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. | â–ª Check pairwise compatibility by placing GFP and RFP under the regulation of each input switch device | S4 |
â–ª Perform mutagenesis on promoter or DNA-binding protein to identify orthogonal pairs. | |||
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. | â–ª 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. | â–ª Characterise different permutation of tandem promoter OR gate to identify the optimal genetic architecture. | 3A, 3C | |
â–ª 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. | |||
Layering AND-OR-NOT into XOR gate | Insufficient repression: Insufficient transcription repressors are generated by upstream genetic circuit to stop transcription elongation, level mismatch. | â–ª Reduce repressors required in NOT gate by designing repressor binding sites such that they are immediately downstream of transcription start site. | 5 |
â–ª 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. |