Abstract
Bacteria’s ability to specifically sense small molecules in their environment and trigger metabolic responses in accordance is an invaluable biotechnological resource. While many transcription factors (TFs) mediating these processes have been studied, only a handful has been leveraged for molecular biology applications. To expand this panel of biotechnologically important sensors here we present a strategy for the construction and testing of chimeric TF libraries, based on the fusion of highly soluble periplasmic binding proteins (PBPs) with DNA-binding domains (DBDs). We validated this strategy by constructing and functionally testing two unique sense-and-response regulators for benzoate, an environmentally and industrially relevant metabolite. This work will enable the development of tailored biosensors for synthetic regulatory circuits.
ABBREVIATION LIST
- 3D
- Three-dimensional
- ApmR
- Resistance to ampicillin/carbenicillin
- aTc
- Anhydrotetracycline
- CmR
- Resistance to chloramphenicol
- CRISPR
- Clustered Regularly Interspaced Short Palindromic Repeats
- Ct
- C-terminal
- DBD
- DNA-Binding Domain
- dCas9
- Defective Cas9 protein, able to bind DNA and RNA but lacking nuclease activity
- eLCR
- enhanced LCR
- FACS
- Fluorescence-Associated Cell Sorting
- GGBP
- glucose-binding protein (E. coli)
- GmR
- Resistance to gentamycin
- IPTG
- Isopropyl β-D-1-thiogalactopyranoside
- KmR
- Resistance to kanamycin
- LCR
- Ligase Chain Reaction
- LNK
- linker
- nSP
- SBD that does Not contain its Signal Peptide
- Nt
- n-terminal
- PBP
- Periplasmic Binding Protein
- PBS
- Phosphate Buffered Saline
- RBS
- Ribosome Binding Site
- SBD
- Substrate-Binding Domain
- OD
- Oligomerization Domain
- PID
- Percentage of Identity
- SP
- Signal Peptide
- TALENs
- Transcription activator-like effector nuclease
- TF
- Transcription factor