Biology subjects

Larsson, E. M.

Publications and source records attributed to Larsson, E. M..

5 recordsLinked to original sources

bioRxiv2024

A Field-Deployable Arsenic Sensor Integrating Bacillus Megaterium with CMOS Technology

Bacteria innately monitor their environment by dynamically regulating gene expression to respond to fluctuating conditions. Through synthetic biology, we can harness this natural capability to design cell-based sensors. Bacillus megaterium, a soil bacterium, stands out due to its remarkable heavy metal tolerance and sporulation ability, making it an ideal candidate for heavy metal detection with low transportation costs. However, challenges persist: the synthetic biology toolkit for this strain is underdeveloped and conventional whole-cell sensors necessitate specialized laboratory equipment to read the output. In our study, we genetically modified B. megaterium for arsenic detection, establishing a detection threshold below the EPA recommendation of 10 ppb for drinking water in both vegetative cell form and spore form. Additionally, we integrated both engineered B. megaterium living cells and spores with CMOS chip for field-deployable arsenic detection. We show that the limit of detection of our integrated sensor is applicable in soil and air arsenic contamination testing. As a proof of concept, this work paves the way for deploying our sensor in resource-limited settings, ensuring real-time arsenic detection in challenging environments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/604150v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1940e83org.highwire.dtl.DTLVardef@144da0aorg.highwire.dtl.DTLVardef@146e906org.highwire.dtl.DTLVardef@6cb8c1_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Annotation-free prediction of microbial dioxygen utilization

Aerobes require dioxygen (O2) to grow; anaerobes do not. But nearly all microbes -- aerobes, anaerobes, and facultative organisms alike -- express enzymes whose substrates include O2, if only for detoxification. This presents a challenge when trying to assess which organisms are aerobic from genomic data alone. This challenge can be overcome by noting that O2 utilization has wide-ranging effects on microbes: aerobes typically have larger genomes, encode more O2-utilizing enzymes, and tend to use different amino acids in their proteins. Here we show that these effects permit high-quality prediction of O2 utilization from genome sequences, with several models displaying >70% balanced accuracy on a ternary classification task wherein blind guessing is only 33.3% accurate. Since genome annotation is compute-intensive and relies on many assumptions, we asked if annotation-free methods also perform well. We discovered that simple and efficient models based entirely on genome sequence content -- e.g. triplets of amino acids -- perform about as well as intensive annotation-based algorithms, enabling the rapid processing of global-scale sequence data to predict aerobic physiology. To demonstrate the utility of efficient physiological predictions we estimated the prevalence of aerobes and anaerobes along a well-studied O2 gradient in the Black Sea, finding strong quantitative correspondence between local chemistry (O2:sulfide concentration ratio) and the composition of microbial communities. We therefore suggest that statistical methods like ours can be used to estimate, or "sense," pivotal features of the environment from DNA sequencing data. ImportanceWe now have access to sequence data from a wide variety of natural environments. These data document a bewildering diversity of microbes, many known only from their genomes. Physiology -- an organisms capacity to engage metabolically with its environment -- may provide a more useful lens than taxonomy for understanding microbial communities. As an example of this broader principle, we developed algorithms that accurately predict microbial dioxygen utilization directly from genome sequences without first annotating genes, e.g. by considering only the amino acids in protein sequences. Annotation-free algorithms enabled rapid characterization of natural samples, demonstrating a quantitative correspondence between sequences and local O2 levels. These results suggest that DNA sequencing can be repurposed as a multi-pronged chemical sensor, estimating concentrations of O2 and other key facets of complex natural settings.

microbiology↗

bioRxiv2023

Engineering the soil bacterium Pseudomonas synxantha 2-79 into a ratiometric bioreporter for phosphorus limitation

Microbial bioreporters hold promise for addressing challenges in medical and environmental applications. However, the difficulty of ensuring their stable persistence and function within the target environment remains a challenge. One strategy is to integrate information about the host strain and target environment into the design-build-test cycle of the bioreporter itself. Here, we present a case study for such an environmentally-motivated design process by engineering the wheat commensal bacterium Pseudomonas synxantha 2-79 into a ratiometric bioreporter for phosphorus limitation. Comparative analysis showed that an exogenous P-responsive promoter outperformed its native counterparts. This reporter can selectively sense and report phosphorus limitation at plant-relevant concentrations of 25-100 {micro}M without cross-activation from carbon or nitrogen limitation or high cell densities. Its performance is robust over a field-relevant pH range (5.8-8), and it responds only to inorganic phosphorus, even in the presence of common soil organic P. Finally, we used fluorescein-calibrated flow cytometry to assess whether the reporters performance in shaken liquid culture predict its performance in soil, finding that although the reporter is still functional at the bulk level, its variability in performance increases when grown in a soil slurry as compared to planktonic culture, with a fraction of the population not expressing the reporter proteins. Together, our environmentally-aware design process provides an example of how laboratory bioengineering efforts can generate microbes with greater promise to function reliably in their applied contexts.

synthetic biology↗

bioRxiv2023

Development of cell-free transcription-translation systems in three soil Pseudomonads

In vitro transcription-translation (TX-TL) can enable faster engineering of biological systems. This speed-up can be significant, especially in difficult-to-transform chassis. This work shows the successful development of TX-TL systems using three soil-derived wild-type Pseudomonads known to promote plant growth: Pseudomonas synxantha, Pseudomonas chlororaphis, and Pseudomonas aureofaciens. One, P. synxantha, was further characterized. A lysate test of P. synxantha showed a maximum protein yield of 2.5 M at 125 proteins per DNA template and a maximum protein synthesis rate of 20 nM/min. A set of different constitutive promoters driving mNeonGreen expression were tested in TX-TL and integrated into the genome, showing similar normalized strengths for in vivo and in vitro fluorescence. This correspondence between the TX-TL derived promoter strength and the in vivo promoter strength indicates these lysate-based cell-free systems can be used to characterize and engineer biological parts without genome integration, enabling a faster designbuild-test cycle.

synthetic biology↗

bioRxiv2019

Construction of an inducible amyloid expression circuit in Bacillus megaterium: A case study with CsgA and TasA

Environmental applications of synthetic biology such as water remediation require engineered strains to function robustly in a fluctuating and potentially hostile environment. The construction of synthetic biofilm formation circuits could potentially alleviate this issue by promoting cell survival. Towards this end, we construct a xylose-inducible system for the expression of the functional amyloids CsgA and TasA in the soil bacterium Bacillus megaterium. We find that although both amyloids are expressed, only TasA is successfully exported from the cells. Furthermore, expression of CsgA results in a significant growth penalty for the cells while expression of TasA does not. Finally, we show that TasA expression conveys a small but detectable increase in cells adhesion to nickel beads. These results suggest that TasA is a promising candidate for future work on synthetic biofilm formation in B. megaterium.

synthetic biology↗