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Abstract Foundational techniques in molecular biology—such as cloning genes, tagging biomolecules for purification or identification, and overexpressing recombinant proteins—rely on introducing non-native or synthetic DNA sequences into organisms. These sequences may be recognized by the transcription and translation machinery in their new context in unintended ways. The cryptic gene expression that sometimes results has been shown to produce genetic instability and mask experimental signals. Computational tools have been developed to predict individual types of gene expression elements, but it can be difficult for researchers to contextualize their collective output. Here, we introduce CryptKeeper, a software pipeline that visualizes predictions of Escherichia coli gene expression signals and estimates the translational burden possible from a DNA sequence. We investigate several published examples where cryptic gene expression in E. coli interfered with experiments. CryptKeeper accurately postdicts unwanted gene expression from both eukaryotic virus infectious clones and individual proteins that led to genetic instability. It also identifies off-target gene expression elements that resulted in truncations that confounded protein purification. Incorporating negative design using CryptKeeper into reverse genetics and synthetic biology workflows can help to mitigate cloning challenges and avoid unexplained failures and complications that arise from unintentional gene expression. 

Abstract Adenosine-to-inosine (A-to-I) messenger RNA (mRNA) editing can affect the sequence and function of translated proteins and has been extensively investigated in eukaryotes. However, the prevalence of A-to-I mRNA editing in bacteria, its governing regulatory principles, and its biological significance are poorly understood. Here, we show that A-to-I mRNA editing occurs in hundreds of transcripts across dozens of gammaproteobacterial species, with most edits predicted to recode protein sequences. Furthermore, we reveal conserved regulatory determinants controlling editing across gammaproteobacterial species. Using Acinetobacter baylyi as a model, we show that mutating TadA, the mediating enzyme, reduces editing across all sites. Conversely, overexpressing TadA resulted in the editing of >300 transcripts, attesting to the editing potential of TadA. Notably, we show for the first time, at the protein level, that normal levels of A-to-I mRNA editing lead to wild-type bacteria expressing two protein isoforms from a single gene. Finally, we show that a TadA mutant with deficient editing activity does not grow at high temperatures, suggesting that RNA editing has a functional role in bacteria. Our work reveals that A-to-I mRNA editing in bacteria is widespread and has the potential to reshape the bacterial transcriptome and proteome. 

Abstract 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 affectedEscherichia coligrowth 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 ofE. coliby >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. 

Abstract BackgroundPathogens face strong selection from host immune responses, yet many host populations support pervasive pathogen populations. We investigated this puzzle in a model system ofBartonellaand rodents from Israel’s northwestern Negev Desert. We chose to study this system because, in this region, 75–100% of rodents are infected withBartonellaat any given time, despite an efficient immunological response. In this region,Bartonellaspecies circulate in three rodent species, and we tested the hypothesis that at least one of these hosts exhibits a waning immune response toBartonella, which allows reinfections. MethodsWe inoculated captive animals of all three rodent species with the sameBartonellastrain, and we quantified the bacterial dynamics andBartonella-specific immunoglobulin G antibody kinetics over a period of 139 days after the primary inoculation, and then for 60 days following reinoculation with the same strain. ResultsContrary to our hypothesis, we found a strong, long-lasting immunoglobulin G antibody response, with protective immunological memory in all three rodent species. That response prevented reinfection upon exposure of the rodents to the sameBartonellastrain. ConclusionsThis study constitutes an initial step toward understanding how the interplay between traits ofBartonellaand their hosts influences the epidemiological dynamics of these pathogens in nature. Graphical Abstract