Claire Komives

Growth of Bacillus methanolicus in seawater-based media

Bacillus methanolicus has been proposed as a biocatalyst for the low cost production of commodity chemicals. The organism can use methanol as sole carbon and energy source, and it grows aerobically at elevated temperatures. Methanol can be made available from off-shore conversion of natural gas to methanol, through gas-to-liquid technology. Growth of the organism in seawater-based medium would further reduce the costs of chemical production performed near an off-shore natural gas source. The growth of strain PB1 (ATCC 51375) in shake flask experiments with trypticase soy broth medium showed minimal salt-inhibition at the concentration of NaCl in seawater. The ability of B. methanolicus PB1 to grow in Pacific Ocean water using methanol as a carbon and energy source was also tested. Following a simple adaptation procedure, PB1 was able to grow on methanol in semi-defined medium with 100% seawater with good growth yields and similar growth rates compared with those achieved on media prepared in deionized water.

Expression of recombinant green fluorescent protein in Bacillus methanolicus

Microbial biocatalysts are used in a wide range of industries to produce large scale quantities of proteins, amino acids, and commodity chemicals. While the majority of these processes use glucose or other low‐cost sugars as the substrate, Bacillus methanolicus is one example of a biocatalyst that has shown sustained growth on methanol as a carbon source at elevated temperature (50–53°C optimum) resulting in reduced feed and utility costs. Specifically, the complete chemical process enabled by this approach takes methane from natural gas, and following a low‐cost conversion to methanol, can be used for the production of high value products. In this study, production of recombinant green fluorescent protein (GFPuv) by B. methanolicus is explored. A plasmid was constructed that incorporates the methanol dehydrogenase (mdh) promoter of B. methanolicus MGA3 together with the GFPuv gene. The plasmid, pNW33N, was shown to be effective for expression in other Bacillus strains, although not previously in B. methanolicus. A published electroporation protocol for transformation of B. methanolicus was modified to result in expression of GFP using plasmid pNW33N‐mdh‐GFPuv (pNmG). Transformation was confirmed by both agarose gel electrophoresis and by observation of green fluorescence under UV light exposure. The mass yield of cells and protein were measured in shake flask experiments. The optimum concentration of methanol for protein production was found to be at 200 mM. Higher concentrations than 200 mM resulted in slightly higher biomass production but lower amounts of recombinant protein.

Opossum peptide that can neutralize rattlesnake venom is expressed in Escherichia coli

An eleven amino acid ribosomal peptide was shown to completely neutralize Western Diamondback Rattlesnake (Crotalus atrox) venom in mice when a lethal dose of the venom was pre‐incubated with the peptide prior to intravenous injection. We have expressed the peptide as a concatenated chain of peptides and cleaved them apart from an immobilized metal affinity column using a protease. After ultrafiltration steps, the mixture was shown to partially neutralize rattlesnake venom in mice. Preliminary experiments are described here that suggest a potential life‐saving therapy could be developed. To date, no recombinant therapies targeting cytotoxic envenomation have been reported.

Process for production and purification of lethal toxin neutralizing factor (LTNF) from E. coli and its economic analysis: Process for producing rLTNF and its economic analysis

BACKGROUND Purification of peptides offers unique challenges with respect to obtaining the desired process yield and selectivity. Lethal Toxin Neutralizing Factor (LTNF) is a peptide that is known to neutralize snake venom in mice when the peptide is preincubated with the venom prior to intravenous injection. A process for producing highly purified recombinant LTNF has been developed. The process has been modelled in SuperPro designer using laboratory data for a plant capable of producing 10 Kg of purified rLTNF. Economic analysis has been performed for manufacturing 3 ton of purified rLTNF. RESULTS The process developed produces peptide in the form of concatemer that has been specifically designed to accumulate as insoluble inclusion bodies (IB) during expression in E. coli. A cation exchange chromatography step has been developed to capture the rLTNF concatemer at 140 g/L dynamic binding capacity. Further, the purified concatemer is cleaved completely into monomeric rLTNF using alpha-chymotrypsin enzyme. Finally, a reversed phase high performance liquid chromatography has been designed to purify rLTNF with a recovery of more than 90% and purity greater than 98%. The overall process recovery is 78±2% resulting in 3.36 g of purified product per batch. Techno-economic evaluation of the process has been performed to demonstrate its economic feasibility against currently marketed antivenom products. CONCLUSIONS The developed process is able to produce purified rLTNF with 78±2% recovery. The study shows that recombinant technology can be used to produce rLTNF cost effectively and shows potential as a substitute for currently available antivenoms against snakebite.

Inquiry-Based Laboratory for Teaching Students Design-of-Experiments

The paper explores the new technology commercialization model being followed at The University of Akron (UA).UA has implemented the National Science Foundation (NSF)Innovation ICorpsTMmodel since 2013. This paper describes how the NSF I-Corps model has changed the innovation culture at UA and allowed the academic faculty to explore the business potential of their intellectual property.Differences in implementation across several I-Corps Sites are explored. The lessons from UAs experience show that the I-Corps Sites program has been a resounding success and helped faculty and students to become more entrepreneurial in exploring the commercial value and demand drivers of technologies invented at UA.Students are more attracted towards and enthusiastic about modern teaching techniques. This paper provides a new concept in the design of a coursework thus enabling the teachers to enhance the teaching and learning process. The course work, laboratory and course projects are linked through proper planning. It is observed that there is an increase in the degree of involvement of the students in the course and they move from doing demonstrations to structured enquiry and also open ended enquiry. This new framework can also address some of the ABET criteria effectively. Results show positive response from the students towards this new technique.The real time experience of Signals and Systems has been embedded in the form of mathematical expressions. The learners fi nd diffi culty in applying these mathematical concepts in a real time scenario. This paper presents the details of effective learning technique attempted for the Signals and Systems course at the under graduate engineering program using mind mapping. Mind mapping as a technique tests the students ability for interactive learning to improve retention and reduce revision time. This paper discusses about the effects of digital/ paper-based mind mapping over conventional teaching method to shift an teaching centric to learning centric. This learning not only focuses on retention of concepts but also caters to the generation of ideas required for solving an engineering problem, which in turn improves the writing skills of the students. Findings showed that there was a signifi cant positive difference in students academic achievement and attitude towards learning the subject through the paper/digital based mind mapping.