Efficient natural plasmid transformation of Vibrio natriegens enables zero-capital molecular biology.

The fast-growing microbe Vibrio natriegens is capable of natural transformation where it draws DNA in from media via an active process under physiological conditions. Using an engineered strain with a genomic copy of the master competence regulator tfoX from Vibrio cholerae in combination with a new minimal competence media (MCM) that uses acetate as an energy source, we demonstrate naturally competent cells which are created, transformed, and recovered entirely in the same media, without exchange or addition of fresh media. Cells are naturally competent to plasmids, recombination with linear DNA, and cotransformation of both to select for scarless and markerless genomic edits. The entire process is simple and inexpensive, requiring no capital equipment for an entirely room temperature process (zero capital protocol, 104 cfu/µg), or just an incubator (high-efficiency protocol, 105-6 cfu/µg). These cells retain their naturally competent state when frozen and are transformable immediately upon thawing like a typical chemical or electrochemical competent cell. Since the optimized transformation protocol requires only 50 min of hands-on time, and V. natriegens grows quickly even on plates, a transformation started at 9 AM yields abundant culturable single colonies by 5 PM. Further, because all stages of transformation occur in the same media, and the process can be arbitrarily scaled in volume, this natural competence strain and media could be ideal for automated directed evolution applications. As a result, naturally competent V. natriegens could compete with Escherichia coli as an excellent chassis for low-cost and highly scalable synthetic biology.

Efficiency estimates for electromicrobial production of branched-chain hydrocarbons.

In electromicrobial production (EMP), electricity is used as microbial energy to produce complex molecules starting from simple compounds like CO2. The aviation industry requires sustainable fuel alternatives that can meet demands for high-altitude performance and modern emissions standards. EMP of jet fuel components provides a unique opportunity to generate fuel blends compatible with modern engines producing net-neutral emissions. Branched-chain hydrocarbons modulate the boiling and freezing points of liquid fuels at high altitudes. In this study, we analyze the pathways necessary to generate branched-chain hydrocarbons in vivo utilizing extracellular electron uptake (EEU) and H2-oxidation for electron delivery, the Calvin cycle for CO2-fixation and the aldehyde deformolating oxygenase decarboxylation pathway. We find the maximum electrical-to-fuel energy conversion efficiencies to be 40.0-4.4+0.6% and 39.8-4.5+0.7%. For a model blend containing straight-chain, branched-chain, and terpenoid components, increasing the fraction of branched-chain alkanes from zero to 47% only lowers the electrical energy conversion efficiency from 40.1-4.5+0.7% to 39.5-4.6+0.7%.

Multiple Rounds of In Vivo Random Mutagenesis and Selection in Vibrio natriegens Result in Substantial Increases in REE Binding Capacity.

Rare earth elements (REE) are essential ingredients in many modern technologies, yet their purification remains either environmentally harmful or economically unviable. Adsorption, or biosorption, of REE onto bacterial cell membranes offers a sustainable alternative to traditional solvent extraction methods. But in order for biosorption-based REE purification to compete economically, the capacity and specificity of biosorption sites must be enhanced. Although there have been some recent advances in characterizing the genetics of REE-biosorption, the variety and complexity of bacterial membrane surface sites make targeted genetic engineering difficult. Here, we propose using multiple rounds of in vivo random mutagenesis induced by the MP6 plasmid combined with plate-throughput REE-biosorption screening to improve a microbe's capacity and selectivity for biosorbing REE. We engineered a strain of Vibrio natriegens capable of biosorbing 210% more dysprosium compared to the wild-type and produced selectivity improvements of up to 50% between the lightest (lanthanum) and heaviest (lutetium) REE. We believe that mutations we observed in ABC transporters as well as a nonessential protein in the BAM outer membrane ß-barrel protein insertion complex likely contribute to some─but almost certainly not all─of the biosorption changes we observed. Given the ease of finding significant biosorption mutants, these results highlight just how many genes likely contribute to biosorption as well as the power of random mutagenesis in identifying genes of interest and optimizing a biological system for a task.

Upper limit efficiency estimates for electromicrobial production of drop-in jet fuels.

Microbes which participate in extracellular electron uptake (EEU) or H2 oxidation have the ability to manufacture organic compounds using electricity as the primary source of metabolic energy. So-called electromicrobial production could be valuable to efficiently synthesize drop-in jet fuels using renewable energy. Here, we calculate the upper limit electrical-to-fuel conversion efficiency for a model jet fuel blend containing 85% straight-chain alkanes and 15% terpenoids. When using the Calvin cycle for carbon-fixation, the energy conversion efficiency is 37.8-4.3+1.8% when using EEU for electron delivery and 40.1-4.6+0.7% when using H2 oxidation. The production efficiency can be raised to 44.2-3.7+0.5% when using the Formolase formate-assimilation pathway, and to 49.2-2.1+0.3% with the Wood-Ljungdahl pathway. This efficiency can be further raised by swapping the well-known Aldehyde Deformolating Oxygenase (ADO) termination pathway with the recently discovered Fatty Acid Photodecarboxylase (FAP) pathway. If these systems were supplied with electricity from a maximally-efficient silicon solar photovoltaic, even the least efficient pathway exceeds the maximum solar-to-fuel efficiency of all known forms of photosynthesis.

Overcoming Leak Sensitivity in CRISPRi Circuits Using Antisense RNA Sequestration and Regulatory Feedback.

The controlled binding of the catalytically dead CRISPR nuclease (dCas) to DNA can be used to create complex, programmable transcriptional genetic circuits, a fundamental goal of synthetic biology. This approach, called CRISPR interference (CRISPRi), is advantageous over existing methods because the programmable nature of CRISPR proteins in principle enables the simultaneous regulation of many different targets without crosstalk. However, the performance of dCas-based genetic circuits is limited by both the sensitivity to leaky repression within CRISPRi logic gates and retroactive effects due to a shared pool of dCas proteins. By utilizing antisense RNAs (asRNAs) to sequester gRNA transcripts as well as CRISPRi feedback to self-regulate asRNA production, we demonstrate a mechanism that suppresses unwanted repression by CRISPRi and improves logical gene circuit function in Escherichia coli. This improvement is particularly pronounced during stationary expression when CRISPRi circuits do not achieve the expected regulatory dynamics. Furthermore, the use of dual CRISPRi/asRNA inverters restores the logical performance of layered circuits such as a double inverter. By studying circuit induction at the single-cell level in microfluidic channels, we provide insight into the dynamics of antisense sequestration of gRNA and regulatory feedback on dCas-based repression and derepression. These results demonstrate how CRISPRi inverters can be improved for use in more complex genetic circuitry without sacrificing the programmability and orthogonality of dCas proteins.

Massively parallel CRISPRi assays reveal concealed thermodynamic determinants of dCas12a binding.

The versatility of CRISPR-Cas endonucleases as a tool for biomedical research has led to diverse applications in gene editing, programmable transcriptional control, and nucleic acid detection. Most CRISPR-Cas systems, however, suffer from off-target effects and unpredictable nonspecific binding that negatively impact their reliability and broader applicability. To better evaluate the impact of mismatches on DNA target recognition and binding, we develop a massively parallel CRISPR interference (CRISPRi) assay to measure the binding energy between tens of thousands of CRISPR RNA (crRNA) and target DNA sequences. By developing a general thermodynamic model of CRISPR-Cas binding dynamics, our results unravel a comprehensive map of the energetic landscape of nuclease-dead Cas12a (dCas12a) from Francisella novicida as it inspects and binds to its DNA target. Our results reveal concealed thermodynamic factors affecting dCas12a DNA binding, which should guide the design and optimization of crRNA that limits off-target effects, including the crucial role of an extended protospacer adjacent motif (PAM) sequence and the impact of the specific base composition of crRNA-DNA mismatches. Our generalizable approach should also provide a mechanistic understanding of target recognition and DNA binding when applied to other CRISPR-Cas systems.