Abstract
The rational design of complex biological systems through the interconnection of single functional building blocks is hampered by many unpredictability sources; this is mainly due to the tangled context-dependency behavior of those parts once placed into an intrinsically complex living system. Among others, the finite amount of translational resources in prokaryotic cells leads to load effects in heterologous protein expression. As a result, hidden interactions among protein synthesis rates arise, leading to unexpected and counterintuitive behaviors. To face this issue in rational design of synthetic circuits in bacterial cells, CRISPR interference is here evaluated as genetic logic inverters with low translational resource usage, compared with traditional transcriptional regulators. This system has been studied and characterized in several circuit configurations. Each module composing the circuit architecture has been optimized in order to meet the desired specifications, and its reduced metabolic load has been eventually demonstrated via in-vivo assays.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- CRISPRi
- Clustered Regularly Interspaced Short Palindromic Repeats interference
- GFP
- Green Fluorescent Protein
- HSL
- N-oxohexanoyl-L-homoserine lactone
- IPTG
- Isopropyl-β-D-1-thiogalactopyranoside
- OD600
- optical density at 600 nm
- PCR
- polymerase chain reaction
- RBS
- ribosome binding site
- RFP
- Red Fluorescent Protein
- rpm
- rotation per minute