All previous theoretical research must be backed up by evidence proving the viability of the proposed project. On this page, we will show you how we designed the proposed methodologies to show that Agrocapsi will work in the real world by demonstrating feasibility at an early stage.
Agrocapsi was created from a desire to provide a preventative treatment option to the innumerable farmers who are afflicted by wilt all around the world. Our proposal includes antimicrobial peptides as well as siRNA technology, so every step of the process is important.
Peptide production is one of our project's core phases and the first in the timeline. This stage has been worked on for months, but it is not the only one that bears the weight of the proof of concept of our product's performance. Other methodologies that contribute to the characterization and validation of the effectiveness of each active ingredient in the final formulation are also important. The processes discussed below are planned to be carried out to achieve the production of an effective solution.
Once the proteins of interest are produced, they must be purified using Immobilized Metal Affinity Chromatography (IMAC). Our constructs were created specifically for this purpose by inserting a 6X-His tag fragment into the sequence.
IMAC is a useful method for purifying synthetic peptides with an N-terminal metal-binding amino acid such as histidine, tryptophan, or cysteine, specially when such residues are not present in other parts of the molecule (Linderberg, 2009).
This can be the method of choice for insoluble proteins because it does not rely on biological function. It is also one of the few affinity chromatography technologies available that can function in denaturing conditions (Charlton & Zachariou, 2008). This chromatography is very helpful for our project because our two antimicrobial molecules are secreted into the periplasmic space and are not initially soluble. Additionally, a lot of research has demonstrated the design expression systems in E. coli with His-tag fusion proteins to be purified by this system (Hwang, et al., 2001; Meiyalaghan et. al., 2014). Knowing that this method is widely used and has proven good results, we design our sequences with its respective polyhistidine tag.
The metal ion is immobilized by a chelating agent attached to a stationary support, with the metal ion captured by said immobilized chelator forming an immobilized metal chelate complex (IMCC). Despite a wide range of alternatives, the most commonly used chelators for such applications are iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA) (Charlton & Zachariou, 2008). Taking into account the advantages of this technique as well as our needs due to the characteristics of our molecules, we decided to design the purification protocol shown below.
Balance the column at the temperature to be worked. This can be at room temperature or at 4 °C.
Prepare the sample by combining the protein extract with the equilibration buffer. The total volume must be equal to the volume of two resin beds.
Remove the tab from the bottom of the HisPur Ni-NTA Spin Column by gently rotating it. Place the column in a centrifuge tube. Note: Use 1.5, 15 or 50 mL centrifuge tubes for the 0.2, 1 and 3 mL columns respectively.
Centrifuge column at 700 ×g for two minutes to remove the buffer.
Equilibrate column with equilibration buffer with volume of two resin beds. Allow the buffer to enter the resin bed.
Centrifuge column at 700 ×g for two minutes to remove buffer.
Add the protein extract to the column and allow it to enter the resin bed. Note: For better binding, the sample can be incubated for 30 min at room temperature or at 4°C over a shaker.
Centrifuge column at 700 ×g for two minutes and collect the continuous flow into a centrifuge tube.
Wash the resin with wash buffer with a volume equivalent to two resin beds. Centrifuge at 700 ×g for two minutes and collect the fraction in a centrifuge tube. Repeat two more times and collect the fraction in separate tubes. Note: Additional washes can be done if desired. Monitor washes by measuring absorbance at 280 nm.
Elute His-tagged proteins from the resin by adding elution buffer with a volume of one resin bed. Centrifuge at 700 × g for two minutes and collect the fraction in a centrifuge tube. Repeat two more times and collect the fraction in separate tubes. Note: If gravity-flow is done add elution buffer with a volume of two resin beds.
Evaluate the elution of the protein by monitoring the absorbance of the different fractions at 280 nm or by Coomassie Plus assay (Bradford), Reagent (Product No. 23238). The eluted protein can be analyzed by SDS-PAGE.
The analysis of the antimicrobial activity of each of the proteins obtained is the second critical stage of the proof of concept. This test was designed to assess the effectiveness of our product against P. capsici; however, in response to stakeholder feedback, we expanded the inhibitory spectrum of our product to include other phytopathogenic fungi such as Fusarium oxysporum and Fusarium solani. The protocols below are adaptable for any of the strains against which containment measurements will be taken.
The inhibition assay will be carried out in two ways, as shown below.
One option for the analysis of the antimicrobial activity is by preparing a spore inhibition essay with resazurin. This is a non-toxic redox dye that has been used with fungi and bacteria. The oxidized, nonfluorescent form of this reagent has a blue color; however, when it is reduced it forms a pink color in response to cell metabolism. The results can be measured by fluorescence through a spectrophotometrically or a fluorometrically equipment (Vega, et al., 2012).
Because resazurin assays have been widely used to analyze the cell viability of mammalian, bacterial, and fungal cells, this would be a good option for testing the efficacy of our peptides (Chadha & Kale, 2015). This is an assay that is primarily used to determine the minimum inhibitory concentration (MIC) (Monteiro, et al., 2012), so it is of great interest for our project because it will be one of the first stages of evaluation, allowing us to know the minimum amount of the molecules of interest that our product should contain in order to have a cidal effect on the pathogen. With that said, we designed the following protocol:
Mount 8 wells per extraction in the 96-well flat-bottom plate. This assay aims to find the ideal amount of protein extract for a significant inhibition. Therefore, dilutions will be performed. Each row corresponds to a serially diluted extraction, adding from 1:1, 1:2, 1:4 and 1:8 µL of extract.
Figure 1. Protein extract dilutions
Add 90 µL of PDA medium, 50 µL of fungal spores in Tween 20, 40 µL of PBS, and 20 µL of resazurin to wells that have dilutions of the protein solution to complete the mixture for a final volume of 200 µL.
It's also advised to use untransformed bacteria as a negative control. It is important to respect the fungal suspension's concentrations and dilutions. Specifically, the negative control in this experiment would consist of a mixture of 50 µL fungal suspension, 90 µL PDA medium, 40 µL PBS, and 20 µL resazurin. A mixture of 50 µL mefenoxam, 90 µL PDA medium, 40 µL PBS, and 20 µL resazurin will be used as the positive control.
A negative contamination control should be taken into account, which means that only PDA medium, resazurin, and PBS should be seeded. The negative control in this experiment would specifically be a mixture of 90 µL PDA medium, 90 µL PBS, and 20 µL resazurin.
Also an extract control is taken into account, in which no bacterial solution is added. This control would specifically be a mixture of 90 µL of PDA medium, 50 µL of the appropriate 1:1 protein extract, 40 µL of PBS, and 20 µL of resazurin diluted in 50 µL of sterile water for this experiment.
The detection range of the resazurin should be taken into consideration while performing absorbance measurements.
Finally, data are quantified by detecting fluorescence 24 hours after mounting (excitation: 570 nm, emission: 615 nm).
Figure 2. Setup of the spore inhibition experiment
We also planted the elaboration of an IC50 essay. The half-maximal inhibitory concentration, or IC50, is one of the most commonly used indices to determine a drug's efficacy. This criterion denotes the concentration at which a biological process is half inhibited (Aykul & Martinez, 2016).
This technique has been used as a parameter in several experiments to determine the inhibitory potency against Phytophthora capsici (Wang, et al., 2021; Lee, et al., 2019; Wang, et al., 2018; Chen, et al., 2019; Ali, et al., 2015).
As a result, as part of our proof-of-concept tests, we decided to develop the following protocol.
Here we would dissolve the purified extract of the peptide in a culture medium before it solidifies. Once the medium is done, an inoculum of a previous cultive of P. capsici will be put in the middle of the agar, and measurements of the diameter would be made for 10 days.
Next an ANOVA with α=0.05 would be done so the IC50 could be determined
Calculate the log10 of the concentrations
Transform the % of inhibition with Finney´s table
Table 1. Finney´s table for transformation of percentage of mortality to probit values
Graphic the profits vs. the log10 of the concentration and calculate the regression equation
Calculate the probit 50% (solve x with y= 5) and determine LC50 (10x).
We would expect:
To have the proteins purified so that we could further characterize and study each of them. Furthermore, it would be extremely useful for scalability because it would provide data on efficacy as well as costs, allowing us to determine whether it is a financially and functionally viable alternative.
Determine the minimum concentration of molecules required to inhibit pathogen growth. This will allow us to formulate our product precisely.
To qualitatively depict the molecules' effect on the oomycete.
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