Team:Lund/Poster

Poster: Lund

Protecto, LUND 2020

Authors

Presented by team iGEM Lund 2020.

Isak Ingerholt, Simon Enbom, Mattias Akke, Emelie Jansson, iGEM student members, department of Pure and Applied Biochemistry, Lund University, Lund, Sweden.

Abstract

Our project aims to create a novel biopesticide to fight Phytophthora infestans, one of the most hazardous plant pathogens to have ever existed. The pathogen is spread world wide, causing the “late blight disease” in both potatoes and tomatoes. It can cause massive crop loss and the heavy use of pesticides needed to prevent infection results in negative environmental effects in addition to economic costs. Our project, “PROTECTO”, is an effort to create an original biopesticide based on antimicrobial peptides to fight this problematic pathogen. We have added several interesting antimicrobial peptide sequences, and composite parts to express these, to the part registry. During the project we had contact with researchers for guidance and discussed GMO ethics through the project “SynthEthics”. Based on a previous iGEM team’s work, we also created a machine learning algorithm capable of generating new antimicrobial peptides.

Introduction and Achievements

Introduction

The aim of our project is to develop a new type of biopesticide that can be used to treat the hazardous potato pathogen Phytophthora infestans. This will be achieved by recombinantly producing antimicrobial peptides with inhibitory properties, which will be the base of the pesticide. Along with this, we have continuous contact with researchers, we discuss GMO ethics within the “SynthEthics” project and we work on implementation of a machine learning algorithm capable of generating new antimicrobial peptide sequences.

Project Achievements

1. Contributed to the iGEM part registry by adding 8 basic parts of antimicrobial peptides with proven toxicity towards various microbes, including 16 composite parts for expression.

2. Expression of AMP-fusion proteins, the base of the biopesticide prototype.

3. Developed a machine learning algorithm capable of generating novel antimicrobial peptides.

Inspiration

In Sweden we have a university in agriculture called SLU (Sweden's university of agriculture). After contacting researchers from SLU, they referred us to lists of their scientific articles which they thought could be of interest for us. That is where we first got to know the potato pathogen Phytophthora infestans, which caught our interest. We realized that it causes major problems in agriculture in terms of crop loss, environmental issues and economical losses.

P. infestans is a worldwide problem, and it is also a local problem in Sweden. We found it to be an issue easy to understand for the public, and the possibility for guidance from the researchers at SLU inspired us to work on this project. We decided to dig deeper to see if we could come up with any ideas on how to solve the problems caused by the pathogen.

We found that one previous team had worked with the pathogen, the 2015 Taiwanese team from NYMU university. We decided to continue and expand upon the work they had started with recombinantly producing one antimicrobial peptide to create a novel biopesticide. Our idea is to produce a cocktail of antimicrobial peptides, and treat potato plants with them.

Problem

Phytohphora infestans, more commonly known as late blight, is an oomycete that causes rot in infected tubers and leaves of potatoes. Instances of the disease were first recorded in the 1840s [1], and have since led to millions of deaths through starvation, forced migrations and caused huge economical damage. It was responsible for the great Irish famine during the late 1840s, causing Ireland's population to fall over 20% due to starvation and emigration [2].The pathogen was even researched to be used as a biological weapon by Canada, France, the United states and the Sovjet union during the 1940s-1950s [3].

As of now, we have learnt to deal with the pathogen with some success, but there are currently no treatments for already ongoing infection, causing a need for heavy use of pesticides in preventative purposes. It is estimated that 6.7 billion dollars are lost annually due to crop loss and treatment, on a global scale [4]. In Sweden, potato farming accounts for 2% of the total crop area yet 21% of the pesticide usage [5]. Taken together, there is a clear need for sustainable, effective and economically viable new pesticides.

Idea

Our idea to fight P. infestans is to create a novel biopesticide which is easily degraded for environmental purposes, highly toxic towards the pathogen, hard to develop resistance towards and at the same time economically viable. Searching the scientific literature we found eight different antimicrobial peptides with varying origins and modes of action, which all have been proven to inhibit or kill P. infestans. Previously these peptides were retrieved by extraction from plants and animals, a highly inefficient process. We instead suggest expressing these peptides using Escherichia coli BL21(DE3) as an expression system.

After successful expression, the produced peptides will be mixed to form a cocktail pesticide which can be sprayed directly onto the plants. Using a mix of peptides will prevent P. infestans from becoming resistant to the pesticide as well as increase the effectiveness. Upon ending up in the soil, the peptides will be easily degraded by other forms of life, leaving no lasting environmental effects. Through a specially engineered expression system described in Project Design, we also believe that we can make the pesticide economically viable.

Project Design

Composite parts were designed to express antimicrobial peptides fused to one of the fusion partners Thioredoxin (Trx) or Glutathione S-transferase (GST). Both fusion partners have been shown to help in protein expression and folding [6]. A protease (Enterokinase) cleavage site is added between the fusion partner and the AMP sequence, to enable cleavage to a free AMP, see figure 1. 16 composite parts were designed to express 8 different AMPs with both fusion partners. Another composite part was created to express enterokinase in a similar fashion, based on previous work [7].

Figure 1. A flowchart for the process from first industrial step to the final result. The parts which our project deals with are marked by a rectangle. Other processes may apply.

Composite parts are inserted into the pET-11a vector and expressed in the expression system Escherichia coli BL21(DE3). After successful expression, the fusion proteins will be digested by enterokinase to free the AMPs. This will firstly be achieved by using single-plasmid systems, expressing each AMP and the enterokinase separately. Free active AMPs will go through a series of bioassays, testing their inhibitory effects on P. infestans. This is done by adding isolated P. infestans together with AMPs to nutrient medium, isolated potato leaves and whole potato plants, where growth of the pathogen will be observed.

Figure 1. A flowchart for the process from first industrial step to the final result. The parts which our project deals with are marked by a rectangle. Other processes may apply.

Upon successful completion of the bioassays, a double-plasmid system can be implemented by having both the AMP-plasmid and the Enterokinase-plasmid in the same cell, see figure x. This will require one less step in the production process as the AMP-fusion partners will be digested within the expression system. Our hope is that this will aid in economical aspects of producing the final product.

Team

Simon Enbom: Team leader, project design et c., Biotechnology Master student.

Isak Ingerholt: Project design and wet lab, Biotechnology Bachelor student.

Ruban Cornelius: Wet lab and safety, Biotechnology Master student.

Robert Skoglund: Finance and graphical design. Mathematics Bachelor student.

Amr Ghazal: Team leader and human practices, Mathematics Bachelor student.

Emelie Jansson: Project design and wet lab, Biotechnology Bachelor student.

Chinnu Ann Jaison: Wet lab and safety, Biotechnology Master student.

Mattias Akke: Modelling, Nanotechnology Bachelor student.

Modelling

There are not many anti-oomycetial peptides that have been exhaustively researched. We therefore developed a classification model called STAAC that can evaluate the antimicrobial properties of peptides. The objective when designing STAAC was to generate new, previously unseen peptides that would suit our project of producing anti-oomycete peptides in bacteria. This means that the peptides should have strong antifungal and therefore also anti-oomycete properties, while at the same time having weak antibacterial properties. STAAC is trained to maximize the harmonic mean (HM) of the precision of anti-fungal peptides (AFP) classification and the recall of non-antifungal, antimicrobial peptides (NoAFP-AMP). Overall, STAAC can classify unseen peptides with an accuracy of 88 %, AFPs with a precision of 88 %, and NoAFP-AMPs with a recall of 65 %. We would like to stress that there are models out there with much better accuracy and precision if one would like to answer the binary question: “Is this peptide antifungal?”. Indeed, when we converted our model to answer this binary question, we received a precision of 93.5 % and an overall accuracy of 94 %.

By combining our classification model STAAC with a genetic algorithm, we were able to generate 10 new peptides with predicted high anti-fungal and low anti-bacterial properties. Unfortunately, due to limited time and resources, we were unable to test the new peptides experimentally.

Human Practices

Björn Andersson and Maja Brus-Szkalej are researchers at SLU who both have a lot of, but different, experience working with P. infestans. Maja comes from a background in molecular biology while Björn is a field pathologist. They both helped us early on in the project by letting us discuss our project with them and they gave us a lot of insight into the pathology and workings of P. infestans, which positively influenced our project. Maja had continuous contact with us throughout the whole project, and even suggested that we could come to SLU to perform our bioassays, but unfortunately we ended up not having enough time.

We also reached out to local potato farmers, the owners of Gränsbo Potatoes, whom despite the pandemic let us come visit to see their farmlands. They gave us their perspective on having to adapt to P. infestans and told us that as long as they sprayed the potatoes with pesticides each week they were not worried, however it did cost them a lot of money. We learned how to spot late blight with the eye and it was a nice experience and insightful to discuss our project with the first hand users.

Last year, Erik Hartman from the iGEM Lund 2019 team founded SynthEthics, a project meant to discuss GMO ethics and appropriate laws revolving GMOs. We had several meetings with Erik to discuss different ethical dilemmas and what we had to be thinking about regarding our project. We also participated in an online lecture hosted by SynthEthics for high school students.

Results

We have added eight antimicrobial peptide sequences which have all shown toxic properties towards P. infestans. Additionally, two composite parts per AMP were added to the registry for expression.

We successfully transformed E. coli BL21(DE3) with seven of our constructs expressing the AMPs, as well as one additional control construct, see constructs in table 1. The transformation was validated by Sanger sequencing of the coding region for the positive transformants, showing a 100% identity with our designed constructs.

Protein expression was successful for seven of the constructs presented in table 1, where the sizes of the expressed proteins are also given to aid in analyzing the SDS-page seen in figure 3.

Figure 1. SDS-page of expressed proteins, red arrows point towards respective constructs.

Future work

The next step is to successfully express Enterokinase and show that we can cleave the protease cleavage site of the expressed AMP-fusion proteins. This will yield free, active AMPs and bioassays will be started to test their effectivity on inhibiting P. infestans. The most potent AMPs will be mixed to form a cocktail of peptides, which will be the first prototype of our product. Different mixes of AMPs will be tested to find the best combinations. Upon successful bioassays, the two-plasmid system will be implemented. We’re also looking to perform bioassays with our STAAC-generated AMPs to validate their antimicrobial properties.

References and Acknowledgements

Acknowledgements

We greatly want to thank the staff of the department for Pure and Applied Biochemistry at LTH. They helped us with tips and taught us about various lab instruments, thanks to Simon Christensen, Liselotte Andersson, Karin Kettisen, Nelida Leiva Eriksson and Alfredo Zambrano Rodriguez.

We also want to thank the previous iGEM students Maeusz Piotr Szewczyk, Savvina Leontakianakou and Erik Hartman for their guidance and contributions.

Lasty, we want to thank Johan Svensson Bonde, our fantastic supervisor who made all of this possible. With his patient, calm and methodical approach we learnt so much from him.

References

Reader, John (17 March 2008), "The Fungus That Conquered Europe", New York Times

Kinealy, Christine (1994), This Great Calamity, Gill & Macmillan, ISBN 0-7171-1881-9

"Chemical and Biological Weapons: Possession and Programs Past and Present", James Martin Center for Nonproliferation Studies, Middlebury College, April 9, 2002

A National Project on Tomato & Potato Late Blight. (n.d.). Retrieved November 5, 2020, from http://usablight.org/

M. Brus-Szkalej (2019), The biology and ecology of Phytophthora infestans (Doctoral dissertation, Institutionen för växtskyddsbiologi, Sveriges LantbruksUniversitet, Alnarp, Sweden), Retrieved from SLU Epsilon (Accession ID 16444)

Ting Deng, 2017. The heterologous expression strategies of antimicrobial peptides in microbial systems. Protein expression and purification 140:52-59. https://doi.org/10.1016/j.pep.2017.08.003

Liu-Di Yuan, Zi-Chun Hua, 2002. Expression, purification, and characterization of a biologically active bovine enterokinase catalytic subunit in Escherichia coli. Protein expression and purification 25: 300-304. https://doi.org/10.1016/S1046-5928