Team:NUS Singapore/Demonstrate
Overview
The necessity to prolong the viability and activity of engineered cells is a significant part of synthetic biology. However, the development of technologies to enable sustained activity have not kept up with the advances made in engineering cells. Many cell-based applications still adopt long established methods such as cold chains or lyophilization for storage. The major limitation to such methods is that they do not provide prolonged function in situ. Inspired by a need to find a method that can truly prolong lifespan in terms of both shelf-life as well as in situ activity, we looked into engineering cells themselves to prolong their functional lifespan. Thus, our platform technology - E.co LIVE - was born.
Firstly, we demonstrate below the two methods we have used to control cell growth and protein production in bacteria, hence improving their functional lifespan. Secondly, we show evidence of the modularity of our method, allowing it to potentially be a platform technology for synthetic biologists. Thirdly, we illustrate our designs enabling the preservation of our genetic engineering in the cell, while simultaneously preventing its unwanted spread into the environment. Lastly, we detail our work in developing a user-friendly software, E.co Grow, to enable the easy usage of our platform technology.
Growth Switch
In our first method - a growth switch - we show that it is possible to control growth arrest and growth resumption in E. coli using the growth switch developed in the project. As the system is designed to be modular, these can be controlled with various inputs such as chemical inducers and blue light. On top of that, our growth switch indirectly regulates protein production when cell growth is controlled, providing users with another level of control over protein production.
We demonstrated that our growth switch can improve the functional lifespan of engineered cells by using measuring protein production in the form of bioluminescence. In addition, we adopted an existing genetic circuit in an engineered cell meant to sense for the presence of bacteria to demonstrate the flexibility and power of our technology (Saeidi et al., 2011). We show that cells with our growth switch activated retain the ability to produce sufficient amounts of reporter proteins even after 10 days, compared to normal-growing cells. We hypothesized that the growth-arrested cells can resume their functionality (i.e protein production) due to accumulation of unused cellular reserves (e.g ATP). To validate our hypothesis, we performed preliminary ATP measurements and discovered that the growth-arrested cells indeed accumulate more ATP as compared to normal growing cells.
Fig. 1: Demonstration of prolonged functional lifespan in our engineered cells (induced) compared to control cells (uninduced) for (a) inducible luminescence production and (b) in situ biosensor application. Growth-arrested cells induced with HicA toxin expression produced relatively higher amount of proteins even after 10 days, which was not observed in uninduced normal-growing cells.
Finally, to tie everything together and gain insights into the system for the next step of experimental design and fine tuning, we constructed a model based on our experimental data to predict how varying different parameters can affect the growth and protein production profile of the cells under different conditions. This model provided us with optimal inducer concentrations, which we utilized for the demonstration of our work.Growth Knob
In our second method - a growth knob - we show that by targeting the glucose transporter in cells, we can control the cellular glucose uptake rate. This allows us to manipulate the growth rate of cells and ultimately achieve different rates of protein production.
As our proof-of-concept, we show that increased expression of SgrS, a regulator of the glucose transporter subunit, decreases cell growth rate and prolongs inducible RFP protein production. In other words, cells induced with SgrS were able to produce proteins for a longer period of time compared to uninduced control cells, albeit at a lower level. Since protein production is coupled to growth, a reduced growth rate would imply a lower protein production rate. We hypothesized that less glucose would be consumed when cell growth is reduced, providing cells with longer-lasting nutrient reserves for growth and protein production. To validate our hypothesis, we performed glucose measurements and the results suggests that the amount of glucose remaining in the spent medium is inversely correlated with the absolute growth of cells. More experiments need to be performed for further validation.
Fig. 2: With a higher SgrS expression (induced by aTc), we observe (a) a reduced protein production rate in aTc-induced cells (compared to uninduced control cells) as well as (b) a lower glucose uptake. Taken together, both results suggest the possibility of unused glucose accumulation to enable prolonged duration of protein production, thereby prolonging the functional lifespan of engineered cells.
Similarly, we also constructed a model based on our experimental data to further predict how different parameters will change the growth and protein production profile of the cells under different conditions.Dual Plasmid Retention System
Besides the growth control systems, preliminary work was done to test the concept of an unconventional biocontainment cum plasmid retention approach - a dual plasmid retention system. This system was developed with the goal of avoiding antibiotic use in our final E.co LIVE system. We have successfully demonstrated the ability of one plasmid (selected by antibiotic) at retaining its complementary plasmid, despite the absence of an antibiotic selection for the latter plasmid. Due to time constraints, we were unable to proceed with downstream characterization of this system. Nevertheless, we are pleased to show the feasibility of our concept, which was carefully planned by speaking to various experts like Prof. Chris Barnes and Dr. Alex Fedorec.
E.co Grow
With E.co LIVE, we strive to provide a comprehensive platform technology for users by incorporating the Design-Build-Test-Learn (DBTL) cycle. For the models which were constructed based on our experimental data, we turned them into an interactive and intuitive software to provide users with predictions of their cell growth and protein production. This allows users to learn from our model before designing and building of their genetic circuits for testing. By adhering to the DBTL principles, we envision our tool to be a reliable and useful aspect in experimental planning and execution for our users.
References
Saeidi, N., Wong, C. K., Lo, T. M., Nguyen, H. X., Ling, H., Leong, S. S. J., … Chang, M. W. (2011). Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen. Molecular Systems Biology. https://doi.org/10.1038/msb.2011.55