Team:TPR China/Proof Of Concept

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Proof of Concept

I. The project design:

We know from the paper published by Academician Kang Le that 4-Vinylanisole (4VA) has been proved to be the aggregation pheromone in locusts, it has a significant attraction for locusts to migrate. With this in mind, we hope to use engineered bacteria to produce 4VA to artificially gather locusts and attract them to specific areas for exterminating. Because locust swarms are highly mobile: this not only increases the area affected, but also makes it more difficult to kill. Therefore, the first part of our project was to set up a trap: to artificially gather locusts by producing 4VA artificially.
In other literatures, we also found a avoidance pheromone-- phenylacetonitrile (PAN). This is a toxic and smelly volatile nitrile. Due to PAN's avoidance effect on locusts, the 4VA aggregation effect will be weakened, and a large number of locusts will accumulate toxicity in the region when swarms gather (besides the toxicity of PAN itself, HCN generated from PAN by stimulated locusts is also highly toxic). To solve this problem, the second part of our project will use engineered bacteria to produce nitrile hydrolases to degrade PAN.

However, continuous high expression of nitrile hydrolase gene in engineering bacteria not only brings growth burden, but also is not economical, and there is also the risk of malignant mutation caused by excessive growth. Therefore, we looked for a broad-spectrum small molecule Sensor of aromatic compounds and tried to respond to PAN. To optimize the PAN degradation design of the second part of our project.

II. Proof of our project:

1. PAN

(1) NIT

a) SDS-PAGE verification of nitrilase
We performed SDS-PAGE verification on the fermentation broth of the engineered strain after induction, and the results are shown below.
Fig.1 The SDS-PAGE result of fermentation broth
Electrophoresis results show that our sample has a specific band around 31KD compared to the negative control, this proves that the engineered bacteria we constructed have expressed nitrilase protein.
b) Nitrilase Enzyme Activity
The nitrilase has PAN as substrate to degrade PAN into phenylacetic acid and ammonium. Ammonium and Nash reagents can produce reddish-brown precipitation. The shade is proportional to the concentration of NH4+ produced by PAN degradation.
i) Standard curve
We plotted the standard curve of the ammonium concentration. The experimental sample's NH4+ concentration can thus be calculated from the standard concentration equation.
Fig.2 Standard curve of NH4+
ii) Nitrilase Enzyme Activity
The activity of nitrilase was verified by detecting the ammonium ions produced by the degradation of PAN by the bacterial liquid. We define U as the amount of PAN that can be degraded per microliter per hour.
Fig.3 Experimental phenomena of enzyme activity test
Fig.4 Enzymatic activity of Nitrilase
As can be seen from Fig. 4, the U value of pTac_LaNIT is 0.6115, which is 1.27 times that of the negative control;the U value of pTac_AdNIT is 0.6205, which is 1.3 times that of the negative control.These data indicate that our engineered bacteria have high activity of nitrilase.
(2) Sensor

a) The fluorescence of the sensor induced by small molecules

Fig.5 The fluorescence of the sensor induced by small molecules
Fig.6 The fluorescence of the Nahr- sensor induced by small molecules
Through the corresponding small molecule sensing experiment, except Dmpr sensor, all our sensors have great induction effects on its corresponding aromatic small molecules. Among which NahR-sfGFP was the best! It is 63 times of the negative control.And here we can see that Xyls-sfGFP is 18 times of the Xyls-GFP( BBa_K1031911). And what surprises us is that Paax sensor also has a good response effect to small molecules, which has not been detected before.
b) The fluorescence of the sensor induced by PAN

Fig.19 The fluorescence of the sensor induced by PAN
And here is the fluorescence of different sensors induced by PAN.This data shows the same result that NahR-sfGFP responded best.

2. 4VA

(1) Background

We are informed that Doctor Le Kang's team published a new paper in Nature magazine(1), which established the status of 4VA as the only gathering pheromone of locusts. Our project is using 4VA engineered bacteria to attract locusts, and putting PAN-degrading engineered bacteria to strengthen the locusts’ aggregation. Finally, further biological measures are taken to remove the locusts within the area.
(2) Design

a) Enzyme: the synthesis of 4VA:
In the synthesis of 4VA, we tried to search for known and common small molecules which have the similar chemical structure to 4VA. Through several step by step enzymatic reactions, we formulated a complete metabolic pathway and obtained 4VA eventually. We searched for lots of papers, and finally found three enzymes, which are TAL, PAD and AIMT. Firstly, we will use tyrosine ammonia lyase gene (tal) from Saccharothrix Espanaensis to convert L-Tyrosine to 4-Coumaric acid. clickThen 4-Hydroxystyrene is produced by the catalysis enzyme phenolic acid decarboxylase gene (pad) from Bacillus.
However, we still need a final reaction to replace the hydroxy group with methoxy group. Luckily, AIMT is an enzyme that could play the role. Thus, 4VA is successfully produced from these chemical reactions.
The above is our design, and we hope to test its expression and functional activity in the future. For the expression and fermentation test, we want to culture bacteria, using substrate tyrosine to ferment 4VA. Finally, the fermentation products are tested by GC.
For the functional activity test, we designed a locust attraction experiment. clickWe will prepare the fermentation broth of the engineered bacteria and the negative control group including only expression vector skeleton of bacteria, and the photographic apparatus.
b) The cooperation with CAU:
The equipment we developed with the CAU is basically a trap that is surrounded by net. At the bottom of the equipment, there is a "bait box" that could hold the 4VA and Nitrilase engineered bacteria to attract locusts and dissolve PAN. Once the locust has been attracted to the equipment, the bacteria designed by the team CAU will kill those locusts.
c) Model:
Beside of the equipment we build with the team CAU, we also came up with a model that shows the functioning area of the 4VA and Nitrilase that has been produced by bacteria in order to encounter large scale locust plague. With this "pit" shaped model, we could have a pretty good idea of where we should put our engineered bacteria and thus let it play its role.
Considering the difficulty of applying our engineering strain in real scene, live experiment you know, we turn to establish the model to predict: with engineering strain prepared, how great our attractive pit acts on attracting locusts, its actually not a pit literally just a metaphor you know.
In this part we use Gaussian to simulate diffusion of 4VA. Given the attractive factor to different concentration of 4VA then we obtain the model of our initial attractive pit. In this figure our model shows how effective it is within this region.

III. Conclusion

From the above, it can be seen that the engineering bacteria we constructed to produce nitrile hydrolase can indeed degrade PAN effectively, and the sensor we designed can indeed respond to PAN. The 4VA generated by the 4VA synthesis pathway we constructed can effectively gather locusts after the verification of model. Therefore, the work of our project does provide a good and feasible method to solve the locust plague.
(1) Guo, X., Yu, Q., Chen, D., Wei, J., Yang, P., Yu, J., … Kang, L. (2020). 4-Vinylanisole is an aggregation pheromone in locusts. Nature, 584(7822), 584–588.