Abstract
Over the last decade, the combining of newly developed molecular tools for DNA editing with engineering principles has allowed the creation of complex cellular devices, usually based on complex genetic circuits, for many different purposes. However, when the technological evolution of genetic circuitry is compared with previous technologies such as electronic circuitry, clear limitations regarding the technological scalability of genetic circuitry are observed due to the lack of predictability. To overcome this problem, it is necessary to create new theoretical frameworks for designing genetic circuits in a feasible and reliable manner, taking into account those limitations. Among a number of such limitations, the so-called genetic burden is one of the main constraints. Surprisingly, despite its relevance, little attention has been paid to genetic burden, and it is often not considered when designing genetic circuits. In this study, a new general mathematical formalism is presented, describing the effects of genetic burden on gene expression. The mathematical analysis shows that alterations in gene expression due to genetic burden can be qualitatively described independently of the specific genetic features of the system under consideration. The mathematical model was experimentally tested in different genetic circuits. The experimental evidence coincides with the expected behaviors described by the model in complex scenarios. For instance, observed modulations in the expression levels of constitutive genes in response to changes in the levels of external inducers of gene expression that do not directly modulate them, or the emergence of limitations in gene overexpression, can be understood in terms of genetic burden. The present mathematical formalism provides a useful general framework for gene circuit design that will help to advance synthetic biological systems.
Highlights
Synthetic biology is a multidisciplinary field of research that seeks to create new biological systems, or to redesign systems that already exist in nature
The final protein concentration depends on the genetic mechanism used for protein synthesis, which is described in the numerator, but it is limited by its own expression and by the expression of the other genes present in the host cell, which is described in the denominator, where the expression of each gene contributes to the total metabolic burden
It should be noted that the limitation in red fluorescent protein (RFP) overexpression observed in Figure 5 is consistent with the results shown in Figure 2C, in which an increase in the number of copies of RFP is not translated into a linear increase in RFP levels, but the levels are lower than expected from such an increase
Summary
Synthetic biology is a multidisciplinary field of research that seeks to create new biological systems, or to redesign systems that already exist in nature. In this study we present a new general mathematical formalization and experimental validation describing how expression levels of foreign genes, at steady-state, are modulated by the negative effect associated with their coexistence with the rest of the genes in the cell, i.e., the genetic burden. Can be combined to obtain the final concentration of mRNAk (see Supplementary Information for a complete mathematical derivation) mRNAk = Υk · 1+ In this equation, factor Υk depends on the total transcriptional resources available to transcribe the N foreign genes and on the mRNAk degradation rate. The final protein concentration depends on the genetic mechanism used for protein synthesis, which is described in the numerator, but it is limited by its own expression and by the expression of the other genes present in the host cell, which is described in the denominator, where the expression of each gene contributes to the total metabolic burden.
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