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 DecoderConstitutive PromoterRBSOmpAGFPTerminator

Plac + PtetRBSOmpARFPTerminator

PtetRBSOmpFCFPTerminator

PlacRBSOmpFYFPTerminator

PlacMicA

PtetMicA

Plac + PtetMicF

• Put both regulators and target on high copy plasmids

• You have two GFPs? thanks• Compare regulation to riboregulators

(BBa_J01008 BBa_J01010 BBa_J01080 BBa_J01086) 

Tranformation/Precipitation of Heavy Metals - WhyHeavy Metals are toxic to life, particularly in high concentrationsDisease CausingAlleviate contamination of water supplyHelp conserve habitable environmentWe'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.

Cell Surface EngineeringMake 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/soilBiomining

Problem: This seems too simple, we'd like to think of a way to take it further than just simply collecting metals.... ideas??

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

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

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?

Arsenic

The resistance pathway uses reduction however, it doesn't involve reducing As to it's elemental form Resistance Pathway:ArsR: Regulatory RepressorArsB: Efflux PumpArsC: Arsenate Reductase

Alternatively, periplasmic oxidase and reductase exist

CadmiumResistance 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 periplasmdue to protons being pumped out

Nickel

• yohMocodes for nickel and cobalt resistance.  opresent in E.colio825 nucleotides longostronger promotor should result in better

resistance.• NreB and NrsD

opresent in the microorganism R. metallidurans

ocould provide exclusive resistance to Ni.  

Gold

Gold resistance is primarily conferred by 3 genes in Salmonella:• golT, golS and golB

o golT - P-type ATPase efflux proteino golS - Gold-dependent transcription factor for golTSBo 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

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

Silver

Silver resistance has been identified : SilCBA operon: efflux system

Present in E. coli and S. Typhimirium

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? 

Questions