Difference between revisions of "Team:SDSZ China/Experiment B"

Quorum sensing is a strategy developed among bacteria that respond to the fluctuation of bacteria density in the environment and alters the expression of certain genes. In natural environments, bacteria can control various physiological activities such as bio-illumination, conjugation and so on.

A bacterial QS system (see fig.1)is mediated by what known as an autoinducer, or a signaling molecule. In low cell density, the autoinducer secreted by individual cells have extremely low density in the extracellular environment and are unable to be recognized by receptors and cause significant functional changes in other cells because of the low rate of diffusion. Yet as cell population continues to grow, more autoinducers are secreted and will eventually reach the threshold density. By which point the density is significant enough to activate receptors and began their impact upon cell functions.

As we have already learned the mechanisms of the system, it’s parts can be used in genetic engineering and help us sort out problems such as low production efficiency.

To optimize the production of chitosan, we decide to employ the Quorum-Sensing System, which can regulate the gene expression in a density-dependent manner. The density-dependent property of QS System can be extremely beneficial to us since we want the bacteria to express heterogeneous gene CDA and produce chitosan when the bacteria density reaches a relatively high level. As the total amount of energy inside the bacterial cell is constant, when bacteria are growing, their CDA expression will be reduced, while bacterial proliferation will be hindered when they allocate more energy in CDA expression. Therefore, Quorum-Sensing system can act as a switch to us: when the density is low, the CDA is inactive, allowing the bacteria to put into more energy in growth and reproduce; in contrast, when the density reaches a threshold, the CDA is activated, leading to a higher expression level of CDA.

When we first designed our model, we considered the QS system as a convenient way of initializing the CDA transcription under appropriate bacteria concentration. The concentration is measured and we use the data to determine if the threshold density is reached. As soon as it is ready for CDA transcription to start, we introduce the AHL autoinducers to the system thus activating the transcription process. After that the system will regulate on its own.

In fermentation, we need to constantly measure the density of the bacteria to determine whether the threshold density (quorum) is reached or not. As a result, fermenting with the regulation of Quorum-Sensing can be time-consuming. To eliminate the need of waiting and constantly measuring, we decide to use IPTG concentration to decide the threshold density directly and the bacteria would switch the stage of expression automatically.

Soma, Yuki, et al. "Metabolic flux redirection from a central metabolic pathway toward a synthetic pathway using a metabolic toggle switch." Metabolic engineering 23 (2014): 175-184.
Soma, Yuki, and Taizo Hanai. "Self-induced metabolic state switching by a tunable cell density sensor for microbial isopropanol production." Metabolic engineering 30 (2015): 7-15.
Lutz, Rolf, and Hermann Bujard. "Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements." Nucleic acids research 25.6 (1997): 1203-1210.