RESUMO
Engineered DNA will slow the growth of a host cell if it redirects limiting resources or otherwise interferes with homeostasis. Escape mutants that alleviate this burden can rapidly evolve and take over cell populations, making genetic engineering less reliable and predictable. Synthetic biologists often use genetic parts encoded on plasmids, but their burden is rarely characterized. We measured how 301 BioBrick plasmids affected Escherichia coli growth and found that 59 (19.6%) were burdensome, primarily because they depleted the limited gene expression resources of host cells. Overall, no BioBricks reduced the growth rate of E. coli by >45%, which agreed with a population genetic model that predicts such plasmids should be unclonable. We made this model available online for education ( https://barricklab.org/burden-model ) and added our burden measurements to the iGEM Registry. Our results establish a fundamental limit on what DNA constructs and genetic modifications can be successfully engineered into cells.
Assuntos
Escherichia coli , Engenharia Genética , Plasmídeos , Biologia Sintética , Biologia Sintética/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Plasmídeos/genética , Engenharia Genética/métodos , Modelos GenéticosRESUMO
The TTG2 transcription factor of Arabidopsis regulates a set of epidermal traits, including the differentiation of leaf trichomes, flavonoid pigment production in cells of the inner testa (or seed coat) layer and mucilage production in specialized cells of the outer testa layer. Despite the fact that TTG2 has been known for over twenty years as an important regulator of multiple developmental pathways, little has been discovered about the downstream mechanisms by which TTG2 co-regulates these epidermal features. In this study, we present evidence of phosphoinositide lipid signaling as a mechanism for the regulation of TTG2-dependent epidermal pathways. Overexpression of the AtPLC1 gene rescues the trichome and seed coat phenotypes of the ttg2-1 mutant plant. Moreover, in the case of seed coat color rescue, AtPLC1 overexpression restored expression of the TTG2 flavonoid pathway target genes, TT12 and TT13/AHA10. Consistent with these observations, a dominant AtPLC1 T-DNA insertion allele (plc1-1D) promotes trichome development in both wild-type and ttg2-3 plants. Also, AtPLC1 promoter:GUS analysis shows expression in trichomes and this expression appears dependent on TTG2. Taken together, the discovery of a genetic interaction between TTG2 and AtPLC1 suggests a role for phosphoinositide signaling in the regulation of trichome development, flavonoid pigment biosynthesis and the differentiation of mucilage-producing cells of the seed coat. This finding provides new avenues for future research at the intersection of the TTG2-dependent developmental pathways and the numerous molecular and cellular phenomena influenced by phospholipid signaling.
Assuntos
Proteínas de Arabidopsis , Regulação da Expressão Gênica de Plantas , Fosfoinositídeo Fosfolipase C , Epiderme Vegetal , Transdução de Sinais , Fatores de Transcrição , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Flavonoides/metabolismo , Mutação , Fenótipo , Fosfatidilinositóis/metabolismo , Epiderme Vegetal/metabolismo , Epiderme Vegetal/genética , Epiderme Vegetal/citologia , Sementes/genética , Sementes/metabolismo , Sementes/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Tricomas/genética , Tricomas/metabolismo , Tricomas/crescimento & desenvolvimento , Fosfoinositídeo Fosfolipase C/genética , Fosfoinositídeo Fosfolipase C/metabolismoRESUMO
Engineered DNA will slow the growth of a host cell if it redirects limiting resources or otherwise interferes with homeostasis. Populations of engineered cells can rapidly become dominated by "escape mutants" that evolve to alleviate this burden by inactivating the intended function. Synthetic biologists working with bacteria rely on genetic parts and devices encoded on plasmids, but the burden of different engineered DNA sequences is rarely characterized. We measured how 301 BioBricks on high-copy plasmids affected the growth rate of Escherichia coli. Of these, 59 (19.6%) negatively impacted growth. The burden imposed by engineered DNA is commonly associated with diverting ribosomes or other gene expression factors away from producing endogenous genes that are essential for cellular replication. In line with this expectation, BioBricks exhibiting burden were more likely to contain highly active constitutive promoters and strong ribosome binding sites. By monitoring how much each BioBrick reduced expression of a chromosomal GFP reporter, we found that the burden of most, but not all, BioBricks could be wholly explained by diversion of gene expression resources. Overall, no BioBricks reduced the growth rate of E. coli by >45%, which agreed with a population genetic model that predicts such plasmids should be "unclonable" because escape mutants will take over during growth of a bacterial colony or small laboratory culture from a transformed cell. We made this model available as an interactive web tool for synthetic biology education and added our burden measurements to the iGEM Registry descriptions of each BioBrick.