Sustainable protein regeneration in encapsulated materials.

Zhu et al. introduce MELG (materials engineered by living grafting), combining engineered microbes with non-living scaffolds for functional protein regeneration within. These MELGs can be used for long-term controlled release, enzyme-mediated biocatalysis, and DNA purification. This approach offers enhanced functionality and durability in bioactive materials compared to traditional non-living counterparts.

Expanding the genetic programmability of Lactiplantibacillus plantarum.

Lactobacilli are ubiquitous in nature and symbiotically provide health benefits for countless organisms including humans, animals and plants. They are vital for the fermented food industry and are being extensively explored for healthcare applications. For all these reasons, there is considerable interest in enhancing and controlling their capabilities through the engineering of genetic modules and circuits. One of the most robust and reliable microbial chassis for these synthetic biology applications is the widely used Lactiplantibacillus plantarum species. However, the genetic toolkit needed to advance its applicability remains poorly equipped. This mini-review highlights the genetic parts that have been discovered to achieve food-grade recombinant protein production and speculates on lessons learned from these studies for L. plantarum engineering. Furthermore, strategies to identify, create and optimize genetic parts for real-time regulation of gene expression and enhancement of biosafety are also suggested.

Plasmonic stimulation of gold nanorods for the photothermal control of engineered living materials.

Engineered living materials (ELMs) encapsulate microorganisms within polymeric matrices for biosensing, drug delivery, capturing viruses, and bioremediation. It is often desirable to control their function remotely and in real time and so the microorganisms are often genetically engineered to respond to external stimuli. Here, we combine thermogenetically engineered microorganisms with inorganic nanostructures to sensitize an ELM to near infrared light. For this, we use plasmonic gold nanorods (AuNR) that have a strong absorption maximum at 808 nm, a wavelength where human tissue is relatively transparent. These are combined with Pluronic-based hydrogel to generate a nanocomposite gel that can convert incident near infrared light into heat locally. We perform transient temperature measurements and find a photothermal conversion efficiency of 47 %. Steady-state temperature profiles from local photothermal heating are quantified using infrared photothermal imaging and correlated with measurements inside the gel to reconstruct spatial temperature profiles. Bilayer geometries are used to combine AuNR and bacteria-containing gel layers to mimic core-shell ELMs. The thermoplasmonic heating of an AuNR-containing hydrogel layer that is exposed to infrared light diffuses to the separate but connected hydrogel layer with bacteria and stimulates them to produce a fluorescent protein. By tuning the intensity of the incident light, it is possible to activate either the entire bacterial population or only a localized region.

Novel genetic modules encoding high-level antibiotic-free protein expression in probiotic lactobacilli.

Lactobacilli are ubiquitous in nature, often beneficially associated with animals as commensals and probiotics, and are extensively used in food fermentation. Due to this close-knit association, there is considerable interest to engineer them for healthcare applications in both humans and animals, for which high-performance and versatile genetic parts are greatly desired. For the first time, we describe two genetic modules in Lactiplantibacillus plantarum that achieve high-level gene expression using plasmids that can be retained without antibiotics, bacteriocins or genomic manipulations. These include (i) a promoter, PtlpA , from a phylogenetically distant bacterium, Salmonella typhimurium, which drives up to 5-fold higher level of gene expression compared to previously reported promoters and (ii) multiple toxin-antitoxin systems as a self-contained and easy-to-implement plasmid retention strategy that facilitates the engineering of tuneable transient genetically modified organisms. These modules and the fundamental factors underlying their functionality that are described in this work will greatly contribute to expanding the genetic programmability of lactobacilli for healthcare applications.

In vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1.

Lactobacilli are gram-positive bacteria that are growing in importance for the healthcare industry and genetically engineering them as living therapeutics is highly sought after. However, progress in this field is hindered since most strains are difficult to genetically manipulate, partly due to their complex and thick cell walls limiting our capability to transform them with exogenous DNA. To overcome this, large amounts of DNA (>1 µg) are normally required to successfully transform these bacteria. An intermediate host, like E. coli, is often used to amplify recombinant DNA to such amounts although this approach poses unwanted drawbacks such as an increase in plasmid size, different methylation patterns and the limitation of introducing only genes compatible with the intermediate host. In this work, we have developed a direct cloning method based on in-vitro assembly and PCR amplification to yield recombinant DNA in significant quantities for successful transformation in L. plantarum WCFS1. The advantage of this method is demonstrated in terms of shorter experimental duration and the possibility to introduce a gene incompatible with E. coli into L. plantarum WCFS1.

Transcription factor based whole-cell biosensor for specific and sensitive detection of sodium dodecyl sulfate.

Extensive use of Sodium Dodecyl Sulfate (SDS) in households, agricultural operations, and industries is leading to its subsequent disposal in waterways. There is an apprehension of the adverse effect of such detergents on various living organisms. Thus, an efficient, specific, and simple detection method to monitor SDS reliably in the environment is needed. We used sdsB1 activator protein and SDS-responsive promoter of sdsA1 gene along with Green Fluorescent Protein (GFP) to construct a novel SDS biosensor in Pseudomonas aeruginosa chassis. The GFP intensity of the biosensor showed a linear relationship (R2 = 0.99) from 0.4 to 62.5 ppm of SDS with a detection limit of 0.1 ppm. This biosensor is highly specific for SDS and has minimal interference from other detergents, metals, and inorganic ions. The biosensor showed a satisfactory and reproducible recovery rate for the detection of SDS in real samples. Overall, this is a low cost, easy-to-use, selective, and reliable biosensor for monitoring SDS in the environment.