Alongside application projects, we have identified a series of projects which can be done to improve and expand upon the core foundational technology behind the PixCell system.
Construction of an Improved Patterning Circuit
A simple improvement of the PixCell patterning circuit could be made with the addition of an inverter switch meaning an oxidising potential causes expression of GFP and repression of RFP. This would allow for improved characterisation of the system’s “leakiness” as well as providing more visually appealing images to use for public engagement.
Pyocyanin Production in E. coli
Pyocyanin was the main redox-cycling molecule we tested with our system which effectively carried a signal from an electrode to a genetic circuit. Although, pyocyanin is incredibly expensive due to it being purified from its host organism: Pseudomonas aeruginosa (Mavrodi et al., 2001). To make cells naturally responsive to electrical inputs the Pyocyanin synthesis operon could be cloned into E. coli. Furthermore, deletions of the final genes in the pathway would allow for selection of intermediates in the pathway which also have redox-cycling activities but have reduced toxicity.
Engineering Electrogenetic Strains
Throughout our project we used DJ901, an E. coli strain which had the soxRS regulon deleted. This prevents bacterial stress responses from reducing the redox-cycling molecules which would otherwise activate our circuit following an electrical pulse. Identifying these genes and creating knockout strains could increase the sensitivity of the cell to electronic signals. Furthermore, alternative redox-protectant genes could be added to the strains to ensure signal remains high without significantly impacting cell growth.
An Integrated Potentiostat-Electrode Array
Our current system utilised a commercial measuring potentiostat which we required for electrochemical characterisation. An improved system could be created where the Arduino controller, potentiostat and array all integrated on a single PCB. Potentiostats can be made for less than $80. This would make the cost of this portable, remotely controlled device at less than $250.
Porting the System to Other Species
Although E. coli is a model organism for synthetic biology work, when it comes to making applications the specialised machinery of other cells outcompete it. The conservation of the SoxR/pSoxS system across many bacterial species suggests that porting this electrogenetic to other bacterial species is entirely feasible (Sheplock et al., 2013). These could include B. subtilis for biomaterial production or various cyanobacterial species for use in microbial fuel cell and biophotovoltaic control. Detoxified mutants could also be created to allow for porting into eukaryotic species.
Combining with Electrogenetic Output Systems
By combining our electrogenetic “input” device with an electrogenetic “output” device would allow for in silico feedback be used for ultra-precise control of gene expression. One candidate “output” system is the electroconductive pilli of Geobacter which allow for the cells to form conductive biofilms (Holmes et al., 2016). Altering pilli structure and function with a gene circuit controlled by the PixCell system would allow for true integration of in silico feedback with genetic circuitry.
Testing Aerobicity of System
Although the our results showing sulfite has no effect on GFP fluorescence suggests the cells are in aerobic environments, this could be further proved experimentally. The chemical conditions of our electrogenetic system could be tested on cells containing mutations that prevent NADPH reduction in anaerobic conditions. If the cells survive then this confirms they are in an aerobic condition.