Bringing light to bacterial electrophysiology – bacterial optogenetics

Project Outline

Background

Recent publications in high-impact journals indicate growing interest in bacterial electrophysiology (1–4). Studies, including by our group, have shown that bacterial membrane potential can exhibit non-linear spiking and rhythmic oscillations which function as bioelectrical signals during antibiotic exposure, cellular differentiation and biofilm formation (5).

The ability to control bacterial membrane potential, with precision in space and time, will provide a new window to control and engineer bacterial functions. It could also open a new avenue in synthetic biology and engineering biology to control physiological dynamics of cells. However, bacterial electrophysiology is a newly emerging field and there has been limited developments of membrane-potential modulation technologies.

Project Aim

This PhD project will develop genetically encoded optical modulation technology for bacterial electrophysiology. The project will also use a photoswitch azobenzene for modulating bacterial membrane potential. Using the optical modulation tools, we aim to alter biofilm formation, spore formation, antibiotic susceptibility and growth rates.

Methods

The project will offer the interdisciplinary training opportunity for experimental and analytical skills in biophysics, neuroscience and microbiology. We will apply the optogenetic techniques in neuroscience for the application with bacteria. The key methods to be used include molecular biology, microbiology, fluorescence microscopy, patterned optical stimulation, computational modelling, and quantitative image analysis (Python and imageJ). The project offers an aspiring student to pioneer the field of bacterial electrophysiology and bioelectricity.

References

1. K. Kikuchi, et al., Electrochemical potential enables dormant spores to integrate environmental signals. Science (80-. ). 378, 43–49 (2022).
2. A. Prindle, et al., Ion channels enable electrical communication in bacterial communities. Nature 527, 59–63 (2015).
3. X. Jin, et al., Sensitive bacterial V m sensors revealed the excitability of bacterial V m and its role in antibiotic tolerance. Proc. Natl. Acad. Sci. 120, 2017 (2023).
4. J. P. Stratford, et al., Electrically induced bacterial membrane-potential dynamics correspond to cellular proliferation capacity. PNAS 116, 9552–9557 (2019).
5. J. M. Benarroch, M. Asally, The Microbiologist’s Guide to Membrane Potential Dynamics. Trends Microbiol. 28, 304–314 (2020).

Techniques

• Molecular cloning
• Time-lapse Fluorescence microscopy
• Optical stimulation
• Plate reader assays
• Biofilm assay
• Sporulation assay
• Quantitative image analysis
• Computational modelling

Electrogenetics – engineering living electronic transducer protein

Project Outline

Background

Synthetic Biology uses biological parts to construct circuits that are analogous to electronic circuits. Beyond the conceptual analogy, directly linking biological and electronic circuits could give rise to a bio-electronics hybrid systems to achieve tasks. The convergence of synthetic biology and bioelectronics is beginning to be explored (1–3).

Project Aim

This project will develop transducer proteins that convert changes in membrane potential to enzymatic activities and gene expression. You will explore three protein engineering designs that leverage i) Voltage-sensitive and ii) Potassium sensitive protein conformation change and iii) membrane-potential dependent transporter protein.

Methods

The project will combine synthetic biology, fluorescence microscopy and electrophysiology. The key methods to be used include molecular biology, microbiology, fluorescence microscopy, patterned optical stimulation, electrical stimulation, and quantitative image analysis (Python and imageJ).

References

1. Y. Zhang, L. H. H. Hsu, X. Jiang, Living electronics. Nano Res. 13, 1205–1213 (2020).
2. J. Selberg, M. Gomez, M. Rolandi, The Potential for Convergence between Synthetic Biology and Bioelectronics. Cell Syst. 7, 231–244 (2018).
3. J. T. Atkinson, M. S. Chavez, C. M. Niman, M. Y. El-Naggar, Living electronics: A catalogue of engineered living electronic components. Microb. Biotechnol. 16, 507–533 (2023).

Techniques

• Molecular cloning
• Time-lapse Fluorescence microscopy
• Optical stimulation
• Electrical stimulation
• Quantitative image analysis

Molecular mechanism of bacterial electrical signalling

Project Outline

Background

Recent publications in high-impact journals indicate growing interest in bacterial electrophysiology (1–4). Studies, including by our group, have shown that bacterial membrane potential can exhibit non-linear spiking and rhythmic oscillations which function as bioelectrical signals during antibiotic exposure, cellular differentiation and biofilm formation (5).

Bacterial electrophysiology is an exciting cutting-edge research topic that holds a potential to become a novel bioelectrical paradigm to understanding and engineering of biology. It could provide new bioelectrical approaches to address important societal challenges, such as biofilms and antibiotic resistance.

However, bacterial electrophysiology is still a largely uncharted territory in science, and little is known about their molecular, cellular and biophysical mechanisms.

Project Aim

This PhD project aims to gain molecular and biophysical insights to bacterial electrophysiology by patch clamp assay of bacterial ion channels. Focusing on the model Gram-positive bacterium Bacillus subtilis, we have identified 25 putative ion channel genes by a bioinformatic analysis. You will characterise the roles of ion channels in B. subtilis cells using mutant strains. In particular, you will systematically characterise biofilm formation, spore formation, antibiotic susceptibility and growth rate of the deletion mutant strains. For the ion channels that show interesting phenotypes, you will perform electrophysiological assays to characterise their biophysical properties.

Methods

The project will provide an interdisciplinary training opportunity for developing experimental and analytical skills in biophysics, neuroscience and microbiology. The key methods to be used include molecular biology, patch clamp, microbiology, fluorescence microscopy, computational modelling, and quantitative image analysis (using Python and imageJ). The project offers an aspiring student the chance to pioneer the field of bacterial electrophysiology and bioelectricity.

References

1. K. Kikuchi, et al., Electrochemical potential enables dormant spores to integrate environmental signals. Science (80-. ). 378, 43–49 (2022).
2. A. Prindle, et al., Ion channels enable electrical communication in bacterial communities. Nature 527, 59–63 (2015).
3. X. Jin, et al., Sensitive bacterial V m sensors revealed the excitability of bacterial V m and its role in antibiotic tolerance. Proc. Natl. Acad. Sci. 120, 2017 (2023).
4. J. P. Stratford, et al., Electrically induced bacterial membrane-potential dynamics correspond to cellular proliferation capacity. PNAS 116, 9552–9557 (2019).
5. J. M. Benarroch, M. Asally, The Microbiologist’s Guide to Membrane Potential Dynamics. Trends Microbiol. 28, 304–314 (2020).

Techniques

• Molecular cloning
• Time-lapse Fluorescence microscopy
• Patch clamp
• Plate reader assays
• Biofilm assay
• Sporulation assay
• Quantitative image analysis
• Computational modelling