Engineering principles for synthetic biology : from concept to practice

Synthetic Biology is a relatively new and multi-faceted interdisciplinary emergent field of research that combines biology with technology in novel and exciting ways. One of its main branches aims to see living systems engineered in a rational and straightforward bottom-up approach, like in other engineering disciplines. The inherent complex nature of living systems turns them into a difficult and challenging substrate where to apply common engineering principles such as standardization, abstraction and modularity. Efforts to overcome these limitations and adapt such principles for working upon living systems have been devoted, though yet with relative success. The aim of this Thesis is to critically explore what is Synthetic Biology and how far it is from a veritable engineering discipline. In this Thesis, we first present a review that thoroughly explores and discusses this scenario. Then, we present two works that shall contribute to this ambitious and hard goal. First, within the context of standardization, we address the need for better genetic parts characterization by providing an example of a biologically grounded framework inspired by classical enzymology theory. Second, and in relation with the principle of modularity, we provide a theoretical framework, in this case inspired by the Ohm’s law of electric theory, that describes the unintended coupling of the coexisting genetic loads within a given host cell due to sharing a limited common pool of machinery and resources. Together, both works contribute, on one hand, to increase our understanding of the organizing principles of living systems, and on the other hand, to improve how engineering principles are applied to synthetic circuit design. Finally, these works emphasize the need to find better experimentally backed-up theoretical frameworks or models that should allow us to jump from the current time-consuming, trial-error and ad hoc Synthetic Biology to a well-established engineering discipline as fruitful and efficient with the living systems realm as other engineering disciplines are.La Biologia Sintètica és un camp de recerca emergent relativament nou i multi-facètic que combina la biologia amb la tecnologia de formes innovadores i emocionants. Una de les seves principals branques té com a objectiu aconseguir ingenieritzar els sistemes vius des d’abaix de manera racional i senzilla, tal com passa en altres tipus d’enginyeria. La naturalesa inherentment complexa dels éssers vius els converteix en un substrat difícil sobre el qual aplicar principis d’enginyeria com l’abstracció, l’estandardització i la modularitat. S’han dedicat esforços per superar aquestes limitacions i adaptar aquests principis perquè funcionin sobre sistemes vius, tot i que encara que amb un èxit relatiu. L’objectiu d’aquesta tesi és explorar críticament què és la Biologia Sintètica i quan lluny està de ser una veritable enginyeria. En aquesta tesi, primer presentem un article de revisió que explora i discuteix a fons aquest escenari. Després presentem dos treballs que han de contribuir a aquest ambiciós i difícil objectiu. En primer lloc, en el context de l’estandardització, adrecem la necessitat d’una millor caracterització de les parts genètiques oferint un exemple de marc teòric amb fonaments biològics que esta inspirat en teoria enzimològica clàssica. En segon lloc, i relacionat amb el principi de modularitat, oferim un marc teòric, aquest cop inspirat en la llei de Ohm de la teoria elèctrica, que descriu l’aparellament no intencionat de les carregues genètiques coexistents dins d’una cèl.lula hoste qualssevol degut al fet de compartir un conjunt comú limitat de recursos i maquinària cel•lular. Ambdós treballs contribueixen, per un cantó, a incrementar el nostre coneixement sobre els principis d’organització dels éssers vius, i per l’altre, a millorar com s’apliquen els principis d’enginyeria pel disseny de circuits sintètics. Finalment, aquests treballs emfatitzen la necessitat de trobar millors marcs teòrics o models recolzats experimentalment que haurien de permetre’ns fer un salt des de l’actual Biologia Sintètica ad hoc, farregosa i basada en assaig-error, a un tipus d’enginyeria ben establerta que pugui ser tan profitosa i eficient en el reialme dels éssers vius com ho són les altres enginyeries

Dealing with the genetic load in bacterial synthetic biology circuits: convergences with the Ohm's law

Synthetic biology seeks to envision living cells as a matter of engineering. However, increasing evidence suggests that the genetic load imposed by the incorporation of synthetic devices in a living organism introduces a sort of unpredictability in the design process. As a result, individual part characterization is not enough to predict the behavior of designed circuits and thus, a costly trial-error process is eventually required. In this work, we provide a new theoretical framework for the predictive treatment of the genetic load. We mathematically and experimentally demonstrate that dependences among genes follow a quantitatively predictable behavior. Our theory predicts the observed reduction of the expression of a given synthetic gene when an extra genetic load is introduced in the circuit. The theory also explains that such dependence qualitatively differs when the extra load is added either by transcriptional or translational modifications. We finally show that the limitation of the cellular resources for gene expression leads to a mathematical formulation that converges to an expression analogous to the Ohm's law for electric circuits. Similitudes and divergences with this law are outlined. Our work provides a suitable framework with predictive character for the design process of complex genetic devices in synthetic biology.Fundación Botín, Banco de Santander through its Santander Universities Global Division [BES-2010-038940]; ERC SYNCOM [291294]; Spanish Ministry of Economy and Competitiveness [MINECO SAF2014-59284-Rand FEDER]. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [MINECO SAF2014-59284-R and FEDER]

A bottom-up characterization of transfer functions for synthetic biology designs: lessons from enzymology

Within the field of synthetic biology, a rational design of genetic parts should include a causal understanding of their input-output responses-the so-called transfer function-and how to tune them. However, a commonly adopted strategy is to fit data to Hill-shaped curves without considering the underlying molecular mechanisms. Here we provide a novel mathematical formalization that allows prediction of the global behavior of a synthetic device by considering the actual information from the involved biological parts. This is achieved by adopting an enzymology-like framework, where transfer functions are described in terms of their input affinity constant and maximal response. As a proof of concept, we characterize a set of Lux homoserine-lactone-inducible genetic devices with different levels of Lux receptor and signal molecule. Our model fits the experimental results and predicts the impact of the receptor's ribosome-binding site strength, as a tunable parameter that affects gene expression. The evolutionary implications are outlined.Fundacion Botín, Banco de Santander through its Santander Universities Global Division [BES-2010-038940 to/nR.M., C.R.C.]; ERC SYNCOM [291294 to M.C.B.]; FPI MINECO fellowship [to S.D.N.]. Funding for open access charge: ERC SYNCOM [291294]