Burdensome gene expression promotes heterogeneity in cellular response that can be mitigated up to a tipping point

The activation of burdensome gene
circuits is marked by significant cell-to-
cell heterogeneity in induction levels.
Whether applied in bioprocessing,
cellular ecology, or biomedical contexts,
there is considerable interest into
comprehending and mitigating this
heterogeneity.This study focuses on the E. coli BL21 T7
expression system as a representative
example of a burdensome gene circuit.
Utilizing a quantitative measure of
heterogeneity, entropy, we observed that
periodic pulsing of the inducer could
homogenize the cell-to-cell expression
levels of the T7 expression system in the
context of a continuous cultivation. We
examined the population's rebound to a
heterogeneous structure and proposed
that this heterogeneity serves as a survival
strategy, allowing the population to evade
washout from its environment. Indeed,
harsh induction drastically reduce the
growth rate of cells and thus we propose
that the population avoids washout via a
mechanism that we define as ‘entropy
compensation’ where a fraction of cell that
did not activate the gene circuit rapidly
takes over. To explore the limits of this
survival strategy, we increased the pulsing
frequency, aiming to push the overall
induction level to a point where washout
becomes unavoidable. Intriguingly, despite
demonstrating the system's tunability with
respect to pulse frequency, no washout
occurred. Instead, a new sub-population
with a lower induction level emerged and
gradually
dominated.
Subsequent
sequencing revealed that this population
harbored a mutation weakening the
expression strength. In summary, our work
highlights the instability of the BL21 T7
expression system, showcases its ability to
adapt through heterogeneity as a survival
strategy and reveals a genetic escape
when trying to mitigate phenotypic
heterogeneity and push up induction.