. The opinions expressed by this iGEM team are those of the team members and do not necessarily represent those of Merck Sharp & Dohme Limited, nor its Affiliates.