RESUMEN
Engineered living materials (ELMs) exhibit desirable characteristics of the living component, including growth and repair, and responsiveness to external stimuli. Escherichia coli (E. coli) are a promising constituent of ELMs because they are very tractable to genetic engineering, produce heterologous proteins readily, and grow exponentially. However, seasonal variation in ambient temperature presents a challenge in deploying ELMs outside of a laboratory environment because E. coli growth rate is impaired both below and above 37 °C. Here, a genetic circuit is developed that controls the expression of a light-absorptive chromophore in response to changes in temperature. It is demonstrated that at temperatures below 36 °C, the engineered E. coli increase in pigmentation, causing an increase in sample temperature and growth rate above non-pigmented counterparts in a model planar ELM. On the other hand, at above 36 °C, they decrease in pigmentation, protecting the growth compared to bacteria with temperature-independent high pigmentation. Integrating the temperature-responsive circuit into an ELM has the potential to improve living material performance by optimizing growth and protein production in the face of seasonal temperature changes.
Asunto(s)
Escherichia coli , Ingeniería Genética , Temperatura , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas/metabolismoRESUMEN
Temperature is a versatile input signal for the control of engineered cellular functions. Sharp induction of gene expression with heat has been established using bacteria- and phage-derived temperature-sensitive transcriptional repressors with tunable switching temperatures. However, few temperature-sensitive transcriptional activators have been reported that enable direct gene induction with cooling. Such activators would expand the application space for temperature control. In this technical note, we show that temperature-dependent versions of the Lambda phage repressor CI can serve as tunable cold-actuated transactivators. Natively, CI serves as both a repressor and activator of transcription. Previously, thermolabile mutants of CI, known as the TcI family, were used to repress the cognate promoters PR and PL. We hypothesized that TcI mutants can also serve as temperature-sensitive activators of transcription at CI's natural PRM promoter, creating cold-inducible operons with a tunable response to temperature. Indeed, we demonstrate temperature-responsive activation by two variants of TcI with set points at 35.5 and 38.5 °C in E. coli. In addition, we show that TcI can serve as both an activator and a repressor of different genes in the same genetic circuit, leading to opposite thermal responses. Transcriptional activation by TcI expands the toolbox for control of cellular function using globally or locally applied thermal inputs.
Asunto(s)
Bacteriófago lambda , Escherichia coli , Bacteriófago lambda/genética , Escherichia coli/genética , Proteínas Represoras , Temperatura , Transcripción Genética/genética , Activación Transcripcional/genética , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Mycobacterium tuberculosis remains a leading cause of death worldwide, especially among individuals infected with HIV. Whereas phylogenetic analysis has revealed M. tuberculosis spread throughout history and in local outbreaks, much less is understood about its dissemination within the body. Here we report genomic analysis of 2,693 samples collected post mortem from lung and extrapulmonary biopsies of 44 subjects in KwaZulu-Natal, South Africa, who received minimal antitubercular treatment and most of whom were HIV seropositive. We found that purifying selection occurred within individual patients, without the need for patient-to-patient transmission. Despite negative selection, mycobacteria diversified within individuals to form sublineages that co-existed for years. These sublineages, as well as distinct strains from mixed infections, were differentially distributed throughout the lung, suggesting temporary barriers to pathogen migration. As a consequence, samples taken from the upper airway often captured only a fraction of the population diversity, challenging current methods of outbreak tracing and resistance diagnostics. Phylogenetic analysis indicated that dissemination from the lungs to extrapulmonary sites was as frequent as between lung sites, supporting the idea of similar migration routes within and between organs, at least in subjects with HIV. Genomic diversity therefore provides a record of pathogen diversification and repeated dissemination across the body.