Unlikely Remediator: Engineering D. radiodurans for toxic metal cleanup Morgan Etheridge
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.
• 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.
• As detoxification occurs…– Toxic effects on the outer cell wall?– Toxic effects on the membranes responsible for
cell transport (and growth)?
References
• Brim, H., McFarlan, S.C., Fredrickson, JK., Minton, K.W., Zhai, M., Wackett, L.P., Daly, M.J. 1999. Engineering Deinococcus radiodurans for metal remediation inradioactive mixed waste environments. Nature Biotechnology: 18. 85 – 90.
• Battista, J.R. 1997. Against all odds: The survival strategies of Deinococcus radiodurans. Annual Review of Microbiology: 51. 203 – 224.
• Wong, P.C., Wong, K., Foot, H. 2003. Organic data memory: Using the DNA approach. Communications of the ACM: 46. 95 – 98.