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.

As our bacteria are programmed to be effective chitin processors and chitosan producers inadequate environments, we hope that during the life cycles of cells and growth of the clone, this function can be executed at its best conditions and do not impose negative effects on the bacteria itself.

Development of a clone includes two major phases which correspond to the different parts of a logistic growth curve: A mass-propagating phase and a slower growth phase (as the population approaches k). In an ideal situation, our bacteria should be able to multiply quickly while the population is relatively smaller thus reach the second phase in the shortest time by which point it is ready to deliver a steady output. To achieve that goal, we need to make sure that when clones are just beginning to develop only the functions necessary to the cell is enabled. In other words, we do not want the cell to continue expressing CDA in low concentration for it will be extremely ineffective and only increases energy consumption without countable output. After the clone is fully developed it is ready to initiate the chitin production functions and—because of massive numbers of cells—generate a considerable output.

Using the QS system here saves a lot of effort on monitoring the growth of the clone—which would be consuming since we are talking about an industrialized process with machines conducting the fermentation process. Instead of manually inducing the expression of CDA genes under appropriate circumstances, we let the bacteria themselves to decide. –An advantage that is only rendered possible when we are using self-regulatory, flexible organisms.

We have chosen protein from the luxI and luxR protein family to build our parts, which is the most common family of acyl homoserine lactone (AHL) autoinducer-receptor system. The luxI gene encodes for an autoinducer synthase that catalyzes the formation of the acyl homoserine lactone from the acyl-S-adenosylmethionine intermediate, [1] which is then secreted and bound to transcription factors encoded by the luxR gene. Being activated, a luxR complex can bind the promoter with a DNA binding region near its N-terminal and initiate the transcription process of downstream CDA genes.

In our construction (see fig.2), the system is regulated by both lac operon and AHL signaling pathway. Our PluxlacO promoter can both respond to lacI repressors and luxI regulator. Under the presence of IPTG, which is the key to both the quorum sensing system and expression of our CDA protein, repression is removed and the transcription of both luxI and luxR is initiated. The two plasmid vectors, pTA1109 and pTA1083, serve as a translation regulator carrier and target gene carrier, respectively.

Under low cell density, transcription of the CDA gene on pTA1083 is repressed by inactivated promotor and all unwanted background expression is repressed. After the threshold concentration is passed, the presence of AHL promotes both the transcription of CDA genes as well as the luxI gene—which in turn produces more AHL in intracellular space and creates a positive feedback loop that eventually raises the transcription level of CDA genes rapidly and dramatically.

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.