Development and Optimization of Electroporation Protocol for Extracting Biotechnologically Relevant Molecules from Bacterial Cells (2014-2017) |
| Principal investigator: | Prof. Damijan Miklavcic, Faculty of Electrical Eng., University of Ljubljana |
| Partner: | Dr. Matjaz Peterka, Center of Excellence for Biosensors, Instrumentation and Process Control (COBIK), Ljubljana |
| Funding: | Slovenian Research Agency (ARRS), Slovenia |
| Code: | J7-6783 |
Research project funded by the Slovenian Research Agency.
- Member of University of Ljubljana
University of Ljubljana, Faculty of Electrical Engineering
- Code
- J7-6783
- Project
- Development and optimization of electroporation protocol for extracting biotechnologically relevant molecules from bacterial cells
- Period
- 1.7.2014-30.6.2017
- Amount of financing
- In 2017 0,40 FTE
- Head
- Research activity
- Interdisciplinary research
- Research Organisation
- Abstract
Proteins extracted from bacteria have proved of great value in industry and medicine. Established processes to extract proteins from bacteria (e.g. mechanical disintegration, enzyme lysis) often include mechanical forces or chemicals, which can be detrimental to protein’s 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 viable protein extraction tool from bacteria.
When a cell is exposed to electric pulses of sufficient strength, a 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 or exit the cell. Recently, electroporation was also shown to have great potential for extracting molecules from various microorganisms.
In the project, we explored the possibility to develop an efficient method for extraction of proteins 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 was also demonstrated. The most relevant parameters affecting the efficiency of protein extraction by means of electroporation were studied: pulse parameters (amplitude, duration, number, pulse repetition frequency), growth phase of bacteria and exposure temperature. Our method was compared to mechanical disintegration with glass beads and sonification. The main advantages of the proposed method were shown to be: significantly less contaminants (especially gDNA), shorter extraction time (less than a second), reduced lysate volume, no toxic chemicals used for extraction and in-line setup.
We developed a pulse generator that enables continuous pulse treatment and designed a continuous flow electroporation treatment chamber and determined optimized electrode position and spacing for efficient extraction of proteins by means of electroporation – a laboratory flow-through system for extraction of proteins by means of electroporation was developed, achieving Technology Readiness Level 4.
Objectives of the project were: (1) establish and optimize the experimental protocol to extract proteins by means of electroporation from bacteria in a batch system, (2) establish and optimize the experimental protocol to extract proteins by means of electroporation from bacteria in a laboratory scale flow-through system and (3) compare flow-through extraction by means of electroporation with mechanical disintegration with glass beads and sonification in terms of protein yield, presence of endotoxins and gDNA, productivity, cost and extraction time.
- Researchers
- Karel Flisar (02204): 2014-2016
- Saša Haberl Meglič (29041): 2014-2017
- Nikolaja Janež (33406): 2014-2017
- Tadej Kotnik (15675): 2014-2017
- Damijan Miklavčič (10268): 2014-2017
- Nataša Pavšelj (20822): 2014-2016
- Matjaž Peterka (16327): 2014-2017
- Urška Zelenko (32914): from 2016
- Barbara Zorec (34299): from 2016
- The phases of the project and their realization
WP1
In the scope of this WP we first focused on size specific molecule extraction by means of electroporation. E. coli cells were exposed to a variety of electric pulses (different amplitude, duration, number and pulse repetition frequency) using square wave electric pulse generator. We determined which molecules are extracted from bacteria at specific pulse parameters.
The results were presented:
- at the international conference Mipro 2014 in a presentation entitled “Extraction by means of electroporation of biotechnologically relevant molecules from bacterial cells”, author Haberl Meglič S.
- at the international conference 2nd World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies 2017 in a presentation entitled “Electroextraction of large biomolecules is efficient but underestimated in protein and DNA production”, authors Peterka M, Janež N, Haberl Meglič S, Miklavčič D.
- patent application A method for isolation of plasmin DNA (PDNA) from cell cultures, patent : PCT/EP2014/050084, authors Peterka M, Jarc M, Štrancar A, Haberl Meglič S, Miklavčič D.
In the second part of WP1 we focused on extraction of proteins from E. coli by means of electroporation in a batch system, where we optimized pulse parameters, temperature and bacterial growth phase in order to extract maximum amount of proteins and preserve bacterial viability. We found that longer pulses (1 ms) with lower pulse amplitude (5 kV/cm) and 4°C temperature performed best. At these conditions maximum amount of proteins were extracted.
The results were presented:
- graduate thesis by Marolt T. entitled “Influence of electric pulse parameters on electroextraction of proteins from E.coli”, 2014.
- at the international conference 16th European Congress on Biotechnology 2014 in a presentation entitled “Protein extraction by means of electroporation and bacterial viability of E. coli”, authors Haberl Meglič S, Marolt T, Miklavčič D.
- at the international conference 2nd PEF School 2015 in a presentation entitled “Protein extraction by means of electroporation and bacterial viability of E. coli”, authors Haberl Meglič S, Marolt T, Miklavčič D.
- in paper: Haberl Meglič S, Marolt T, Miklavčič D. Protein extraction by means of electroporation from E. coli with preserved viability, The journal of membrane biology, 248:893-901, 2015.
- at the international conference 1nd World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies 2015 in a presentation entitled “The effect of temperature on protein extraction by electroporation and on bacterial viability, authors Haberl Meglič S, Levičnik E, Luengo E, Raso J, Miklavčič D.
- at the international conference 30th EFFOST International Conference 2016 in a presentation entitled “The effect of temperature and growth phase on protein extraction from bacterial cells by means of electroporation”, authors Haberl Meglič S, Levičnik E, Miklavčič D.
- at the international conference XXIII International Symposium on Bioelectrochemistry and Bioenergetics 2015 in an invited lecture entitled “Optimization of electroporation protocol for extracting proteins and plasmid DNA from E. coli”, author Haberl Meglič S.
- in paper: Haberl Meglič S, Levičnik E, Luengo E, Raso J, Miklavčič D. The effect of temperature and bacterial growth phase on protein extraction by means of electroporation, Bioelectrochemistry, 112:77-82, 2016.
WP2
In WP2 we first developed a laboratory scale flow-through system for extraction of proteins by means of electroporation. Prototype pulse generator that enables continuous pulse treatment and adequate electric pulse chamber were developed.
The results were presented:
- in paper: Flisar K, Haberl Meglič S, Morelj J, Golob J, Miklavčič D. Testing a prototype pulse generator for a continuous flow system and its use for E. coli inactivation and microalgae lipid extraction, Bioelectrochemistry, 100:44-51, 2014
- doctoral thesis by Flisar K. entitled “High energy device for reversible and irreversible electroporation of the cell membrane”, 2017.
In the second part of WP2 we focused on optimization of extraction by means of electroporation in a laboratory scale flow-through system in terms of temperature. We found that temperature does not have large influence on protein extraction. We believe that maybe cooling or heating of flow through tubes and electrodes is needed in order to obtain comparable results to batch system, where cooled or heated cuvettes were used.
The results were presented:
- at the international conference 3rd PEF School 2016 in a presentation entitled “The influence of temperature on protein extraction from E. coli in a flow through system”, authors Haberl Meglič S, Levičnik E, Flisar K, Miklavčič D.
WP3
In the scope of this WP we compared extraction by means of electroporation with mechanical disintegration (glass bead homogenization and sonification) in terms of extracted protein quantity. Presence of contaminants (endotoxins, gDNA), cost and time needed for the extraction were estimated and compared. We found that early exponential bacterial growth phase and temperature 37°C performed best. At these conditions maximum amount of proteins were extracted by means of electroporation and less endotoxins and gDNA were present in the final sample. Furthermore, we found that with extraction by means of electroporation less contaminants (especially gDNA) were present in final sample in comparison to glass bead homogenization and sonification.
The results were presented:
- in paper: Kotnik T, Frey W, Sack M, Haberl Meglič S, Peterka M, Miklavčič D. Electroporation-based applications in biotechnology, Trends in biotechnology, 33:480-488, 2015
- in book chapter: Haberl Meglič S, Kotnik T. Electroporation-based applications in biotechnology, Handbook of electroporation, Springer, pp.1-17, 2016.
- in book chapter: Haberl Meglič S. Pulsed electric fields-assisted extraction of molecules from bacterial and yeast cells, Handbook of electroporation, Springer, pp.1-19, 2016.
- at the international conference 2nd World Congress on Electroporation and Pulsed Electric Fields in Biology, Medicine and Food & Environmental Technologies 2017 in a presentation entitled “Comparing two methods for protein extraction from bacterial cells-electroporation and glass bead homogenization”, authors Haberl Meglič S, Miklavčič D
- Citations for bibliographic records