Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts

doi: 10.3389/fbioe.2021.727584

Figure Lengend Snippet: Optimized in-house cell-free reactions compared to commercial alternatives. (A) Left: Schematic representation of testing the performance of home-made or commercial cell-free systems using the sfGFP constitutive reporter. Right: Example of the endpoint sfGFP reaction and negative control (without input DNA) in a home-made cell-free system supplemented with maltodextrin energy source. Tubes were photographed under white light or blue light plus an amber filter that allows visualizing the sfGFP fluorescence. (B) Endpoint sfGFP fluorescence (plasmid DNA at 9 nM final concentration) was measured in four different cell extracts (Batch A, B, C, D) supplemented with either maltodextrin and polyphosphates (light blue) or 3-PGA (green) as energy source. Grey dots represent the arithmetic mean of three measurements performed on each batch, and error bars represent standard deviations of the means of the four batches tested (N = 4). t -test for paired measurements was performed and statistically significance was found between the two groups ( p -value = 0.03, shown by *). Assumptions of the paired t -test were verified using the Shapiro-Wilk test for normality of the differences between energy sources for a given batch ( p -value = 0.97), and Levene test for homoscedasticity of the 3-PGA and maltodextrin data sets ( p -value = 0.25). (C) sfGFP production dynamics from plasmid DNA (9 nM final concentration) in reactions performed at 29°C using NEB PURExpress and Promega S30 T7 High Yield commercial kits along with four optimized in-house cell-free reactions (Batch A, B, C, D) using maltodextrin and polyphosphates as the energy source. Error bars represent the standard deviations of three independent replicates, dots are centered at the arithmetic mean for each time point.

Article Snippet: Plasmid DNA input was produced by midi prepping an overnight culture of 200 ml LB with the appropriate strain (Promega, A2492) and cleaned again using PCR cleanup (Promega, A6754).

Techniques: Negative Control, Fluorescence, Plasmid Preparation, Concentration Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts

doi: 10.3389/fbioe.2021.727584

Figure Lengend Snippet: Performance of ZIKV toehold sensors in low-cost cell-free lysate reactions. (A) Schematic representation of toehold-mediated RNA sensing. (B) Dynamics of the RNA sensing reactions performed with ZIKV toehold sensor 8 (0.7 nM plasmid DNA) and 27 (2 nM plasmid DNA), regulating the expression of the full-length LacZ in home-made cell extracts and PURExpress cell-free reactions. Error bars represent the standard deviations of three independent experiments, dots are centered at the arithmetic mean for each time point. (C) Example of the endpoint visualization of the experiments after 4 hours of incubation at 29°C. (D) Endpoint measurement of RNA sensing reactions performed with ZIKV sensor 27 and trigger 27 in a range of concentrations with and without NASBA isothermal amplification. Gray dots represent data from six independent measurements performed from two independent NASBA amplifications performed on different days. Black error bars correspond to standard deviations of these six measurements.

Article Snippet: Plasmid DNA input was produced by midi prepping an overnight culture of 200 ml LB with the appropriate strain (Promega, A2492) and cleaned again using PCR cleanup (Promega, A6754).

Techniques: Plasmid Preparation, Expressing, Incubation, Amplification