Team:Lund/Design

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Proposed Implementation

Project Design

This page discusses the design of the project and the theory behind it.

Background

Phytopththora infestans, or late blight, is the pathogen causing potato late blight. In order to combat it, heavy preventative use of pesticides is required, and potatoes account for high pesticide use per crop area. In order to minimize the impact of the pesticides used, sustainable ones are necessary.The best current pesticide, oxathiapiprolin is excellent as compared to the historic ones, but P. infestans has a plastic genome and is prone to developing resistance, and as such a pipeline of sustainable pesticides is necessary. 

Design Process

A potent, sustainable and efficiently produced antimicrobial agent is required to compete with current pesticides. Antimicrobial peptides either inhibit or kill the susceptible microorganisms, and are part of plants' and animals’ immune defense. We believe that these can be taken from one organism, transferred to another, and then serve to protect the previously vulnerable plant, much like humans requiring antibodies for their immune system to work properly. We decided to try to create a simple SynBio system to achieve this. 

Expression System

Our constructs are designed to be cloned into the pET-11a vector and expressed in E. coli BL21 (DE3), and should have a strong expression after induction with IPTG. The pET-11a vector contains the T7 promoter (code) and terminator (code) as parts. However, other chassis and plasmids can likely also be used with good success.

Designing the composite parts

The composite parts follow the same pattern, having a His-tag, fusion partner, protease cleavage site and lastly the AMP coding region, depicted in figure 1. For enterokinase, the His-tag is instead located at the C-terminal. which can be seen in figure 2.

The purpose of the His-tag is to enable protein purification for later analysis. The fusion partners are either Thioredoxin 1 or Glutathione S-Transferase, which should both increase solubility and expression rates. Thioredoxin 1 could also help in catalyzing cysteine bridges of the AMPs. The Enterokinase-cleavage site is added to enable separation of the fusion partner from the AMP/Ek through proteolytic cleavage with enterokinase. Lastly, the AMP and Enterokinase parts are the main functional units of the constructs.

Figure 1: The components of the AMP insert. Fusion partner is either Trx A or GST, Cleavage site is for enterokinase and AMPs is either AMP.

Figure 2: The components of the EK insert. Fusion partner is either Trx A or GST, Cleavage site is for enterokinase and EK is enterokinase, light chain.

Activating the AMPs

After successfully expressing the constructs, the AMP-fusion proteins will have to be digested by enterokinase to free the AMPs from their fusion partners. Enterokinase is expressed using a fusion partner as well, but will self-digest through autocatalyzed cleavage and become a fully active enterokinase. Enterokinase will be mixed with the individual cell lysates of the AMPs, and enterokinase will then digest the AMP-fusion proteins as seen in figure 3. This will result in free, active AMPs that are ready for application.

Figure 3: presenting how expressed Trx-Enterokinase will first self-digest and then enterokinase will continue digesting the AMP-fusion proteins.

Double or single plasmid system

The first step for expressing the AMP-fusion proteins and Trx-Enterokinase will be to express them in separate cells as shown in figure 4. Once this is successful, activation of the AMPs can be performed as described above. However, we have a more delicate solution that could increase efficiency and allow for a single cell to produce already active AMPs, without having to mix the cell lysates, which is described below.

Figure 4: presenting the single plasmid systems, expressing AMPs and Enterokinase individually.

The second step, after initial expression of the constructs, would be to implement a double plasmid system to express both the AMP-fusion protein and Trx-Enterokinase within the same cell, see figure 5. pET-11a would still be used for the AMPs but to avoid competition of replication machinery, a plasmid with a different Ori would be used for Enterokinase. The details of this system are however not fully complete, due to unforeseen circumstances.

Figure 5: presenting the double plasmid system, expressing AMP and Enterokinase within the same cell on different plasmids.

Testing effectivity of the AMPs

Testing of the AMPs is meant to give an first indication to whether the cell lysates inhibit P. infestans or not, and testing will not yield any quantitative results. Having the cell lysates containing the AMPs, growth inhibition tests will be performed on P. infestans in three different ways. The AMPs will be tested individually to determine which AMPs that will be more likely to make up an effective cocktail. After individual testing, tests can be performed for cocktails containing several AMPs.

The tests described below will be done on three different kinds of potatoes: Minerva, King Edward and Desirée, which we will grow ourselves.

Inhibition of P. infestans growing on nutrient agar in petri dishes.

Isolates of P. infestans will be incubated on nutrient agar in petri dishes with the presence of cell lysates. Results of growth inhibition will be examined by eye-observations and microscopy.

Inhibition of P. infestans growing on potato leaf in petri dishes.

Liquid cultivations of P. infestans will be sprayed and incubated on single isolated potato leaves in petri dishes. Individual cell lysates will be sprayed on leaves prior to the incubation as this would more resemble the real life use of the pesticide. Growth inhibition will be observed by the eye.

Inhibition of P. infestans infecting a whole potato plant.

If growth inhibition tests 1 and 2 show positive results, the AMP will also be tested on infection of a whole plant. The cell lysate will be sprayed on the plant prior to spraying liquid cultivation of P. infestans on the plant and incubating it. Growth inhibition will be observed by the eye.

Explaining our choice of antimicrobial peptides

After conducting a literature study to find peptides known to disrupt P. infestans growth, we rated all the peptides based on different criteria. These were for example toxicity towards the pathogen, towards the chassi producing the peptides, towards the potato plant and what class of AMP that the peptide belonged to, as this likely determines the inhibitory mechanism of the peptide. The peptides we selected were suitable according to all of our criteria.

To see how it all played out, head over to the Results-page!

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