iGEM 2025 — Team lethbridgehs

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{% block title %}Notebook{% endblock %}
{% block lead %}Wet lab work{% endblock %}
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## Notebook

We unfortunately were unable to do much wet lab work this year but all of our experiments are recorded [here](https://static.igem.wiki/teams/5677/wetlab/wet-lab-notebook-2025.pdf).

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{% block lead %}RhizoRetention's engineering journey{% endblock %}
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## Introduction

The initial goal of Rhizoretention was to engineer rhizobacteria to assist canola in the toleration of drought. This would be through increased production of essential plant hormones and regulatory molecules in the chosen plant. Each following iteration of our project improved and specified the delivery mechanisms, host bacteria, and molecular targets. By using the engineering design cycle, we refined our approach from the very first conception to our current project’s design.

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## Iteration 1

[![First attempt at RhizoRetention](https://static.igem.wiki/teams/5677/engineering/iteration-1-engineering.webp)](https://static.igem.wiki/teams/5677/engineering/group-3-goats-igem-tfc-2025.pdf)

Our team’s first design involved engineering rhizobacteria to produce five essential plant hormones: ABA, proline, trehalose, auxins, and polyamines. The ABA produced by the bacteria would trigger the plant to close its stomata, reducing water loss through transpiration. The proline and trehalose would accumulate in the canola plant cells, helping them retain water and prevent damage from dehydration. The auxins would stimulate the growth of deeper and more extensive roots, and polyamines would protect the plant’s cells from stress and damage. Genes encoding enzymes for these molecules would be inserted into the bacteria’s DNA. The engineered rhizobacteria would be introduced to the roots of plants using inoculation. Each bacterium would be equipped with a natural kill switch to self-destruct after a specific time or upon detecting a specific stimulus. With this design underway, our team sought the feedback of multiple experts through the Tech Futures Challenge (TFC) competition, where we presented our ideas. While the feedback was mostly positive, the judges expressed concerns about feasibility of producing this many hormones as well as about the biosafety of the proposed inoculation method of our project. Using these insights, our team came up with our second iteration.

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## Iteration 2

Pivoting from complex multi-pathway engineering in this second iteration, our team explored a simplified approach, focusing primarily on ABA production. For possible bacteriums, we identified E. coli as a candidate capable of expressing parts of the ABA biosynthetic pathway, particularly the zeaxanthin epoxidase enzyme. Other candidate bacteria such as Bacillus spp. and Azospirillum brasilense were considered for their root-colonizing ability. From our feedback, our team also decided to shift to a hydrogel-based water retention seed coating for both germination support and bacterial delivery instead of our first proposed delivery system. Inspired by MIT’s gel-like mucilage-mimicking coating, we selected sodium alginate for the outer layer due to its water retention properties. This encapsulation method involves alginate beads created via endogenous emulsification and nutrients like chitosan were planned to be added to sustain bacterial viability. Our team also began exploring the integration of RNA interference (RNAi), aiming to knock down plant genes that contribute to drought sensitivity. To further improve upon our project, the team reached out and consulted multiple experts, who reviewed what we had accomplished and gave us feedback. These reviewers asked whether canola might have different stomata specialized for gas exchange versus water regulation, and how this could influence plant respiration under drought conditions. They also recommended exploring other pathways regulated by abscisic acid (ABA), as well as understanding any potential side effects when manipulating drought-response genes. Additionally, we were encouraged to investigate whether ABA production could be naturally triggered by drought stress and how this might interact with our RNAi strategy. This feedback guided us to further refine our testing framework and to explore a more comprehensive analysis of stress-response pathways in canola.

![RNAi Pathway](https://static.igem.wiki/teams/5677/engineering/rnaipath.webp)
![Plan overview](https://static.igem.wiki/teams/5677/engineering/planoverview.webp)

## Iteration 3

In our most current iteration of RhizoRetention, we plan to engineer the root-colonizing bacterium Arthrobacter globiformis to produce small interfering RNA (siRNA) designed to silence the bHLH61 gene. Previous work in the model plant Arabidopsis thaliana has demonstrated that overexpression of the transcription factor bHLH61 reduces drought tolerance, whereas plants with mutations in this gene exhibit improved drought resistance. Since an 86% homologous gene is present in canola, we hypothesize that targeted knockdown of bHLH61 expression can enhance drought tolerance in this crop.The bacteria will be incorporated into a seed coating, allowing them to establish themselves in the rhizosphere of developing canola plants. Once colonized, the bacteria will deliver double-stranded RNA (dsRNA) to the host, triggering RNAi and suppressing expression of the drought-sensitivity gene. In addition, the bacteria are expected to stimulate host stress response pathways, providing further protection under adverse conditions.
To avoid unnecessary energy expenditure and ensure precise regulation, the siRNA expression cassette will be driven by the heat-inducible dnaK promoter. This promoter is activated during elevated temperatures associated with drought and heat stress, ensuring that siRNA is produced only when plants are most vulnerable. This stress-responsive system provides a targeted, sustainable approach to enhancing drought tolerance in canola, supporting food security in a changing climate. Once we had firmly established this design, our team authored a paper through Biotreks, where scientific experts of their respective fields reviewed our writings and provided feedback.
Through the Biotreks platform, our team was provided valuable feedback and insights through detailed comments and questions. During the process, reviewers highlighted the need for deeper research into how bHLH61 functions in drought-response pathways while also encouraging exploring additional drought-related genes. The feedback also emphasized considering safe bacterial concentrations in seed coatings to avoid disrupting the soil microbiome and recommended clear testing strategies. Lastly, reviewers noted that methods should also be explored for greater efficiency and stability. With the help of the judge’s feedback and our testing progress (below) , we continue to modify our project to allow it to function in the most effective way possible

![siRNA sequences](https://static.igem.wiki/teams/5677/engineering/sirnasequences.webp)
![proof of concept plasmid](https://static.igem.wiki/teams/5677/engineering/proofocplasmid.webp)
![Wetlab results](https://static.igem.wiki/teams/5677/engineering/wetlab.webp)

As a proof of concept for our current project, our team designed an engineered plasmid (pPSLA) which contains the heat-inducible dnaK promoter, RBS, tsPurple coding sequence and the sense, loop, antisense sequences for siRNA production. To test the functionality of our proposed design, we plan to insert this plasmid into RNase III-deficient E.coli and measure its tsPurple expression under different heat stress conditions in order to verify dnaK control conditions and parameters, and then compare our results to our kinetic models. As of now, our team has performed insertion mutagenesis PCR on the parent pSB1C3-tsPurple plasmid (using NEB Q5 mutagenesis protocol). We used the above primers to introduce two insertions into our parent plasmid. The PCR products were run on 1% agarose and compared to a wildtype plasmid, resulting in inconclusive results prompting further testing.

![Growth test](https://static.igem.wiki/teams/5677/engineering/growthtest.webp)

We also grew Arabidopsis thaliana lines carrying either GA-YFP, ER-YFP or SEC-RFP or AFVY-RFP reporter genes for proof-of-concept models for gene knockdown experiments, where successful silencing would result in a loss of fluorescence. Since these seeds were from older stocks, our first goal was to germinate and grow the plants to confirm their viability before proceeding with RNAi-based assays. The RFP variants grew the best and will be used for future experiments.

Through iterative design, our project evolved from general hormone production to targeted RNAi delivery using a native soil bacterium and hydrogel seed coating. This progression reflects our efforts to balance the biological complexity of our goals with real-world feasibility.

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# Engineering Soil Bacteria, *Arthrobacter globiformis*, to Produce siRNA Under Drought Conditions Using a Heat-Inducible Promoter

## Systems Level

Our project aims to increase drought tolerance in *Brassica napus* (canola) by deploying engineered *Arthrobacter globiformis* as a delivery vehicle for siRNAs. A heat-inducible promoter will regulate siRNA production, ensuring that gene silencing is activated only under environmental stress conditions such as elevated soil temperatures during drought. The target gene, **bHLH61**, negatively regulates drought tolerance, and its knockdown is predicted to improve crop resilience.

To avoid antibiotic markers in the engineered bacteria, plasmid maintenance will rely on a non-antibiotic selection system based on thymidine auxotrophy. The engineered bacteria will be applied via seed coatings, allowing for colonization of the rhizosphere and localized siRNA delivery to plant roots, while minimizing environmental dispersal.

## Device Level

A **pUB110 plasmid** (or similar) compatible with Gram-positive soil bacteria will encode our genetic circuit: a heat-inducible promoter (dnaK) upstream of the siRNA hairpin targeting bHLH61, and a functional thyA expression cassette that complements a chromosomal thyA knockout in the host. Only cells that retain the plasmid express thyA and can synthesize dTMP, creating plasmid dependence without antibiotics. The heat-inducible promoter restricts siRNA transcription to drought-associated temperature conditions.

Seed coatings, such as alginate or pectin hydrogel, will be optimized to preserve bacterial viability and control initial exposure to the soil environment.

## Parts Level

* **Host organism:** *Arthrobacter globiformis*, engineered with chromosomal knockout of thyA (thymidylate synthase) and RNase III to maintain siRNA synthesis.
* **Promoter for siRNA:** Heat-inducible promoter (dnaK) driving transcription of the siRNA hairpin.
* **siRNA cassette:** Sense–loop–antisense hairpin targeting bHLH61.
* **Plasmid backbone:** pUB110 and derivatives with ssoU-type single-stranded origin for robust replication in Gram-positive bacteria.
* **Seed coating matrix:** Hydrogel (e.g., alginate/pectin) optimized for water retention and microbial viability.

## Next Steps

While we would like to build on our design of bacteria-mediated RNAi in canola, it has not been carried out yet as it involves multiple complex steps beyond the scope of our team. Our biggest hurdles are constructing a thyA auxotrophic *Arthrobacter globiformis* strain and knocking out RNase III. These steps require more time and skills than currently available. Therefore, we decided to pursue an alternative route to test some of our project ideas.

# Proof-of-Concept using Arabidopsis and E. coli

## Systems Level

Our proof-of-concept will test whether bacterial systems can produce functional siRNAs that induce gene silencing in plants. RNase III-deficient *Escherichia coli* HT115 will be engineered to produce siRNAs against both RFP (in transgenic Arabidopsis) and bHLH61 (endogenous target). The bacterial production of siRNAs will be monitored via tsPurple expression. siRNAs purified from bacterial cultures will be applied to *Arabidopsis thaliana* seedlings to evaluate uptake and knockdown efficiency.

## Device Level

Our plasmid construct places a hairpin RNA sequence targeting RFP or bHLH61 downstream of the tsPurple reporter gene. Successful transcription produces both the chromoprotein and siRNA hairpin, linking reporter presence to siRNA output. siRNAs can be purified from *E. coli* cultures and applied to Arabidopsis by foliar spraying or root soaking. Knockdown will be measured by reduced RFP fluorescence in transgenic lines and phenotypic stress-response assays in wild-type plants targeting bHLH61. We will also test dnaK promoter activation by measuring tsPurple expression under different heat conditions.

## Parts Level

* **Promoter:** T7 promoter (BBa\_I712074) for strong constitutive transcription in E. coli or dnaK promoter (BBa\_J100387) for heat-inducible conditions.
* **Reporter:** tsPurple chromoprotein (BBa\_K1033906) linked to siRNA transcription to visualize siRNA production.
* **siRNA Constructs:** Hairpin RNAs targeting RFP (AF168419.2) and bHLH61 (AT5G10570) in Arabidopsis.
* **Backbone:** pSB1C3 plasmid for high-copy expression in E. coli.
* **Host:** E. coli HT115 (RNase III deficient for stable hairpin RNA accumulation).

## Testing

Our experimental protocols can be found [here](https://static.igem.wiki/teams/5677/wetlab/igemhs-protocols2025.pdf). The first goal was to insert the siRNA sequences into the tsPurple plasmid using PCR; however, we were unable to verify successful insertion and must troubleshoot.

* pPSLA-RFPsiRNA\_fwd TGTTTCAAGAGAACAAGAATTGGGACAACTCCtactagtagcggccgctg
* pPSLA-RFPsiRNA\_rev CTTGTTCTCTTGAAACAAGAATTGGGACAACTCCttattacgtcgctttttc
* pPSLA-bHLH61siRNA\_fwd CTCATCAAGAGTGAGTCCATTCCATGTCCATCtactagtagcggccgctg
* pPSLA-bHLH61siRNA\_rev CTCACTCTTGATGAGTCCATTCCATGTCCATCttattacgtcgctttttc
* DnaK\_pro\_fwd CCTATTACAGACTCACACTTTAACCCGTCAACTTTGGTCTGCAAAGCGGGGATAATGTCTGAGTGCGCCCTACTAGAGACCTTAGGAGGTAAACATATGGCG
* DnaK\_pro\_rev TTGACGGGTTAAAGTGTGAGTCTGTAATAGGGGCGAAACGTCTGGTTTCAACTGCCCAATTTCAGTCGGTAAACTCTAGAAGCGGCCGCGAATTCCAG

Concurrently, four Arabidopsis variants (GA-YFP, ER-YFP, SEC-RFP, AFVY-RFP) were grown to test viability (kind donation from Dr. Elizabeth Schultz, University of Lethbridge). Plants were placed in a growth chamber with a 22/8 hour light/dark cycle at 22°C. The RFP variants grew best.

## Next Steps

We will continue plasmid engineering to insert the siRNA sequences and exchange promoters. Then we will test dnaK heat activation by quantifying tsPurple expression under different heat conditions. Additionally, we aim to produce large amounts of siRNA in E. coli, purify it, and apply it to Arabidopsis seedlings. These findings will support downstream engineering of field-deployable bacteria.

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## System Design

![Plasmid with promoter primers](https://static.igem.wiki/teams/5677/results/psb1c3-tspurple-map-promoter-primers.webp)

One of our big issues to overcome was how to trigger RNAi production for gene silencing in response to drought conditions. We didn't want bHLH61 knockdown to be occuring all the time, therefore we researched heat-inducible promoters in the iGEM registry and found  [iGEM Part BBa\_J100387](https://registry.igem.org/parts/bba-j100387). We used this sequence to create primers (BBa\_25RWK0XJ and BBa\_25ESH2UM) to replace the constitutive promoter on pSB1C3 with the DnaK promoter making our new plasmid heat sensitive and hopefully siRNA production occuring only under high heat conditions.

## Wet Lab Results

![Agarose gel of PCR and MP](https://static.igem.wiki/teams/5677/results/agarose-gel-igem-2025.webp)

Wet lab work focused on engineering the pSB1C3-tsPurple backbone to have both the DnaK promoter and siRNA sequences. First, plasmid DNA was successfully isolated by mini prep(196 µg/mL). Using NEB Q5 mutagenesis, siRNA sequences targeting GFP or bHLH61 were introduced. Agarose gel analysis indicated that the expected 100 bp size increase was not observed, suggesting the mutagenesis step requires troubleshooting.

Experimental protocol and lab book can be found [here](https://2025.igem.wiki/lethbridgehs/notebook).

![Plasmid with siRNA primers](https://static.igem.wiki/teams/5677/results/psb1c3-tspurple-map-sirna-primers.webp)

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## Plant Growth

![First attempt at RhizoRetention](https://static.igem.wiki/teams/5677/results/arabidopsis.webp)

We started by germinating *Arabidopsis thaliana* lines carrying GA-YFP, ER-YFP, SEC-RFP, and AFVY-RFP reporter genes to establish proof-of-concept models for RNAi knockdown. Of these, the SEC-RFP and AFVY-RFP variants showed the most consistent growth and will be used for future silencing assays.

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## Next Steps

Our immediate priority is troubleshooting the siRNA mutagenesis step, repeating PCR with optimized primer design and cycling conditions to confirm insertion of promoter and siRNA sequences into the pSB1C3-tsPurple backbone. Once constructs are validated by agarose gel and sequencing, they will be transformed into E. coli for functional testing of the heat-inducible dnaK promoter using the tsPurple reporter. Parallel experiments will use in vitro transcribed siRNA on Arabidopsis reporter lines to establish proof-of-concept gene knockdown assays. Fluorescence imaging and qRT-PCR will quantify silencing efficiency. Following successful validation in Arabidopsis, we will move to testing bHLH61-targeting siRNAs in canola seedlings. In addition, development of an auxotrophic Arthrobacter globiformis chassis strain will enable safe delivery of engineered constructs in seed coatings. Future work will also expand the computational models by incorporating plant growth and drought-stress parameters to predict field-level performance, ultimately guiding the design of a drought-tolerant canola system.

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