Suicide Switch Test
Negative Control
Induced modified Bax
Team:NAU-CHINA/Demonstrate
Demonstration
Theoretical basis
Peptides are electrostatically attracted and accumulate at numerous sites covering the surface of the membrane, which will eventually lead to the formation of transmembrane pores. The cell died of the content outflow.[1]
Project scheme
Through the yeast factory, we can achieve mass production of a good deal of antifungal peptides, these eukaryotic expression of antimicrobial peptides, can be used for biological control of wheat scab (you can see more information by clicking HERE!) . At the same time, we pay close attention and strict supervision to the downstream wheat processing products. We can utilize the hardware(you can see more information by clicking HERE!) of our project to carry out wheat processing products in the residual DON and ZEN detection, if the content is beyond the criteria of the national standard, we can then utilize the hardware to carry out the degradation of toxins, so that the food DON and ZEN content can be greatly reduced.
Response Module verification
Through the graph of zone of inhibition experiment we can see that compared with negative control treated with sterile water, different concentrations of Cecropin A, Plant Defensin Corn 1 and MtDef4 can show the inhibition and killing of Fusarium graminearum spores, in higher concentrations of the Cecropin A and MtDef4, the antimicrobial peptides can show a similar or even more bactericidal activity compared with positive control—Carbendazim, which is dissolved in acetic acid at a final concentration of 100ppm.
Fig.1 The inhibition zone results show the three antifungal peptide can exhibit a great antifungal activity, and the area of the inhibition zone is dose dependent. We choose to utilize sterile water as our negative control and 100ppm carbendazim dissolved in acetic acid as positive control. All the three antifungal peptides are dissolved in sterile water. We test the cell viability under the action of a series of different concentrations of antifungal peptide. Among them we can conclude the MtDef4 possess the best antifungal activity while PDC1 possess a inferior antifungal activity. Significant differences versus vehicle were assessed by student’s t test: *=p<0.05, **=p<0.01, ***=p<0.001, ****=<0.0001.
Conclusion
It is undeniable that, the bactericidal activity of Carbendazim largely depends on the cytotoxicity of acetic acid. Besides, due to the use of carbendazim, Fusarium graminearum is prone to produce drug resistance. After Human practice investigation, we acquire the information that every year the government and hundreds of researchers will be force to rennovate new chemicals to prevent wheat scab. you can check out HERE! .
By contrast, antifungal peptides are prone to have less side effects[1]:
1. The half-life of the polypeptide is shorter, and its decomposition product is nitrogen-containing organic matter, which can be reused by the crop.
2. The above two families of peptides can specifically form pores in the surface of microbial cell membrane, so that the outflow of cell plasma contents cause cell death, this specific sterilization mechanism makes the microbial tough to mutate resistance, and they can also coordinate with other antimicrobial peptides, enzymes or even traditional antibiotics, because the antimicrobial peptide can increase the permeability of the cell membrane, it can play a synergistic effect without cross-resistance
3. They function on multiple targets within the cell. If the pathogen mutate any target within, it eventually will not have a significant impact on antimicrobial peptides sterilization function.
4. Antibacterial peptides inhibitory concentration and sterilization concentration is very close, which makes them ideal fast fungicide.
Sensing Module verification
Fig. 2 A schematic representation of our construction of sensing module.
Fig. 3 (A) Quantification of ZEN by Saccharomyces cerevisiae-based sensor. Fluorescence intensity spectroscopy for Saccharomyces cerevisiae treated with different doses of ZEN: 1, 2, 5, 7, 10, 15, 30, 40, 50 and 100 ng/mL. The curve of fluorescence intensity versus various concentrations of ZEN. (B) Gene switch activity monitored via GFP expression and measured using flow cytometry. The GFP channel histograms are shown.
Ranging from 15 to 100 ng/mL, fluorescence intensities exhibited good linear relationships with ZEN concentrations, with correlation coefficients of 0.994. The limit of detection (LOD) calculated from Fig. 4B were 61.72 ng/mL, according to the formula: LOD = 3 s/m, where s represents the blank sample standard deviation (n = 2) and m represents the slope of the related ZEN calibration curve.
Degradation Module verification
Fig. 4 The degradation and acetylation efficiency of ZHD101 and AYT1 for ZEN and DON respectively.
We successfully test the effectiveness of the genetic switch which is controlled by blue light to regulate the expression of ZHD101 and AYT1, you can check out HERE!. The ability of acetylation of DON can reach as high as 66.23% at 14h. And the ability of degradation of ZEN can reach 100% at 14h.
Improvement on previous part hbax:
Fig. 5 10μl galactose aliquot was treated with SD-URA-glucose(2%) plate periodically. The yeasts colonies were observed in a series of time gradients. It is evident that the amount of alive yeasts with pYES2-Bax was evidently decreased after the induction by galactose, and almost disappeared after 24 hours, while the negative control colony still stayed alive.
To test the effects of Bax, we insert the coding sequences of Bax into vectors pYES2 (URA as marker), subsequent to Gal1 promoter, so that Bax will be induced by galactose. We transformed the recombined plasmids pYES2-Bax into yeasts at the same time the pYES2 plasmid as a negative control.The yeasts containing pYES2-Bax or pYES2 were cultured to log phase in liquid URA-deficient SD media with 2% glucose as carbon sources, and were then dropped into liquid SD-URA-glucose(2%) media respectively and cultivated for one day. 10μl galactose aliquot was dropped on SD-URA-glucose(2%) plate periodically. The results are showed in Figure.1: As time passed, the amount of alive yeasts with pYES2-Bax was evidently decreased after the induction by galactose, and almost disappeared after 24 hours. In contrast, The yeasts with pYES2 as a negative control hardly reduced.
