Team:NAU-CHINA/Demonstrate
Demonstrate
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OVERVIEW
Based on our design, anti-Plasmodium peptides should be secreted into the midgut of mosquitoes at a suitable concentration, giving the best killing effect to the Plasmodium. Our verification is mainly divided into three parts.:
1. The efficiency of the HasA secretion system.
2. The efficiency of expression and cleavage of TEVp and anti-Plasmodium fusion protein.
3. Effect of different promotor strength ratio of TEVp and anti-Plasmodium fusion protein on cleavage efficiency.
VERIFICATION OF SECRETING EFFICIENCY
Our engineered bacteria should secrete anti-Plasmodium peptides into extracellular environment and let them play roles in the midgut of mosquitoes, so it is necessary to verify the secretion efficiency. The ideal strain involved in our project is Serratia marcescence since this strain can colonize in the mosquito very well[1]. However, since we did not know the genetic manipulation conditions of S. marcescens at the beginning, we chose to verify whether HlyA secretion system can work well in Escherichia coli. After several experiments, we found that HlyA secretion system cannot work as we desired. Fortunately, we found out the genetic manipulation methods of S. marcescens, and finally developed a way to make competent cells and transform plasmids by electroporation. In September, we started to verify the secreting efficiency of HasA secretion system in S. marcescence. In consideration that fluorescence intensity is easy to measure and intuitive, we verified the secreting efficiency through the following method: firstly constructed a plasmid containing rfp and hasA, then transformed it into S. marcescence. We also set a negative control: constructed a plasmid containing only rfp then transformed it into S. marcescence. We sampled positive strains continuously for 72 hours on a four-hour gradient, then measured the red fluorescence intensity of the supernatant to reflect the secretion effect.
The above experiment shows that HasA secretion system can work efficiently in S. marcescence and meet our requirements for secreting anti-Plasmodium peptides.
VERIFICATION OF THE EFFICIENCY OF THE EXPRESSION AND CLEAVAGE OF FINAL PLASMID
Before we know the conditions of the electroporation of S. marcescence, we carried out experiments in E. coli. During this period, we found that HlyA secretion system in parts bank cannot secret related protein effectively in DH5α, so we decided to verify the efficiency of expression and cleavage of TEVp and anti-Plasmodium fusion protein intracellularly. We constructed a plasmid which can express anti-Plasmodium fusion protein and TEVp simultaneously to verify the intracellular effect, besides, anti-Plasmodium fusion protein and TEVp are both with His-tag for purification and identification. Then we transformed this plasmid into BL21. After culturing for a period, we conducted ultrasonic disruption and purified target protein. It was predicted that TEVp’s cleavage would not be so complete that TEVp's bands and at most nine different sizes of protein bands would appear. We verified the condition of expression and cleavage through western blotting. Pre-experiment data shows the existence of cleavage, so we carried out several formal experiments. During the period of formal experiments, we got an unexpected discovery. We purified the protein from fresh bacteria inoculum and which placed at 4℃ for 3 days, and found out a difference between their Western Blotting results.
The above experiments showed that the plasmid we constructed can effectively express TEVp and anti-Plasmodium fusion protein and perform cleavage, besides, TEVp can perform good cleavage efficiency even at 4°C.
COMPARISON AMONG THREE PROMOTOR COMBINATIONSt
We modeled the expression and cleavage of TEVp and anti-Plasmodium fusion proteins. Our model suggests that the ratio of expressed TEVp and anti-Plasmodium fusion protein will affect the efficiency of cleavage, thus affecting the concentration of single effectors and anti-Plasmodium effect. There exists an optimal ratio which maximizes the concentration of single effectors.
The expression ratio of TEVp and anti-Plasmodium fusion protein is regulated by RBS and the promoter together. Since it is difficult to change the RBS in our plasmid, we regulated the expression of TEVp and anti-Plasmodium fusion protein only by changing the promoters.
In order to verify the deduction of our model, we selected three promoter combinations J23100&J23111, J23100&J23106, J23100&J23110 with strength ratio are 1.7, 2.3 and 3.5 respectively to carry out the experiment. We constructed three plasmids corresponding to the three combinations, then transformed these three plasmids into S. marcescence. After culturing for 60 h, we collected the supernatants and then detected it by Western Blotting. The result shows that the efficiency of expression and cleavage performs best with the promotor combination J23100&J23106 (strength ratio = 2.3). It confirms the deduction of our model. When the promoter combination is J23100&J23110 with ratio 3.5 the performance of the efficiency of expression and cleavage is the poorest, which means the optimal strength ratio is between 1.7 and 3.5.
The experimental result above shows that the efficiency of expression and cleavage of TEVp and anti-Plasmodium peptides is consistent with the deduction of the model. The reasonable prediction of the optimal strength ratio of the promoters from our model is very important for us to select the promoter combination for the circuit design to improve the antimalarial effect of the engineered bacteria.
Under our RBS, our model suggests that the optimal promoter strength ratio roptimal (roptimal = fusion promoter strength / TEVp promoter strength) is greater than 1.7.
In order to verify the deduction of our model, we selected three promoter combinations J23100&J23111, J23100&J23106, J23100&J23110 with strength ratio are 1.7, 2.3 and 3.5 respectively to carry out the experiment. We constructed three plasmids corresponding to the three combinations, then transformed these three plasmids into S. marcescence. After culturing positive strains continuously for 60 hours, and then we tried to detect it by Western Blotting. The result shows that the efficiency of expression and cleavage performs best with the promotor combination J23100&J23106 (strength ratio = 2.3). It confirms the deduction of our model. When the promoter combination is J23100&J23110 with ratio 3.5 the performance of the efficiency of expression and cleavage is the poorest, which means the optimal strength ratio is between 1.7 and 3.5.
The experimental result above shows that the efficiency of expression and cleavage of TEVp and anti-Plasmodium peptides is consistent with the deduction of the model. The reasonable prediction of the optimal strength ratio of the promoters from our model is very important for us to select the promoter combination for the circuit design to improve the antimalarial effect of the engineered bacteria.
MASS SPECTROMETRY ANALYSIS OF HIS-ADDLP
In order to further confirm that the cleavage did happen in our MARS, we collected the supernatant and purified with Ni-NTA column. We centrifuged the purified protein with 10 kDa ultrafiltration tube and took the final products into mass spectrometry. The results showed that His-AdDLP is detected, indicating the happening of cleavage.
[1] Sibao Wang,André L. A. Dos-Santos,Wei Huang,Kun Connie Liu,Mohammad Ali Oshaghi,Ge Wei,Peter Agre,Marcelo Jacobs-Lorena. Driving mosquito refractoriness to Plasmodium falciparum with engineered symbiotic bacteria[J]. Science,2017,357(6358).