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iGEM week 2: 6/1 - 6/4

iGEM week 2: 6/1 - 6/4. Overview. Bacterial Decoder Resistance Precipitation Cell Surface Engineering Magnetite Possible Metals: Mercury Arsenic Cadmium Nickel (Gold) Zinc (Silver). What metals are feasible to work with?. Bacterial Decoder. Constitutive Promoter RBS OmpA GFP

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iGEM week 2: 6/1 - 6/4

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  1. iGEM week 2: 6/1 - 6/4

  2. Overview • Bacterial Decoder • Resistance • Precipitation • Cell Surface Engineering • Magnetite Possible Metals: • Mercury • Arsenic • Cadmium • Nickel • (Gold) • Zinc • (Silver) What metals are feasible to work with?

  3. Bacterial Decoder Constitutive Promoter RBS OmpA GFP Terminator Ptet RBS OmpF CFP Terminator Plac RBS OmpF YFP Terminator Plac + Ptet RBS OmpA RFP Terminator Plac MicA Ptet MicA Plac + Ptet MicF • Put both regulators and target on high copy plasmids • You have two GFPs? thanks • Compare regulation to riboregulators (BBa_J01008BBa_J01010BBa_J01080BBa_J01086)

  4. Tranformation/Precipitation of Heavy Metals - Why Heavy Metals are toxic to life, particularly in high concentrations Disease Causing Alleviate contamination of water supply Help conserve habitable environment We'll eventually run out of space to dig for storage In terms of manufacturing, precipitation of these metals may lead to bionanofabrication applications. • quantum dots, single-electron transistors, fuel cells,  fluorescent labelling,  DNA/RNA detection, biomedical diagnostic devices, biosensors, nanocomputers, drug and gene transport systems and carbon nanotubes Making bacteria resistant to these metals is the theoretical first step towards a more efficient water cleaning system or manufacturing system.

  5. Cell Surface Engineering Make a chimeric protein containing a metallothionein Fuse cell membrane protein with metallothionein Cell will display MTs on surface, and can collect soluble metals that way Applications: Bioremediation of heavy metals in water/soil Biomining Problem: This seems too simple, we'd like to think of a way to take it further than just simply collecting metals.... ideas??

  6. Valls, M, Gonzalez-Duarte R, Atrian S, Lorenzo V (1998) Bioaccumulation of heavy metals with protein fusions of metallothionein to bacterial OMPs. Biochimie 80: 855-861

  7. Marc Valls, Sílvia Atrian, Víctor de Lorenzo & Luis A. Fernández Nature Biotechnology 18, 661 - 665 (2000)

  8. Hg • Resistance could be accomplished using: MerP, MerT, and MerA (mercuric reductase) • This process involves the reduction of Hg+2 to Hg0 • Export? • 2ppb limit for       drinking water  • Could we work with       mercury?

  9. Arsenic The resistance pathway uses reduction however, it doesn't involve reducing As to it's elemental form Resistance Pathway: ArsR: Regulatory Repressor ArsB: Efflux PumpArsC: Arsenate Reductase Alternatively, periplasmic oxidase and reductase exist

  10. Cadmium Resistance is well defined, however the components involved in reduction are less clear. May be related to resistance. metals accumulate in the periplasm where they form metal bicarbonates and carbonates that crystallize on cellular bound metals. alkaline pH in periplasm due to protons being  pumped out

  11. yohM codes for nickel and cobalt resistance.   present in E.coli 825 nucleotides long stronger promotor should result in better resistance. NreB and NrsD present in the microorganism R. metallidurans could provide exclusive resistance to Ni.   Nickel

  12. Gold Gold resistance is primarily conferred by 3 genes in Salmonella: • golT, golS and golB • golT - P-type ATPase efflux protein • golS - Gold-dependent transcription factor for golTSB • golB - Gold-binding protein Gold reduction occurs by reducing Au(II) to Au(0) •   Several bacteria can naturally do this •   Pathway and proteins unknown for all of them Expensive! • $80/gram

  13. Zinc                     There is a zinc metallothionein gene ZmtA that we could possibly use for cell surface engineering. It comes from Synechococcus elongatus PCC 7942. It is a gram-negative bacteria. There is also a repressor for this gene, ZmtB. There is a zinc, cobalt, and lead efflux system called ZntA that is in Escherichia coli str. K-12 substr. MG1655. Lastly is a zinc-responsive transcriptional regulator ZntR Escherichia coli str. K-12 substr. MG1655. Microbial precipitation of zinc is done through complexing zinc with sulfur, not reducing it to elemental zinc  • Mechanism unknown

  14. Silver Silver resistance has been identified : SilCBA operon: efflux system Present in E. coli and S. Typhimirium

  15. Magnetite • Fe(II)O + Fe(III)2O3 • Out ~25 proteins involved in magnetosome formation in Magnetosprillum magneticum, 5 genes are involved in magnetite production from iron: mms6, and mamGFDC. • No intermediates between iron and magnetite • Iron saturation is required  • 2 genes are involved in the uptake of iron: mamA, mamB • oxygen conditions and pH determine the efficacy of crystalization (optimal at low oxygen/anoxic conditions, high hydrogen partial pressure, slightly reducing conditions) • Is it possible to try and produce magnetite without the magnetosome? 

  16. Questions    

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