While natural systems integrate diverse “digital” signals to precisely specify “analog” gene expression levels, synthetic systems thus far have focused on controlling expression in either a digital or an analog capacity. Our team sought to develop a “digitized-analog” expression system using CRISPR-dCas9, capable of specifying varied exogenous gene expression levels based on different signals. We first developed digital elements by pairing gRNAs with minimal operator promoters and using dCas9 to transactivate. We then created analog elements by multimerizing operator sites to obtain graded activation levels. Finally, we integrated our digital and analog elements into higher-order genetic logic circuits to achieve varying expression responses. We characterized and optimized our system in human cells, enabling synthetic biologists to better control transgene expression for important therapeutic applications.
Each and every one of us begins as a fusion of two cells, instilled with an innate capacity to perform elaborately intricate functions. These small computers have been pumping out megabytes of information in the form of protein synthesis since the minute you were conceived. They do this without a programmer or an engineer. As Dr. Timothy Lu and colleagues have stated, “Living cells implement ... both analogue- and digital-like processing…” to perform their complex tasks (Rubens et al, Nat Comms, 2016)
As synthetic biologists, we aim to replicate these complex behaviors to produce a wide array of technologies. These technologies include in vivo and in vitro diagnostics (Slomovic, Pardee, & Collins, 2015), control systems for metabolic engineering (Wu et al. 2016), and therapies to combat genetic disorders and cancer (Khalil & Collins, 2010). While we are coming closer and closer each day to accomplishing these advancements (in particular Rubens et al. 2016 successfully developed logic circuits with the capacity to act as a Digital to Analog converter among other complex designs) there is still work that needs to be done in our field to achieve more sophisticated circuitry.
This need is most prevalent in the mammalian environment in which there are far fewer tools to complete these tasks. This has many benefits for synthetic biologists. Primarily, proving that a system functions and is non-toxic in mammalian cells is a major hurdle for any work that will eventually be used for human applications. Secondly, certain products of metabolic engineering can only be produced in the mammalian cellular environment. A classic example is antibodies, which can only form properly under certain protein folding events that can be initiated in mammalian cells only.
With this in mind, we set out on our project…
Rubens, J. R., Selvaggio, G., & Lu, T. K. (2016). Synthetic mixed-signal computation in living cells. Nature Communications Nat Comms, 7, 11658. doi:10.1038/ncomms 11658.2016.02.010
Khalil, A. S., & Collins, J. J. (2010). Synthetic biology: Applications come of age. Nature Reviews Genetics, 11(5), 367-379. doi:10.1038/nrg 2775.2016.02.010
Wu, G., Yan, Q., Jones, J. A., Tang, Y. J., Fong, S. S., & Koffas, M. A. (2016). Metabolic Burden: Cornerstones in Synthetic Biology and Metabolic Engineering Applications. Trends in Biotechnology, 34(8), 652-664. doi:10.1016/j.tibtech.2016.02.010
Slomovic, S., Pardee, K., & Collins, J. J. (2015). Synthetic biology devices for in vitro and in vivo diagnostics. Proceedings of the National Academy of Sciences Proc Natl Acad Sci USA, 112(47), 14429-14435. doi:10.1073/pnas.1508521112