15 research outputs found
A frame-shifted gene, which rescued its function by non-natural start codons and its application in constructing synthetic gene circuits
Efficient engineering of chromosomal ribosome binding site libraries in mismatch repair proficient Escherichia coli
Homology-dependent recombination of large synthetic pathways into E. coli genome via λ-Red and CRISPR/Cas9 dependent selection methodology
Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli
Programming mRNA decay to modulate synthetic circuit resource allocation
Synthetic circuits embedded in host cells compete with cellular processes for limited intracellular resources. Here we show how funnelling of cellular resources, after global transcriptome degradation by the sequence-dependent endoribonuclease MazF, to a synthetic circuit can increase production. Target genes are protected from MazF activity by recoding the gene sequence to eliminate recognition sites, while preserving the amino acid sequence. The expression of a protected fluorescent reporter and flux of a high-value metabolite are significantly enhanced using this genome-scale control strategy. Proteomics measurements discover a host factor in need of protection to improve resource redistribution activity. A computational model demonstrates that the MazF mRNA-decay feedback loop enables proportional control of MazF in an optimal operating regime. Transcriptional profiling of MazF-induced cells elucidates the dynamic shifts in transcript abundance and discovers regulatory design elements. Altogether, our results suggest that manipulation of cellular resource allocation is a key control parameter for synthetic circuit design