Engineered signal-coupled inducible promoters: measuring the apparent RNA-polymerase resource budget.

Inducible promoters are a central regulatory component in synthetic biology, metabolic engineering, and protein production for laboratory and commercial uses. Many of these applications utilize two or more exogenous promoters, imposing a currently unquantifiable metabolic burden on the living system. Here, we engineered a collection of inducible promoters (regulated by LacI-based transcription factors) that maximize the free-state of endogenous RNA polymerase (RNAP). We leveraged this collection of inducible promotors to construct simple two-channel logical controls that enabled us to measure metabolic burden - as it relates to RNAP resource partitioning. The two-channel genetic circuits utilized sets of signal-coupled transcription factors that regulate cognate inducible promoters in a coordinated logical fashion. With this fundamental genetic architecture, we evaluated the performance of each inducible promoter as discrete operations, and as coupled systems to evaluate and quantify the effects of resource partitioning. Obtaining the ability to systematically and accurately measure the apparent RNA-polymerase resource budget will enable researchers to design more robust genetic circuits, with significantly higher fidelity. Moreover, this study presents a workflow that can be used to better understand how living systems adapt RNAP resources, via the complementary pairing of constitutive and regulated promoters that vary in strength.

Cost-effective batch production process of scFv antibody in Escherichia coli.

BACKGROUND: Cost-effective production of antibody (Ab) fragments is of great interests of many pharmaceutical industries, in large part due to their high usages in research, diagnosis and therapy. Thus, the production of Abs necessitates accomplishment of the optimal strategies. OBJECTIVE: In this study, based on the induction start time using arabinose, we implemented a novel strategy for the cost-effective production of single chain variable fragment (scFv) in Escherichia coli (E. coli). METHODS: Complex and minimum media were used to investigate the batch fermentation in 50 mL batch tubes to find the optimum conditions for the production of a scFv in the Escherichia coli HB2151. RESULTS: Arabinose was used as an appropriate economical alternative of isopropyl ß-D-1-thiogalactopyranoside (IPTG) for the production of scFv antibody. The optimum concentration of arabinose as an inducer was 0.1% (w/w), while below this point the scFv production yield (YP/X) decreased significantly. The start time of the induction of E. coli HB2151 cells was adjusted to the stationary phase of the growth, and the results showed higher specific scFv production yields up to 0.9 mg scFv/g biomass in the minimum media. The optimum induction duration times for the complex and minimum media were about 12 and 24 hours, respectively. CONCLUSIONS: We propose this method to possibly be used for the large-scale production of recombinant proteins/peptides such as scFv and Fab antibodies.

Cost-benefit tradeoffs in engineered lac operons.

Cells must balance the cost and benefit of protein expression to optimize organismal fitness. The lac operon of the bacterium Escherichia coli has been a model for quantifying the physiological impact of costly protein production and for elucidating the resulting regulatory mechanisms. We report quantitative fitness measurements in 27 redesigned operons that suggested that protein production is not the primary origin of fitness costs. Instead, we discovered that the lac permease activity, which relates linearly to cost, is the major physiological burden to the cell. These findings explain control points in the lac operon that minimize the cost of lac permease activity, not protein expression. Characterizing similar relationships in other systems will be important to map the impact of cost/benefit tradeoffs on cell physiology and regulation.