Figure 9
FAST detection of gene:gfp11 fusions of increasing length synthetized using a commercial CFPS. (A) Synthesis was performed under the conditions described in Materials and Methods using the PURE express kit. At the end of the incubation time, 400 pmol of MBP:GFP1-10fast were added to each reaction tube, and fluorescence was monitored after 1.5 and 3.5 h. The error bars represent the standard error calculated from triplicates. In all experiments, fluorescence measured in control tubes that did not contain mRNA was subtracted. (B) 4–15% PAGE (Mini-PROTEAN TGX BIO-RAD) analysis of 5 µL taken from duplicate samples before the MBP:GFP1-10fast addition. The bands corresponding to the synthetized proteins are indicated with red dots. The full uncropped gel picture is shown in Fig. S11. Note that HU has a certain propensity to form oligomers. However, the denaturing conditions of the electrophoresis reduce this risk. (C) Increase in fluorescence resulting from the addition of the indicated concentration of MBP:GFP1-10fast mixed with 5 µL of CFPS reaction carried out with PURE express and pUT7cspA:gfp11. The complementation reaction was performed at 25 °C for 3.5 h. (D) Fluorescence emitted by aliquots taken from fractions collected during the purification by affinity chromatography of the MBP:GFP1-10fast:GFP11-HupB complex. Fraction 1: flow through; fraction 2: wash; fractions 3–10: elution. The profiles refer to the MBP:GFP1-10fast:GFP11-HupB complexe formed in two duplicate reactions as described in Materials and Methods.
