Gene Circuit Performance Characterization and Resource Usage in a Cell-Free “Breadboard”

Abstract

Alternative performance metricsFor the purposes of characterization of a particular biocircuit testing environment, in vitro or in vivo, there are any number of performance metrics that may be used. We have chosen to use integrated mRNA and final protein concentration since they intuitively represent the total transcriptional and translational capacity of a system. Other metrics, such as the mRNA and protein production rates and end times, are complementary to those used in this work and may be particularly informative depending on the specific circuit or system requirement.In Figs. S16 and S17 we show the maximum deGFP and MGapt production rates as a function of reporter concentration under different conditions. Using these measures, stark differences between simple one-stage gene expression and expression from the two-stage T7 cascade can be seen; for example, at low reporter concentrations, the maximum production rate of deGFP in the cascade is considerably larger than the one-stage rate, even when the strongest promoter is used. The cascade deGFP rates are also relatively flat with respect to reporter concentration. There is also a clear effect of NTPs on peak rates when a cascade is used versus simple expression. In the former case, additional NTPs provide for a significant increase in the maximum protein and mRNA production rates, whereas NTPs have little to no effect on maximum production rates for the Pr-deGFP-MGapt construct.The deGFP production end time t end,T L is shown for all conditions in Fig. S18. t end,T L is between 330 and 350 minutes for simple expression in the 'linear' regime, but as high as ∼580 minutes in some of the conditions and concentrations tested. The end time is a particularly good measure of system capacity when extended performance is required.

Streptomyces venezuelae TX‐TL – a next generation cell‐free synthetic biology tool

“…One potential route for the further development of S. venezuelae and the characterization of its genetic parts is the introduction of an in vitro transcription-translation system (TX-TL). TX-TL has recently been developed as a highly adaptable tool for bottom-up synthetic biology and is based on a whole-cell extract [6][7][8][9] to synthesize recombinant proteins from the chemical building blocks of life.…”

Translation Inhibition and Resource Balance in the Cell-Free Gene Expression System

“…We use a plasmid described in Siegal-Gaskin et al, kindly provided to us by the Murray lab, with a builtin RNA aptamer (35-base MGapt sequence, which contains a binding pocket for malachite green (MG) dye and a fluorescent protein for accomplishing the simultaneous measurement of RNA and protein [7]. The result of a typical experiment is shown in Figure 1.…”

“…Using both the RNA and protein profiles, we extract time-dependent translation rates, following methods similar to those appearing in Siegal-Gaskin et al from 2014 [7]. We assume that GFP (g) and MG (m) are related to protein (p) and RNA (r) expression as follows:…”

“…Not only do various ion concentrations need to be accurately prescribed, but even resources like NTPs have to be optimized carefully for significant expression to occur. Our understanding of the resource requirements of gene expression is challenged by the observation that gene expression decreases with NTP concentration, once NTP is increased beyond a certain threshold [7]. This phenomenon is unexpected, since higher amounts of NTPs imply a greater source of energy available for transcription and translation.…”

“…This phenomenon is unexpected, since higher amounts of NTPs imply a greater source of energy available for transcription and translation. Siegal-Gaskin et al [7] speculate that increased NTP levels enhance transcriptional activity, and thus exhaust most of the present NTPs. As a result, very little resources remain for translation to proceed, explaining the reduction of gene expression.…”