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

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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. </p>
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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.  
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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. </p>
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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.
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<h2>INITIAL MODEL</h2>
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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.
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<h2>EXPERIMENTAL DESIGN</h2>
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<h2>Improved Design </h2>
 
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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.  
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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.  
 
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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. </p>
 
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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.</p>
 
  
  

Revision as of 22:16, 17 October 2018

iGem SDSZ_China 2018
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ABOUT QUORUM SENSING

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.

OUR DESIGN

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.

...

INITIAL MODEL

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.

Improved Design

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.

References

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.

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