Correction: Bottom-Up Engineering of Biological Systems through Standard Bricks: A Modularity Study on Basic Parts and Devices

Crosstalk between fluorescent proteins spectra might be an issue regarding systems containing both GFP-and RFP-expressing devices. In order to validate whether this phenomenon was significantly present with the acquisition system used in this study (i.e., the Infinite F200 microplate reader, Tecan), at least in the tested conditions for the combined gene expression cassettes, an ad-hoc experiment was performed. TOP10 recombinant cultures bearing J23118 with GFP32, (the only GFP-expressing construct in the combined cassettes study), P LlacO1 with RFP34 and P lacIQ with RFP34 (the strongest and the weakest RFP-expressing constructs), all in the low copy vector pSB4C5, were grown as described in the Methods section in the main text. Cultures bearing P LlacO1 with RFP34 were induced with 1 mM of IPTG. All the cultures were assayed in the microplate reader (gain=80) as described in the Methods section in the main text. In this experiment, red fluorescence was measured for J23118 with GFP32 and P lacIQ with RFP34, while green fluorescence was measured for J23118 with GFP and P LlacO1 with RFP34. After a proper background subtraction (see main text, Data analysis section), the 100· (green fluorescence) ratios, estimating the maximum percent contribution that GFP and RFP could give to red and green fluorescence measurements respectively, were computed. No detectable RFP contribution could be observed in the green fluorescence acquisitions, while the resulting ratio was 1.4% when red fluorescence of GFP-expressing cells was acquired. This can be considered as reasonably low crosstalk value. The first design of the interconnected system included a logic inverter with a strong RBS (BioBrick BBa B0034) upstream of the tetR gene. Such system, however, always remained in the OFF state even when the uninduced P lux promoter, whose basic activity was very low, was assembled upstream. Only the promoterless logic inverter gave a high output when tested (data not shown). This result is consistent with previous findings in which such logic inverter was tested in similar conditions [15]. In order to construct a logic inverter that could switch from the ON state to the OFF state in a range of RPUs between 0.05 and 2, which is exhibited by a number of easy-to-retrieve promoters from the Registry of Standard Biological Parts [14], the RBS upstream of tetR was changed. A much weaker candidate was chosen (BioBrick BBa B0031). It gave the expected effect (see main text), as the switch point occurred when the input RPUs …