Sporosarcina pasteurii is famous for turning urea into limestone-like minerals, a reaction that can seal cracks in concrete, capture carbon, and clean up heavy-metal spills. Yet scientists still cannot easily add new genes to this rugged, alkaline bacterium, limiting its potential. Sonoporation—brief pulses of low-frequency ultrasound that open tiny, reversible pores in the cell wall—has emerged as a simple, low-cost way to deliver DNA into other microbes, matching or out-performing electroporation without expensive gear or harsh chemicals. Recent studies reported transformation efficiencies up to 3 × 10⁵ colonies per microgram of DNA in E. coli and successful gene delivery into tough, Gram-positive thermophiles with no loss of cell viability .
My broad objective is to adapt ultrasound-mediated DNA delivery to S. pasteurii and establish a reliable, bench-top protocol that anyone can use. I believe that carefully tuned ultrasound will transiently puncture S. pasteurii’s thick cell wall just long enough for plasmid DNA to enter, after which the wall reseals and the cell grows normally. I hope to optimize sonication parameters (sound power, pulse length, growth phase, and magnesium levels) for maximum transformation efficiency and cell survival and show function by inserting a fluorescent reporter and a biomineralization-enhancing gene, then measuring light output and calcium-carbonate formation.
Cultures of S. pasteurii will be chilled, mixed with plasmid DNA, and exposed to controlled 20 kHz ultrasonic bursts in solution. Surviving cells will be plated on selective media, counted, and potentially screened for fluorescence. By delivering a practical genetic “doorway” into S. pasteurii, this work will unlock greener building materials and new environmental clean-up tools.
Wang, B.-P., Yuan, Y.-M., Yang, S., Xu, Y., Liao, C.-Y., & Niu, F.-X. (2023). Ultrasound-Mediated DNA Transformation of Bacteria. Processes, 11(7), Article 7. https://doi.org/10.3390/pr11072163
Sporosarcina pasteurii is an alkaliphilic, urease-rich bacterium that converts urea and calcium ions into calcite. This microbially induced calcium-carbonate precipitation (MICP) underpins emerging “living” technologies for crack-sealing concrete, heavy-metal immobilisation and low-carbon building materials. Recent omics work shows that S. pasteurii couples high urease expression with a highly negative cell-surface charge and rigid envelope, providing ideal nucleation sites for calcite but also making the cell wall unusually dense and refractory to DNA uptake. Because of this barrier, genetic studies have largely been confined to plasmid shuttle experiments in E. coli and other bacteria, leaving key questions and a lot of its potential effectively out of reach. A robust transformation method would let engineers add fluorescence reporters, tailor metabolic fluxes, install biosafety circuits, and more, dramatically widening the bacterium’s biotechnological toolbox.
Ultrasound-mediated DNA delivery (sonoporation) offers a promising workaround. Low-frequency (20–40 kHz) ultrasound generates transient cavitation bubbles that create reversible nanometre-scale pores in bacterial envelopes, allowing plasmids to slip inside before the membrane reseals. Song et al. first demonstrated the concept in Gram-negative Pseudomonas and E. coli, achieving up to 9.8 × 10⁻⁶ transformants cell⁻¹, four-fold higher than electroporation, without requiring ion-free buffers or expensive pulse generators. Building on this, Wang et al. systematically mapped the influence of power, duty cycle, growth phase and divalent cations, and achieved 3.2 × 10⁵ CFU µg⁻¹ DNA while directly visualizing reversible pore formation by SEM. Crucially, their study showed the technique is largely species-agnostic and works even in yeast, hinting that cell-wall thickness may not be a fundamental limitation.
Evidence that ultrasound can breach thick Gram-positive envelopes comes from Lin et al., who delivered a 6 kb shuttle vector into the thermophilic anaerobe Thermoanaerobacter sp. X514 at 6 × 10² CFU µg⁻¹ while retaining high viability and plasmid integrity. Despite these advances, no peer-reviewed report yet documents stable sonoporative transformation of S. pasteurii. Optimizing cavitation energy under alkaline conditions, protecting incoming DNA through methylation, and managing osmotic stress therefore constitute the central technical gaps this project addresses.
By critically integrating lessons from broad-host sonoporation studies with the unique physiological features of S. pasteurii, our work will close a longstanding barrier to genetic analysis in this bacterium and unlock finely tuned, next-generation biocementation strategies.\
The overarching goal of this project is to create a rigorously tested, ultrasound-mediated DNA-delivery platform for Sporosarcina pasteurii , thereby eliminating the single largest experimental barrier to the mechanistic dissection and rational engineering of microbially induced calcium-carbonate precipitation (MICP). Sonoporation produces transient cavitation micro-jets that perforate even recalcitrant Gram-positive envelopes, as demonstrated in Thermoanaerobacter sp. X514, where a 20 s, 40 kHz pulse yielded ~6 × 10² CFU µg⁻¹ of plasmid without compromising viability. More recently, an optimized protocol in E. coli achieved 3.2 × 10⁵ CFU µg⁻¹ and visualized reversible pore formation by scanning-electron microscopy, confirming that the process is largely species-agnostic and mediated by acoustic cavitation. By calibrating acoustic power, duty cycle and ionic composition to SP’s alkali-tolerant physiology, I aim to attain comparable efficiencies, enabling stable integration of reporter cassettes, metabolic rewiring constructs and biosafety circuits. Successful implementation would convert SP from a “black-box” biocement factory into a genetically tractable chassis for systematic studies on nucleation kinetics, urease regulation and carbonate polymorph selection, areas that have so far relied on phenomenological observations rather than molecular experimentation.
From a systems-level standpoint, cement manufacture accounts for ~8 % of anthropogenic CO₂ emissions. Self-healing concretes that exploit SP-driven MICP could curb these emissions by extending service life, lowering clinker demand and mineralizing atmospheric CO₂ in situ. A universally accessible genetic toolkit will accelerate translation from proof-of-concept to industrial deployment by allowing (i) kinetics-tuned urease pathways for rapid crack sealing, (ii) heterologous carbonic anhydrases for enhanced CO₂ sequestration, and (iii) programmable fluorescence or electrochemical reporters for non-destructive structural health monitoring. Beyond civil infrastructure, the methodology establishes a transferable blueprint for sonic gene delivery into other alkaline or thick-walled environmental isolates, broadening the repertoire of organisms that can be drafted into carbon-negative materials, heavy-metal immobilisation or subsurface geo-engineering. In sum, achieving the proposed aims will elevate SP from an industrial curiosity to a fully programmable bio-mineralization platform, with ripple effects across microbial genetics, materials science and global CO₂-mitigation technologies.
Imagine buildings that seal their own cracks, seawalls that harden in response to rising tides, and mine tailings that lock toxic metals into stone. Sporosarcina pasteurii can already do these things in the lab, but today I can’t easily re-engineer it for speed, safety, or new functions because its thick Gram-positive wall blocks most DNA-delivery tools. By adapting sonoporation, I aim to create the first gateway into genetic engineering of S. pasteurii. Once that door is open, researchers will be free to Introduce genes for faster calcite precipitation, CO₂-mineralizing enzymes, or built-in fluorescent “health monitors,” transforming a niche laboratory strain into a versatile, living construction material.
This project pioneers the use of low-frequency, cavitation-tuned ultrasound as a precision DNA-delivery tool for the alkaline, thick-walled bacterium Sporosarcina pasteurii, for which no robust transformation method currently exists. By coupling real-time acoustofluidic monitoring with plasmids that carry orthogonal kill-switches and biomineralization reporters, it transforms sonoporation from a largely empirical technique into a chassis-specific engineering platform that can be deployed on an inexpensive benchtop transducer rather than specialized electroporators or labor-intensive protoplast workflows. The ability to program S. pasteurii in situ with designer calcite-forming pathways and fluorescence-based health monitors challenges the prevailing view that recalcitrant environmental isolates are genetically intractable, and it opens a new frontier of “programmable lithic microbiomes” for carbon-negative construction and subsurface remediation.