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Diep, P.

Publications and source records attributed to Diep, P..

11 recordsLinked to original sources

TreeGazer: Prospecting Protein Sequence-Function Landscapes via Phylogenetic Structure

Building diverse and informative protein sequence datasets is critical for understanding how function varies across sequence space. Because only a small fraction of sequences in a dataset can typically be experimentally characterised, strategies for selecting what sequences to characterise should maximise the information gained from each experiment. Here, we present TreeGazer, a phylogeny-informed framework that combines Bayesian optimisation with the topology of a tree to guide sequence selection. TreeGazer balances exploitation of sequences predicted to exhibit favourable properties against exploration of regions higher model uncertainty. Unlike existing approaches that apply Bayesian optimisation for sequence selection, TreeGazer does not rely on black-box models and instead uses latent representations of property distributions that are directly tied to phylogenetic structure. Modelling properties in this way enables biologically interpretable predictions and uncertainty estimates. Across two simulated selection campaigns, TreeGazer consistently selected sequences that produced datasets more representative of the underlying property distribution than alternative strategies that used protein language models. TreeGazer also performed effectively in low-data settings, where tree-guided selection enabled accurate identification of functional transitions across clades. TreeGazer can be run on conventional laptop computers while still providing equivalent or superior performance to embedding-based approaches. These results demonstrate that phylogenetic structure is a powerful and underutilised prior for guiding informative sequence selection.

bioinformatics↗

Iron limitation alters diatom carbon flow through shifts in microbiome exometabolite consumption

Iron is required for photosynthesis, and thus affects biogeochemical cycling in widespread regions where its availability is limited. Turnover of aquatic photosynthetically-derived carbon is largely constrained by bacterial activity, but we lack a mechanistic understanding of how iron limitation influences this activity. We examined a bacterial enrichment community dependent on carbon from the diatom Phaeodactylum tricornutum to investigate how iron limitation alters the flow of carbon to bacteria, and exometabolite and community composition. Using stable isotope tracing, we quantified diatom exudate incorporation with single-cell-resolution. We identified a population of bacteria under iron limitation with high metabolic activity yet low incorporation of newly-fixed diatom carbon, indicating a shift in metabolism relative to the iron-replete control. Ultra-high-resolution exometabolomics revealed bacterial consumption of aromatics, lipid-like compounds, and purines and pyrimidines occurred under iron-limitation, when these compounds also exhibited increased exudation. We identified gene pathways for utilization of these compounds in taxa with increased abundance under iron limitation which may be responsible for carbon flow shifts. These results provide a mechanistic link between iron-limitation driven shifts in exudate composition and flow of carbon to the microbiome. This has important implications for predicting carbon flow in surface oceans and manipulating algal-bacterial interactions in engineered systems.

microbiology↗

A family portrait of lanmodulin selectivity for enhanced rare-earth separations

Proteins offer a molecular design space to create bespoke ligands for the separation of critical metals, like rare earth elements (REs). However, data-intensive approaches to fine-tune metalloprotein selectivity are bottlenecked by the low-throughput nature of existing characterization methods. Here we invented an assay called SpyTag-Catcher Immobilization of Lanmodulin for Assaying Metal-Binding Selectivity (SpyCI-LAMBS) to measure metalloprotein selectivity en masse. This 96-format workflow was used to study the selectivity of 621 lanmodulin (LanM) orthologs for 15 REs, revealing eight distinct selectivity profiles based on sequence-to-function analyses. We discovered >200 LanMs with dampened selectivity for low-value LaIII relative to the prototypical LanM. This includes a LanM that can perform a challenging one-stage separation of PrIII from LaIII with up to >99.9 mol% purity and 83% yield. SpyCI-LAMBS is a powerful tool that can rapidly collect high-fidelity selectivity data to inform metal ion separations and machine learning-assisted metalloprotein design.

bioengineering↗

Synthetic communities as a model for determining interactions between a biofertilizer chassis organism and native microbial consortia

Biofertilizers are critical for sustainable agriculture since they can replace ecologically disruptive chemical fertilizers while improving the trajectory of soil and plant health. Yet, to continue improving deployment, the persistence of designer biofertilizers within native soil consortia must be elucidated and enhanced. Here, we describe a high-throughput, modular, and automation-friendly in vitro approach to screen for biofertilizer organism persistence within soil-derived consortia after co-cultivation with stable synthetic soil microbial communities (SynComs) obtained through a top-down cultivation process. We profiled [~]1200 SynComs isolated from various soil sources and cultivated in divergent media types, and detected significant phylogenetic diversity (e.g., Shannon index > 4) and richness (Observed richness > 400) across these communities. We observed high reproducibility in SynCom community structure from common soil and media types, which provided a testbed for assessing biofertilizer persistence within representative native consortia. Furthermore, we demonstrate the screening method described herein can be coupled with microbial engineering to efficiently identify soil-derived SynComs where an engineered biofertilizer organism (i.e. Bacillus subtilis) persists. Additionally, our approach enables an analysis of the ecological impact of B. subtilis inoculation on SynCom structure and profile alterations in community diversity and richness (or lack thereof) associated with the presence of a genetically modified model bacterium. Ultimately, this work establishes a modular pipeline that could be integrated into a variety of microbiology/microbiome-relevant workflows or related applications that would benefit from assessing persistence and interaction of a specific organism of interest with native consortia.

systems biology↗

In vitro quantification of metal ion specificity of CcNikZ-II in the presence of competing metal ions

Many proteins bind transition metal ions as cofactors to carry out their biological functions. Despite binding affinities for divalent transition metal ions being predominantly dictated by the Irving-Williams series for wild-type proteins, in vivo metal ion binding specificity is ensured by intracellular mechanisms that regulate free metal ion concentrations. However, a growing area of biotechnology research considers the use of metal-binding proteins in vitro to purify specific metal ions from wastewater, where specificity is dictated by the proteins metal binding affinities. A goal of metalloprotein engineering is to modulate these affinities to improve a proteins specificity towards a particular metal; however, the quantitative relationship between the affinities and the equilibrium metal-bound protein fractions depends on the underlying binding kinetics. Here we demonstrate a high-throughput intrinsic tryptophan fluorescence quenching method to validate kinetic models in multi-metal solutions for CcNikZ-II, a nickel-binding protein from Clostridium carboxidivorans. Using our validated models, we quantify the relationship between binding affinity and specificity in different classes of metal-binding models for CcNikZ-II. We further demonstrate that principles for improving specificity through changes in binding affinity are qualitatively different depending on the competing metals, highlighting the power of mechanistic models to guide metalloprotein engineering targets.

biochemistry↗

Ni(II) binding affinity and specificity of solute binding proteins: the importance of the double His motif and variable loop revealed by structural and mutational studies

Extracytoplasmic solute binding proteins (SBPs) are molecular shuttles involved in the cellular uptake of various small molecules and metal ions including Ni(II). Our previous study with the Ni(II) binding proteins (NiBPs) CjNikZ from Campylobacter jejuni and CcNikZ-II from Clostridium carboxidivorans demonstrated they were able to bind Ni(II) at low micromolar affinity without the need for additional chelators. Here, we determined the crystal structure of apo CcNikZ-II, which revealed a Ni(II) binding site comprised of the highly conserved double His (HH-)prong (His511, His512) and a short variable (v-)loop nearby (Thr59-Thr64, TEDKYT). Alanine scanning mutagenesis of the CcNikZ-II Ni(II) binding site identified Glu60 and His511 as essential for high affinity binding to Ni(II). Phylogenetic analysis of >4000 SBP sequences demonstrated the presence of two clusters of proteins containing the HH-prong with CcNikZ-II and CjNikZ. To provide insights into the role of the double His-prong and v-loop sequence in Ni(II) binding of NiBPs, nine purified CcNikZ-II homologues containing the HH-prong and v-loop were screened using an automated screening workflow. Metal binding assays with purified homologous NiBPs revealed high Ni(II) binding affinity without requirement for chelators indicating that the double His prong represents a signature motif for the presence of Ni(II) binding activity in SBPs. The engineered CcNikZ-II variants with the wild type v-loop (TEDKYT) replaced with v-loops from NiBPs with higher affinity showed up to an order of magnitude higher affinity for Ni(II). In addition, the v-loop appears to play a role in metal ion specificity as purified wild type and engineered NiBPs with different v-loop sequences showed distinct metal profiles. This work paves way for metalloprotein engineering of NiBPs towards biocatalytic and metal recovery applications.

biochemistry↗

Engineered nickel bioaccumulation in Escherichia coli by NikABCDE transporter and metallothionein overexpression

Mine wastewater often contains dissolved metals at concentrations too low to be economically extracted by existing technologies, yet too high for environmental discharge. The most common treatment is chemical precipitation of the dissolved metals using limestone and subsequent disposal of the sludge in tailing impoundments. While it is a cost-effective solution to meet regulatory standards, it represents a lost opportunity. In this study, we engineered Escherichia coli to overexpress its native NikABCDE transporter and a heterologous metallothionein to capture nickel at concentrations in local effluent streams. We found the engineered strain had a 7-fold improvement in the bioaccumulation performance for nickel compared to controls, but also observed a drastic decrease in cell viability due to metabolic burden or inducer (IPTG) toxicity. Growth kinetic analysis revealed the IPTG concentrations used based on past studies lead to growth inhibition, thus delineating future avenues for optimization of the engineered strain and its growth conditions to perform in more complex environments.

bioengineering↗

REVOLVER: a low-cost automated protein purifier based on parallel preparative gravity column workflows

Protein purification is a ubiquitous operation in biochemistry and life sciences and represents a key step to producing purified proteins for research (understanding how proteins work) and various applications. The need for scalable and parallel protein purification systems keeps growing due to the increase in throughput in the production of recombinant proteins and in the ever-growing scale of biochemistry research. Therefore, automating the process to handle multiple samples in parallel with minimal human intervention is highly desirable; yet only a handful of such tools have been developed, all of which are closed source and expensive. To address this challenge, we present REVOLVER, a 3D-printed programmable and automatic protein purification system based on gravity-column workflows and controlled by Arduino boards that can be built for under $130 USD. REVOLVER completes a full protein purification process with almost no human intervention and yields results equivalent to those obtained by an experienced biochemist when purifying a real-world protein sample. We further present and describe MULTI-VOLVER, a scalable version of the REVOLVER that allows for parallel purification of up to six samples and can be built for under $250 USD. Both systems will be useful to accelerate protein purification and ultimately link them to bio-foundries for protein characterization and engineering. Specifications Table O_TBL View this table: org.highwire.dtl.DTLVardef@13fff66org.highwire.dtl.DTLVardef@98aa2forg.highwire.dtl.DTLVardef@cd2e70org.highwire.dtl.DTLVardef@a3bd86org.highwire.dtl.DTLVardef@1efc67d_HPS_FORMAT_FIGEXP M_TBL C_TBL

biochemistry↗

Automation assisted anaerobic phenotyping for metabolic engineering

Microorganisms can be metabolically engineered to produce a wide range of commercially important chemicals. Advancements in computational strategies for strain design and synthetic biological techniques to construct the designed strains have facilitated the generation of large libraries of potential candidates for chemical production. Consequently, there is a need for a high-throughput, laboratory scale techniques to characterize and screen these candidates to select strains for further investigation in large scale fermentation processes. Several small-scale fermentation techniques, in conjunction with laboratory automation have enhanced the throughput of enzyme and strain phenotyping experiments. However, such high throughput experimentation typically entails large operational costs and generate massive amounts of laboratory plastic waste. In this work, we develop an eco-friendly automation workflow that effectively calibrates and decontaminates fixed-tip liquid handling systems to reduce tip waste. We also investigate inexpensive methods to establish anaerobic conditions in microplates for high-throughput anaerobic phenotyping. To validate our phenotyping platform, we perform two case studies - an anaerobic enzyme screen, and a microbial phenotypic screen. We used our automation platform to investigate conditions under which several strains of E. coli exhibit the same phenotypes in 0.5 L bioreactors and in our scaled-down fermentation platform. Further, we propose the use of dimensionality reduction through t-distributed stochastic neighbours embedding in conjunction with our phenotyping platform to serve as an effective scale-down model for bioreactor phenotypes. By integrating an in-house data-analysis pipeline, we were able to accelerate the test phase of the design-build-test-learn cycle of metabolic engineering.

bioengineering↗

Challenges in Undergraduate Synthetic Biology Training: Insights from a Canadian iGEM Student Perspective

The last two decades have seen vigorous activity in synthetic biology research and ever-increasing applications of synthetic biology technologies. However, pedagogical research on synthetic biology is scarce, especially when compared to some scientific and engineering disciplines. Within Canada, there are only three universities that formally teach synthetic biology programs; two of which are at the undergraduate level. Many Canadian undergraduate students are instead introduced to synthetic biology through participation in the annual International Genetically Engineered Machine (iGEM) competition where they work in design teams to conceive of and execute a synthetic biology project that they present at an international jamboree. We surveyed the Canadian landscape of synthetic biology education through the experience of students from the Canadian iGEM teams of 2019. Using a thematic codebook analysis, we gathered insights to generate recommendations that could empower future iGEM team operations and inform educators about best practices in teaching undergraduate synthetic biology.

scientific communication and education↗

A microplate screen for metal-binding activity based on a nickel-binding protein from Clostridium carboxidivorans

Solute-binding proteins (SBPs) from ATP-binding cassette (ABC) transporters play crucial roles across all forms of life in transporting compounds against chemical gradients. Some SBPs have evolved to scavenge metal substrates from the environment with nanomolar and micromolar affinities (KD). There exist well established techniques like isothermal titration calorimetry for thoroughly studying these metalloprotein interactions with metal ions, but they are low-throughput. For protein libraries comprised of many metalloprotein homologues and mutants, and for collections of buffer conditions and potential ligands, the throughput of these techniques is paramount. In this study, we describe an improved method termed the microITFQ-LTA and validated it using CjNikZ, a well-characterized nickel-specific SBP (Ni-BP) from Campylobacter jejuni. We then demonstrated how the microITFQ-LTA can be designed to screen through a small collection of buffers and ligands to elucidate the binding profile of a putative Ni-BP from Clostridium carboxidivorans that we call CcSBPII. Through this study, we showed CcSBPII can bind to various metal ions with KD ranged over 3 orders of magnitude. In the presence of L-histidine, CcSBPII could bind to Ni2+ over 2000-fold more tightly, which was 11.6-fold tighter than CjNikZ given the same ligand. HighlightsO_LIan improved version of a high-throughput screen (microITFQ-LTA) is described for multiplexed elucidation of metalloprotein binding profiles C_LIO_LIvalidation was accomplished with the previously characterized CjNikZ; testing was accomplished with an uncharacterized homologue herein named CcSBPII C_LIO_LICcSBPII is shown to bind to multiple transition metal ions with a large range of affinities, and potentially overcome mismetallation using a simple histidine metallophore C_LI

biochemistry↗