Exeter/6 June 2013

Dr. Clive Butler spoke to us about synthetic nano-cages using natural bacterial peptides, highlighting the aspects of synthetic biology that can make a project or research topic both novel and ground-breaking.

Developing synthetic nano-cages using natural bacterial peptides

Respiratory products and energy generation

• Carbon substrate is oxidised, its electron used to drive synthesis of ATP (oxidative phosphorylation reducing oxygen to form water, pumping protons across a membrane)

• Nitrates as an alternative electron acceptor, can also use sulphur compounds, DMSO, extracellular metals, etc

Bacterial respiration of selenite (SeO42-)

• Selenium waste generated in petroleum extraction, fossil fuel extraction, etc

• SeO42- has a high redox potential

• Se0 is a red precipitate, and generally non-toxic, so a good final form for bioremediation. But having a build-up of a solid in a cell can be a problem. Could methylate into a volatile form to diffuse away

Selenate reductase

• Periplasmic, has 3 subunits (SerA, SerB, SerC) and includes iron, molybdenum and haem for electron transfer

• Reduces selenate to selenite (SeO32-) which is soluble but TOXIC.

Fate of selenite

• Need to remove from the cell, but is it kicked out as selenite and reduced outside of the cell to Se0? Or something different?

• Cells do need Se for selenocysteine and active sites in vital enzymes. Can they selectively absorb some, or absorb all then kick out unrequired Se?

• There are particles of Se seen within the cell (picture on slide) but how do they get lumps of Se across a very tightly regulated membrane?

• Look at where deposits were using microscopy and a growth curve – in cells exposed to Se, can see areas of darkness inside of cells, but at a certain point the solutions of bacteria turn red and the dark areas vanish from the cell

• Use SEM to look at the outsides of the cells, confirming that Se is excreted by the cells without breaking the membrane

• Particles were only a few angstroms across, so putting through a 0.2um filter to removes the bacteria but allow Se particles to run through. All appear to be a similar size/diameter (150 angstrom)

• Can also run on a gel, they move to the +ve electrode, so could be associated with something which gives a net negative charge

Se-nanosphere

• Looking at particle, no evidence of a lipid bi-layer

• View particles in real time, observe Brownian motion and track movement to work out radius. Results – 131 ± 27nm

• In E. cloacae, particles have a much wider range of sizes and appear to “grow”.

• To see if any biological matter associated with the Se, use SDS gels to separate the “proteins”. Ran against cells growth on nitrate as a negative control. The gel was expected with to have lots of proteins, or no proteins, but one strong band was seen on the gel. The cells had not been lysed (would give a massive column of bands).

• The protein isn’t used in aerobic conditions, when selenate reductase is down-regulated

• Cut out protein band from gel, use LC-MS to extract protein, then determine peptide sequence. Can also use N-terminal sequencing.

• Run against BLAST, no hits. Nothing the right size and sequence. Use Illumina sequencer to get genome.

• Find a gene (for a 94.5kDa protein) which matches the N-terminal sequencing. No obvious leader peptide, so we know it doesn’t go into the periplasmic membrane.

• Only uses a small number of amino acids to reduce waste.

• pI of 3.65, which matches the negative charge behaviour observed earlier

• Similar behaviour found with a nitrogen sulphate growth medium; black spots of sulphate seem on cell cross sections. Gene for associated protein HIGHLY CONSERVED to protein seen with Se.

• Ratio of Se:SefA is ~300:1, so lots of Se per protein.

Synthetic biology

• Try and get E. coli to produce Se dark spots. They can make them, but don’t appear to be excreted or have size limitation.

• Find that it forms large complexes of at least 10 subunits

• Using SefA, get organised balls of Se. No SefA, get untidy, general excretion of Se. But balls still don’t have limited sizes.

• Put His tag on one end of SefA, and another batch with a His tag on BOTH ends of SefA.

Getting Se across the membrane

• How is the ball of Se getting across the membrane? Is it breaking the membrane?

• Now looking a potential formation of a pore in the membrane

• Does it have to be Se in the cage? Other metal, drugs, etc. We know attaching a His tag interferes with assembly of the cage, so have to be careful how much we change

Future

• Could we make an E. coli make something useful, then wrap it up in these proteins and kick them out, so removing needs for purification?

E. coli doesn’t like making cyctochrome c under aerobic conditions. Might be easier to just stick with Shawenella

Look for alternative carbon substrates for E. coli Glycerol is a 20% product for 1st generation biofuel synthesis. What can we do with the glycerol? Carbon substrate for E. coli?

Take me back to the notebook.