Team:Chalmers-Gothenburg/Proof Of Concept
Proof Of Concept
How we proved our system worked
Proof of concept
Our project had the aim of creating a programmable cell factory for the production of fatty acids. That would allow us to control the fatty acid profiles in a tuneable fashion. Although we were unable to produce and regulate the fatty acid production in our wet lab work, we are confident that our work is substantial enough as a proof of concept.
The first critical point of our project was to integrate and test out the bacterial FAS system, that would allow us to regulate the expression of discrete, mono-functional thioesterases. Unfortunately, we were unable to integrate all of the eight required genes into the yeast strain, thus, not being able to show the results ourselves. However, in the study that inspired our project for integrating the bacterial FAS system in yeast (Fernandez‐Moya et al., 2015), they were able to show that the system works in Saccharomyces cerevisiae. Hence, it is with great confidence we can say that the same system would work in our project had we managed to integrate the genes as intended.
In addition, we successfully benchmarked all three chemical induction systems using fluorescent proteins carried on individual plasmids. Each system was verified individually, and in combination with the other two systems. As noted, we did not manage to fully integrate the fully functional bacterial FAS system into the yeast genome, so the thioesterase plasmids we designed were not tested.
The first critical point of our project was to integrate and test out the bacterial FAS system, that would allow us to regulate the expression of discrete, mono-functional thioesterases. Unfortunately, we were unable to integrate all of the eight required genes into the yeast strain, thus, not being able to show the results ourselves. However, in the study that inspired our project for integrating the bacterial FAS system in yeast (Fernandez‐Moya et al., 2015), they were able to show that the system works in Saccharomyces cerevisiae. Hence, it is with great confidence we can say that the same system would work in our project had we managed to integrate the genes as intended.
In addition, we successfully benchmarked all three chemical induction systems using fluorescent proteins carried on individual plasmids. Each system was verified individually, and in combination with the other two systems. As noted, we did not manage to fully integrate the fully functional bacterial FAS system into the yeast genome, so the thioesterase plasmids we designed were not tested.
All three plasmids were benchmarked for all combinations to analyse potential effects the systems could have on each other (which is explained in more detail in our Results wiki page). The most important question that we needed to answer in order to prove that our system is functional was if we are able to regulate the thioesterase expression based on our induction system.
A summary of the results from single plasmid benchmarking are given in the following figures where different concentrations of inducers were added. For all three systems we observed a positive correlation between inducer concentration and fluorescent output implying that our systems are concentration dependent and inducible. The right violin plot shows the expression of RFP where the intensity is shown to increase with increasing concentration of copper sulphate (0 µM to 500 µM), while not so much when 1000 µM is added. Indicating that there is a maximum concentration for a given output. The plot in the middle shows the GFP linked system induced by estradiol which displays a greater dynamic range for different inducer concentrations. The left plot shows similar tendencies for the tetracycline systems with a slight increase in intensity when the inducer concentration is increased. This proves that the expression of the induction systems changes when the inducer concentration is changed.
A summary of the results from single plasmid benchmarking are given in the following figures where different concentrations of inducers were added. For all three systems we observed a positive correlation between inducer concentration and fluorescent output implying that our systems are concentration dependent and inducible. The right violin plot shows the expression of RFP where the intensity is shown to increase with increasing concentration of copper sulphate (0 µM to 500 µM), while not so much when 1000 µM is added. Indicating that there is a maximum concentration for a given output. The plot in the middle shows the GFP linked system induced by estradiol which displays a greater dynamic range for different inducer concentrations. The left plot shows similar tendencies for the tetracycline systems with a slight increase in intensity when the inducer concentration is increased. This proves that the expression of the induction systems changes when the inducer concentration is changed.
In conclusion, by showing that the signal intensities change for the different induction systems it is reasonable to assume that the concept of thioesterase expression regulation also is valid to work. Previous research by Fernandez‐Moya et al., 2015 in combination with the data from our benchmarking efforts, help to validate that the key concepts of our design are workable and suggests that a tuneable fatty acid profile are achievable through the use of this system.
References
Fernandez‐Moya, R., Leber, C., Cardenas, J., & Da Silva, N. A. (2015). Functional replacement of the Saccharomyces cerevisiae fatty acid synthase with a bacterial type II system allows flexible product profiles. Biotechnology and Bioengineering, 112(12), 2618–2623.