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Table 1 Failure modes and engineering solutions for the design and building of layered genetic circuits in a single (bacterial) cell

From: Layering genetic circuits to build a single cell, bacterial half adder

Device Failure mode Engineering solution Fig./[Ref.]
Input switches Genetic crosstalk: Input switch devices crosstalk with one another. ▪ Check pairwise compatibility by placing GFP and RFP under the regulation of each input switch device S4
▪ Perform mutagenesis on promoter or DNA-binding protein to identify orthogonal pairs. Refs. [28, 29, 31, 50]
AND gate Stoichiometric mismatch: Amount of AND gate’s transcription activators are disproportionately matched, resulting in ‘leaky’ AND gate. ▪ Characterise the expression profile of input genetic switches with different RBSs and input the resultant transfer function equations into a steady state AND gate computational model. Match AND gate sub-modules to obtain stoichiometric balance using this forward engineering approach. S11
DNA supercoiling: σ54 AND gate promoter is turned on by the DNA supercoil effects of upstream σ54 promoter. ▪ Insulate σ54 promoters using different plasmid vectors. S5
OR gate Stoichiometric mismatch: Outputs from input device I and II are disproportionately matched, resulting in skewed OR gate. ▪ Characterise the expression profile of input genetic switches with different RBSs and input the resultant transfer function equations into a steady state OR gate computational model. Match OR gate sub-modules to obtain stoichiometric balance using this forward engineering approach. S12, S13
Transcription interference: Tandem promoter OR gate design fails due to downstream DNA sequence acting as a repressor to upstream promoter. ▪ Characterise different permutation of tandem promoter OR gate to identify the optimal genetic architecture. 3A, 3C
▪ Separate OR gate promoters into distinct expression cassettes. 3A, 3C
Layering OR-NOT into NIMPLY gate Insufficient repression: Placing single repressor binding site downstream of inducible promoter cannot fully repress gene expression. ▪ Increase repression efficiency by introducing additional repressor binding sites to the NOT gate. Note that the introduction of extra repressor binding sites may also lead to extensive 5′UTR effects. 4A, 4C
▪ Attenuate expression ‘leakiness’ by using weaker RBS for the NOT gate 4B
Translation interference: Placing repressor binding sites downstream of inducible promoter creates extensive 5′UTR structural effects. ▪ Perform mutagenesis to relieve RNA hairpin structures at selected sites. S6
▪ Use RNA processing tools to remove undesired 5′UTR sequences. Refs. [5, 20]
Layering AND-OR-NOT into XOR gate Insufficient repression: Insufficient transcription repressors are generated by upstream genetic circuit to stop transcription elongation, level mismatch. ▪ Reduce repressors required in NOT gate by designing repressor binding sites such that they are immediately downstream of transcription start site. 5
▪ Increase production of repressor in the AND gate by expressing transcription repressors in high copy plasmid. 2D, 5
Translation interference: Placing repressor binding sites downstream of OR gate tandem promoter creates extensive 5′UTR structural effects. ▪ Separate OR gate promoters into distinct expression cassettes. 5
▪ Use RNA processing tools to remove undesired 5′UTR sequences. Ref [5, 20]