Reviewer
Laura Molina-García
  • Research Associate, University College London
Research fields
  • Microbiology
Evaluation of Plasmid Stability by Negative Selection in Gram-negative Bacteria
Authors:  Damián Lobato Márquez and Laura Molina García, date: 05/05/2017, view: 11575, Q&A: 0
Plasmid stability can be measured using antibiotic-resistance plasmid derivatives by positive selection. However, highly stable plasmids are below the sensitivity range of these assays. To solve this problem we describe a novel, highly sensitive method to measure plasmid stability based on the selection of plasmid-free cells following elimination of plasmid-containing cells. The assay proposed here is based on an aph-parE cassette. When synthesized in the cell, the ParE toxin induces cell death. ParE synthesis is controlled by a rhamnose-inducible promoter. When bacteria carrying the aph-parE module are grown in media containing rhamnose as the only carbon source, ParE is synthesized and plasmid-containing cells are eliminated. Kanamycin resistance (aph) is further used to confirm the absence of the plasmid in rhamnose grown bacteria.
Semi-denaturing Detergent Agarose Gel Electrophoresis (SDD-AGE)
Authors:  Laura Molina-García and Fátima Gasset-Rosa, date: 11/20/2014, view: 22614, Q&A: 2
Pathological proteins in neurodegenerative diseases suffer a conformational change to a misfolded amyloid state. Such pathological event leads to the aggregation of these proteins that indefinitely propagates as an altered form of itself, and harbor prion-like properties (Wickner, 1994; Prusiner, 2012). In addition to diseases, prions can also have beneficial adaptive roles in lower eukaryotes (in fungi and yeast) (Eaglestone et al., 1999; True et al., 2004; Coustou et al., 1999). Besides separating polymers from their precursor soluble monomers, another particular difficulty of the study of amyloid proteins is to resolve the heterogeneity of the aggregates, since these usually exhibit a variable degree of polymorphism. Semi-denaturating detergent agarose gel electrophoresis (SDD-AGE) is a technique that takes advantage of both the property of prions and prion-like polymers to be highly resistant to solubilization by SDS detergent, and the large pores sizes of agarose, that allow the resolution of high molecular weight complexes. In this method, we describe in detail how this technique can be used to characterize heterogeneous aggregation in bacteria and yeast (Gasset-Rosa et al., 2014; Molina-García and Giraldo, 2014), and further be applied to study the aggregation pattern of proteins that become prone to aggregation through genetic manipulation.
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