Both cells are strains of E. Coli, but they responded differently to circuit regulation. A healthy cell is described as "small and cylindrical". The MC4100Z1 E. Coli cells used in figure 3's experiments underwent abnormal elongation (filamentation) from their exposure to the killer protein, which may explain the escape of culture 3 from the circuit. Top10F' cells were immune to this response, and therefore did not escape regulation.

Here the authors noticed that changes in cell density correlated with their appearance, or morphology. For instance, when the bacteria had a small population, they were small and cylindrical (healthy-looking), and their population grew rapidly without problems. However, as their numbers increased, they looked worse and started expressing more killer proteins that led to a decline in density.

Macroscale experimentation not utilizing chemostat dilutions but still containing circuit regulation maintained their populations for approximately 2-3 days, but the microchemostat was able to maintain populations for upwards of 20 days.

By shrinking the volume of the bioreactor, the authors significantly reduced the mutation rate, allowing for observation of genetically similar populations for a longer time. This allowed for precise control of engineered bacterial populations over hundreds of hours without losing regulation control, which was not possible to achieve with larger reactors.

The killer protein took time to be diluted and degraded, so that it continued to filament and kill the cells. This "lag" in signaling is present in many biological systems and is why oscillations occur rather than population immediately reaching a steady-state.

As depicted in Figure 1A, the authors developed a device to grow bacteria without forming biofilms, which are communities that adhere to surfaces and produce a protective slime layer. They achieved this by isolating a section of the bioreactor, eliminating and cleansing the cells within, and then reopening the section.

Pfizer has teamed up with another company to utilize microfluidics to study how drugs impact our bodies and how our body's environment affects a drug. This partnership enables rapid and efficient examination of these interactions before progressing to human trials.

Read more about it here: Pfizer

The smaller the population, the less likely mutations are to occur and spread. Conversely, maintaining a large population increases mutation probability. The authors were puzzled by the loss of regulation in the cells, as smaller populations should theoretically minimize the impact of mutations on the synthetic gene circuit, thus preventing loss of regulation.

As seen in cultures 1-3, the populations with the circuit showed a cyclic increase and decrease until they reached a concentration lower than that of unregulated circuits.

All three cultures eventually escaped the population control circuit, with Culture 3 exhibiting sustained oscillations for the longest duration, up to 186 hours.

Bacterial cultures lacking or with a disabled population-control circuit quickly reached and maintained a large population, whereas those with an active circuit showed unstable population dynamics before stabilizing at a lower level. As the population increased, more growth-limiting signals were released, causing a decline in population size and signal release. Subsequently, the population increased again, repeating the cyclic behavior until reaching an equilibrium between cell growth and death from the released signals.

As the dilution rate (how often cells are removed from the bioreactor) increases, the bacteria concentration decreases. Likewise, when the quantity of nutrients (bacto-tryptone) provided to the bacteria are reduced, their concentration also decreases.

Here, they use another strain of E. Coli which is more tightly regulated, leading to stronger growth regulation (i.e. does not escape the circuit regulation). However, from the cultures that were turned on at various time points (Curves 2 through 6), only culture 4 was able to sustain the oscillatory growth behavior. This observation suggests potential malfunctions within the synthetic circuit.

When the bacteria were first added to the growth media, they appeared healthy. This changed as the population grew.

The nonadhesive coatings mentioned earlier slightly reduce bacteria adhesion and biofilm formation, but channels were still invaded within approximately two days.

The author's method of periodically closing off a section of the loop, cleaning it thoroughly, and reintroducing bacterial growth medium before resuming flow was effective for long-term experiments.

Calico Labs has developed a Miniature-chemostat Aging Device (MAD) platform to understand aging better. They accomplish this by characterizing the genomic and biochemical properties of aging yeast, an established model for eukaryotic cellular aging.

Read more about this at BioSpace: https://www.biospace.com/article/the-search-for-the-fountain-of-longevity-not-youth-/

This aspect presents a significant knowledge gap for this research that remains elusive. As depicted in Figure 4, only one bacterial culture maintained a low concentration upon reactivation of the population-limiting circuit. The authors state that this behavior cannot simply be explained by mutation rate.

A recent study found an alternative way to decrease bacteria populations, by turning on a system that makes them self-destruct when attacked by a virus to protect the other bacterial cells.

Read more at Mount Sinai: https://www.mountsinai.org/about/newsroom/2024/new-approach-to-tackling-bacterial-infections-identified

Balagadde and his team of researchers at K-RITH - the first microfluidic research facility in Africa - are developing related systems to increase access to affordable healthcare.

Read more about his current work at pharma: https://www.outsourcing-pharma.com/Article/2016/11/15/Lab-on-a-chip-diagnostics-to-enable-affordable-research#