Unlikely Remediator: Engineering D. radiodurans for toxic metal cleanup Morgan Etheridge.

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Presentation transcript:

Unlikely Remediator: Engineering D. radiodurans for toxic metal cleanup Morgan Etheridge

merA

Bacterial mercuric reductase Reduces Hg(II) + NADPH  NADP + + H + +Hg(0) 22,23

MD735 Strain

MD736

MD737

Determining mer operon copy number and mercury resistance (1) ~600 ng genomic DNA from each strain was electrophoresed in agarose gel

Determining mer operon copy number and mercury resistance (1) ~600 ng genomic DNA from each strain was electrophoresed in agarose gel (2) gel was blotted and hybridized to a radiolabeled 1.5-kb EcoRI-BglII fragment of pMD726, containing part of merA and all of merD.

Determining mer operon copy number and mercury resistance (1) ~600 ng genomic DNA from each strain was electrophoresed in agarose gel (2) gel was blotted and hybridized to a radiolabeled 1.5-kb EcoRI-BglII fragment of pMD726, containing part of merA and all of merD. (3) The number of disintegration counts in each of the hybridizing bands was determined and used to calculate the mer copy number per cell.

# merA copies per cell R10 MD7351 MD76710 MD73610 MD737150

Growth curves for each of the strains were determined by inoculating ∼ 5 x 106 cells of each into growth medium containing 50 μM Merbromin or 50 μM HgCl2.

Merbromin

Order of resistance BL308 > (MD737, MD736) > MD735 > MD767 > R1

There is a lag in cell growth…What does that tell us?

As detoxification occurs… – Toxic effects on the outer cell wall? – Toxic effects on the membranes responsible for cell transport (and growth)?

Wild-type D. radiodurans did not grow in HgCl 2 …Why?

Hg(II) + NADPH  NADP + + H + +Hg(0) 22,23

Multiple Resistance MD764

An unlikely hard drive?

References Brim, H., McFarlan, S.C., Fredrickson, JK., Minton, K.W., Zhai, M., Wackett, L.P.,Daly, M.J Engineering Deinococcus radiodurans for metal remediation inradioactive mixed waste environments. Nature Biotechnology: – 90. Battista, J.R Against all odds: The survival strategies of Deinococcus radiodurans.Annual Review of Microbiology: – 224. Wong, P.C., Wong, K., Foot, H Organic data memory: Using the DNA approach. Communications of the ACM: – 98.