Natural Products and Pharmacological Properties of Symbiotic Bacillota (Firmicutes) of Marine Macroalgae.

The shift from the terrestrial to the marine environment to discover natural products has given rise to novel bioactive compounds, some of which have been approved for human medicine. However, the ocean, which makes up nearly three-quarters of the Earth's surface, contains macro- and microorganisms whose natural products are yet to be explored. Among these underexplored marine organisms are macroalgae and their symbiotic microbes, such as Bacillota, a phylum of mostly Gram-positive bacteria previously known as Firmicutes. Macroalgae-associated Bacillota often produce chemical compounds that protect them and their hosts from competitive and harmful rivals. Here, we summarised the natural products made by macroalgae-associated Bacillota and their pharmacological properties. We discovered that these Bacillota are efficient producers of novel biologically active molecules. However, only a few macroalgae had been investigated for chemical constituents of their Bacillota: nine brown, five red and one green algae. Thus, Bacillota, especially from the marine habitat, should be investigated for potential pharmaceutical leads. Moreover, additional diverse biological assays for the isolated molecules of macroalgae Bacillota should be implemented to expand their bioactivity profiles, as only antibacterial properties were tested for most compounds.

Topologies of synthetic gene circuit for optimal fold change activation.

Computations widely exist in biological systems for functional regulations. Recently, incoherent feedforward loop and integral feedback controller have been implemented into Escherichia coli to achieve a robust adaptation. Here, we demonstrate that an indirect coherent feedforward loop and mutual inhibition designs can experimentally improve the fold change of promoters, by reducing the basal level while keeping the maximum activity high. We applied both designs to six different promoters in E. coli, starting with synthetic inducible promoters as a proof-of-principle. Then, we examined native promoters that are either functionally specific or systemically involved in complex pathways such as oxidative stress and SOS response. Both designs include a cascade having a repressor and a construct of either transcriptional interference or antisense transcription. In all six promoters, an improvement of up to ten times in the fold change activation was observed. Theoretically, our unitless models show that when regulation strength matches promoter basal level, an optimal fold change can be achieved. We expect that this methodology can be applied in various biological systems for biotechnology and therapeutic applications.

A Whole-Cell Bacterial Biosensor for Blood Markers Detection in Urine.

The early detection of blood in urine (hematuria) can play a crucial role in the treatment of serious diseases (e.g., infections, kidney disease, schistosomiasis, and cancer). Therefore, the development of low-cost portable biosensors for blood detection in urine has become necessary. Here, we designed an ultrasensitive whole-cell bacterial biosensor interfaced with an optoelectronic measurement module for heme detection in urine. Heme is a red blood cells (RBCs) component that is liberated from lysed cells. The bacterial biosensor includes Escherichia coli cells carrying a heme-sensitive synthetic promoter integrated with a luciferase reporter (luxCDABE) from Photorhabdus luminescens. To improve the bacterial biosensor performance, we re-engineered the genetic structure of luxCDABE operon by splitting it into two parts (luxCDE and luxAB). The luxCDE genes were regulated by the heme-sensitive promoter, and the luxAB genes were regulated by either constitutive or inducible promoters. We examined the genetic circuit's performance in synthetic urine diluent supplied with heme and in human urine supplied with lysed blood. Finally, we interfaced the bacterial biosensor with a light detection setup based on a commercial optical measurement single-photon avalanche photodiode (SPAD). The whole-cell biosensor was tested in human urine with lysed blood, demonstrating a low-cost, portable, and easy-to-use hematuria detection with an ON-to-OFF ratio of 6.5-fold for blood levels from 5 × 104 to 5 × 105 RBC per mL of human urine.

Synthetic metabolic computation in a bioluminescence-sensing system.

Bioluminescence is visible light produced and emitted by living cells using various biological systems (e.g. luxCDABE cassette). Today, this phenomenon is widely exploited in biological research, biotechnology and medical applications as a quantitative technique for the detection of biological signals. However, this technique has mostly been used to detect a single input only. In this work, we re-engineered the complex genetic structure of luxCDABE cassette to build a biological unit that can detect multi-inputs, process the cellular information and report the computation results. We first split the luxCDABE operon into several parts to create a genetic circuit that can compute a soft minimum in living cells. Then, we used the new design to implement an AND logic function with better performance as compared to AND logic functions based on protein-protein interactions. Furthermore, by controlling the reverse reaction of the luxCDABE cassette independently from the forward reaction, we built a comparator with a programmable detection threshold. Finally, we applied the redesigned cassette to build an incoherent feedforward loop that reduced the unwanted crosstalk between stress-responsive promoters (recA, katG). This work demonstrates the construction of genetic circuits that combine regulations of gene expression with metabolic pathways, for sensing and computing in living cells.