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. 2021 May 1;49(9):5393–5406. doi: 10.1093/nar/gkab253

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© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — Implementation of ICF and DNF designs in living cells. (A) Utilization of transcriptional interference to mimic subtraction. The P_PUT activates GFP signal. The P_lux reverse promoter is located opposite to P_PUT and upstream to gfp gene repressing GFP signal. The first unidirectional terminator is in the same orientation as P_PUT and downstream to gfp gene. The second unidirectional terminator is in the same orientation as P_lux and upstream to P_PUT. The terminator is represented by a highlighted letter T. The RBS is marked by a blue rectangle. The riboj sequence is inserted upstream of the RBS which is marked by a circle (59). The LuxR transcription activator and mCherry are expressed under P_tetO promoter, encoded on MCP. When no TetR is expressed, P_tetO acts as a constitutive promoter. Both LuxR and mCherry genes have their own RBS sequences. The unit Terminator__RC-P_PUT-P_{lux_RC}-GFP-Terminator is encoded on HCP. The block diagram describes the operation of OL circuit, where the output is regulated both by the input and inhibitor. (B) Utilizing antisense transcription to mimic subtraction. The P_lux promoter is oriented in reverse to P_PUT and downstream to gfp gene repressing GFP signal. The first unidirectional terminator was placed in the same orientation to P_PUT and downstream to gfp gene. The second unidirectional terminator was placed in the same orientation to P_lux and upstream to P_PUT. The LuxR activator and mCherry are expressed by P_tetO promoter encoded on MCP. Both LuxR and mCherry genes have their own RBS sequences. The unit Terminator__RC-P_PUT-GFP-P_{lux_RC}-Terminator is encoded on HCP. The block diagram describes the operation of OL, where both input and inhibitor regulate the output level. (C) Implementation of an inverting switch using TetR repressor. The P_PUT controls the expression of TetR, which represses the activity of P_tetO. The small molecule aTc binds TetR to release the repression of P_tetO. The P_tetO-mCherry-Terminator construct was placed on MCP, while the P_PUT-TetR-Terminator construct was cloned on LCP in order to match their copy numbers in ICF and DNF circuits. The mCherry gene was further replaced by LuxR gene to be integrated in ICF and DNF circuits. The block diagram describes the operation of an inverting switch circuit. (D) Implementation of ICF circuit by combining a transcriptional interference unit with TetR inverting switch. Here TetR is controlled only by P_PUT. (E) Implementation of a DNF circuit by combining transcriptional interference unit with TetR inverting switch. Here TetR is controlled by both P_PUT and P_lux promoters. (F) Implementation of ICF circuit by combining an antisense transcription unit with TetR inverting switch. Here TetR is controlled only by P_PUT. (G) Implementation of a DNF circuit by combining an antisense transcription with TetR inverting switch. Here TetR is controlled by both promoters P_PUT and P_lux.