Results and discussion

nitrate-sensitive promoter characterization

Fig. results of nitrate induction are presented. It is obvious that nitrate can induce GFP expression. Additionally, Pyear (nitrate-sensitive promoter) respond ultra-sensitively when nitrate concentration ranges from approximately 0-2 ug/L. And after concentration of 3.5ug/L,GFP expression maintains at nearly 2800 RFU which showcase its maximal initiation rate of promoter. Top10 brand control is presented. We can clearly see the differences.

phosphate-sensitive promoter characterization

Two phosphate sensitive promoters are induced by low-phosphate condition below 100 uM. However, not significant exciting fluorescence can be detected , though we can observe gradually increasing fluorescence with gradually decreasing phosphate concentration in the medium. We suspected that it is the endogenous stochastic noise that leads to the relatively low exciting fluorescence because a major of cells didn’t respond. Additionally, low transcription rate of the promoters may be another factor that leads to low GFP expression.

Preliminary test of phosphate-sensitive promoter

Fig. preliminary test of two phosphate-sensitve promoters,Pugp and PphoB is shown above.The first diagram shows that Pugp(represented in blue) and PphoB(represented in red) can be induced gradually by phosphate whose concentration ranges from 0 to 80 uM.The second one shows sharp decrease of fluorescence when phosphate concentration below 4uM. The third one shows the logarithm of all the data induced range from 0-400uM,which indicates the clear trend

Future work

Phosphate- and nitrate-sensitive promoters have been characterized. It seems that both of them are in need of modification to enhance TCS robustness and reduce stochastic noise. Besides as expected, our experiment has proved that original nitrate-sensitive promoter needs to be increased the responding sensitivity to match our requirement. Our experiment is still in progress and we have confidence to complete this arduous but exciting task.
For the construction of Phosphate accumulated device. We will link the two parts with promoter and terminator to test the ability of recombination strain to accumulate inorganic phosphate (Pi).We would like to monitor the change of Pi in the medium by a nation standardized method provided by Algology and algae culture laboratory.
Here we introduce the other potential methods we conceived to fine-tune our sensors

Construct chimeric chemoreceptor

It is interesting that chimeric chemoreceptor (HK protein fusion) coupled with non-cognate RR would give TCS some new characterizations like rewiring bacteria behaviors or change of responding sensitivity. And it dawns on me that we could construct a chimeric protein that joins the ligand-binding, trans-membrane and linker domains of the NarX sensor kinase to the signaling and adaptation domains of another chemoreceptor of Escherichia coli to change the sensitivity to nitrate. A number of functional chimeric chemoreceptors have been constructed and we found Scott M. Ward[6] has successfully constructed a NarX-Tar chemoreceptor that could respond with thresholds of 10-5 M for nitrate which enhanced the sensitivity more than 100 times. That seems more feasible to accurately predict the red tide.

RBS selection for appropriate expression rate

The last approach we have considered is to exchange RBS for fine-tuning of the translation rate of the downstream genes. In our case, exchanging of RBS could directly influence the concentrations of sRNA in cell. The corresponding translation rates of RBS library interpreted with RBS calculator(introduced by Peking iGEM team) would be integrated to our model and be screened for several candidate RBSs. Next we would verify those RBSs by experiments.