Burden-driven feedback control of gene expression

Abstract

Cells use feedback regulation to ensure robust growth despite fluctuating demands for resources and differing environmental conditions. However, the expression of foreign proteins from engineered constructs is an unnatural burden that cells are not adapted for. Here we combined RNA-seq with an in vivo assay to identify the major transcriptional changes that occur in Escherichia coli when inducible synthetic constructs are expressed. We observed that native promoters related to the heat-shock response activated expression rapidly in response to synthetic expression, regardless of the construct. Using these promoters, we built a dCas9-based feedback-regulation system that automatically adjusts the expression of a synthetic construct in response to burden. Cells equipped with this general-use controller maintained their capacity for native gene expression to ensure robust growth and thus outperformed unregulated cells in terms of protein yield in batch production. This engineered feedback is to our knowledge the first example of a universal, burden-based biomolecular control system and is modular, tunable and portable.

Absolute quantification of translational regulation and burden using combined sequencing approaches

“…Interestingly, the expression of all of these are under σ 32 regulation which is the most common regulatory mode to counteract heat stress. σ 32 upregulation is often observed by expressing synthetic constructs, although the precise mechanism of σ 32 activation is not known (Ceroni et al , ). In our case, the incompletely synthesized polypeptides from the stalled ribosomes on the PK‐LacZ mRNA are most likely partially folded or misfolded and generate misfolding stress similar to the heat shock response.…”

“…This problem arises because the parts may be required to function near these sharp transition points allowing for minor perturbations to cause large deviations in expression that then propagates to the output of the circuit. The only way to guarantee the robustness of such systems is to either measure every internal state to ensure parts are not functioning near these transition points (Gorochowski et al , ), or to implement feedback control within the circuit itself for self‐regulation (Ceroni et al , ). The ability to monitor every element in a circuit also makes our approach valuable when elucidating the root cause of failures.…”

Analytical solution for a hybrid Logistic‐Monod cell growth model in batch and continuous stirred tank reactor culture

“…With the proposed Logistic‐Monod equation, the product formation kinetics could also be solved, when the above models are coupled with the Gaden equation (Gaden, ). By introducing the carrying capacity concept into the classical Monod equation, this hybrid Logistic‐Monod Model should also find applications in gene circuits engineering to study growth‐dependent gene expression pattern (Aris et al, ) or burden effects that are related with gene overexpression (Ceroni, Algar, Stan, & Ellis, ; Ceroni et al, ). More important, we present the analytical solutions for this hybrid Logistic‐Monod growth model in both batch and CSTR culture.…”

Dynamic control of endogenous metabolism with combinatorial logic circuits

“…Foremost is the problem of toxicity and stability. Even mediumsized synthetic circuits (≥4 regulators) can slow growth (Sleight et al, 2010;Chen et al, 2013;Xu et al, 2014;Ceroni et al, 2018). This can cause instability in the form of plasmid loss or mutations to the genome (Fernandez-Rodriguez et al, 2015).…”