Integrase-mediated differentiation circuits improve evolutionary stability of burdensome and toxic functions in E. coli
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PROVIDER: PRJEB56582 | ENA |
REPOSITORIES: ENA
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Project description:Advances in synthetic biology, bioengineering, and computation allow us to rapidly and reliably program cells with increasingly complex and useful functions. However, because the functions we engineer cells to perform are typically burdensome to cell growth, they can be rapidly lost due to the processes of mutation and natural selection. Here, we show that a strategy of terminal differentiation improves the evolutionary stability of burdensome functions in a general manner by realizing a reproductive and metabolic division of labor. To implement this strategy, we develop a genetic differentiation circuit in Escherichia coli using unidirectional integrase-recombination. With terminal differentiation, differentiated cells uniquely express burdensome functions driven by the orthogonal T7 RNA polymerase, but their capacity to proliferate is limited to prevent the propagation of advantageous loss-of-function mutations that inevitably occur. We demonstrate computationally and experimentally that terminal differentiation increases duration and yield of high-burden expression and that its evolutionary stability can be improved with strategic redundancy. Further, we show this strategy can even be applied to toxic functions. Overall, this study provides an effective, generalizable approach for protecting burdensome engineered functions from evolutionary degradation.
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Project description:One problem with synthetic genes in genetically engineered organisms is that these foreign DNAs will eventually lose their functions over evolutionary time in absence of selective pressures. This general limitation can restrain the long-term study and industrial application of synthetic genetic circuits. Previous studies have shown that because of their crucial regulatory functions, prokaryotic promoters in synthetic genetic circuits are especially vulnerable to mutations. In this study, we rationally designed robust bidirectional promoters (BDPs), which are self-protected through the complementarity of their overlapping forward and backward promoter sequences on DNA duplex. When the transcription of a target non-essential gene (e.g. green fluorescent protein) was coupled to the transcription of an essential gene (e.g. antibiotic resistance gene) through the BDP, the evolutionary half-time of the gene of interest increases 4-10 times, depending on the strain and experimental conditions used. This design of using BDPs to increase the mutational stability of genetic circuits can be potentially applied to synthetic biology applications in general.
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Project description:To understand the impact of alternative translation initiation on a proteome, we performed the first study on protein turnover using positional proteomics and ribosome profiling to distinguish between N-terminal proteoforms of individual genes. Overall, we monitored the stability of 1,941 human N-terminal proteoforms, including 147 N-terminal proteoform pairs that originate from alternative translation initiation, alternative splicing or incomplete processing of the initiator methionine.
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Project description:To understand the impact of alternative translation initiation on a proteome, we performed the first large-scale study of protein turnover rates in which we distinguish between N-terminal proteoforms pointing to translation initiation events. Using pulsed SILAC combined with N-terminal COFRADIC we monitored the stability of 1,941human N-terminal proteoforms, including 147 proteoform pairs with heterogeneous N-termini originating from the same gene that result from alternative translation initiation and incomplete processing of the initiator methionine. N-terminally truncated proteoforms were on average less abundant than canonical proteoforms, many had different stabilities and exhibited both faster and slower turnover rates compared to their canonical counterparts. These differences in stability did not depend on the length of truncation but on individual protein characteristics. In silico simulation of N-terminal proteoforms in macromolecular complexes revealed possible consequences for complex integrity such as replacement of unstable canonical subunits. The extent of intrinsic disorder in N-terminal protein structures correlated with turnover times, indicating that a change in the structural flexibility of protein N-termini in truncated proteoforms might impact proteoform stability. Interestingly, removal of the initiator methionine by methionine aminopeptidases reduced the stability of processed proteoforms while susceptibility for N-terminal acetylation, another common co-translational modification, did not seem to impact on turnover rates. Taken together, our findings reveal differences in protein stability between N-terminal proteoforms and point to a role for alternative translation initiation and co-translational initiator methionine removal in the overall regulation of proteome homeostasis.
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Project description:Diverse applications rely on engineering microbes to carry and express foreign transgenes. This engineered baggage rarely benefits the microbe and is thus prone to rapid evolutionary loss when the microbe is propagated. For applications where a transgene must be maintained for extended periods of growth, slowing the rate of transgene evolution is critical and can be achieved by reducing either the rate of mutation or the strength of selection. Because the benefits realized by changing these quantities will not usually be equal, it is important to know which will yield the greatest improvement to the evolutionary half-life of the engineering. Here, we provide a method for jointly estimating the mutation rate of transgene loss and the strength of selection favoring these transgene-free, revertant individuals. The method requires data from serial transfer experiments in which the frequency of engineered genomes is monitored periodically. Simple mathematical models are developed that use these estimates to predict the half-life of the engineered transgene and provide quantitative predictions for how alterations to mutation and selection will influence longevity. The estimation method and predictive tools have been implemented as an interactive web application, MuSe.
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Project description:To understand the impact of alternative translation initiation on a proteome, we performed the first large-scale study of protein turnover rates in which we distinguish between N-terminal proteoforms pointing to translation initiation events. Using pulsed SILAC combined with N-terminal COFRADIC we monitored the stability of 1,941human N-terminal proteoforms, including 147 proteoform pairs with heterogeneous N-termini originating from the same gene that result from alternative translation initiation and incomplete processing of the initiator methionine. N-terminally truncated proteoforms were on average less abundant than canonical proteoforms, many had different stabilities and exhibited both faster and slower turnover rates compared to their canonical counterparts. These differences in stability did not depend on the length of truncation but on individual protein characteristics. In silico simulation of N-terminal proteoforms in macromolecular complexes revealed possible consequences for complex integrity such as replacement of unstable canonical subunits. The extent of intrinsic disorder in N-terminal protein structures correlated with turnover times, indicating that a change in the structural flexibility of protein N-termini in truncated proteoforms might impact proteoform stability. Interestingly, removal of the initiator methionine by methionine aminopeptidases reduced the stability of processed proteoforms while susceptibility for N-terminal acetylation, another common co-translational modification, did not seem to impact on turnover rates. Taken together, our findings reveal differences in protein stability between N-terminal proteoforms and point to a role for alternative translation initiation and co-translational initiator methionine removal in the overall regulation of proteome homeostasis.
2013-03-14 | PXD002091 | Pride