Characterization of Gene Circuit Parts Based on Multiobjective Optimization by Using Standard Calibrated Measurements

Abstract

Standardization and characterization of biological parts is necessary for the further development of bottom‐up synthetic biology. Herein, an easy‐to‐use methodology that embodies both a calibration procedure and a multiobjective optimization approach is proposed to characterize biological parts. The calibration procedure generates values for specific fluorescence per cell expressed as standard units of molecules of equivalent fluorescein per particle. The use of absolute standard units enhances the characterization of model parameters for biological parts by bringing measurements and estimations results from different sources into a common domain, so they can be integrated and compared faithfully. The multiobjective optimization procedure exploits these concepts by estimating the values of the model parameters, which represent biological parts of interest, while considering a varied range of experimental and circuit contexts. Thus, multiobjective optimization provides a robust characterization of them. The proposed calibration and characterization methodology can be used as a guide for good practices in dry and wet laboratories; thus allowing not only portability between models, but is also useful for generating libraries of tested and well‐characterized biological parts.

Meeting Measurement Precision Requirements for Effective Engineering of Genetic Regulatory Networks

“…There is also a gap between calibration in terms of equivalent dye molecules and the ability to interpret these numbers in terms of the actual fluorescent protein used, as illustrated by the high degree of variation between channels in Figure 6(c−d). Shifting units from equivalent dye molecules to protein molecules (e.g., by adaptation of methods based on spectral information 32 or protein purification 33 ) would also address the problem of channel-to-channel conversion, though in some circumstances this can also be handled by multicolor controls, as has been previously shown. 5,34 Beyond flow cytometry, it will be important establish that other measurement modalities can also achieve the requisite level of measurement precision.…”

FPCountR: Absolute protein quantification using fluorescence measurements

“…This is strongly supported by the results of Volkmer and colleagues (Volkmer and Heinemann, 2011) whose data suggests this variation is within ∼2-fold across a wide range of growth conditions, but also by others who have shown that as cell volumes increase, their OD-specific cell counts decrease by approximately the same magnitude (Schaechter et al ., 1958; Basan et al ., 2015). Units of concentration may also be more meaningful for reaction modelling since ultimately it is molecular concentration that is critical for binding and kinetics (Bloom et al ., 2014; Siegal-Gaskins et al ., 2014; Boada et al ., 2019).…”

“…This is only possible if RFUs from both FPs can be separately converted into molecular units of protein. Some have attempted to tackle this by attempting to predict the relative brightness of FPs using theoretical values (Boada et al, 2019;Vignoni et al, 2019), but such calibrations make a number of unvalidated assumptions, for instance about translation rate equivalence across constructs, that require rigorous testing before they can be adopted.…”

Multicolor plate reader fluorescence calibration

“…Note that results are not particularly sensitive to the tolerance constant of 1.5 (used also in ( 4 ) and ( 3 )), as the absolute maximum pipetting error β found for any sequence is just over 10%. Note also that a potential alternative model substitutive instrument non-linearity for pipetting bias can be found in ( 6 ).…”