Measurements of Gene Expression at Steady State Improve the Predictability of Part Assembly

Mathematical modeling of genetic circuits generally assumes that gene expression is at steady state when measurements are performed. However, conventional methods of measurement do not necessarily guarantee that this assumption is satisfied. In this study, we reveal a bi-plateau mode of gene expression at the single-cell level in bacterial batch cultures. The first plateau is dynamically active, where gene expression is at steady state; the second plateau, however, is dynamically inactive. We further demonstrate that the predictability of assembled genetic circuits in the first plateau (steady state) is much higher than that in the second plateau where conventional measurements are often performed. By taking the nature of steady state into consideration, our method of measurement promises to directly capture the intrinsic property of biological parts/circuits regardless of circuit–host or circuit–environment interactions

Semirational Approach for Ultrahigh Poly(3-hydroxybutyrate) Accumulation in <i>Escherichia coli</i> by Combining One-Step Library Construction and High-Throughput Screening

As a product of a multistep enzymatic reaction, accumulation of poly­(3-hydroxybutyrate) (PHB) in <i>Escherichia coli</i> (<i>E. coli</i>) can be achieved by overexpression of the PHB synthesis pathway from a native producer involving three genes <i>phbC</i>, <i>phbA</i>, and <i>phbB</i>. Pathway optimization by adjusting expression levels of the three genes can influence properties of the final product. Here, we reported a semirational approach for highly efficient PHB pathway optimization in <i>E. coli</i> based on a <i>phbCAB</i> operon cloned from the native producer <i>Ralstonia entropha</i> (<i>R. entropha</i>). Rationally designed ribosomal binding site (RBS) libraries with defined strengths for each of the three genes were constructed based on high or low copy number plasmids in a one-pot reaction by an oligo-linker mediated assembly (OLMA) method. Strains with desired properties were evaluated and selected by three different methodologies, including visual selection, high-throughput screening, and detailed in-depth analysis. Applying this approach, strains accumulating 0%–92% PHB contents in cell dry weight (CDW) were achieved. PHB with various weight-average molecular weights (<i>M</i>_<i>w</i>) of 2.7–6.8 × 10⁶ were also efficiently produced in relatively high contents. These results suggest that the semirational approach combining library design, construction, and proper screening is an efficient way to optimize PHB and other multienzyme pathways