Periodic Reporting for period 4 - CyberGenetics (Cybergenetics: Theory and Design Tools for Biomolecular Control Systems) Reporting period: 2022-02-01 to 2022-12-31 Summary of the context and overall objectives of the project In this project we developd new theory and design tools for the real-time control of living cells. The control systems designed using these tools precisely and robustly steer the dynamic behavior of living cells in real time to achieve desired objectives. Cells could be controlled either collectively at the population level, or individually as single cells. The control systems achieving this regulation are realized either on a digital computer that is interfaced with living cells, or using newly genetic circuits that are genetically engineered into the cells where they are designed to function as molecular control systems. Our methods explicitly address the numerous challenges brought about by the special environment of the cell including nonlinearity, stochasticity, cell-to-cell variability, and metabolic burden. The theory and methods developed in this project thus enable the systematic, rational, and effective feedback control of living cells at the gene level, and lay the foundation for a new field which we call “Cybergenetics”. They also open new research directions in the areas of control theory and estimation. We have on demonstrated our methods on several cybergenetic control systems, each addressing an important application in biotechnology or therapeutics. In one application, the controller uses light to precisely regulate gene expression in bioreactor to enhance bioproduction. In a second application, multiple feedback controllers effectively regulate in parallel a large number of single cells using light. Perhaps the most significant applications of our results involve genetic engineering into living cells robust dynamic molecular control systems that implement integral control to precisely and robustly regulate cell behavior. One of our envisioned application of these advanced controller technologies is the real-time monitoring of dysregulated physiological variables and the corresponding expression of biological effectors to bring these variables back to their normal levels, with important implications for the field of cell therapy. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We have advanced the field of Cybergenetics significantly since the beginning of the project. Most notably, we have developed the theory for designing molecular control systems that achieve a special property called “robust perfect adaptation”. This property means that our specially designed molecular controllers are guaranteed to bring cellular variables of interest to a desired value and maintain it there, in spite of the large uncertainty and randomness inside living cells and other disturbances. Our theory also gives us all controllers that will have this property, so we can select a suitable one that can achieve additional desirable objectives. The theory tells us that such controllers must implement the mathematical process of integration using molecular calculations. With this in mind, we have succeeded in the lab in building and testing the first ever such control system in a living cell. Such molecular control systems should have several applications in industrial biotechnology and medical therapy. We have then proceeded to characterize all controllers that achieve these adaptation, properties and developed a new Internal Model Principle for characterizing them. We then went on to develop the necessary methods for designing and building more advanced control systems with desirable dynamic properties and then we demonstrated how these controllers can be genetically engineered using special proteins called inteins. In short, more than any other group in the work, we have significantly advanced the theory and methods for building genetic feedback control systems.We have also taken a parallel approach for controlling living cells—this time using a digital computer to carry out the controlling. The computer measures the behavior of living cells in real time, and then based on a sophisticated control algorithm commands a light emitting diode to shine light with a carefully chosen intensity on the cells of interest, which have been especially engineered to respond to this light. By continuously measuring the cellular response and correcting it with light, the computer is able to control precisely the dynamic behavior of these cells. We have advanced this type of control in two ways. In the first, we have instrumented bioreactors so we can control cell populations with light and computer feedback in an effort to make bioproduction much more efficient. In the second approach, we have engineered devices that allow us to shine light on single cells. The resulting platform, called Cyberloop, allows single cells to be controlled independently under the microscope. This has many applications in stem cell differentiation, tissue engineering, and basic biology. As we have developed these platforms, we have also developed the necessary theoretical foundations that allow us to understand how to best use them to achieve robust regulation with good performance.The work carried out in this project has been widely disseminated through journal articles, conference and workshop presentations, and numerous lectures. We have also filed for two patents to protect the IP developed. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) When we first started this project, the field of Cybergenetics had barely started. All the developments mentioned above advance the field of Cybergenetics beyond the state-of-the-art. This is as much true of the control theory as it is for the advanced applications of that theory. One of the defining features of our work is that it integrates theory and biological applications very tightly—something that is rarely done in the field of synthetic biology. Indeed the theory is both in the service of the applications and a driver of them. This has required building interdisciplinarity from the start. It has also pointed to a new and effective way of carrying out biological design in synthetic biology— a rational design approach that is guided by theory and rests on its solid foundations. cellularcontrol.png
