Projects / Programmes
Development and optimization of electroporation protocol for extracting biotechnologically relevant molecules from bacterial cells
| Code |
Science |
Field |
Subfield |
| 7.00.00 |
Interdisciplinary research |
|
|
| Code |
Science |
Field |
| T490 |
Technological sciences |
Biotechnology |
| Code |
Science |
Field |
| 2.06 |
Engineering and Technology |
Medical engineering
|
Extraction by means of electroporation, protein, plasmid DNA, batch system, laboratory scale flow-through system, Escherichia coli
Researchers (8)
Organisations (2)
Abstract
Proteins and plasmid DNA (pDNA) extracted from bacteria have proved of great value in industry and medicine. Established processes to extract these molecules (e.g. mechanical disintegration – proteins, alkaline lysis – pDNA) often include mechanical forces or chemicals, which can be detrimental to their structure and integrity. Furthermore, complete disruption of the cell releases large amount of contaminants, particularly endotoxins and genomic DNA (gDNA), thus additional purification steps are required. Consequently the cost of molecule purification at large scales can reach up to 80% of the production costs. This provides strong motivation for development of new, economically more viable protein and pDNA extraction tools from bacteria.
When a cell is exposed to electric pulses of sufficient strength, transmembrane voltage is induced, which, when exceeding a certain threshold value, renders the membrane permeable – electroporation. Thus molecules to which membrane is otherwise impermeable can enter the cell. Recently, electroporation was also shown to have great potential for extracting molecules from various microorganisms.
In the project, we will develop an efficient method for extraction of proteins and pDNA from bacteria (extraction by means of electroporation), providing a basis for significant reduction of downstream processing with low investment costs. Due to avoidance of using harmful chemicals for extraction, a significant improvement of the environmental footprint is also expected. The most relevant parameters affecting the efficiency of protein and pDNA extraction by means of electroporation will be studied: pulse parameters (amplitude, polarity, duration, number and pulse repetition frequency), growth phase of bacteria, composition of the medium and exposure and post-exposure temperature. Our method will be compared to mechanical disintegration and alkaline lysis. The main advantages of the proposed method are expected to be: better yield, significantly less contaminants (endotoxins, gDNA), shorter extraction time (less than a second), reduced lysate volume, no toxic chemicals used for extraction purposes and in-line setup.
Based on our expertise and knowledge, we will build mathematical models of electric field distribution in order to design a continuous flow electroporation treatment chamber and to determine optimized electrode position and spacing for efficient extraction of proteins and pDNA by means of electroporation. Thus a laboratory flow-through system for extraction of proteins and pDNA by means of electroporation will be developed, achieving Technology Readiness Level 4. We will also assess the feasibility to extract proteins in first and pDNA in second step (a two-step extraction), from the same bacteria, which has not been tested yet. Namely, proteins are potentially undesired product in pDNA extract and by removing a vast majority of proteins in first step, obtained pDNA in second step is expected to have less protein contamination.
Objectives of the project: (1) establish and optimize the experimental protocol to extract proteins and pDNA by means of electroporation from bacteria in a batch system, (2) establish and optimize the experimental protocol to extract proteins (also specific periplasmic protein) and pDNA by means of electroporation from bacteria in a laboratory scale flow-through system, (3) compare flow-through extraction by means of electroporation with mechanical disintegration and alkaline lysis in terms of protein and pDNA yield, respectively, presence of endotoxins and gDNA, productivity, concentration and activity of specific periplasmic protein, cost and extraction time and (4) assess the feasibility to selectively extract proteins and pDNA by means of electroporation from the same bacteria in a two-step laboratory scale flow-through system. Throughout the project we will seek identification and protection of intellectual property and opportunities for exploitation of re
Significance for science
The production of molecules in bacteria, e.g. proteins for various applications in biotechnology and medicine has gained on its significance in last years. Methods aiming to extract such molecules on large production scales however still have serious drawbacks, e.g. using of chemicals not desired in final product or total mechanical disintegration of cell. The need to purify the target molecule from cellular debris is thus present, which has an important impact on the economics of whole process, causing up to 80% of the production costs. Therefore, in the scope of the project a novel extraction technique from bacteria – extraction by means of electroporation, where proteins are extracted has been developed without using environmentally-questionable chemicals or totally mechanically disintegrate bacterial cell. Thus novel extraction technique has much better environmental footprint and cost effectiveness compared to other methods (e.g. mechanical disintegration). Furthermore, extraction by means of electroporation has also enabled plasmid DNA extraction and patent has been filed. A method has also been successfully optimized in terms of pulse parameters, temperature and bacterial growth phase. This makes possible for researchers to improve the time and cost effectiveness of new experiments in the area of extraction by means of electroporation. Developed method has also been upgraded and optimized to treat larger volumes – extracting proteins by means of electroporation in a laboratory scale flow-through system and thus enabling new possibilities to treat larger volumes of various microorganisms (e.g. different bacteria, yeasts, microalgae etc.) or diverse liquids with electroporation in order to obtain various intracellular molecules for food or pharmaceutical industry, inactivate microorganisms in liquid food etc. Newly developed and optimized extraction technique by means of electroporation will for other researchers in the field shorten development cycles and reduce costs associated with the development of novel application and will significantly enhance research in the area of electroporation and further the expansion of the field itself.
Significance for the country
The result of the present research project has solidified the position of Slovenia as one of the leading countries in the area of electroporation research. Namely, extraction of proteins by means of electroporation from bacteria in a flow-through process, achieving technology readiness level 4 is of international importance, since the production of molecules in bacteria, e.g. proteins for various applications in biotechnology and medicine has gained on its significance in last years. Furthermore, due to results of the present research project scientists from Serbia and Poland had seek our expertise and laboratory scale flow-through process for their research. The knowledge gained in the research project has already been included in the teaching process at the University of Ljubljana in the scope of Masters’ (finished graduate thesis; COBISS ID 11883860) and PhD (finished doctoral thesis; COBISS ID 10655060) level studies. Furthermore, developed laboratory scale flow-through process was presented at the yearly organized postgraduate course “Electroporation based Technologies and Treatments” which has been attended by PhD students and researches from the field of electroporation from all over the world. The results have also been presented at international conferences and published in SCI-ranked scientific journals. Furthermore, the review of electroporation applications in biotechnology which was written in the scope of this project and published in prestige journal Trends in Biotechnology with high impact factor (5-year impact factor was 13,382) was also awarded by ARRS as Excellent in Science in 2016. This contributed to the further increase in scientific prominence of the research group, as well as of the Republic of Slovenia as a whole.
Most important scientific results
Annual report
2014,
2015,
final report
Most important socioeconomically and culturally relevant results
Annual report
2014,
2015,
final report
