Journal: Life
Article Title: A Programmable Finite-Replicated Organism Framework for Balanced Safety and Functionality
doi: 10.3390/life15091381
Figure Lengend Snippet: Escape frequency analysis of each suicide module and finite replication dynamics of SerS.F213 FROs. ( a ) Suicide module design schematic featuring dual TAG insertions in essential genes. With the gene editing strategy, the E. coli essential genes ( dnaA , murG , and serS ) were inserted with two TAGs, forming the suicide module (Suicide Module A). ( b ) Quantitative escape frequency measurement expressed as CFU ratio ±Cl2Y supplementation (Mean ± SD, N = 7 biological replicates). Escape frequency was quantified as the ratio of escape mutant colony-forming units (CFUs) to total viable CFUs. ( c ) Engineering strategy for SerS.F213 FROs through rescue module insertion. Two plasmids expressing the ncAA orthogonal translation system and storage protein RFP carrying eight TAGs were constructed. When the exogenous supply of ncAA is interrupted, the storage protein RFP with TAGs degrades and releases the expression of genes necessary for ncAA supply. ( d ) The finite replication characteristics of SerS.F213 FROs. Left panel: growth generation quantification (top) and generational distribution (bottom). The SerS.F213-engineered FROs demonstrated finite proliferation, sustaining four generations of growth before growth arrest, while rescue-module-deficient controls (NC) showed unrestricted proliferation (mean ± SD; **** p < 0.0001, two-tailed Student t -test). Right panel: Microscopic imaging demonstrates finite replication phenotypes in serS -modified FRO. The bacterial growth was observed and filmed every 0, 1, 3, 6, 9, and 12 h.
Article Snippet: The E. coli C321.ΔA exp strain (Addgene #87359) (Lajoie MJ, 2013) was cultured in Luria–Bertani medium (10 g/L NaCl, 10 g/L tryptone, 5 g/L yeast extract) supplemented with antibiotics: ampicillin (100 μg/mL), kanamycin (50 μg/mL), chloramphenicol (25 μg/mL), and spectinomycin (50 μg/mL).
Techniques: Mutagenesis, Expressing, Construct, Two Tailed Test, Imaging, Modification
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