A. James Clark School of Engineering
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Item Effect of electrical energy on the efficacy of biofilm treatment using the bioelectric effect(Nature Publishing Group, 2015-09-23) Kim, Young Wook; Subramanian, Sowmya; Gerasopoulos, Konstantinos; Ben-Yoav, Hadar; Wu, Hsuan-Chen; Quan, David; Carter, Karen; Meyer, Mariana T.; Bentley, William E.; Ghodssi, RezaBACKGROUND/OBJECTIVES: The use of electric fields in combination with small doses of antibiotics for enhanced treatment of biofilms is termed the ‘bioelectric effect’ (BE). Different mechanisms of action for the AC and DC fields have been reported in the literature over the last two decades. In this work, we conduct the first study on the correlation between the electrical energy and the treatment efficacy of the bioelectric effect on Escherichia coli K-12 W3110 biofilms. METHODS: A thorough study was performed through the application of alternating (AC), direct (DC) and superimposed (SP) potentials of different amplitudes on mature E. coli biofilms. The electric fields were applied in combination with the antibiotic gentamicin (10 μg/ml) over a course of 24 h, after the biofilms had matured for 24 h. The biofilms were analysed using the crystal violet assay, the colony-forming unit method and fluorescence microscopy. RESULTS: Results show that there is no statistical difference in treatment efficacy between the DC-, AC- and SP-based BE treatment of equivalent energies (analysis of variance (ANOVA) P > 0.05) for voltages < 1 V. We also demonstrate that the efficacy of the BE treatment as measured by the crystal violet staining method and colony-forming unit assay is proportional to the electrical energy applied (ANOVA P < 0.05). We further verify that the treatment efficacy varies linearly with the energy of the BE treatment (r2 = 0.984). Our results thus suggest that the energy of the electrical signal is the primary factor in determining the efficacy of the BE treatment, at potentials less than the media electrolysis voltage. CONCLUSIONS: Our results demonstrate that the energy of the electrical signal, and not the type of electrical signal (AC or DC or SP), is the key to determine the efficacy of the BE treatment. We anticipate that this observation will pave the way for further understanding of the mechanism of action of the BE treatment method and may open new doors to the use of electric fields in the treatment of bacterial biofilms.Item Harnessing the Potential of the Escherichia coli RpoS Phenotype via an Inducible Small RNA Regulatory Platform(2011) Carter, Karen; Bentley, William E; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Recent recognition of the pervasiveness of non-coding RNAs, in both prokaryotic and eukaryotic systems, has prompted metabolic engineers to reevaluate the role of RNAs in a traditionally protein dominated realm. More specifically, bacterial trans-encoded sRNAs have been implicated in the regulation of genes in several critical pathways from quorum sensing to stress responses. The task of responding to stressful conditions, as well as stationary phase, in a comprehensive manner falls to the Escherichia coli global stress regulator, RpoS. Genes transcribed by RpoS are involved in motility, biofilm formation and nutrient limitations. One of the challenges modulating RpoS control is its polymorphic nature. We think this can be addressed using an inducible sRNA regulatory platform. Recent studies have confirmed RpoS to be post-transcriptionally regulated by at least four sRNAs: three activators, DsrA, RprA and ArcZ, and one repressor OxyS. Each of these senses different stress conditions, allowing RpoS synthesis to increase or decrease in response to various stressors. This work investigates the potential of a genetically engineered interchangeable small RNA based gene regulation platform as a switch to affect the expression profiles and metabolic behavior of RpoS. RprA and OxyS were put under the control of an arabinose inducible promoter to test the ability to increase/decrease RpoS protein levels and subsequent changes in RpoS-dependent genes. We then assessed gene expression and phenotypic changes using RT-PCR, Western blotting, microarray and motility and biofilm assays. Positive modulation of RpoS using the pRprA platform resulted in a 2-fold decrease in motility in Top10 cells. This difference in motility improved biofilm formation levels up to 12-fold when compared to direct overexpression of RpoS protein. The positive effect of biofilm formation was further supported by the upregulation of other genes essential for biofilms. Conversely, negative modulation of RpoS using the pOxyS platform resulted in an increase in the transcription of the motility gene, flhD. Both systems were capable of positively and negatively regulating bacterial RpoS protective genes. The ability to deliberately and purposefully control RpoS protective genes, in conjunction with motility and biofilm formation, can potentially have broad impact on biotechnology applications.Item USING ANTISENSE MESSENGER RNA TO DOWNREGULATE LON MEDIATED PROTEOLYSIS IN ESCHERICHIA COLI(2003-12-18) Carter, Karen; Bentley, William E; Chemical EngineeringThe advent of metabolic engineering has instigated the introduction of foreign heterologous proteins into host cells, such as Escherichia coli. However, the metabolic burden incurred by the host cell to produce the desired recombinant protein elicits a cellular stress response that can result in reduced yields and degradation of the desired protein. In, lon is one of the major proteases responsible for this abnormal protein degradation, including recombinant proteins. Consequently, a variety of antisense strategies have been examined and shown to effectively control endogenous gene expression and function in E. coli. For this investigation we explored using a 300 base pair sequence of the 5' coding region of E. coli lon gene, including the start codon, cloned into both the pSE420 and pTO plasmids in the antisense (reverse) orientation. We examined the ability of lon antisense RNA to inhibit the production of endogenous lon protease and increase the protein yield and activity of a model recombinant protein, organophosphorus hydrolase (OPH). Results indicate that the overproduction of lon antisense did effectively downregulate the production of endogenous lon. In addition, cultures induced for lon antisense also revealed higher OPH protein levels in the first hour of production and a 7-fold higher activity.