iGEM 2020 — Team Groningen

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Groningen/Notebook

* [![Rootpatch logo](https://static.igem.org/mediawiki/2020/e/ed/T--Groningen--RootPatch_navbar.png)](https://2020.igem.org/Team%3AGroningen)

* Project

  [Description](https://2020.igem.org/Team%3AGroningen/Description)
  [Implementation](https://2020.igem.org/Team%3AGroningen/Implementation)
  [Engineering](https://2020.igem.org/Team%3AGroningen/Engineering)
  [Parts](https://2020.igem.org/Team%3AGroningen/Parts)
  [Model](https://2020.igem.org/Team%3AGroningen/Model)
  [Notebook](https://2020.igem.org/Team%3AGroningen/Notebook)
* People

  [Team](https://2020.igem.org/Team%3AGroningen/Team)
  [Attributions](https://2020.igem.org/Team%3AGroningen/Attributions)
  [Collaborations](https://2020.igem.org/Team%3AGroningen/Collaborations)
  [Contributions](https://2020.igem.org/Team%3AGroningen/Contribution)
* Human Practices

  [Integrated Human Practices](https://2020.igem.org/Team%3AGroningen/Human_Practices)
  [Education](https://2020.igem.org/Team%3AGroningen/Education)
  [Outreach](https://2020.igem.org/Team%3AGroningen/Education#Outreach)
  [Benelux Meetup](https://2020.igem.org/Team%3AGroningen/ViBM)
  [In the Media](https://2020.igem.org/Team%3AGroningen/Education#InTheMedia)
  [Animations](https://2020.igem.org/Team%3AGroningen/Animations)
* [Sponsors](https://2020.igem.org/Team%3AGroningen#Sponsors&Partners)

![](https://static.igem.org/mediawiki/2020/9/9e/T--Groningen--NotebookBanner.png)

[Week 1](#week1)
[Week 2](#week2)
[Week 3](#week3)
[Week 4](#week4)
[Week 5](#week5)
[Week 6](#week6)
[Week 7](#week7)
[Week 8](#week8)
[Week 9](#week9)
[Week 10](#week10)
[Week 11](#week11)
[Week 12](#week12)
[Week 13](#week13)
[Week 14](#week14)
[Week 15](#week15)
[Week 16](#week16)
[Week 17](#week17)
[Week 18](#week18)
[Week 19](#week19)
[Week 20](#week20)
[Week 21](#week21)
[Week 22](#week22)
[Week 23](#week23)
[Week 24](#week24)
[Week 25](#week25)
[Week 26](#week26)
[Week 27](#week27)
[Week 28](#week28)
[Week 29](#week29)
[Week 30](#week30)
[Week 31](#week31)
[Week 32](#week32)
[Week 33](#week33)
[Week 34](#week34)
[Week 35](#week35)
[Week 36](#week36)
[Week 37](#week37)
[Week 38](#week38)

**Week 1:**

The first team meeting took place this week. All members of the team suggested ideas for our iGEM project.

**Week 2:**

We finalized on two ideas: Saving coffee beans from a pathogen or combating parasitic nematodes. Both of these ideas were further investigated and were pitched to the supervisors.

**Week 3:**

Amongst the two ideas - Combating cyst nematodes was picked for our iGEM 2020 project. The topic was divided amongst all the team members, who looked into the different research aspects of this topic.

**Week 4:**

A detailed plan, was presented to the supervisors. We received feedback on how to proceed further and information on what facilities are available for our proposed experiments.

**Week 5:**

In the beginning of March, we heard the news about the COVID-19 virus and the possibility of a lockdown. To continue our weekly meeting through the pandemic, the idea of weekly Zoom meetings was proposed. Along with that, we continued to work and further refine our project ideas.

**Week 6:**

The team was divided into groups to work on specific parts of the project. A sponsor team was also setup. They looked up possible companies for sponsoring our team. By this time, Coronavirus is started to be taken seriously by governments around the world.

**Week 7:**

A lockdown was announced in the Netherlands. To adapt to this situation, our project was significantly altered to account for the fact that we could not enter the lab this year. The team brainstormed about which experiments we could conduct *in silico*.

**Week 8:**

A COVID-19-proof project idea was pitched. Different modeling ideas were considered and team members were assigned to investigate each idea. For the outreach, we decided on making a Massive Open Online Course (MOOC).

**Week 9:**

The dry lab team refined the population model. Three smaller model ideas were also proposed, among which was the biofilm feasibility analysis that would later become a big part of the dry lab.

**Week 10:**

In this week, we ran into some problems. The idea to put DAPG in RootPatch is cast into doubt due to its possibility to harm the biofilm formation. Also, since the potato planting season commenced, we started looking into a field trip to a potato farm to learn more about the problem.

**Week 11:**

The team decided to discard the DAPG idea after consultation with the HLB lab. We brainstormed ideas for overcoming obstacles to making a functional kill switch.

**Week 12:**

The computational models were refined based on what questions we want them to answer and how they fit into the whole project. The kill switch was improved to also trigger when potatoes are harvested.

**Week 13:**

A new team member - Ronja joined the dry lab team to help with modeling. The outreach team planned to the MOOC with the help of Science LinX.

**Week 14:**

The wet lab team changed organism to *B. mycoides* in response to feedback from Oscar Kuipers. Meanwhile, we started planning for hosting the Benelux Meetup.

**Week 15:**

The Benelux Meetup plan was further refined. We also set up strategies for the social media page for our project. The outreach team partnered up with NEMO for the MOOC.

**Week 16:**

The dry lab team shifted the Biofilm model from a micro-level approach to a macro-level approach based on feedback from Jordi van Gestel. *B. mycoides* was definitively chosen as our organism by the wet lab team based on the feedback from Jan Spoelder.

**Week 17:**

Every team worked on their part of the project. No major changes to the project were made in this week.

**Week 18:**

The team made a lot of content for the Benelux Meetup. No other major developments were made this week.

**Week 19:**

Our team had an interview with the UKrant about our project. The dry lab team successfully finished the rate equations for the models. Meanwhile, the outreach team fixed on topics for the MOOC.

**Week 20:**

This week, the dry lab team continued with developing the equations for the models. Wet lab and outreach further worked on their parts of the project. Along with this, the team was also planning the BeNelux Meetup.

**Week 21:**

The outreach team worked on circumventing issues with video recording for the MOOC. Transcriptomics data was used to find genes dependent on solanine by the dry lab team.

**Week 22:**

As the lockdown starts being lifted, we made efforts to find a physical working/meeting space. The dry lab team shifted the research questions of the biofilm model from qualitative to quantitative-based on feedback from Sander van Doorn.

**Week 23:**

The outreach team downscaled the MOOC to two 45 minute lessons due to time considerations. The team had talks with iGEM BITS Pilani-Goa about a possible partnership; we gave them advice on modeling.

**Week 24:**

New team members (Arne, Alex and Job) joined as the art team and are responsible for visual design. We designed a 3D printed trophy for the Benelux Meetup.

**Week 25:**

The team started working on the 2-minute video for the jamboree. We also finalized the planning and successfully hosted the Benelux Meetup.

**Week 26:**

This week we visited the HLB lab which has expertise in the *Globodera pallida*. We started working on the storyboard of the 2 minute video. The wet lab team wrote protocols of the experiments and the sensitivity analysis of the biofilm model was started by the dry lab team.

**Week 27:**

Equations for the dry lab are finished, both models started sensitivity analysis. We started discussing the work for the collaboration with NOVA Lisbon. Protocols and the lesson series were still being developed.

**Week 28:**

The team started working on hosting a webinar for the Global Meetup with NOVA Lisbon. The rendering of the 2-minute video shots began in this week as well. Dry lab, wet lab and outreach continued to work on their projects without significant alterations.

**Week 29:**

A webinar was hosted at the Global Meetup and the team spirit poster was made. An experimental layout was made for the Wiki. We started working on the 20-minute presentation for the jamboree.

**Week 30:**

The dry lab team concluded the analysis of the biofilm model. We also established contact with RIVM about the safety implications of our project. The outreach team finalized a plan for the recording of videos for the lesson series.

**Week 31:**

All team members started writing their Wiki text. The dry lab team continued to do the final analysis of the population model in this week.

**Week 32:**

The outreach team finalized scripts for the lesson series. Dry lab results are in; writing conclusions for the graphs. The first version of the storyboard for the 20-minute video was written.

**Week 33:**

The storyboard for the 20-minute video was revised and recording space was set. The outreach team started recording videos for the lesson series. Also, a model for team NOVA was developed.

**Week 34:**

The voiceover and final touches were made to the 2-minute promotional video and submitted to the iGEM PeerTube portal. Track selection was finalised to “Food & Nutrition”. Team roster was completed

**Week 35:**

Jamboree registration was completed. New sub-teams like Review and Video team were made since most parts of the dry lab, wet lab and writing for the Wiki were done. A schedule was set up for the Video team for writing the script, shooting and editing the 20-min presentation video.

**Week 36:**

Project title and abstract was finalised. The Video team was busy writing the field scenes of the presentation video.

**Week 37:**

The field scenes were shot at the Agrico Research facility. The script for the studio scenes was written and finalised. Lesson 1 of the outreach program was uploaded online.

**Week 38:**

The studio scenes were shot at the Academie MInerva studios and are being edited. Lesson 2 of the outreach program was uploaded online.

---

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Groningen/Engineering

* [![Rootpatch logo](https://static.igem.org/mediawiki/2020/e/ed/T--Groningen--RootPatch_navbar.png)](https://2020.igem.org/Team%3AGroningen)

* Project

  [Description](https://2020.igem.org/Team%3AGroningen/Description)
  [Implementation](https://2020.igem.org/Team%3AGroningen/Implementation)
  [Engineering](https://2020.igem.org/Team%3AGroningen/Engineering)
  [Parts](https://2020.igem.org/Team%3AGroningen/Parts)
  [Model](https://2020.igem.org/Team%3AGroningen/Model)
  [Notebook](https://2020.igem.org/Team%3AGroningen/Notebook)
* People

  [Team](https://2020.igem.org/Team%3AGroningen/Team)
  [Attributions](https://2020.igem.org/Team%3AGroningen/Attributions)
  [Collaborations](https://2020.igem.org/Team%3AGroningen/Collaborations)
  [Contributions](https://2020.igem.org/Team%3AGroningen/Contribution)
* Human Practices

  [Integrated Human Practices](https://2020.igem.org/Team%3AGroningen/Human_Practices)
  [Education](https://2020.igem.org/Team%3AGroningen/Education)
  [Outreach](https://2020.igem.org/Team%3AGroningen/Education#Outreach)
  [Benelux Meetup](https://2020.igem.org/Team%3AGroningen/ViBM)
  [In the Media](https://2020.igem.org/Team%3AGroningen/Education#InTheMedia)
  [Animations](https://2020.igem.org/Team%3AGroningen/Animations)
* [Sponsors](https://2020.igem.org/Team%3AGroningen#Sponsors&Partners)

![](https://static.igem.org/mediawiki/2020/9/94/T--Groningen--EngineeringBackground8.png)

---

×

**Choosing our host**

The main idea of RootPatch is to engineer a bacterium that can efficiently express and secrete neuropeptide-like proteins (NLPs) that will repel the parasitic nematodes. Several factors may get in the way of this plan.

First of all, survivability of the host bacterium is key. If the bacterium is unable to survive in the harsh conditions in the soil, even a high NLP production will not be sufficient to act on all the nematodes. In order to give RootPatch a higher chance of success, we need our host bacterium to have the following characteristics:

**▪ The bacterium is a natural soil isolate from the potato rhizosphere.** This will give it a competitive advantage over non-native bacteria to the potato rhizosphere . The soil is a tough environment for bacteria and hence bacterial competition may be the biggest challenge for RootPatch to succeed. Our [dry lab model](https://2020.igem.org/Team%3AGroningen/Model) and several of our [stakeholders](https://2020.igem.org/Team%3AGroningen/Human_Practices) have indicated this as well.

**▪ The bacterium needs to be able to proliferate at both high humidity and low humidity**. According to our [dry lab model](https://2020.igem.org/Team%3AGroningen/Model), the lower the minimum water activity at which the bacteria in RootPatch can grow, the higher the chance of survival throughout the potato season. This minimal water activity is a characteristic that is typical for bacterial species but it has not been measured for our potential host bacterium.

**▪ The bacterium is capable of sporulation.** This will give it a higher survival probability under harsh conditions but will also guarantee a better shelf life of the eventual product. More about this issue can be found in the potato “Choosing the method of application”.

**▪ The bacterium is capable of forming a biofilm layer at the roots.** This will give RootPatch structural strength against severe environmental conditions such as heavy rainfall. However, this property is also questionable and should be further researched, likely bacteria in a biofilm alter their metabolic activity and that could significantly impact neuropeptide production (Pisithkul et al., 2019; Vlamakis et al., 2013).

Secondly, we need a bacterium that is good at producing and secreting peptides at high quantities to create the desired NLP environment around the roots. A bacterium that can be easily genetically modified is therefore of importance. Most wild-type bacteria from the soil are difficult to genetically manipulate.

Many different bacteria could be chosen as a host. In the end, only lab and field tests will tell which bacterium checks all the boxes: amenable to genetic engineering, suitable for high production of the neuropeptides, and high survival rate in the soil after having been genetically modified. We suggest four bacteria that could potentially be used for RootPatch. *Bacillus mycoides* is our first choice since, on paper, it shows the best characteristics. While reading through the other parts of this RootMap, you will notice that some of the parts are designed for *Bacillus mycoides*, as it is our primary target host bacterium.

***Bacillus mycoides* M2E15**

![](https://static.igem.org/mediawiki/2020/thumb/2/23/T--Groningen--EngineeringMycoides1.png/800px-T--Groningen--EngineeringMycoides1.png)

*Bacillus mycoides* M2E15 is an endophytic soil bacterium that has been isolated in high abundance from the potato rhizosphere (Yi et al., 2018). Because it appears to be dominant in the potato rhizosphere, we believe it has a competitive advantage over other bacteria spp. and could, therefore, give RootPatch a higher probability of survival throughout the season. In addition, it can be genetically engineered, is able to sporulate and is capable of forming biofilm-like layers (Yi et al., 2018). The minimal water activity has never been investigated for *Bacillus mycoides* and remains to be tested.

We plan to introduce the gene coding for the NLP in the chromosome of *Bacillus mycoides* using the CRISPR-Cas9 technique. In order to achieve this, we will use the pYCR vector and the one-step CRISPR-based integration approach that has been described by Yi and colleagues in 2018. This technology has proven to be successful in this wildtype *Bacillus* species. The gene will be expressed under the strong constitutive promoter Ppta (Frenzel et al., 2018). Click on [this link](https://static.igem.org/mediawiki/2020/e/e6/T--Groningen--CloningPDF2610.pdf) for more detailed information about this cloning strategy.

---

×

**Choosing the secretion method**

After ensuring the strong expression of the neuropeptide, we need to confirm that it will be secreted into the environment. Proteins destined to be secreted by bacteria are usually equipped with an N-terminal secretion signal. Selecting the proper secretion-signal is a crucial step in optimizing protein secretion yield while other characteristics of the protein may also play a role. Our main strategy is to employ a secretion tag known to drive efficient protein secretion in a variety of Gram-positive hosts. As an alternative, we describe a library of five secretory signals that appear to be promising for NLP secretion.

![](https://static.igem.org/mediawiki/2020/thumb/6/61/T--Groningen--secretion2.png/799px-T--Groningen--secretion2.png)

**Usp45 signal peptide**

Usp45 is the major secreted protein of the lactic acid bacterium *Lactococcus lactis*. It is equipped with a signal peptide that has been extensively used for secreting various heterologous peptides not only in *Lactococcus lactis* but also in other Gram-positive bacteria (Borrero et al., 2011; Marciniak et al., 2012). Because we suggest using alternative hosts for NLP synthesis, we decided to employ this signal sequence as our main strategy. However, although it has been described as a universal signal peptide, we do not have experimental data on its efficiency in *Bacilllus mycoides*. We also have to keep in mind that the NLP14a peptide is significantly smaller than the proteins that have been secreted with the help of the Usp45 secretion signal.

---

**Alternative secretion signals**

In case the Usp45 signal peptide is not efficient for secreting neuropeptides, we suggest to use several alternative signal peptides that might induce strong and consistent secretion of the neuropeptides.

Phr are small peptides secreted by *Bacillus subtilis* that regulate cell density. Although the amino acid sequences of the peptides are quite different, their lengths are comparable and similar to that of the NLPs (between 5 and 30 amino acid residues) (Tjalsma et al, 2000). Based on this reasoning we suggest using different signal sequences of the Phr class of peptides as they may turn out to be suitable options. As a last alternative, we propose using a signal peptide prediction algorithm on secreted proteins of all the suggested host bacteria.

For a high-throughput method of screening the feasibility of the signal sequences in secreting the NLP, we suggest using a dot blot assay with an antibody that targets the His-tag which is attached to the C-terminus of the NLP sequence for the purpose of this experiment. This is a fast immunological technique that will hopefully allow identifying the most suitable signal peptide from the ones suggested.

**Table of the possible secretion signals**

| **Name** | **Host organism** | **Amino acid residue sequence** | **Reference** |
| --- | --- | --- | --- |
| Usp45 | *Lactococcus lactis* | MKKKIISAILMSTVILSAAAPLSGVYA | van Asseldonk et al., 1990 |
| PhrH | *Bacillus subtilis* | MPIKKKVMMCLAVTLVFGSMSFPTLTNSG | Bolotin & Borchert, 1997 |
| PhoD Tat type signal | *Bacillus subtilis* | MPKQAVPVKWEVAKDEHFRKIVRKGTEMAKP SLAHSVHVEADGLEPNKVYYYRFKT | Eder et al., 1996 |
| AmyE | *Bacillus megaterium* | MKGKKWTALALTLPLAASLSTGVHA | Jong et al., 2016 |
| AmyE | *Bacillus mycoides* | MFAKRFKTSLLPLFAGFLLLFHLVLAG | van Asseldonk et al., 1990 |

---

**Required Protocols**

[Cloning protocol](https://www.protocols.io/view/pycr-cloning-strategy-bnvxme7n)
[Dot blot protocol](https://www.protocols.io/view/nlp-screening-dot-blot-bkbfksjn)

---

**References**

Borrero, J., Jiménez, J. J., Gútiez, L., Herranz, C., Cintas, L. M., & Hernández, P. E. (2011). Use of the usp45 lactococcal secretion signal sequence to drive the secretion and functional expression of enterococcal bacteriocins in Lactococcus lactis. Applied Microbiology and Biotechnology, 89(1), 131–143. <https://doi.org/10.1007/s00253-010-2849-z>

Marciniak, B. C., Trip, H., van-der Veek, P. J., & Kuipers, O. P. (2012). Comparative transcriptional analysis of Bacillus subtilis cells overproducing either secreted proteins, lipoproteins, or membrane proteins. Microbial Cell Factories, 11, 1–13. <https://doi.org/10.1186/1475-2859-11-66>

Tjalsma, H., Bolhuis, A., Jongbloed, JDH., Bron, S., & van Dijl, JM. (2000). Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiology and Molecular Biology Reviews, 64(3), 515-547. [https://doi.org/(...)BR.64.3.515-547.2000](https://doi.org/10.1128/MMBR.64.3.515-547.2000)

×

**Choosing the integration method**

The engineering strategy we have chosen is to integrate the gene for NLP synthesis in the genome of our host bacteria using CRISPR-Cas9 technology. We will describe this strategy for *Bacillus mycoides*, our preferred host by characterizing the main parts of the pYCR system. As an alternative, we suggest using a different chromosomal integration site.

**The α-amylase gene as integration site**

![](https://static.igem.org/mediawiki/2020/thumb/b/b8/T--Groningen--pYCRNLP14a.png/800px-T--Groningen--pYCRNLP14a.png)

**The NLP14a site in the pYCR plasmid which contains the repair fragment and Cas9 protein.**

Yi and colleagues designed a CRISPR-Cas9 system for *Bacillus mycoides* starting with plasmid pJOE originally established for *Bacillus subtilis* (Yi et al., 2018). The vector they constructed, pYCR, allows performing a one-step integration as the plasmid contains both the *cas9* gene and the required DNA repair fragment. The method has a transformation efficiency of 77% (Yi et al., 2018). We designed the spacer sequences required for the system using Benchling and opted for the α-amylase gene as the site for integration. The neuropeptide gene is used as a ‘’repair fragment’’ and the expression of this gene will be controlled by the strong constitutive promoter *pPta*. For detection purposes, a 6 residue His-tag was introduced at the C-terminus of the NLP sequence. In parallel, we also created a plasmid that encodes the neuropeptide without the His-tag to test whether the stretch of 6 histidine residues influences the biological activity of the peptide. For more detailed information regarding the cloning strategy, [click here](https://static.igem.org/mediawiki/2020/e/e6/T--Groningen--CloningPDF2610.pdf).

---

**Alternative integration site**

Even though replacing the α-amylase gene with other sequences has never been reported to inhibit the growth of the bacteria in the laboratory, we do not have experimental evidence that this will also be the case in nature. It has also been shown that different integration sites may yield different levels of expression (Jeong et al., 2018). Common sites for chromosomal integration in *Bacillus subtilis* are nonessential genes such as *lacA, gltA, pyrD, sacA* and *thrC*. All of these are parts of amino acid or sugar metabolism. Nevertheless, we suggest using *gltA* (glutamate synthase gene). The potato rhizosphere is rich in glutamate so we expect that by deleting this gene, the survivability of *Bacillus mycoides* will not be affected. The removal of the glutamate synthase gene may also improve the containment of the bacteria in RootPatch as is described in the potato “*Safety mechanism*”.

---

**Required Protocols**

[Cloning protocol](https://www.protocols.io/view/pycr-cloning-strategy-bnvxme7n)

---

**References**

Jeong, D. E., So, Y., Park, S. Y., Park, S. H., & Choi, S. K. (2018). Random knock-in expression system for high yield production of heterologous protein in Bacillus subtilis. Journal of biotechnology, 266, 50–58. <https://doi.org/10.1016/j.jbiotec.2017.12.007>

Yi, Y., Li, Z., Song, C., & Kuipers, O. P. (2018). Exploring plant-microbe interactions of the rhizobacteria Bacillus subtilis and Bacillus mycoides by use of the CRISPR-Cas9 system. Environmental Microbiology, 20(12), 4245–4260. <https://doi.org/10.1111/1462-2920.14305>

×

**Choosing the neuropeptide**

The core of RootPatch is the utilization of neuropeptides to influence the behavior of parasitic nematodes. This approach is based on the pioneering study by Warnock and colleagues in 2017. These authors showed that by presenting the proper neuropeptides in the environment of nematodes, one can alter their chemoattraction towards root exudate. Several potential candidates from the class of NLPs were examined. The study shows that NLPs can be used to reverse the chemoattraction of the parasitic nematode of interest (*Globodera pallida*) towards root exudate; whereas in a normal situation, the nematode is attracted by the exudate, it now avoids it.

From a metabolic point of view, NLP appears to be a great choice for RootPatch because its production does not require energy-consuming and complicated post-translational modifications. Thus, the metabolic burden of NLP synthesis and secretion by the bacteria that we are planning to employ, should not greatly affect the survivability of RootPatch. However, the NLPs are rather unexplored and there are still a lot of questions around this very interesting class of peptides. Most likely this type of neuropeptide will bind to G-protein-coupled receptors of the anterior neurons of the nematodes.

To learn more about this pioneering study from Warnock and colleagues, what neuropeptides are and how they affect the nervous system, [visit the project description page](https://2020.igem.org/Team%3AGroningen/Description).

We will explore several NLPs by testing their efficiency in a simple chemosensory assay. Thus, we will be able to find out how well the different NLPs affect nematode behavior and also which peptide concentrations are required for such an effect. It is important to understand in which concentration range the NLPs, produced by the bacteria in RootPatch, will be effective. Our main neuropeptide candidate is NLP14a as it showed the highest potential in creating the root exudate avoidance behavior (see Figure 1). In case this NLP does not produce the desired effect in the nematodes, we propose two other peptide candidates: NLP21e and NLP15c.

![](https://static.igem.org/mediawiki/2020/thumb/d/da/T--Groningen--Dalzellexp.png/800px-T--Groningen--Dalzellexp.png)

**Results from Warnock and colleagues in 2017 showing the disruption of normal chemotaxis behavior of *Globodera pallida* second-stage juveniles by exogenous neuropeptide-like proteins (NLPs).** 100 juveniles were incubated with the selected NLPs, and subsequently released to an agar plate divided into three parts; agar mixed with root exudate, water control, and a normal stroke of agar in the middle. This was repeated 10 times for each neuropeptide. After 3 hours their location was used to determine the chemotaxis score.

**NLP14a**

[

](https://static.igem.org/mediawiki/2020/a/a1/T--Groningen--NLP14Avideo3.mp4)

**One of the predicted structures for NLP14a with its amino acid residue sequence below.** Note that due to the short length, the peptide probably does not have a fixed structure. Structure is predicted using the PEP-FOLD server (Shen et al., 2014).

[NLP14a](http://parts.igem.org/Part%3ABBa_K3507001) is a peptide of 13 amino acid residues, which is predicted from the genome of the *Globodera pallida* (see Figure 2). The work of Warnock et al. shows that this NLP has the highest potential in creating the avoidance behavior towards the root exudate in *Globodera pallida* in nature (see Figure 1). For our proof-of-concept, it is important to replicate these findings to make sure that they are valid, but also to investigate whether this NLP may have additional side-effects on other nematodes in the soil. Therefore, we plan not only to perform this experiment on *Globodera pallida*, but also on other non-parasitic nematodes such as *Caenorhabditis elegans*.

---

**NLP21e or NLP15c**

NLP21e and NLP15c are two NLPs consisting of, respectively, 21 and 14 amino acid residues (see Figure 3). Both NLPs also showed potential avoidance effects. Should NLP14a fail (e.g. in recombinant extracellular production by our host bacteria or in *in vivo* efficiency) these two neuropeptides may be possible alternatives that can be explored.

[

](https://static.igem.org/mediawiki/2020/2/2b/T--Groningen--NLP1521video.mp4)

**Predicted structures for NLP21e & NLP15c with their amino acid residue sequence below.** Note that due to the short length, the peptides probably do not have a fixed structure. Structure is predicted using the PEP-FOLD server (Shen et al., 2014).

---

**Required Protocols**

[Chemosensory assay](https://www.protocols.io/view/chemosensory-assay-bjwckpaw)

---

**References**

Warnock, N. D., Wilson, L., Patten, C., Fleming, C. C., Maule, A. G., & Dalzell, J. J. (2017). Nematode neuropeptides as transgenic nematicides. PLoS Pathogens, 13(2).<https://doi.org/10.1371/journal.ppat.1006237>

Shen Y, Maupetit J, Derreumaux P, Tufféry P. (2014). Improved PEP-FOLD approach for peptide and miniprotein structure prediction. J. Chem. Theor. Comput. 20;10(10):4745-4758. <http://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD>

×

**Choosing the promoter**

The NLPs in RootPatch will be secreted into the soil by a bacterium that is described in the “*Creating our chassis*” pathway. While this production may work very well in the laboratory, there are no guarantees that it is sufficient for application in nature. Many environmental factors such as rain and temperature, but also possible proteases in the soil may influence the fate of the NLPs. For RootPatch to be effective, it needs to produce a stable concentration of NLPs around the root system of the potato plant. This means that the breakdown of the neuropeptide should not be too fast and the NLPs should not diffuse too far away from the roots. Because for an iGEM project, it is almost impossible to get to the stage of a contained field trial, we propose two alternative experiments that could give indications as to how the NLPs will behave in a soil environment:

▪ Soil thin layer chromatography is a technique that can give a relative indication of diffusion of peptides, protein, and other compounds in soils. By comparing the diffusion with two root exudate compounds (glucose, which diffuses far away from the roots, and glutamic acid, which does not diffuse far) with the diffusion of the NLPs in this experiment, we can get an indication of its diffusion in nature. Check out the protocol given below for more information.

▪ Using His-tag purification of soil samples from the potato rhizosphere, we can investigate the rate of breakdown of NLPs by proteases in the soil. By taking random time point samples from the soil that is inoculated with the NLPs, purifying them using a nickel column, and performing SDS polyacrylamide gel electrophoresis, we can determine the breakdown of the His-tagged NLPs over time. Since the quantity of the neuropeptides in the sample may be very low, western blot hybridization may be necessary with anti-his tag antibodies to detect a proper signal.

![](https://static.igem.org/mediawiki/2020/thumb/6/66/T--Groningen--NLPdiffusion.png/800px-T--Groningen--NLPdiffusion.png)

**When the breakdown of NLPs is too fast and/or they diffuse too far**, we need to adjust the setup of RootPatch. These problems could be solved by changing the promoter driving NLP gene expression. Higher production would lead to a higher NLP concentration in the soil, which could counteract the fast breakdown and high diffusion rate.

**The breakdown by proteases could also be too slow and the NLPs may barely diffuse at all**. This could lead to a very high concentration of NLPs and that might have undesired effects in the nematodes (this will be pointed out by the chemosensory assay in the "choosing the neuropeptide" step). In this case we should probably aim for a promoter that leads to a lower peptide production.

Our primary approach is to use the [*Ppta*](http://parts.igem.org/Part%3ABBa_K3507004) promoter because it is strong, stable and has already been used in *Bacillus mycoides*. If this promoter leads to an undesirable protein concentration, we propose a promoter from a list of promoters that could potentially work better.

***Ppta* promoter**

The *Ppta* promoter of the housekeeping phosphotransacetylase gene *pta* from *Parageobacillus thermoglucosidasius* DSM 2542 is a strong constitutive promoter that has been used before in *Bacillus mycoides* to produce GFP (Yi et al,. 2018; Frenzel et al., 2018). Since it leads to a high rate of transcription in *Bacillus mycoides*, this will be our first recommendation for a promoter that should drive NLP gene expression.

**Testing out different promoters**

If the *Ppta* promoter does not work in a proper way (see above), we suggest that one of a number of other promoters may be suitable for NLP production in RootPatch (see the Table below).

**Table of the possible promoters**

| **Name** | **Nucleic acid sequence** | **Reference** |
| --- | --- | --- |
| *Ppta* | TTTGAATTTGATTGGAAGAAGAGTATGCTA | Presecan-siedel et al., 1999 |
| P43 | ATTTTACATTTTTAGAAATGGGCGTGAAAAAAAGCGCGCGATTATGTAAAATATAA | Wang & Doi, 1984 |
| Psigx | AATAGCCAACATTAATAAAATTTAAGGATATGTTAATATAAATTCCCTT | Song et al., 2016 |
| PgroES | ATCTTATCACTTGAAATTGGAAGGGAGATTCTTTATTATAAGAATTGTG | Song et al., 2016 |
| PdltA | TATGTATGGTTTTCACACCGCGAATACCGGTTCATATATTTATAACGATT | Song et al., 2016 |

---

**Required Protocols**

[Cloning protocol](https://www.protocols.io/view/pycr-cloning-strategy-bnvxme7n)
[Dot blot protocol](https://www.protocols.io/view/nlp-screening-dot-blot-bkbfksjn)

[Soil thin-layer chromatography](https://www.protocols.io/view/soil-thin-layer-chromatography-bkcaksse)
[Soil sample purification by His column](https://www.protocols.io/view/his-tag-purification-bnw5mfg6)
[Visualization of NLP-degradation using SDS polyacrylamide gel](https://www.protocols.io/view/sds-page-for-very-small-protein-bkktkuwn)

---

**References**

Frenzel, E., Legebeke, J., Van Stralen, A., Van Kranenburg, R., & Kuipers, O. P. (2018). In vivo selection of sfGFP variants with improved and reliable functionality in industrially important thermophilic bacteria. Biotechnology for Biofuels, 11(1), 1–19.<https://doi.org/10.1186/s13068-017-1008-5>

Yi, Y., Li, Z., Song, C., & Kuipers, O. P. (2018). Exploring plant-microbe interactions of the rhizobacteria Bacillus subtilis and Bacillus mycoides by use of the CRISPR-Cas9 system. Environmental Microbiology, 20(12), 4245–4260.<https://doi.org/10.1111/1462-2920.14305>

×

**Choosing the method of application**

The goal for RootPatch is to grow along with the root system of the potato plant, protecting it throughout the entire potato season. As our [dry lab model](https://2020.igem.org/Team%3AGroningen/Model) has pointed out, the start of the season is the most sensitive period for the bacteria in RootPatch, which makes the method of inoculation crucial. Choosing the correct composition of the inoculant and providing the necessary nutritional support will stimulate the outgrowth and survival of the bacteria. In addition, since the inoculation should, in theory, be applied on a large scale (the potato field), it should not contain high-cost compounds (such as yeast extract and tryptone) that are routinely used in the laboratory, in order to keep the costs of applying RootPatch for farmers low.

There are two main types of inoculants: solid and liquid formulations. Both have their advantages and disadvantages. We suggest using a solid fine granulate formulation as that would better fit the characteristics of our host bacteria and is well suited for large-scale application. If this formulation should be too costly, we propose to shift to a liquid formulation.

![](https://static.igem.org/mediawiki/2020/3/36/T--Groningen--liquidapplication.jpg)

**Farming equipment used to apply granular or liquid fertilizer to the potatoes in the field.** The green spray system in the middle is used to apply liquids to the soil and potato, whereas the tube on the right, is used to apply a granulate. Both methods are possible for RootPatch but a granulate formulation is preferred.

**Granulate formulation**

Our first choice would be to create RootPatch as a powder inoculant. All proposed *Bacillus* RootPatch host species (see the "*Choosing a host*" potato) can form spores, which make them very suitable for this type of application. Because the bacteria are available as spores, the shelf life will be more than 1 year (Martinez-Alvarez et al., 2016; Cabrefiga et al., 2014). In addition, when talking with farmers about our project, they showed the equipment they use to plant the seed potato tubers. That equipment is already set up to apply granulates to the tubers as they often apply granulated fertilizer at the beginning of the season. No additional equipment would therefore be needed to apply RootPatch on a large scale. We suggest using talc as the natural carrier. Talc powder is suitable for long-term storage and has been used previously for creating an inoculant for *Bacillus cereus*, a species close to *Bacillus mycoides* (Martinez-Alvarez et al., 2016). To give nutritional support, carboxymethyl cellulose, calcium carbonate and glucose will be added to the inoculant. The nutritional environment of the root system will trigger the spore germination.

---

**Liquid formulation**

As an alternative to the solid granulated formulation, we suggest to use a liquid formulation. Liquid inoculations are easier to produce and often at lower costs than solid inoculants (Lobo et al., 2019). However, their shelf life is often worse because the bacteria are more susceptible to abiotic stresses such as nutrient depletion (He et al., 2015). Farmers are more accustomed to the application of granulate at the moment of tuber planting. If experiments would point out that re-application of RootPatch is necessary at a given moment in the season, only a liquid formulation would then be possible.

---

**Required Protocols**

[Granulate formulation protocol](https://www.protocols.io/view/granulate-formulation-protocol-bkrpkv5n)
[Counting bacterial density in soil](https://www.protocols.io/edit/measuring-the-amuont-of-bacteria-in-a-soil-sample-bkrckv2w)

---

**References**

Cabrefiga, J., Francés, J., Montesinos, E., Bonaterra, A., 2014. Improvement of a dry formulation of Pseudomonas fluorescens EPS62e for fire blight disease biocontrol by a combination of culture osmoadaptation with a freeze‐drying lyoprotectant. J. Appl. Microbiol. 117, 1122–1131.<https://doi.org/10.1111/jam.12582>

He, Y., Peng, Y., Wu, Z., Han, Y., & Dang, Y. (2015). Survivability of Pseudomonas putida Rs-198 in liquid formulations and evaluation of its growth-promoting abilities on cotton. Journal of Animal and Plant Sciences, 25(3), 180–189.

Lobo, C. B., Juárez Tomás, M. S., Viruel, E., Ferrero, M. A., & Lucca, M. E. (2019). Development of low-cost formulations of plant growth-promoting bacteria to be used as inoculants in beneficial agricultural technologies. Microbiological Research, 219(November 2018), 12–25. <https://doi.org/10.1016/j.micres.2018.10.012>

Martínez-Álvarez, J.C., Castro-Martínez, C., Sánchez-Peña, P., Gutiérrez-Dorado, R., Maldonado-Mendoza, I.E., 2016. Development of a powder formulation based on Bacillus cereus sensu lato strain B25 spores for biological control of Fusarium verticillioides in maize plants. World J. Microbiol. Biotechnol. 32, 75.  [https://doi.org/10. 1007/s11274-015-2000-5](https://doi.org/10.1007/s11274-015-2000-5)

×

**Safety Mechanism**

A very important aspect of RootPatch is safety. After all, RootPatch is a layer of genetically modified bacteria present in nature. What would happen if these bacteria spread and possibly affect other organisms outside the potato field? It is impossible to provide valid answers at the moment; all we can do is to make sure that we never get into such a situation. To ensure this, we aim to develop molecular mechanisms that will make the bacteria dependent on the plant. Whenever a bacterium leaves the root environment of the potato plant, it will not survive. We contemplate using one of the two possible methods to achieve this goal. One is to make our host bacterium auxotrophic for the amino acid tryptophan. The other is to make it dependent on solanine. To improve the efficiency of the safety mechanism, we might even look at combining both approaches. This “potato” will explain how these approaches work.

**Tryptophan auxotrophy**

Root exudates are mixtures of organic compounds secreted by the root systems of plants; they have the ability to attract different bacterial communities. Tryptophan is one of the most abundant organic molecules in the potato root exudate according to mass spectrometry studies when compared to the exudates of other plants (Ochola et al, 2020). Transcriptomics studies of differential gene expression of *Bacillus mycoides* in response to potato root exudate showed downregulation of tryptophan biosynthesis genes (Yi et al, 2018). These findings suggest that tryptophan is one of the molecules that might be used by the potato plant to attract bacterial communities and that *Bacillus mycoides* may take advantage of that. By designing a tryptophan auxotrophic *Bacillus mycoides*, one might be able to still attract the mutant to the potato roots while at the same time restricting its spreading into the environment to a significant degree, as it would die from a shortage of the now essential amino acid.

![](https://static.igem.org/mediawiki/2020/f/f2/T--Groningen--TrpEoperon2.png)

**Figure 1: The *trp* operon.** To make *Bacillus mycoides* auxotrophic, we plan to knock-out the *trpE* gene necessary for the tryptophan synthesis.

In order to create a tryptophan auxotroph, we plan to knock-out the *trpE* gene, the first structural gene of the *trp* operon that is responsible for tryptophan synthesis (Figure 1) (Band et al., 1984). In its absence, *Bacillus mycoides* will rely on the tryptophan secreted by the roots for survival. To delete *trpE*, we intend to use the plasmid pYCR described earlier. We designed guide (g)RNAs that target the *trpE* gene and together with the Cas9 protein inactivate it. More information about the gRNAs and the cloning strategy can be found [here](https://static.igem.org/mediawiki/2020/e/e6/T--Groningen--CloningPDF2610.pdf).

One of the crucial steps for inactivating genes is the efficiency with which this is done by CRISPR-Cas9 in combination with gRNA; this can be quantified by on-target and off-target scores. We have selected gRNAs using a criterium that maximizes on-target activity while minimizing off-target activity. If our first choice of gRNAs turns out to be inefficient, we suggest alternatives that will expectantly introduce the required deletion.

---

**Solanine dependency**

The most abundant organic compound in potato root exudate is solanine, a glycoalkaloid specifically synthesized by potato plants (Koroney et al., 2016; Ochola et al.,2020). We will use this molecule in another approach to make the bacterium dependent on the potato plant root environment. We designed a system that uses solanine as a dependency molecule in the YpcG/YcpF toxin-antitoxin system described in *Bacillus subtilis* by Holberger and colleagues in 2012.

![](https://static.igem.org/mediawiki/2020/3/34/T--Groningen--SolanineDep.png)

**Figure 2: The proposed system for making for Bacillus mycoides dependent on solanine.** A solanine-responsive promoter/operator will bind to solanine and allow the expression of YpcF. YpcG, which shows DNase activity, is continuously expressed in the genome and can be inactivated by YpcF, thereby preventing the death of *Bacillus mycoides*. This mechanism will allow the bacterium to only survive in an environment rich in solanine.

The toxin, [YpcG](http://parts.igem.org/Part%3ABBa_K3507002), exhibits DNase activity and will be continuously expressed in the host bacterium (Eibaz et al., 2015). The antitoxin [YpcF](http://parts.igem.org/Part%3ABBa_K3507003) inactivates YpcG and our aim is to place its gene under the controlled expression of a promoter responsive to the presence of solanine (see Figure 2) (Holberger et al., 2012). By using a toxin with DNase activity, we expect to minimize the spread of recombinant bacteria in the environment, away from the potato plant. More information about, among others, the strategies for cloning can be found [here](https://static.igem.org/mediawiki/2020/e/e6/T--Groningen--CloningPDF2610.pdf).

For this experimental setup to work, the expression of the toxin and antitoxin should be well regulated to prevent one of the two being constantly more abundant. Also, the toxin should be stable and should not be removed too quickly from the bacterial population over time. By integrating the toxin gene into the genome and that of the antitoxin in a plasmid, we aim to minimize this risk.

So far, no promoter has been described that is responsive for solanine. Only a few bacteria species seem to use solanine as a source of carbon. For all of these, the solanine degrading enzymes are constitutively expressed. Because *Bacillus mycoides* is attracted by potato root exudate (which contains a relatively high solanine concentration), we presume that it is already equipped with a solanine-responsive promoter/operator. To find this promoter, we have designed a transcriptomics experiment that will help pinpoint solanine-responsive genes to identify and use a solanine-responsive promoter/operator to drive the toxin-antitoxin containment system.

---

**Combining tryptophan auxotrophy & solanine dependency**

To ensure that RootPatch is well contained in the designated root environment, we suggest combining the described kill switch strategies. Thus, *Bacillus mycoides* will be dependent on both tryptophan and solanine in the potato root exudate. To accomplish this, the antitoxin gene should be placed under the control of the solanine-inducible promoter and this cassette should be integrated in the *trpE* gene, knocking this gene out. By this strategy the resulting bacterium will be both auxotrophic for tryptophan and in need for solanine to suppress the DNase activity of the toxin YpcG.

---

**Required Protocols**

[Cloning protocol for YpcF](https://www.protocols.io/view/cloning-of-bacillus-mycoides-bkuwkwxe)
[Cloning protocol for YpcG](https://www.protocols.io/view/pycr-cloning-strategy-bnvxme7n)

[Transcriptomics experiment](https://www.protocols.io/view/transcriptomics-bme9k3h6)
 [Testing the safety mechanisms](https://www.protocols.io/view/measuring-the-amuont-of-bacteria-in-a-soil-sample-bkrckv2w)

---

**References**

Band, L., H. Shimotsu, and D. J. Henner. 1984. Nucleotide sequence of the Bacillus subtilis trpE and trpD genes. Gene 27:55-65

Elbaz, M., & Ben-Yehuda, S. (2015). Following the fate of bacterial cells experiencing sudden chromosome loss. MBio, 6(3), 1–11. <https://doi.org/10.1128/mBio.00092-15>

Holberger, L. E., Garza-Sánchez, F., Lamoureux, J., Low, D. A., & Hayes, C. S. (2012). A novel family of toxin/antitoxin proteins in Bacillus species. FEBS Letters, 586(2), 132–136. <https://doi.org/10.1016/j.febslet.2011.12.020>

Koroney, A. S., Plasson, C., Pawlak, B., Sidikou, R., Driouich, A., Menu-Bouaouiche, L., & Vicré-Gibouin, M. (2016). Root exudate of solanum tuberosum is enriched in galactose-containing molecules and impacts the growth of pectobacterium atrosepticum. Annals of Botany, 118(4), 797–808.<https://doi.org/10.1093/aob/mcw128>

Ochola, J., Cortada, L., Ng’ang’a, M., Hassanali, A., Coyne, D., & Torto, B. (2020). Mediation of Potato–Potato Cyst Nematode, G. rostochiensis Interaction by Specific Root Exudate Compounds. Frontiers in Plant Science, 11(June). <https://doi.org/10.3389/fpls.2020.00649>

×

**What if RootPatch fails?**

Ultimately, to prove the concept of RootPatch, we will need to perform greenhouse experiments. We could grow potato plants with RootPatch and test its efficiency against nematode infiltration.

But what if the suggested approach, of having a bacterium producing the neuropeptides, does not suffice or is not safe enough? Or even if we achieve proper results with RootPatch, what if the market would not accept a GM microbial solution as, in fact, several of our stakeholders have suggested?

Together with stakeholders we brainstormed possible alternative solutions that still utilize neuropeptides, but are not dependent on GM bacteria to be released in the soil. This “potato” summarizes these alternative solutions that came up during our conversations and describes the various advantages and disadvantages of each.

**Production of NLPs in a bioreactor**

One of the alternative, albeit very labor intensive, option is the *in vitro* production of NLPs and their subsequent application via a liquid solution to the field. *Escherichia coli* could be used as a potential expression host in such a setup because of the relatively low costs of growing it on a large scale (Cardoso et al., 2020; Baolei et al., 2016). After the production step, the NLP should be purified to get rid of the GM *Escherichia coli*. However, to maintain a constant NLP environment around the roots, this would require regular application of the neuropeptide solution to the soil, which would be labor intensive and would demand an extremely high quantity of neuropeptides on a yearly basis.

[

](https://static.igem.org/mediawiki/2020/0/06/T--Groningen--Bioreactor.mp4)

---

**Encapsulation of the NLPs**

Building further on the bioreactor initiative above, the purified neuropeptides could be encapsulated and applied in this form to the field. The advantage of encapsulation is that it could be done in such a way that the neuropeptides are slowly and constantly released. This would significantly lower the number of re-applications to the potato fields. Many different studies have examined encapsulation methods for the release of various agrochemicals and some of these show really promising results (Adisa et al., 2019; Gonzales et al., 2015; Jarosiewicz et al., 2003; Sampathkumar et al., 2020). Although the number of applications per season is lowered, there is still a need for re-application, which makes this method, again, higher in cost and more labor-intensive than when RootPatch could be used.

[

](https://static.igem.org/mediawiki/2020/2/26/T--Groningen--NLPencap.mp4)

---

**Using the potato plant as the production chassis**

[

](https://static.igem.org/mediawiki/2020/f/fe/T--Groningen--GMOplantvideo.png)

Another alternative strategy for using NLPs as a protective agent for potato plants is to let the plants produce the peptides. Plants are very good at secreting peptides, which in some cases even doesn't rely on the usage of signal sequences (Wang et al., 2017). Plants are already being explored as an alternative for protein production in the industry but because of problems with purification, due to many other compounds secreted by plants, they are not yet used commercially (Schillberg et al., 2019). However, for RootPatch, this would not matter. Besides, the potato plant, *Solanum tuberosum*, has already been genetically modified using CRISPR-Cas9 (Butler et al., 2016; Ma J. et al., 2017).

There are some disadvantages to creating a genetically modified potato plant. During our conversations with [stakeholders](https://2020.igem.org/Team%3AGroningen/Human_Practices), it became clear that a GM crop is not a solution that is considered safe by the consumers. The *Innate potato* is a good example for this. This GM potato plant was approved by the US FDA but never found a market since consumers were reluctant to eat something that was genetically modified (Gunther, 2013). Also, there are over three thousand different potato cultivars. To get the approval for the genetic modification of RootPatch for every different cultivar would be an immense challenge. By having a bacterium that coats the roots and that can protect every potato cultivar, deregulation would only be necessary once.

---

**Required Protocols**

[Testing RootPatch](https://www.protocols.io/view/measuring-the-amuont-of-bacteria-in-a-soil-sample-bkrckv2w)

---

**References**

Adisa, I. O., Pullagurala, V. L. R., Peralta-Videa, J. R., Dimkpa, C. O., Elmer, W. H., Gardea-Torresdey, J. L., & White, J. C. (2019). Recent advances in nano-enabled fertilizers and pesticides: A critical review of mechanisms of action. Environmental Science: Nano, 6(7), 2002–2030. <https://doi.org/10.1039/c9en00265k>

Butler, N. M., Baltes, N. J., Voytas, D. F., & Douches, D. S. (2016). Geminivirus-Mediated Genome Editing in Potato (Solanum tuberosum L.) Using Sequence-Specific Nucleases. Frontiers in plant science, 7, 1045. <https://doi.org/10.3389/fpls.2016.01045>

Cardoso, V. M., Campani, G., Santos, M. P., Silva, G. G., Pires, M. C., Gonçalves, V. M., … Zangirolami, T. C. (2020). Cost analysis based on bioreactor cultivation conditions: Production of a soluble recombinant protein using Escherichia coli BL21(DE3). Biotechnology Reports, 26, e00441. <https://doi.org/10.1016/j.btre.2020.e00441>

González, M. E., Cea, M., Medina, J., González, A., Diez, M. C., Cartes, P., … Navia, R. (2015). Evaluation of biodegradable polymers as encapsulating agents for the development of a urea controlled-release fertilizer using biochar as support material. Science of the Total Environment, 505, 446–453. <https://doi.org/10.1016/j.scitotenv.2014.10.014>

Gunther, M. (2013). McDonald’s GMO dilemma: why fries are causing such a fuss. Guardian News and Media Limited.

Jarosiewicz, A., & Tomaszewska, M. (2003). Controlled-release NPK fertilizer encapsulated by polymeric membranes. Journal of Agricultural and Food Chemistry, 51(2), 413–417. <https://doi.org/10.1021/jf020800o>

Jia, B., & Jeon, C. O. (2016). High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives. Open biology, 6(8), 160196. <https://doi.org/10.1098/rsob.160196>

Ma, J., Xiang, H., Donnelly, D. J., Meng, F.-R., Xu, H., Durnford, D., et al. (2017). Genome editing in potato plants by agrobacterium-mediated transient expression of transcription activator-like effector nucleases. Plant Biotechnol. Rep. 11, 249–258. <https://doi.org/10.1007/s11816-017-0448-5>

Sampathkumar, K., Tan, K. X., & Loo, S. C. J. (2020). Developing Nano-Delivery Systems for Agriculture and Food Applications with Nature-Derived Polymers. IScience, 23(5), 101055. <https://doi.org/10.1016/j.isci.2020.101055>

Schillberg, S., Raven, N., Spiegel, H., Rasche, S., & Buntru, M. (2019). Critical analysis of the commercial potential of plants for the production of recombinant proteins. Frontiers in Plant Science, 10(June). <https://doi.org/10.3389/fpls.2019.00720>

Wang, X., Chung, K. P., Lin, W., & Jiang, L. (2017). Protein secretion in plants: Conventional and unconventional pathways and new techniques. Journal of Experimental Botany, 69(1), 21–37. <https://doi.org/10.1093/jxb/erx262>

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Groningen/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Groningen/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)