The role of the cellular economics in the expression of exogenous genes: Towards modularity in synthetic circuit design.

One of the goals of synthetic biology is the design and construction of biological systems capable of performing logic computations, that is, to integrate environmental signals to produce desired outputs. This is a very important step towards further developments of the field, such as the construction of synthetic tissues and organs. To achieve that goal, it is required that engineering principles could be applied into biological systems. This proposal focuses in the principle of modularity as a rule of design in biology. Modularity will allow fast and reliable building of complex synthetic genetic circuits. Large circuits could be easily implemented as the sum of well-known modules that keep the same properties that they exhibit when they are isolated, as it happens in electronics. That is a big challenge for synthetic biologists as the modules are rarely independent from each other. This is mainly because all of them compete for the same pool of cellular resources for the expression of their constituent genes.
As a consequence in the limitations of the availability of resources, the cell can be envisioned as a molecular economical market where many genes compete simultaneously for the same transcriptional and translational machineries. These machineries catalyse, respectively, the synthesis of the RNA and the synthesis of the proteins that ultimately perform functions in the cell. The resources available for gene expression are not infinite and, therefore, depending on how the cellular resources are invested, some genes will benefit from larger fractions of the pool while some others will only have access to very small amounts. This asymmetric distribution or resources is consequently reflected in the different relative abundances of the proteins encoded by genes being expressed at the same time. This uneven distribution affects particularly the performance of artificial genetic circuits and, ultimately, compromise the notion of modularity.
In a comparison with computer science, the synthetic circuits would be the software of the system - a set of scripts designed to perform a function -, whereas the cell machinery would be the hardware needed to accomplish the instructions coded in the exogenous DNA. In this project we aim to identify modifications of the hardware increasing the processing capabilities of the cell so that more complex programs can be run using to that end the model system Escherichia coli. We will analyse in a test circuit the effect of modifications in genes involved in the synthesis and function of the RNA polymerase and ribosomes. We will use for this task a circuit that I developed and applied previously to determine the extent of competition in gene expression using fluorescent reporters. Once the relevant genes for resource allocation have been identified, we will develop strains optimized for circuit implementation. These newly evolved strains will be used to implement complex computations based on genetic circuits encoding transcriptional cascades, oscillators and multi-layered logic gates. To understand the behaviour of these complex systems we will generate mathematical models describing each of the circuits operating in conditions where the cell resources are limited. The models will be used to make predictions about circuit performance, and these predictions will be validated experimentally.
With the results of these investigations we aim to bypass the problem of poor circuit performance due to sharing of resources in the cell, a phenomenon that substantially limits the complexity of the circuits that can be built. In addition, we will test the limits of modular design in bacteria. Achieving modular construction of genetic networks will have a great impact in the way that we can design complex processes for making decisions in living organisms. It will help to transform synthetic biology into a real engineering discipline, paving the path for multiple biotechnological applications.

In this proposal we aim to understand the mechanisms of allocation of transcriptional and translational resources in Escherichia coli looking to optimize the performance of synthetic genetic circuits. We hypothesize that the determinants of how those resources are invested are genetically encoded and, therefore, simple manipulations of the host can greatly improve the dynamic properties of synthetic circuits allowing complex computations.
We propose a combined experimental and theoretical approach where we will determine the role of genes controlling the production and activity of RNA polymerase and ribosomes in the levels of protein production that can be achieved in the cell. We will use to that end a test circuit that I developed previously and measures the coupling in the expression of two fluorescent reporters in single cells. We will test mutants displaying different allocation of resources and we will select those that show and improved capacity for protein production. Once this new strains are characterized, we will use them to implement larger circuits for complex cellular computations. We will first model the behaviour of known synthetic transcriptional cascades, oscillators and multi-layered logic gates under the different conditions given by the newly evolved economies of the selected hosts. We will use for that the parameters provided in the literature and we will add the reactions and conservation laws exclusive of our model. We will validate the theoretical predictions building the circuits and implementing them in the hosts endowed with the new properties. With these experiments we will investigate the limits of modularity in the design and construction of synthetic genetic circuits.
As a result of these investigations the major sources of unaccounted coupling in gene expression will be addressed in the design phase, greatly facilitating the task of engineering large biological networks.

Economic Growth and Development

The Synthetic Biology Roadmap for UK highlights a recent assessment performed by BCC Research. It predicts an increase of one order of magnitude in a period of five years. By 2016 the market will be worth $10.8 bn and will continue growing. The rapid expansion takes place thanks in part to the identification of new niches to develop applications, but also because of technological breakthroughs that could significantly reduce the time and cost of implementation of engineered biological systems. This proposal aims to help keeping the pace of development of the field, filling the current gap in the design of modular synthetic genetic circuits by addressing the question of how resources required for gene expression are allocated in a cell. We envision a broad impact of our work benefiting distinct sectors of society.

Commercial exploitation and industrial partnership

Many biotechnological processes, ranging from large fermentation processes to cellular decision making in synthetic tissues, could be improved if accounting for the findings of the proposal. We will identify companies in the UK with interest in the topic for collaborative endeavours, several of which are accessible in the Surrey area. We have already made contact with the company ReBio (former TMO renewables) that is aware of the problematic issue of resource competition and could adapt our discoveries to its own specific applications in production of metabolites with added value from waste, mainly for synthesis of biopolymers. We expect to obtain results at different stages of the project with potential commercial interest that we will foster through the university technology transfer office. We will increase the impact of this work in industry under the umbrella of the EU program Horizon2020. Bacmine SL (Spain), a company specialized in the synthesis of genetic circuits for tailor made applications, is interested in the concepts detailed in the proposal and willing to test the strains produced.

Improvement to human health

Although the work does not aim to solve a health concern directly, it will improve health indirectly as many applications could be achieved in an affordable way. This research is likely to translate in larger numbers of molecules with pharmacological relevance produced at a lower cost, better synthesis of bioinspired devices and improved environmental quality control.

Public engagement in scientific issues

The development of the field of synthetic biology not only depends on the technical capabilities and scientific achievements. The perception of synthetic biology by society is critical for its success and we will make sure that the ethics and importance of the work are available for the general public, technical specialists and policy makers. The vision of the field highly depends on the implication and knowledge of the audience. For that reason, in this project we also aim to analyse whether all social groups involved share a common vision of the technology, its prospective risks and benefits, and the ethical boundaries that should be set for the targets of the field. We aim to collect sociological data from polls and interviews to assess the perception and elaborate communication strategies targeting different audiences.

Evidence based policy making

With this work we hope to accelerate the development of synthetic biology so that many applications become feasible in a short period of time. To ensure this, we expect a parallel contribution to policy making so that we can anticipate challenges regarding intellectual property of methods, applications and deliverables. In relationship with the previous section, with the public awareness of the approach we also expect to contribute to the implementation of policies according with the demands of society. With the visibility provided by this contribution we also hope to foster the investment in the field from both public and private sources.