iGEM 2024 — Team unilausanne

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![](https://static.igem.wiki/teams/5047/titles-animated/notebopok.png)

Xiphydria longicollis

Fact: Wood wasp larvae can take up to three years to develop inside the wood, eventually breaking through the bark as adults after chewing their way out, (Smith 1976).

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Bootcamp notebook

# *Lactococcus Lactis* Highlights

| Month | *E. coli* RNAse | *L. lactis* RNAse | shRNA producing plasmid |
| --- | --- | --- | --- |

*E. Coli* RNAse deletion

*L. Lactis* RNAse deletion

*L. Lactis* shRNA

*L. Lactis* promoter characterisation

# Yeast System Highlights

| Month | AGO plasmid | DICR plasmid | pTEF1 characterisation |
| --- | --- | --- | --- |

AGO DICR notebook

pTEF Characterisation notebook

shRNA in Yeast notebook

# Dry Lab Highlights

| Month | Identifying gene targets | Image recognition software | Hardware | Model |
| --- | --- | --- | --- | --- |

Software and Hardware notebook

Model notebook

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Right side content here

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![](https://static.igem.wiki/teams/5047/titles-animated/engineering.png)

Araneus diadematus

Fact: Spider "blood" is blue! It is because they have hemocyanin to bind oxygen, unlike humans that have haemoglobin, (Göttler et al. 2021).

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![temporary image of the engineering cycle: design, build, test, learn](https://static.igem.wiki/teams/5047/titles-animated/cycle.png)

[## AGO plasmid](#AGO)
[#### Round 1](#round1)
[Design](#design1)
[Build](#build1)
[Test](#test1)
[Learn](#learn1)
[#### Round 2](#round2)
[Design](#design2)
[Build](#build2)
[Test](#test2)
[Learn](#learn2)
[## *Lactococcus lactis*](#lac)
[#### Round 1](#lac_round1)
[Design](#lac_design1)
[Build](#lac_build1)
[Test](#lac_test1)
[Learn](#lac_learn1)
[#### Round 2](#lac_round2)
[Design](#lac_design2)
[Build](#lac_build2)
[Test](#lac_test2)
[#### References](#ref)

## Engineering the AGO plasmid

The objective of our project is to target Asian hornet larvae using RNA interference, however working on this animal in laboratory conditions is very challenging. So when looking at what past iGEM teams have done, we came across the TUIT Estonian iGEM team from 2023. They used an inventive solution to test RNA interference using *Saccharomyces cerevisiae* as a model. Unfortunately, the team and their PIs did not reply to our repeated request of sharing the corresponding strains with us. Therefore, we aimed to create our own yeast strain that expresses the RNA interference machinery, namely the Argonaute protein and the Dicer protein that are part of the RISC complex. More concretely, we created plasmids containing the different parts necessary for subsequent insertion into the yeast genome. In this section we describe how we managed to clone the plasmids containing the Argonaute protein, following two rounds of the “design-build-test-learn” cycle.

# Round 1

## Design

The assembly of the AGO plasmid requires the promoter (pPGK1), the 3 AGO g-blocks (AGO1, AGO2, AGO3), the terminator (tSIF2) as well as the backbone (pN1-011) which contains spectinomycin resistance.

![Design of the AGO plasmid](https://static.igem.wiki/teams/5047/aline-pictures/ago-design.png)

Figure 1: Design of the AGO plasmid

The assembly of the AGO plasmid requires the promoter (pPGK1), the 3 AGO g-blocks (AGO1, AGO2, AGO3), the terminator (tSIF2) as well as the backbone (pN1-011) which contains spectinomycin resistance.

The first step of our design was to create a plasmid containing the gene that codes for the Argonaute protein (Figure 1), as well as a plasmid containing the gene that codes for Dicer protein (DICR). Next, we planned to combine parts of those two plasmids into one plasmid in order to insert it into the yeast genome. For this “engineering success” section, we focus on the cloning of the plasmids containing the Argonaute protein (AGO). The design and cloning of the DICR plasmid can be found in the **design** and **results** sections, respectively.

More specifically, we designed the following parts to build the AGO plasmid:

* The argonaute (AGO) reading frame is approximately 4kb in length. Which we ordered as 3 separate g-blocks (AGO1, AGO2, AGO3).
* We ordered the terminator (tSIF2) as a g-block, for which we also designed and ordered primers to amplify the fragment so that it would have homology regions with the backbone and the last AGO g-block.
* We designed and ordered primers to amplify the promoter (pPGK1) from the pVW222 plasmid.
* We designed and ordered primers to amplify the backbone pN1-011.

## Build

With all the parts and primers designed, the next step was to amplify the fragments by PCR.

![gel of the AGO fragments](https://static.igem.wiki/teams/5047/aline-pictures/fragments-ag.png)

Figure 2: Gel of amplified AGO fragments

**A:** Gel of amplified fragments pN1-011 (2603bp), pPGK1 (1060bp) and tSIF2 (552bp)

**B:** Gel of amplified pN1-011(2603bp)

We ran the PCR products on a gel (Figure 2A), and we obtained fragments of the right size for pPGK1 however we noticed that the backbone (pN1-011) was not amplified. So we ran another PCR reaction for the backbone lowering the annealing temperature and obtained the right band on the gel (Figure 2B). While running the first gel (Figure 2A) we also noticed that the band for tSIF2 was not very clean, the lower band was at the correct size but the higher band was not supposed to be there. We performed another PCR lowering the annealing temperature and shortening the elongation time, to see if this would mitigate the issue. However we obtained a very similar result even with these changes implemented (Figure 3).

![gel of the tSIF2](https://static.igem.wiki/teams/5047/aline-pictures/tsif-1.png)

Figure 3: Gel of amplified tSIF2 (expected band at 552bp lower band, but we see higher band as well)

Nevertheless, we decided to go ahead with the Gibson assembly, hoping that the fragment with the correct size would be assembled into the plasmid. However, we did not obtain any clones after Gibson assembly and transformation. Therefore,we decided that we needed to purify the tSIF2 from the gel in order to assemble the plasmid correctly.

After reamplifying the fragments needed to perform another Gibson assembly of the AGO plasmid, we purified tSIF2 from the gel. However, after using the gel purification kit the DNA concentration was too low to obtain a successful assembly.

After consulting the instructors we noticed that the AGO g-blocks we had ordered were too diluted, so we ordered some new primers to amplify them. When trying to assemble the plasmid again, we amplified all of the fragments without purifying tSIF2 from the gel and made sure all the fragments were correctly amplified (Figure 4).

![gel of the AGO fragments but better](https://static.igem.wiki/teams/5047/aline-pictures/ago-frag-better.png)

Figure 4: gel of all the fragments needed for AGO plasmid assembly. From left to right pPGK1, AGO1, AGO2, AGO3, tSIF2, pN1-011. All bands were at the expected sizes.

What we also did differently that time, is that we cleaned all of the DNA fragments together and eluted them together into 10µl of water to use for the Gibson assembly. Finally we transformed them into *E. Coli* and successfully managed to get some colonies.

## Test

We performed colony PCRs on several of those colonies and sent them for Sanger sequencing. The most promising colony based on the Sanger sequencing results (Figure 5), we also sent for whole plasmid sequencing (nanopore sequencing).

![sequencing from AGO colony 8](https://static.igem.wiki/teams/5047/aline-pictures/ago-col8-sequence.png)

Figure 5: Sequencing results from colony 8 (forward strand) shows the least amount of mistakes, the reverse sequence is short but correct.

In the results from the whole plasmid sequencing we noticed that tSIF2 had a 32bp insertion (Figure 6A), which we deemed unproblematic for the functioning of the terminator, as the secondary structure of the stem loop was unaltered. However, we also noticed a 15bp deletion in the first AGO g-block, which would be an issue for the functioning of the argonaute protein (Figure 6B).

![Sequencing results from AGO colony 8](https://static.igem.wiki/teams/5047/aline-pictures/ago-seq.png)

Figure 6: Sequencing results from the whole plasmid sequencing of colony number 8.

**A:** 32bp insertion in tSIF2

**B:** 15bp deletion in AGO1 g-block

## Learn

The sequencing results indicated that we had to modify our assembly once more in order to obtain the correct AGO plasmid.

# Round 2

## Design

We planned to insert the correct AGO g-block into the plasmid that already contained the correctly assembled promoter, 2 g-blocks for the AGO gene and the terminator (Figure 7). In the next step, we designed and ordered new primers to amplify the plasmid.

![New design of the AGO plasmid](https://static.igem.wiki/teams/5047/aline-pictures/ago-2nd-round.png)

Figure 7: New AGO plasmid assembly requiring the amplification of the previously assembled plasmid containing a deletion in AGO1 (red line), as well as the amplification of the AGO1 g-block.

## Build

With the new primers ordered, we were able to amplify the fragments needed for the new Gibson assembly. Once we ran the gel we were able to see bands at the correct length (Figure 8), so we then proceeded with the assembly followed by the transformation. The next day we got colonies on which we performed colony PCRs and sent some of them for sequencing.

![gel of the final AGO plasmid](https://static.igem.wiki/teams/5047/aline-pictures/ago-gel-2.png)

Figure 8: Gel of amplified fragments including the new AGO backbone containing the promoter, 2 g-blocks and the terminator, as well as the AGO1 g-block. Both fragments were at the expected size.

**1** backbone: 6777bp

**2** AGO1 g-block: 1275bp

## Test

The results of the sequencing were not very clean, but the part that was correctly sequenced in one of the colonies was the section that was missing in the first assembly. Following these results we decided to perform a miniprep on the final assembled plasmid and we sent it for whole plasmid sequencing (nanopore sequencing).

![sequencing results of the final AGO plasmid](https://static.igem.wiki/teams/5047/aline-pictures/final-seq.png)

Figure 9: Sequencing results from the whole plasmid sequencing of AGO

## Learn

Following these final sequencing results we learnt we had our final assembled AGO plasmid, ready to be inserted into the yeast genome.

## DNA delivery into *Lactococcus lactis*

Our main goal was to engineer a *L. lactis* strain that would be capable of producing specific shRNA against Asian hornet larvae. To achieve this, we set two key subgoals: first, to delete the RNAse III gene from the *L. lactis* genome, as it could degrade the shRNA before it reaches the hornet larvae (Paddison et al. 2002; Court et al. 2013); and second, to clone and insert a plasmid that produces the specific shRNA into *L. lactis*.

Both of these goals required introducing extragenomic DNA into *L. lactis* (Fig. 10). We experienced some challenges in delivering extragenomic DNA into *L. lactis*. By applying two rounds of design-build-test-learn cycle, we were able to overcome these challenges and obtain a protocol to transform extragenomic DNA into *L. lactis*.

![Scheme of Lactococcus transformation](https://static.igem.wiki/teams/5047/lacto/transfo.png)

Figure 10: Our experiments involving *L. lactis*. The steps requiring *L. lactis* transformation are highlighted, showing the importance of this process for our project.

# Round 1: Electroporation

## Design

In our project, we have decided to introduce a shRNA producing plasmid into a *L. lactis* strain that was isolated from the Asian hornet's gut. A gut commensal strain would be better in colonising the hornet's gut, allowing for more shRNA release, and therefore more efficient killing of the Asian hornet larvae. In parallel with the commensal *L. lactis* strain, we also worked on a lab strain of *Lactococcus lactis* subsp. *cremoris* MG1363, that we refer to as "DMF" in our lab notebook, plates and gel pictures. This second strain is a back up, as the gut commensal has never been engineered, whereas the "DMF" strain is often used in lab settings.

As we began the RNAse deletion experiment, we followed a protocol described by Holo and Nes, frequently cited in the literature, for introducing a plasmid into *L. lactis* through electroporation (Holo and Nes 1989).

## Build

Briefly, the Holo and Nes protocol involves growing our *L. lactis* strains overnight in GM17 medium, then refreshing the culture until it reached an optical density (OD600) of 0.2 to 0.7. The cells were then washed by centrifugation with a sucrose solution. After washing, the cells were electroporated and immediately refreshed in SGM17 medium, followed by incubation at 30°C for 1 hour and 30 minutes. Finally, the cells were plated on GM17 agar plates containing erythromycin. We have attempted to insert the plasmid pKD46 containing an ampicillin resistance into *L. lactis* following this approach.
After several unsuccessful attempts using this protocol, we modified our protocol by adding magnesium dichloride and calcium chloride to the recovery medium, inspired by other *L. lactis* transformation protocols (Mulder et al. 2017).

Despite these adjustments, there were no significant improvements. We hypothesised that increasing the plasmid concentration during electroporation might improve transformation efficiency. Initially, we had been using 200 ng of plasmid, but we decided to increase this to 1 μg

When this also failed to improve results, we considered whether the plasmid itself might be the issue—perhaps it was being degraded by *L. lactis* endonucleases. In our last attempt to electro-transform *L. lactis*, we have taken the *Lactococcus lactis* subsp. *cremoris* MG1363 strain that we already used, but that one already harboured a plasmid. We extracted that plasmid via miniprep and attempted to reintroduce it through electroporation, but, once again, the transformation was unsuccessful.

## Test

Our method for testing whether electro-transformation was successful involved plating the bacteria transformed with the pKD46 plasmid on GM17 agar plates containing ampicillin. The plasmid we introduced carried genes for ampicilin resistance, and since *L. lactis* is not naturally resistant to this antibiotic, the appearance of colonies would indicate successful plasmid integration.

However, none of the plates showed any bacterial growth, confirming that our transformation attempts were unsuccessful.

## Learn

Since none of our attempts at electro-transformation seemed to be successful, we have decided to try a different approach: **conjugation.**

# Round 2

## Design

Electroporation uses an electrical current to permeabilize the cell membrane, allowing the bacterial cell to integrate a DNA fragment. Conjugation, on the other hand, transforms cells through a completely different process. In conjugation, the plasmid must first be introduced into an *E. coli* strain capable of facilitating the transfer. This plasmid also needs to carry an oriT (origin of transfer) sequence, which signals that the plasmid is ready for conjugation. Once the *E. coli* strain with the plasmid is plated alongside the target bacterium, the *E. coli* injects the plasmid into the target cell. The *E. coli* strain (strain JKE20, (Harms et al. 2017)) we used could only grow in the presence of diammonium phosphate (DAP), which allowed for the selection of *L. lactis* cells after they have been conjugated.

We decided to first try to integrate the plasmid pMG36E into *L. lactis*, the plasmid we were planning to use as a backbone for the shRNA producing plasmid. As mentioned above, in order for a plasmid to be transferable through conjugation it needs to carry an oriT sequence. Therefore, we needed to introduce an oriT sequence into the pMG36E plasmid before proceeding with the rest of the conjugation protocol.

![Experimental approach to conjugating L. lactis.](https://static.igem.wiki/teams/5047/lacto/pmg.jpeg)

Figure 11: Experimental approach to conjugating *L. lactis*.

## Build

The steps we followed during conjugation appear in Figure 11.

We have begun by inserting an oriT sequence in the pMG36E plasmid through Gibson assembly. Afterwards, the pMG36E-oriT plasmid was introduced into *E. coli* strain JKE201 through electroporation. The transformed *E. coli* cells were grown on LB plates containing DAP as well as erythromycin, as the pMG36E plasmid carries resistance genes to this antibiotic. We checked the insertion of the oriT sequence in our plasmids as well as the fact that the plasmid was integrated into *E. coli* colonies that grew with a colony PCR.

Next, we co-plated the transformed E. coli cells with our *L. lactis* strains—one plate for each strain, with 5 times more *L. lactis* than *E. coli*. The plates used were GM17, supplemented with DAP to support *E. coli* growth. The plates were incubated at 30°C. After a few days, once cells grew, we transferred some of the cells to a new plate containing LB and erythromycin to select *L. lactis* strains that received the pMG36E plasmid.

## Test

The final plate, lacking DAP and containing erythromycin, allowed us to selectively isolate only *L. lactis* cells that had successfully received the plasmid. The absence of DAP prevented E. coli JKE201 from growing, while the presence of erythromycin ensured that only *L. lactis* cells carrying the pMG36E plasmid, which contains erythromycin resistance genes, survived, as *L. lactis* is not naturally resistant to erythromycin (Fig. 12C).

![Erythromycin containing plates with plated L. lactis.](https://static.igem.wiki/teams/5047/lacto/plates.jpeg)

Figure 12: Erythromycin containing plates with plated *L. lactis*.
Transformed *L. lactis* strains after 3 days of growth at 30°C from A) the hornet's gut and B) *Lactococcus lactis* subsp. *cremoris* MG1363, growing on plates with erythromycin, proving that they harbour a plasmid giving them erythromycin resistance. C) Untransformed *Lactococcus lactis* strains after 4 days of growth at 30°C. *Lactococcus lactis* subsp. *cremoris* MG1363 on the left side of the plate and the hornet commensal strain on the right. No colonies are visible on either side.

To be certain that the colonies we saw on our final plates (Fig. 12A and 12B) were indeed *L. lactis*, we did a colony PCR with primers attaching to *L. lactis* genome (Fig. 13).

![Gel of amplified fragments of L. lactis genome](https://static.igem.wiki/teams/5047/lacto/lactogel.jpeg)

Figure 13: Gel of amplified fragments of *L. lactis* genome, with gut commensal *L. lactis* colonies on the left and *Lactococcus lactis* subsp. *cremoris* MG1363 on the right. The expected size is around 1 kb for both *L. lactis* strains.

The primers used for this colony PCR bind to *L. lactis* genome around the RNAse region. We saw bands of the expected size, around 1kb for all colonies that we tested on both *L. lactis* strains.

This final test indicates that the colonies we saw on our LB and erythromycin plates were indeed *L. lactis*, proving that **we successfully transformed *L. lactis* through conjugation.**

### References

* Court, Donald L., Jianhua Gan, Yu-He Liang, Gary X. Shaw, Joseph E. Tropea, Nina Costantino, David S. Waugh, and Xinhua Ji. 2013. “RNase III: Genetics and Function; Structure and Mechanism.” Annual Review of Genetics 47: 405–31. https://doi.org/10.1146/annurev-genet-110711-155618.
* Harms, Alexander, Marius Liesch, Jonas Körner, Maxime Québatte, Philipp Engel, and Christoph Dehio. 2017. “A Bacterial Toxin-Antitoxin Module Is the Origin of Inter-Bacterial and Inter-Kingdom Effectors of Bartonella.” PLOS Genetics 13 (10): e1007077. https://doi.org/10.1371/journal.pgen.1007077.
* Holo, Helge, and Ingolf F. Nes. 1989. “High-Frequency Transformation, by Electroporation, of Lactococcus Lactis Subsp. Cremoris Grown with Glycine in Osmotically Stabilized Media.” Applied and Environmental Microbiology 55 (12): 3119–23.
* Mulder, Joyce, Michiel Wels, Oscar P. Kuipers, Michiel Kleerebezem, and Peter A. Bron. 2017. “Unleashing Natural Competence in Lactococcus Lactis by Induction of the Competence Regulator ComX.” Applied and Environmental Microbiology 83 (20): e01320-17. https://doi.org/10.1128/AEM.01320-17.
* Paddison, Patrick J., Amy A. Caudy, Emily Bernstein, Gregory J. Hannon, and Douglas S. Conklin. 2002. “Short Hairpin RNAs (shRNAs) Induce Sequence-Specific Silencing in Mammalian Cells.” Genes & Development 16 (8): 948–58. https://doi.org/10.1101/gad.981002.

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![](https://static.igem.wiki/teams/5047/titles-animated/experiments.png)

Panorpa communis

Fact: Scorpionflies are neither scorpions nor flies! They are actually closely related to fleas, (Whiting 2002)

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# Protocols

**Gene Deletion:** Technique to remove specific gene segments from DNA.

**Colony PCR:** Method to amplify DNA from bacterial colonies.

**DNA Clean Up:** Process to purify DNA from contaminants.

**Gel Electrophoresis:** Technique for separating DNA fragments by size.

**Gibson Assembly:** Method to join multiple DNA fragments seamlessly.

**Glycerol Stock:** Preservation method for bacterial cultures using glycerol.

**GM17 Media:** Nutrient-rich medium used for culturing lactococcus lactis.

**Making Competent Cells:** Process to prepare cells for DNA uptake.

**Miniprep:** Small-scale procedure to extract plasmid DNA.

**PCR:** Technique to amplify specific DNA sequences.

**Preparing Plates:** Method for creating nutrient agar plates for bacterial growth.

**Transformation (electroporation):** Process to introduce foreign DNA into cells.

# Primers

#### Project

#### Name

#### Purpose

#### Sequence 5'-3'

#### Size

#### Temperature

#### Notes

E.Coli RNAse deletion

PR\_VV\_001

Create linear fragment with HR2

5'- caggt ctgtt tcgtg tgctg aattg ttgac gcattt attta ttggt atcgc attcc gggga tccgt cgacc -3'

pkD13 - E. coli

74°C

E.Coli RNAse deletion

PR\_VV\_002

Create linear fragment with HR1

5'- ttttt tcaac gcctg ttcgg cggca gcctg ctcag cctta cgacg gcttg agtgt aggct ggagc tgctt c -3'

pkD13 - E. coli

79.1°C

E.Coli RNAse deletion

PR\_VV\_003

Check insertion of Kanamycin cassette into genome

5'- tgcgg gaagt gatgg agatt -3'

E. coli genome

55.4°C

E.Coli RNAse deletion

PR\_VV\_004

Check insertion of Kanamycin cassette into genome

5'- ccccg aagtt taagg ttggc -3'

E. coli genome

55.5°C

L. lactis RNAse gene deletion

PR\_VV\_005

50bp overhang with the region upstream rnc (forward)

5'- taagg gataa aacta aaaat taaag agaag tttta agtga aggga dattt tttgt gtggc tggag ctgct tc -3'

pKD13 / 1304 bp

57.2°C

L. lactis RNAse gene deletion

PR\_VV\_006

50bp overhang with the region at the end of rnc (reverse)

5'- tttta ttgca ttttc tgctg ctttt tgttc tgcaa ctttt ttagt ttcat tccgg ggatc cgacc -3'

pKD13 / 1304 bp

60.2°C

L. lactis RNAse gene deletion

PR\_VV\_007

Check rnc presence or removal on the genome

5'- tcatg gcggc tactt taggg -3'

1055 bp if rnc is in place, 1734 bp if rnc has been replaced by the KamR cassette, 512 bp if the KamR cassette has been removed

55.5°C

L. lactis RNAse gene deletion

PR\_VV\_008

Check rnc presence or removal on the genome

5'- gattg ctcgc ccaaa accc -3'

55.5°C

S. cerevisciae GFP reporter plasmid

PR\_VV\_009

PCR pRS314 backbone

5'- gggaa taagg gcgac acgga aatg -3'

pRS314 backbone / 3883 bp

60°C

Pair with PR\_VV\_011

S. cerevisciae GFP reporter plasmid

PR\_VV\_010

PCR pRS314 backbone

5'- cattt ccgtg tcgcc cttat tccc -3'

pRS314 backbone / 2091 bp

60°C

Pair with PR\_VV\_012

S. cerevisciae GFP reporter plasmid

PR\_VV\_011

PCR pRS314 backbone

5'- ctcga gTTAT TTGTA CAATT CATCC ATACC ATGGG -3'

pRS314 backbone / 3883 bp

60°C

Pair with PR\_VV\_009

S. cerevisciae GFP reporter plasmid

PR\_VV\_012

PCR pRS314 backbone

5'- gggac gctcg aaggc tttag atctg gtacc cagct ttttg ttccc tttag tgag -3'

pRS314 backbone / 2091 bp

60°C

Pair with PR\_VV\_010

S. cerevisciae GFP reporter plasmid

PR\_VV\_013

PCR tCYC1 + target site

5'- agatc taaag ccttc gargc gtccc -3'

tCYC1 gblock / 307 bp

60°C

Pair with PR\_VV\_014 / 015 / 016

S. cerevisciae GFP reporter plasmid

PR\_VV\_014

PCR tCYC1 + target site 1

5'- ATGGA TGAAT TGTAC AAATA Actcg agCAC TAGAG TCTAG CTTGA GAtcg cccgt acatt catgt aatta gttat gtcac -3'

tCYC1 gblock / 307 bp

60°C

Pair with PR\_VV\_013

S. cerevisciae GFP reporter plasmid

PR\_VV\_015

PCR tCYC1 + target site v3

5'- ATGGA TGAAT TGTAC AAATA Actcg agAGT CTGTA ACGTC CGCCA Ctcgc ccgta cattc atgta atta gtta tgtca c -3'

tCYC1 gblock / 307 bp

60°C

Pair with PR\_VV\_013

S. cerevisciae GFP reporter plasmid

PR\_VV\_016

PCR tCYC1 + target site v10

5'- ATGGA TGAAT TGTAC AAATA Actcg agTCG CCAAT ATGCG TGTAC Ctcgc ccgta cattc atgta atta gtta tgtca c -3'

tCYC1 gblock / 307 bp

60°C

Pair with PR\_VV\_013

S. cerevisciae GFP reporter plasmid

PR\_VV\_017

Diagnostic PCR for pRS314\_pGAL1-GFP\_target

5'- cagtc acgac gttgt aaaac gacgg -3'

Colony PCR pRS314 / 1642 bp

60°C

Pair with PR\_VV\_018

S. cerevisciae GFP reporter plasmid

PR\_VV\_018

Diagnostic PCR for pRS314\_pGAL1-GFP\_target

5'- gatta cgcca agctc ggaat taacc c -3'

Colony PCR pRS314 / 1642 bp

60°C

Pair with PR\_VV\_017

S. cerevisciae shRNA controls

PR\_VV\_019

PCR pRS426 backbone

5'- tatag ttttt tctcc ttgac gttaa agtat agagg tatat taaca atttt ttttg -3'

pRS426 backbone / 4168 bp

60°C

Pair with PR\_VV\_009

S. cerevisciae shRNA controls

PR\_VV\_023

PCR shRNA sequences

5'- ggata agaaa gcaaa cacct ggcaattcctta -3'

shRNA / ~120 bp

60°C

Pair with PR\_VV\_024

S. cerevisciae shRNA controls

PR\_VV\_024

PCR shRNA sequences

5'- acctc tatact ttaac gtcaa ggaga aaaa actata -3'

shRNA / ~120 bp

55°C

Pair with PR\_VV\_023

S. cerevisciae shRNA controls

PR\_VV\_025

shRNA1 with 9nt loop

5'- acctc tatact ttaac gtcaa ggaga aaaa actat aAACACTAG AGTCTAGCT TGAGATTCA AGAGATCTC AAGCTAGAC TCTAGTGTT taagg aattgc caggtt gcttt tcttatcc -3'

S. cerevisciae shRNA controls

PR\_VV\_026

shRNA1 mut-17

5'- cctct tatact ttaac gtcaa ggaga aaaa actat aAACACTA GAGTCTAG CTTGAGATG ATATGTGCA TCTCAAGC TAGACTC TAGTGTT taagg aattgc caggtt gcttt tcttatc -3'

S. cerevisciae shRNA controls

PR\_VV\_027

shRNA1 mut-25

5'- ctcta tatact ttaac gtcaa ggaga aaaa actat aAACACTA GAGTCTAG CTTGAGA CCTCTCA ACACTGGTC TCAAGC TAGACTCT AGTGTT taagg aattgc caggtt gcttt tcttatc -3'

S. cerevisciae shRNA controls

PR\_VV\_028

shRNAv3 with 9nt loop

5'- acctc tatact ttaac gtcaa ggaga aaaa actat aAAGTG GCGGACGTT ACAGAC TTTCAAGAGA AGTCTGT AACGTCC GCCACTT taagg aattgc caggtt gcttt tcttatcc -3'

S. cerevisciae shRNA controls

PR\_VV\_029

shRNAv3 mut-17

5'- acctc tatact ttaac gtcaa ggaga aaaa actat aAAGTGG CGGACGT TACAGACT TGATATGTGC AAGTCT GTACT CGCACC TTaagg aattgc caggtt gcttt tcttatcc -3'

S. cerevisciae shRNA controls

PR\_VV\_030

shRNAv3 mut-25

5'- ctcta tatact ttaac gtcaa ggaga aaaa actat aAAGTGG CGGACGTTAC AGACTCC TCTCAACAC TGGTCTC AAGACT TAGTG TTtaag gaa ttgccaggt tgctt tttctt atcc -3'

S. cerevisciae shRNA controls

PR\_VV\_031

shRNAv10 with 9nt loop

5'- acctc tatact ttaac gtcaa ggaga aaaa actat aAAGGTACAC GCATATT GGCGATTCAAGAGAT CGCCAATA TGCGTGTAC CTTtaagg aa ttgccaggt ttgct tttctt atcc -3'

S. cerevisciae shRNA controls

PR\_VV\_032

shRNAv10 mut-17

5'- acctc tatact ttaac gtcaa ggaga aaaa actat aAAGGT ACACGC ATATGG CGATG ATA TGTGCAC CAGTACT GTGTTTA TTAA-3'

S. cerevisciae shRNA controls

PR\_VV\_034

Diagnostic PCR

5' - CGAAA AACCG TCTAT CAGGG CGA TGG - 3'

S. cerevisciae shRNA controls

PR\_VV\_035

diagnostic PCR for pRS426\_shRNA

5'- CGCAA TTAA TGTGA GTTAC CTCA CTCAT TAGGC -3'

colony PCR pRS426\_shRNA/~1500 bp

60°C

Pair with PR\_VV\_034

Cloning shRNA in lactococcus

PR\_VV\_036

Open pMG36E backbone with homology arms for PTSIIC-celA promoter

5'- CCAAA ATTGT CATAT AAGTT GAATT CGATC GACCC ATATTT AAAAG C -3'

55.6°C

Paired with PR\_VV\_0037 : 3407 bp

Cloning shRNA in lactococcus

PR\_VV\_037

Open pMG36E backbone with homology arms for rrnBT1-T7TE double terminator

5'- TGGGC CTTTC TGCAG TTTAT AGTCG ACCTG CAGGC ATGC -3'

58.3°C

Cloning shRNA in lactococcus

PR\_VV\_038

Amplify PTSIIC-celA-shRNA-rrnBT1-T7TE

5'- AACTT ATATA GACAA TTTTG GTACA GGAGT C -3'

promoter-shRNA-terminator G-block

55.6°C

Cloning shRNA in lactococcus

PR\_VV\_039

5'- TATAA ACGCA GAAAG GCCCA CC -3'

57.1°C

Create promoter-shRNA-terminator part from long oligos

PR\_VV\_040

Long oligo corresponds to PTSIIC-celA promoter

5'- AACTT ATATA GACAA TTTTG GTACA GGAGT CTTC AAAAG TGGCA CAGAAC CAAAG TGATG GAAAA TAAAG AAAC TGC -3'

Create promoter-shRNA-terminator part from long oligos

PR\_VV\_041

Long oligo corresponds to (almost all) the reverse of rrnBT1-T7TE

5'- TATAA ACGCA GAAAG GCCCA CCCGA AGGTG AGCCG TGTGA TCTAG TAGAG AGCGT TCACC GACAA ACAAC AGATA A -3'

Create promoter-shRNA-terminator part from long oligos

PR\_VV\_042

Long oligo corresponds to shRNA\_1 (control) with homology arms for promoter and terminator (to be used as template for amplification with VV\_040 and 041)

5'- GCTT TACTT TGCCT ATTAG TGC TATAA TGA AAT AACAC TAGAG TCTAG CTGAG ATTCA AGAG ATCTC AAGCT AGACT CTAGT GTTCC AGG -3'

Create promoter-shRNA-terminator part from long oligos

PR\_VV\_043

Long oligo corresponds to shRNA\_v3 with homology arms for promoter and terminator (to be used as template for amplification with VV\_040 and 041)

5'- GCTT TACTT TGCCT ATTAG TGC TATAA TGA AAT AAGT GGCG GACGT TACAG ACTTT CAAGA GAAGT CTGT AACGT CCGC CACTT CCA -3'

Include promoter-RBS-GFP on pKD13 for GFP knock-in

PR\_VV\_044

Open pKD13 backbone, between KamR and one of the FRT sites

5'- ACTGG GCTAT CTGGA CAAG -3'

Include promoter-RBS-GFP on pKD13 for GFP knock-in

PR\_VV\_045

Open pKD13 backbone, between KamR and one of the FRT sites

5'- AGCTG ACATT CATCC GGGG -3'

Include promoter-RBS-GFP on pKD13 for GFP knock-in

PR\_VV\_046

Amplify GFP from pREXC3GH

5'- CCTTG TCCAG ATAGC CCAGT TTATT TGTAG AGCTC ATCCAT GCC -3'

S. cerevisciae intermediate plasmid pN1-011\_AGO1

PR\_VV\_047

PCR pPGK1 promoter

5'- ATAGG ATAGA TTCTG GAAAC TTTAC CGTCC CAGAC GCGA ATTTT TCGA AGAAG TACCT TC -3'

pVW222\_ pRS305\_ pPGK1\_ UbiY.../ 1060 bp

60°C

Pair with PR\_VV\_048

S. cerevisciae intermediate plasmid pN1-011\_AGO1

PR\_VV\_048

PCR pPGK1 promoter

5'- GAGAG TTCTC TTCCG AGTTT GAGGA CATTT GTTTT ATATT TGTTG TAAAA AGTAG ATAAT TACTT CCTTG ATGAT CTG -3'

pVW222\_ pRS305\_ pPGK1\_ UbiY.../ 1060 bp

60°C

Pair with PR\_VV\_047

S. cerevisciae intermediate plasmid pN1-011\_AGO1

PR\_VV\_051

PCR pN1-011 backbone

5'- CCATG GTTC AGCCA AAAAAC TTAAG ACC -3'

pN1-011 / 2603 bp

59°C

Pair with PR\_VV\_052

S. cerevisciae intermediate plasmid pN1-011\_AGO1

PR\_VV\_052

PCR pN1-011 backbone

5'- GGACG GTAAG TTTCC AGAAT CTATC CTAT -3'

pN1-011 / 2603 bp

57°C

Pair with PR\_VV\_051

S. cerevisciae intermediate plasmid pN1-011\_AGO1

PR\_VV\_053

diagnostic PCR1 for pN1-011\_AGO1 & pN1-011\_DCR1

5'- CACAT TTCCC CGAAA AGTGC CACCT -3'

colony PCR1 pN1-011\_AGO1/2317 bp, colony PCR pN1-011\_DCR1/3110 bp

60°C

AGO1: pair with PR\_VV\_055, DCR1: pair with PR\_VV\_054

S. cerevisciae intermediate plasmid pN1-011\_AGO1

PR\_VV\_054

diagnostic PCR1 for pN1-011\_AGO1 & pN1-011\_DCR1

5'- GCTAG TTATT GCTCA GCGGT GGCAG CA -3'

colony PCR2 pN1-011\_AGO1/3451 bp, colony PCR pN1-011\_DCR1/3110 bp

60°C

AGO1: pair with PR\_VV\_056, DCR1: pair with PR\_VV\_053

S. cerevisciae intermediate plasmid pN1-011\_AGO1

PR\_VV\_055

diagnostic PCR2 for pN1-011\_AGO1

5'- ACTGT TTAG GCCCA CAACT TCCTG TC -3'

colony PCR1 pN1-011\_AGO1/2317 bp

60°C

AGO1: pair with PR\_VV\_053

S. cerevisciae intermediate plasmid pN1-011\_AGO1

PR\_VV\_056

diagnostic PCR2 for pN1-011\_AGO1

5'- AGAGC AAAG GAAAT TAATG ACCTT GCTGC -3'

colony PCR2 pN1-011\_AGO1/3451 bp

60°C

AGO1: pair with PR\_VV\_054

S. cerevisciae intermediate plasmid pN1-011\_DCR1

PR\_VV\_057

PCR pTEF2 promoter

5'- ATAGG ATAGA TTCTG GAAACT TTACC GTCCCA AAATG TTTC TACTC CTTTT TTACT CTTCC AGATT TTTC TC -3'

pSP329-SIV-TEFCherrySZ4 / 478 bp

60

pair with PR\_VV\_058

S. cerevisciae intermediate plasmid pN1-011\_DCR1

PR\_VV\_058

PCR pTEF2 promoter

5'-ATGTT TTAG ATAGA GATCG GCGCT TTTTT TCCCT ATTC ATAGC TTTAG ATTAG ATTG CTAT GC-3'

pSP329-SIV-TEFCherrySZ4 / 478 bp

57.7

pair with PR\_VV\_057

S. cerevisciae integrative plasmid pSP599\_AGO1\_DCR1

PR\_VV\_061

PCR pSP599\_pVB9 backbone

5'-CATGG TCATA GCTGT TTCC TGATA ACTTC G-3'

pSP599\_pVB9 / 1982 bp

59.5

pair with PR\_VV\_062

S. cerevisciae integrative plasmid pSP599\_AGO1\_DCR1

PR\_VV\_062

PCR pSP599\_pVB9 backbone

5'-AAAAA TTCGC GTCTG GAGCT CCAAT TCGCC CTAT AGTG AGTC-3'

pSP599\_pVB9 / 1982 bp

60

pair with PR\_VV\_061

S. cerevisciae integrative plasmid pSP599\_AGO1\_DCR1

PR\_VV\_063

PCR AGO1

5'-CGAAT TGGAG CTCC AGAC GCGAA TTTTT TCGA AGAAG TAC-3'

pN1-011\_AGO1 / 5418 bp

58.9

pair with PR\_VV\_064

S. cerevisciae integrative plasmid pSP599\_AGO1\_DCR1

PR\_VV\_066

PCR pSP599\_pVB9 backbone

5'- ACTGG CCGTC GTTTT ACATG GAAAA AC-3'

pSP599\_pVB9 / 2304 bp

60

pair with PR\_VV\_065

S. cerevisciae integrative plasmid pSP599\_AGO1\_DCR1

PR\_VV\_068

PCR DCR1 construct

5'- GAAAAT CTGGA AGAGT AAAAA AGGAG TAGAA ACATTT TGCAT GGTC ATAGC TGTTC CCTG ATAAC TTCG-3'

pN1-011\_DCR1 / 2778 bp

58

pair with PR\_VV\_067

Include promoter-RBS-GFP on pKD13 for GFP knock-in

PR\_VV\_069

Amplify GFP from pREXC3GH

5'- GAAAAT AATAT TTAGG AGAAG GCAtg agcaa aggaga agaact ttttc ac-3'

Include promoter-RBS-GFP on pKD13 for GFP knock-in

PR\_VV\_070

Amplify PTSIIC-celA with homology arm for pKD13 backbone

5'- ACCCC GGATG AATGT CAGCT AACTT ATATA TGACA ATTTT GGTA CAGGA GTC-3'

Include promoter-RBS-GFP on pKD13 for GFP knock-in

PR\_VV\_071

Amplify PTSIIC-celA with homology arm for GFP

5'- GCTCA TGCCT TCTCC TAAAT ATTTA TTTTC ATTAT AGCATT AATAG GCAAG TAAGC-3'

S. cerevisciae intermediate plasmid pN1-011\_AGO1

PR\_VV\_49a

PCR tSIF2 terminator for pN1-011\_AGO1

5'- GTTAC GGA CATC ATGT ATACT ACAT ATAAG CACTG AAGG AGTA CTTG AGGC ATTG C-3'

tSIF2 / 552 bp

60

pair with PR\_VV\_050a

S. cerevisciae intermediate plasmid pN1-011\_AGO1

PR\_VV\_50a

PCR tSIF2 terminator for pN1-011\_AGO1

5'- GTCTT AAGT TTTT TTGG CTGAA CCAT GGCG GCGGT CTTA AGTT TTTT TTGG CTGAA CC-3'

tSIF2 / 552 bp

60

pair with PR\_VV\_049a

S. cerevisciae shRNA controls

PR\_VV\_020a

PCR pRS426 backbone

5'-AGCTG TTTCC TGTG TGA AATT GTTAT CCGC-3'

pRS426 / 2005 bp

60

pair with PR\_VV\_10

S. cerevisciae shRNA controls

PR\_VV\_021a

PCR tSIF2 terminator for pRS426\_shRNA

GCGG ATAA CAAT TTCAC ACAG GAAAC AGCT CGGCG GTCT TAAG TTTTT TTGG CTGAA CC-3'

tSIF2 / 559 bp

60

pair with PR\_VV\_022a

S. cerevisciae shRNA controls

PR\_VV\_022a

PCR tSIF2 terminator for pRS426\_shRNA

5'- TAAGG AATG CCAG GTGT TGCT TTTCT TATCC GCACT GAA GGA GTACT TGAG GCATT GC-3'

tSIF2 / 559 bp

60

pair with PR\_VV\_021a

S. cerevisciae intermediate plasmid pN1-011\_DCR1

PR\_VV\_059a

PCR tSTE2 terminator for pN1-011\_DCR1

5'- GGAGT ACTTG AGGC ATTG CAACA ATCT GTAA AACAT TAAGG ATTT TAAAC CTCC ATAC ATTT ATTCT C-3'

tSTE2 / 629 bp

56.7

pair with PR\_VV\_060a

S. cerevisciae intermediate plasmid pN1-011\_DCR1

PR\_VV\_060a

PCR tSTE2 terminator for pN1-011\_DCR1

5'- GGTCT TAAGT TTTTT TGG CTGAA CCAT GGAA TCAGA AATT GGAG AAAGTC CTGG TTGA ACAAGA-3'

tSTE2 / 629 bp

60

pair with PR\_VV\_059a

S. cerevisciae integrative plasmid pSP599\_AGO1\_DCR1

PR\_VV\_064a

PCR AGO1 construct

5'- CTCA CTAAG GGAACA AAAG CTGG GTACC CGGCG GTCT TAAG TTTTT TTGG CTGAA CC-3'

pN1-011\_AGO1 / 5442 bp

60

pair with PR\_VV\_063

S. cerevisciae integrative plasmid pSP599\_AGO1\_DCR1

PR\_VV\_067a

PCR DCR1 construct

5'- GTTTT TTCC ATGT AAAAC GACG GCCAG TAAT CAGA AATT GGAG AAAGTC CTGG TTGA ACAAGA AAAAG-3'

pN1-011\_DCR1 / 2872 bp

60

pair with PR\_VV\_068

S. cerevisciae GFP reporter plasmid

PR\_VV\_009a

PCR pRS314 backbone (new primer instead of PR\_VV\_009)

5'- GCATC TTTTA CTTTC ACCAG CGTTT CTGG-3'

pRS314

60

pair with PR\_VV\_011

S. cerevisciae shRNA controls

PR\_VV\_075a

PCR pRS426 backbone

5'- CGGCG GCTTC TAATCC GTAC TTCA ATATAG GGTG ATGGT TCAC GTAG TGGG CCATC-3'

pRS426

60

pair with PR\_VV\_009a

S. cerevisciae shRNA controls

PR\_VV\_074a

PCR pGAL1 promoter

5'- CTAT ATTG AAGT ACGG ATTAG AAGCC GCCCG-3'

pRS314 (pGAL1)

60

pair with PR\_VV\_019

S. cerevisciae integrative plasmid pSP599\_AGO1\_DCR1

PR\_VV\_065a

PCR pSP599\_pVB9 backbone

5'- AGCTT TTGTT CCCTT TAGTG AGGGTT AATTT CCTG TAAACT TTATA AATGA AATTC ATA TAG-3'

pSP599

60

pair with PR\_VV\_066

S. cerevisciae shRNA controls

PR\_VV\_022b

PCR tSIF2 gblock

5'- GCACT GAAGG AGTACT TGAGG CATTG-3'

tSIF2 gblock

59.7

pair with PR\_VV\_021a

lacto shRNA

PR\_VV\_081

Forward primer shRNA to check the insertion of fragment

5'- TTGGTT GATAAT GAAC TGTG CTG-3'

lacto shRNA

PR\_VV\_082

Reverse primer shRNA to check the insertion of fragment

5'- CTCCAA ATATC GTAG CGCCG-3'

AGO g-block amplification

PR\_VV\_083

Forward primer AGO1

5'- ATGTC CTC AACT CGGA AGAG AACT CTTC-3'

AGO g-block amplification

PR\_VV\_084

Reverse primer AGO1

5'- GTCAT TAATT TCCTT TGCT CTACT GTTT AGGCC-3'

AGO g-block amplification

PR\_VV\_085

Forward primer AGO2

5'- GACAG GAA GTTC TGTG GGC CTA AAC AG-3'

AGO g-block amplification

PR\_VV\_086

Reverse primer AGO2

5'- AGCTC GCCT TAA GTTC ATCC ATGACT-3'

AGO g-block amplification

PR\_VV\_087

Forward primer AGO3

5'- CAGTC ATGG ATGAA CTTAA GGC GAGC-3'

AGO g-block amplification

PR\_VV\_088a

Reverse primer AGO3

5'- TTATA TGTAG TACAT GATGT CCGT AACATT CTTAT GCAC-3'

pMG36E amplification

PR\_VV\_089

Amplify backbone of pMG36E (with promoter and terminator) Reverse

5'- ATTTTC ATTATA GCATTA ATAGG-3'

pMG36E amplification

PR\_VV\_090

Amplify backbone of pMG36E (with promoter and terminator) Forward

5'- CCAGGC ATCAA ATAAA ACGAA AGG-3'

Reverse of primer 42

PR\_VV\_093

42 reverse

Dilution

lacto shRNA - restriction enzymes tactic

PR\_VV\_094

Amplify promoter + backbone + terminator with overhangs for restriction enzymes

5'- AAACTT TACCG TCCCT CGAGC CAGGC ATCAA ATAAA ACGAA AGG-3'

lacto shRNA - restriction enzymes tactic

PR\_VV\_095

Amplify promoter + backbone + terminator

5'- CCAGAA TCTAT CCTAT GAGCT CATTTT CATTATA GCATTA ATAGG-3'

lacto shRNA - restriction enzymes tactic

PR\_VV\_096

SacI - shRNA - XhoI - v1 - control - Primer Reverse

5'- TCGAG AACAC TAGAG TCTAG CTGAG ATCTC TTGA ATCTC AAGCT AGACT CTAG TGTTG AGCT-3'

lacto shRNA - restriction enzymes tactic

PR\_VV\_097

SacI - shRNA - XhoI - v1 - control - Primer Forward

5'- CAACA CTAG AGTCT AGCT TGAG ATTCA AGAG ATCTC AAGCT AGACT CTAG TGTTC-3'

lacto shRNA - restriction enzymes tactic

PR\_VV\_098

SacI - shRNA - XhoI - v3 - Primer Forward

5'- CAAGT GGCGG ACGTT ACGAG ACTTT CAA GAGAA GTCTG TAACG TCCG CCACTTC-3'

lacto shRNA - restriction enzymes tactic

PR\_VV\_099

SacI - shRNA - XhoI - v3 - Primer Reverse

5'- TCGAG AAGT GGCGG ACGTT ACGAG ACTT CTCTT GAAAG TCTG TAACG TCCG CCACTTG AGCT-3'

Reverse of primer 43

PR\_VV\_100

43 reverse

Dilution

shRNA production in yeast

PR\_VV\_101

shRNA1, F

5'- TAACGT CAAGG AAAAA AACTA TAAAC ACTAG AGTCT AGCTTG AGATT CAAGAG ATCTC AAGCT AGACT CTAG TGTTG CACTG AAGG AGTACT TGAG-3'

97 bp

69.8

shRNA production in yeast

PR\_VV\_102

shRNA1, R

5'- CTCAAG TACTC CTTCA GTGCA ACAC TAACT AGAGT CTAGCT TGAGAT CTCTT GAATC TCAAG CTAGAC TCTAG TGTTT ATAGT TTTTT TCTC CTTGAC GTA-3'

97 bp

69.8

shRNA production in yeast

PR\_VV\_103

shRNA1, mut 17, F

5'- TAACGT CAAGG AAAAA AACTA TAAAC ACTAG AGTCT AGCTTG AGATT GATAT GTGCAT CTCAAG CTAGAC TCTAG TGTTG CACTG AAGG AGTACT TG-3'

97 bp

69.8

shRNA production in yeast

PR\_VV\_104

shRNA, mut 17, R

5'- CAAGT ACTC CTTCA GTGCA ACAC TAACT AGAGT CTAGCT TGAGAT GCACAT ATCAT CTCAAG CTAGAC TCTAG TGTTT ATAGT TTTTT TCTC CTTGAC GTA-3'

97 bp

69.8

shRNA production in yeast

PR\_VV\_105

shRNA, mut 25, F

5'- TAACGT CAAGG AAAAA AACTA TAAAC ACTAG AGTCT AGCTTG AGACCT CTCAAC ACTGG TCTCA AGCTAG ACTCT AGTGTTG CACTG AAGG AGTAC-3'

97 bp

71

shRNA production in yeast

PR\_VV\_106

shRNA, mut 25, R

5'- GTACTC CTTCA GTGCA ACAC TAACT AGAGT CTAGCT TGAGACC AGTGTT GAGAGG TCTCA AGCTAG ACTCT AGTGTTT ATAGT TTTTT TCTC CTTGAC GTA-3'

97 bp

71

shRNA production in yeast

PR\_VV\_107

shRNAv3, F

5'- CTATA CTTTA ACGTC AAGG AAAAA AACTA AAGTGG CGGAC GTTAC AGACT TTCAAG AGAAGT CTGTA ACGTCC GCCACT TGCACT GAAGG AGTA-3'

97 bp

71

shRNA production in yeast

PR\_VV\_108

shRNAv3, R

5'- TACTC CTTCA GTGCA AGTGG CGGAC GTTAC AGACT TCTCT TGAAG TCTGTA ACGTCC GCCACT TATAGT TTTTT TCTC CTTGAC GTA-3'

97 bp

71

shRNA production in yeast

PR\_VV\_109

shRNAv3, mut 17, F

5'- TAACGT CAAGG AAAAA AACTA AAGTGG CGGAC GTTAC AGACT TGAT ATGTGC AAGTCT GTAAC GTCCG CCACT TGCACT GAAGG AGTACT TG-3'

95 bp

71.7

shRNA production in yeast

PR\_VV\_110

shRNAv3, mut 17, R

5'- CAAGT ACTC CTTCA GTGCA AAGTGG CGGAC GTTAC AGACT TGCAC ATATCA AGTCT GTAAC GTCCG CCACT TATAGT TTTTT TCTC CTTGAC GTA-3'

95 bp

71.7

shRNA production in yeast

PR\_VV\_111

shRNAv3, mut 25, F

5'- ATACTT TTAAC GTCAAG GAAAA AACTA AAGTGG CGGAC GTTAC AGACT CCTCT CAAC ACTGG AGTCT GTAAC GTCCG CCACT TGCACT GAAGG AGT-3'

98 bp

72.6

shRNA production in yeast

PR\_VV\_112

shRNAv3, mut 25, R

5'- CTCCTT CAGTG CAAGT GGCGG ACCTG TTAAC ACTG TGTAG GAGGT CTGTA ACGTCC GACCT TATAGT TTTTT TCTC CTTGAC GTA-3'

98 bp

72.6

shRNA production in yeast

PR\_VV\_113

shRNAv10, F

5'- TCTATA CTTTA ACGTC AAGG AAAAA AACTA AAGGT ACACG CATATT GGCGAT TCAAG AGATC GCCAAT ATGCGT GTACCT TGCACT GAAGG AGTA-3'

98 bp

70.1

shRNA production in yeast

PR\_VV\_114

shRNAv10, R

5'- TACTC CTTCA GTGCA AGGTAC ACGCA TATTG GCGAT CTCTT GAATC GCCAAT ATGCGT GTACCT TATAGT TTTTT TCTC CTTGAC GTA-3'

98 bp

70.1

shRNA production in yeast

PR\_VV\_115

shRNAv10, mut 17, F

5'- TAACGT CAAGG AAAAA AACTA AAGGT ACACG CATATT GGCGAT GATAT GTGCAT CGCCA ATATG CGTGTACCT TGCACT GAAGG AGTACT TGA-3'

96 bp

70.7

shRNA production in yeast

PR\_VV\_116

shRNAv10, mut 17, R

5'- TCAAG TACTC CTTCA GTGCA AGGTAC ACACG CATATT GGCGAT GCACAT ATCAT CGCCA ATATG CGTGTACCT Ttatagttttttctccttgacgtta-3'

96 bp

70.7

shRNA production in yeast

PR\_VV\_117

shRNAv10, mut 25, F

5'- TAACGT CAAGG AAAAA AACTA AAGGT ACACG CATATT GGCGAC CCTCT CAAC ACTGG TCGCC AATAT GCGTGTACCT TGCACT GAAGG AGTACT-3'

96 bp

72

shRNA production in yeast

PR\_VV\_118

shRNAv10, mut 25, R

5'- AGTACT CCTTC AGTGCA AGGTAC ACACG CATATT GGCGAC CCTCT CAAC ACTGG TCGCCA ATATG CGTGTACCT Ttatagttttttctccttgacgtta-3'

96 bp

72

conjugation lacto

PR\_VV\_119a

PCR backbone pMG36E + overhang for oriT

5'- cctgc tctgc gaggc tggcc gtatc aggct tgatc cccag taagtc-3'

3467 bp

54.6°C

conjugation lacto

PR\_VV\_120

PCR backbone pMG36E + overhang for oriT

5'- gaagc agggt tatgc agcgg aaaag gaagta catcc gcaa cgtcc ata ctctg-3'

3467 bp

55.4°C

conjugation lacto

PR\_VV\_121

PCR to amplify oriT

5'- tacgg ccagc ctcgc agagc agg-3'

conjugation lacto

PR\_VV\_122

PCR to amplify oriT

5'- ctttt ccgct gcata accct gcttc-3'

Primers for target plasmid

PR\_VV\_011a

PCR pRS314 backbone

5'- ctcga gTTATT TGTAT AGTTC ATCCA TGCCA TGTG-3'

56

Primers for target plasmid

PR\_VV\_014a

PCR tCYC1 + target site 1

5'- ATGGA TGAACT ATACAA ATAAC tcgag CACTAG AGTCT AGCTT GAGAt cgccc ggtaca ttcat gtaat tagtt atgtc ac-3'

60

Primers for target plasmid

PR\_VV\_015a

PCR tCYC1 + target site v3

5'- ATGGA TGAACT ATACAA ATAAC tcgag AGTCT GTAAC GTCC GCCAC tcgcc cgtaca ttcat gtaat tagtt atgtc ac-3'

60

Primers for target plasmid

PR\_VV\_016a

PCR tCYC1 + target site v10

5'- ATGGA TGAACT ATACAA ATAAC tcgag TCGCC AATAT GCGT GTACC tcgcc ggtaca ttcat gtaat tagtt atgtc ac-3'

60

Primers for final AGO + DICR

PR\_VV\_061a

PCR pSP599\_pVB9 backbone

5'- AAGAG ATTTCT ACACA CGATT GAGCA CTGT CTCA TGGTC ATAGC TGTTT CCCT GATAA CTTTC G-3'

Primers for final AGO + DICR

PR\_VV\_066a

PCR pSP599\_pVB9 backbone

5'- AGCAA TCAATA GTGTT CGTAAG TGTAG TCCCT ggaa aaaaaca atagg tcctt aaata tttgt caag-3'

Primers for final AGO + DICR

PR\_VV\_067b

PCR DCR1 construct from pN1-011

5'- GGGAC TACA CTTA CGAA AACTA TTGA TTGCT aatc agaa atttg gaga aagtc ctgg ttgaac-3'

Primers for final AGO + DICR

PR\_VV\_068a

PCR DCR1 construct from pN1-011

5'- AGACA GTGCT CAATC GTGTAG AAATC TCTTC AAAAT GTTTC TACTC CTTTT TTACTC TTCCAG ATTTTC-3'

shRNA plasmid lacto, true targets

PR\_VV\_131

Target sequence hairpin 1, F

5'- GCTT tactt tgcc tatta atgc TATAA Tgaaa atAAT GATGC CTTGG GTGCA ATAGG TGTTC AAGAG ACACC TTATT GCACC CAAGG CATCA TTCc caggc atcaa ataaa acgaa aggc tcagt cga-3'

126 bp

shRNA plasmid lacto, true targets

PR\_VV\_132

Target sequence hairpin 1, R

5'- tcgac tgagc cttt cgttt tattt gatgc ctgg AATGA TGCCT TGGGT GCAAT AGGTT GTCT CTTGA ACACC TTATT GCACC CAAGG CATCA TTat tttc ATTAT Agcat taata taggc aaagt aAAGC-3'

126 bp

shRNA plasmid lacto, true targets

PR\_VV\_133

Target sequence hairpin 2, F

5'- GCTT tactt tgcc tatta atgc TATAA Tgaaa atAAT CGTAC AAATG ATGC TTTGG GTGTT CAAGA GACAC CCAAG GCATC ATTTG TACGA TTcc aggc atcaa ataaa acgaa aggc tcagt cga-3'

126 bp

shRNA plasmid lacto, true targets

PR\_VV\_134

Target sequence hairpin 2, R

5'- tcgac tgagc cttt cgttt tattt gatgc ctgg AATCG TACAA ATGAT GCCTT GGGT GTCT CTCTT GAAC ACCCA AGGC ATCAT TTTG TACGA TTat tttc ATTAT Agcat taata taggc aaagt aAAGC-3'

126 bp

shRNA plasmid lacto, true targets

PR\_VV\_135

Target sequence hairpin 3, F

5'- GCTT tactt tgcc tatta atgc TATAA Tgaaa atAAT GATGC CTTGG GTGCA ATAGG TTTCA AGAGA AACC TATTG CACCC AAGGC ATCAT TTTc caggc atcaa ataaa acgaa aggc tcagt cga-3'

126 bp

shRNA plasmid lacto, true targets

PR\_VV\_136

Target sequence hairpin 3, R

5'- tcgac tgagc cttt cgttt tattt gatgc ctgg AAATG ATGC CTTGG GTGCA ATAGG TTCT CTCTG AAAC CCTAT TGCAC CCAAG GCATC ATTTT attttc ATTAT Agcat taata taggc aaagt aAAGC-3'

126 bp

shRNA plasmid lacto, true targets

PR\_VV\_137

Target sequence hairpin 4, F

5'- GCTT tactt tgcc tatta atgc TATAA Tgaaa atAAT TTGG CGCATT GATA Cgtag tatTT CAAG AGAA TACT ACGT ATCA ATGC CCAA ATTcc aggc atcaa ataaa acgaa aggc tcagt cga-3'

126 bp

shRNA plasmid lacto, true targets

PR\_VV\_138

Target sequence hairpin 4, R

5'- tcgac tgagc cttt cgttt tattt gatgc ctgg AATTT GGCG CATTG ATAC gtagt atTCT CTTG Aata ctac GTAT CAATG CGCC AAATT attttc ATTAT Agcat taata taggc aaagt aAAGC-3'

126 bp

Characterization of Lactococcus lactis promoter PTSIIC-celA via expression of GFP, comparison with J23100 promoter

PR\_VV\_139

Binds on RBS in front of GFP on PN2-006, homology with PR\_VV\_089

5'- cctat taa tgc TATAA Tgaaa tTAAT TTTGT TTAAC TTTA AGAAG GAGA GATA TACA T-3'

Characterization of Lactococcus lactis promoter PTSIIC-celA via expression of GFP, comparison with J23100 promoter

PR\_VV\_140

Binds on the C-term of GFP on PN2-006, homology with PR\_VV\_090

5'- ccttt cgttt tattt gatgc ctgg TTATT AGTC TTC AATCC AATCC AGG-3'

Characterization of Lactococcus lactis promoter PTSIIC-celA via expression of GFP, comparison with J23100 promoter

PR\_VV\_141

Binds on RBS in front of GFP on pBTK503, homology with PR\_VV\_089

5'- cctat taa tgc TATAA Tgaaa atat acag aaac agagg agat attac atat g-3'

Characterization of Lactococcus lactis promoter PTSIIC-celA via expression of GFP, comparison with J23100 promoter

PR\_VV\_142

Binds on the C-term of GFP on pBTK503, homology with PR\_VV\_090

5'- ccttt cgttt tattt gatgc ctgg ttat tTGT atag ttc atcc atgc cgtg-3'

Characterization of Lactococcus lactis promoter PTSIIC-celA via expression of GFP, comparison with J23100 promoter

PR\_VV\_143

Binds just upstream PTSIICcelA promoter on pMG36E-shRNA, with overhang to include J23100 promoter

5'- gctag cac tgta ccta ggac tgag ctag ccgt caag aatt cgat cgac ccata tttaa aagc-3'

Characterization of Lactococcus lactis promoter PTSIIC-celA via expression of GFP, comparison with J23100 promoter

PR\_VV\_144

Binds on RBS in front of GFP on PN2-006, homology with PR\_VV\_143 (J23100)

5'- ttgac ggc tagc tcagt ccta ggtac agtg ctag cTAAT TTTGT TTAAC TTTA AGAAG GAGA GATA TACA T-3'

Characterization of Lactococcus lactis promoter PTSIIC-celA via expression of GFP, comparison with J23100 promoter

PR\_VV\_145

Binds on RBS in front of GFP on pBTK503, homology with PR\_VV\_143 (J23100)

5'- ttgac ggc tagc tcagt ccta ggtac agtg ctag catac agaa acag agg agat attac atat g-3'

Characterization of Lactococcus lactis promoter PTSIIC-celA via expression of GFP, comparison with J23100 promoter

PR\_VV\_146

Binds on GFP without including MarA deg tag, adds the missing stop codon

5'- ccttt cgttt tattt gatgc ctgg TTATT ATTTA TACAG TTCGT CCA TGC CCG -3'

# Plasmids

#### Plasmid

#### Characteristics

#### Source

pDA120

Plasmid containing pCYC + mCherry used as a comparaison point to pTEF1

Serge Pelet (DMF, University of Lausanne)

pDA122

Plasmid containing pADH1 + mCherry used as a comparaison point to pTEF1

Serge Pelet (DMF, University of Lausanne)

pDA95

Plasmid containing pTEF1 + mCherry used for the characterisation of this pTEF1

Serge Pelet (DMF, University of Lausanne)

pVW222

Plasmid containing pPGK1 taken for the regulation of the AGO protein

Serge Pelet (DMF, University of Lausanne)

pSP329

Plasmid containing pTEF1 taken for the regulation of the DICr protein

Serge Pelet (DMF, University of Lausanne)

pSP599

Plasmid used as the backbone for the final AGO + DICR assembly

Serge Pelet (DMF, University of Lausanne)

pMG36E

Used as the backbone for the shRNA producing plasmid in L. lactis

Ordered from Addgene

pREXC3GH

Backup plasmid for the shRNA production in L. lactis

Ordered from Addgene

pCP20

FLP helper plasmid /!\/!\ LOST AT 37°C (incubate at 30°C)

Given to us by the Schaerli Lab

pKD13

Plasmid containing the kanamycin cassette and the FRT sites

Given to us by the Schaerli Lab

pKD46

Red recombinase expression plasmid /!\/!\ LOST AT 37°C (incubate at 30°C), used for the recombination of the FRT sites and inducible by arabinose

Given to us by the Schaerli Lab

pRS314

Plasmid used as the backbone for the target sequence introduction in yeast

Given to us by the Schaerli Lab

pRS426

Plasmid used as the backbone for the shRNA production in yeast

Given to us by the Schaerli Lab

pN1-011 AGO

Plasmid coding for the Argonaute protein

Engineered by us

pN1-011 DICR

Plasmid coding for the Dicer protein

Engineered by us

pMG36 shRNA

Plasmid coding for the production of shRNA in L. lactis

Engineered by us

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}

![](https://static.igem.wiki/teams/5047/titles-animated/resluts.png)

Tipula paludosa

Fact: Often called "mosquito hawks" or "skeeter eaters", crane flies only feed on plants and do not hunt! (Dawson et al. 2002)

{% endblock %}
{% block page\_content %}

# Project Achievements

1. We identified target regions for our RNAi using bioinformatics analysis.

2. We successfully deleted RNAse in *E. coli*.

3. We successfully transformed *L. lactis* through conjugation.

4. We successfully constructed a backbone with a promoter and a terminator to produce shRNA in *L. lactis* and *E. coli*.

5. We successfully characterised the pTEF1 promoter.

6. We successfully created both AGO and DICR plasmids.

7. We successfully implemented the VespAI software.

8. We successfully built a box which can house the necessary electronics.

9. We successfully implemented the software and the hardware.

10. We successfully simulated bee and hornet population dynamics in a model.

#### Projects

[Summary](#summary)
[RNAi target gene identification](#target-gene)
[*E. coli* RNAse deletion](#eRNAse)
[*L. lactis* RNAse deletion](#lRNAse)
[shRNA producing plasmid in *L. lactis*](#shRNA)
[Testing in yeast](#yeast)
[Image recognition software](#image)
[Model](#model)
[References](#ref)

## Summary

The objective of our project was to target Asian hornet larvae using a gut commensal bacterium of the hornet which produces RNA interference. To accomplish this goal, our project consisted of wet lab and dry lab parts. In this section, we summarise the results of our project and the problems we encountered along the way.

Our first objective was to identify which gene to target in the Asian hornet without targeting any local fauna. We developed a bioinformatics pipeline to extract the conserved regions of the target genes and perform off-target analysis. Check our [Software page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27software%27%29%20%7D%7D) page for additional details.

### Goals

**We had 3 main goals:**

1. We wanted to engineer *Lactococcus lactis*, a gut commensal bacterium of the hornet, to produce shRNA which would target an essential developmental gene in the hornets. To produce a sufficient amount of shRNA, we had to delete the RNAse gene as well as introduce a plasmid containing the shRNA production.

2. We wanted to test the efficiency of this shRNA in degrading its target, and to avoid animal testing we wanted to perform these tests in *S. cerevisiae*. To do that we needed to design and assemble plasmids containing the RISC complex (Argonaute and Dicer proteins) and insert them into the genome, since yeast does not naturally express this machinery. Additionally, we wanted to introduce a plasmid coding for the shRNA and a plasmid containing the target of the shRNA downstream of a GFP reporter.

3. We wanted to use well-characterised promoters, to ensure a good expression of the RISC complex. To do that, we needed to characterise a new promoter that wasn’t in the iGEM registry: pTEF1, the promoter of our DICR protein. In that mindset, we set another goal of our lab experiments and tried to characterise this promoter in yeast, with an mCherry reporter, and compared its level of expression to that of 2 other similar promoters, pCYC1, pADH1.

Additionally, we wanted to make sure that our initial project design, namely targeting larvae with a gut commensal bacteria producing RNA interference, was the most efficient solution. To do that, we designed a model that would simulate bee population dynamics confronted to predation by the Asian hornet, and Asian hornet population dynamics confronted to our solution.

Finally, we had another goal in the dry lab. We wanted to make sure that our shRNA would only be accessible to the Asian hornet, since the gene we wanted to target was very similar to other members of the *Vespa* genus. Our idea was to create a box which would contain a protein bait with our bacteria on it and selectively allow Asian hornets (*V. Velutina*) to access it. To do that we had to adapt an image recognition software (VespAI), as well as connect our software to our hardware.

## RNAi Target Gene Identification

The target gene identification process aimed to find a gene specific to *Vespa velutina* (Asian hornet) that could disrupt larval development while avoiding off-target effects in other species. We developed a bioinformatic pipeline to do this analysis (see software page for details). After an initial literature search, we considered several genes, including Vitellogenin Receptor, Cytochrome C (CYC), Vacuolar ATPase (V-ATPase), and Chitin Synthase. However, after further analysis, we selected Chitin Synthase as the optimal RNA interference (RNAi) target (Vontas, Katsavou, and Mavridis 2020; Zhang, Zhang, and Zhu 2010; Lu and Shen 2024; Jin et al. 2015). This was because CYC did not have highly conserved regions within *V. Velutina* population, while Off-target analysis using BLAST showed that the V-ATPase had significant sequence similarity with other insect species, leading to a high risk of off-target effects. In contrast, Chitin Synthase exhibited minimal off-target risks across species such as bees and wasps.

Chitin Synthase plays a critical role in larval development by facilitating exoskeleton formation, making it an ideal candidate for disrupting the Asian hornet's development. Additionally, this gene has already been targeted in the *Anopheles gambiae* parasite to eradicate malaria (Vontas, Katsavou, and Mavridis 2020; Lu and Shen 2024). Through sequence alignment and variant analysis, we identified multiple conserved regions within the *V. velutina* Chitin Synthase gene, which could serve as RNAi target sites.

Further refinement of the RNAi target regions based on GC content and RNA secondary structures led to the identification of three optimal regions within the Chitin Synthase gene (see Figure 1). These regions had favourable GC content (30-40%) and weak secondary structures, making them highly suitable for RNAi treatment (see software page for more details).

![ Identification of target genes in the Asian hornet and potential off-targets.](https://static.igem.wiki/teams/5047/bioinf/chs-targets-v6.png)

**Figure 1:** Identification of target genes in the Asian hornet and potential off-targets. RNAi Target sequences are indicated on the y-axis, while off-target species are given on x-axis. The scores represent the bit scores from BLAST, reflecting the sequence similarity between BLAST hits in off-target species and the RNAi target regions. This score is based on nucleotide matches, mismatches, and sequence length.

In conclusion, we identified Chitin Synthase as the final target gene due to its essential role in larval development, minimal off-target effects, and multiple conserved regions ideal for RNAi. This gene represents a promising target for disrupting *V. velutina* populations through RNAi-based control strategies.

## *E. coli* RNAse deletion

##### Objective:

We wanted to create an *E. coli* strain that lacked an RNAse, since RNAse activity may interfere with shRNA production (Court et al. 2013; Paddison et al. 2002). Though the strain we wanted to use is *L. lactis*, we were first thinking of using *E. coli* as a proof of concept strain, and designed our shRNA producing plasmid with a promoter that works in both organisms.

##### Methodology:

![Strategy to delete the RNAse gene in <i>E. coli</i>](https://static.igem.wiki/teams/5047/lacto/ecolirna.png)

**Figure 2:** Strategy to delete the RNAse gene in *E. coli* with the lambda Red recombination protocol. A kanamycin resistance cassette (KmR) with homology regions to the *E. coli* genome around the RNAse gene is used to delete the RNAse gene. KmR also has FRT sites to take out the kanamycin resistance out of the genome once the RNAse is knocked out.

The genomic deletion was achieved by following the lambda Red recombination protocol, as schematised in Figure 2(Datsenko and Wanner 2000). Briefly, we made an insert with homology regions to *E. coli* genome around the RNAse gene, and also a kanamycin resistance gene – it will be referred to as "KmR cassette". The aim was to make this cassette recombine with the RNAse gene in *E. coli* genome. To do so, the KanR cassette was introduced into *E. coli* alongside with the pkD46 plasmid, through electroporation. This plasmid codes for a recombinase, necessary to induce recombination. The recombination of the KmR cassette essentially knocked out the RNAse gene. The final step was to introduce the pCP20 plasmid encoding for a flippase that would induce recombination of the FRT sites. This last step removes the kanamycin resistance gene from the *E. coli* genome.

##### Results:

We have successfully deleted the RNAse from *E. coli*, as you can see in Figure 3, when we ran a PCR with primers around the RNAse region in our deletion strains the amplified fragment was around 423 bp, corresponding to the length of the scar in the genome left after the recombination of FRT sites. On the contrary, in the wild type strain, the amplified region with these primers is 956 bp long. This proves that we deleted the RNAse in *E. coli*.

![PCR amplification of the region around the RNAse gene in <i>E.coli</i>](https://static.igem.wiki/teams/5047/lacto/ecoligels.jpeg)

**Figure 3:** PCR amplification of the region around the RNAse gene in *E.coli* with deleted RNAse. On the right : same primers as on the left, amplification of the region around the RNAse in wild type *E.coli*.

## *L. lactis* RNAse deletion

##### Objective:

We wanted to create a *V. Velutina* gut commensal *L. lactis* strain without an RNAse, so that we can later transform it with our shRNA-producing plasmid. We want to ensure that the shRNA that the strain will produce will not be degraded by the bacterium.

##### Design:

Our protocol was based on a paper by Gu *et al.* (Gu et al. 2013) where *L. lactis* gene deletion was achieved by adapting the aforementioned lambda Red recombination protocol that we used for *E. coli* gene deletion.

We have attempted to do this on two *L. lactis* strains. The first strain was a kind gift by Ghent University from Peter Vandamme's lab. That strain was extracted from an Asian hornet's gut, and would probably have a higher chance of colonising the gut. Though we would prefer the gut commensal strain, as it has never been used for synthetic biology, we also had a second, more "domesticated" strain, *Lactococcus lactis* subsp. *cremoris* MG1363 that we received through our university.

![PCR amplification of the region around the RNAse gene in lacto](https://static.igem.wiki/teams/5047/lacto/lactorna.png)

**Figure 4:** Steps of RNAse deletion in *L. lactis* with the Red recombination protocol. A kanamycin resistance cassette (KmR), flanked by homology regions matching the *L. lactis* genome near the RNAse gene, is used to delete the RNAse gene. The KmR cassette also includes FRT sites, allowing for the removal of the kanamycin resistance from the genome after the RNAse knockout is achieved.

##### Results:

The gene deletion involved the introduction of several plasmids into *L. lactis*, for example the integration of pkD46 plasmid is the first step of the gene deletion, as it is necessary to induce recombination between the KanR cassette and the *L. lactis* genome (see intermediate step in Figure 4). After consulting literature, we concluded that the most common way of transforming *L. lactis* is through electroporation (Papagianni, Avramidis, and Filioussis 2007; Holo and Nes 1989; Gu et al. 2013).

We have had many attempts to electro-transform *L. lactis*.
Briefly, first we have tried to change the media in which we cultured *L. lactis*, both before and after electroporation. We grew both of our *L. lactis* strains in liquid GM17 media containing varying amounts of glycine to find the optimal concentration for growth for each strain.We also have added sucrose to both mediums, as well as MgCl2 and CaCl2 to the medium used to refresh the cells after the transformation.

Afterwards,we increased the amount of plasmid that we used during electroporation to 1ug.
Finally, we attempted to electro-transform our *Lactococcus* strains with different plasmids: for instance, one had a resistance to a different antibiotic, and another one that we knew has been introduced in the *Lactococcus lactis* subsp. *cremoris* MG1363 strain before.

Despite our numerous attempts, we could not insert any plasmid into *L. lactis* through electroporation. This is why we have decided to try another way of introducing extragenomic DNA into our target bacterium: by conjugation.

##### The conjugation strategy:

![Scheme with steps of <i>L. lactis</i> conjugation strategy.](https://static.igem.wiki/teams/5047/lacto/pmg.jpeg)

**Figure 5:** Scheme with steps of *L. lactis* conjugation strategy. First the oriT is added to the plasmid that will be introduced to *L. lactis* through Gibson assembly. Then, that plasmid is put into *E. coli*, that will introduce it to *L. lactis* when they are co-plated. Finally, to select the *l. lactis* that received the plasmid, cells are plated on LB with erythromycin and with no DAP.

To do the conjugation, we assembled a plasmid by Gibson cloning that we wanted to use as a backbone for shRNA production and added oriT to it, so that it could be transferred through conjugation. oriT is a short sequence necessary for the transfer of DNA from the donor to the recipient cell during conjugation. The plasmid was then transformed into *E. coli* strain JKE201 (Harms et al. 2017) (Figure 5).

*E. coli* and *l. lactis* were put on the same plate together, with 5 times more *L. lactis* cells compared to *E. coli* cells. The plates contained DAP, allowing *E. coli* to grow and transfer the plasmid to *l. lactis* through conjugation.

After 3 days of co-plating, we restreaked the cells from the plate we used for conjugation (LB+DAP) on a new plate containing LB, erythromycin, but without any DAP. As *E. coli* JKE201 strain cannot survive without DAP, and *Lactococcus* is not naturally resistant to erythromycin (Figure6C ), only *Lactococcus* that received the plasmid was selected. Thus, the colonies (Fig 6A and 6B)we saw on LB + erythromycin had integrated the plasmid.

![Erythromycin containing plates with plated <i>L. lactis</i>. ](https://static.igem.wiki/teams/5047/lacto/plates.jpeg)

**Figure 6:** Erythromycin containing plates with plated *L. lactis*.
Transformed *L. lactis* strains after 3 days of growth at 30°C: A. the hornet gut strain and B. *L. lactis* subsp. *cremoris* MG1363, both growing on erythromycin plates, confirming that they carry a plasmid conferring erythromycin resistance. C. Untransformed *L. lactis* strains after 4 days of growth at 30°C, with L.lactis subsp. *cremoris* MG1363 on the left and the hornet commensal strain on the right. No colonies are visible on either side.

To make sure that the transconjugant colonies we saw on our second plate were indeed *Lactococcus* we did a colony PCR with primers that bind to *L. lactis* genome. All colonies had bands of the expected size (around 1 kb) (Figure 7)

![Gel of amplified fragments of <i>L. lactis</i> genome](https://static.igem.wiki/teams/5047/lacto/lactogel.jpeg)

**Figure 7:**Gel of amplified fragments of *L. lactis* genome, with gut commensal *L. lactis* colonies on the left and *Lactococcus lactis* subsp. *cremoris* MG1363 on the right . The expected size is around 1 kb for both *L. lactis* strains.

##### Conclusion and outlooks:

We now have a protocol to insert plasmids into *Lactococcus* using conjugation and if we had more time we would proceed with our initial protocol to delete the RNAse.

## shRNA producing plasmid in *L. lactis*

##### Preemptive bioinformatic and research work:

For the design of the shRNA producing sequence, we identified 20 to 22 nucleotide regions within these genes that follow an AA dinucleotide sequence, as this is believed to optimise RNAi efficiency ("siRNA Design Guidelines | Technical Bulletin #506 | Thermo Fisher Scientific - CH," n.d.). These regions were then used as inverted repeats, separated by a short flexible loop, to generate our shRNAs. To choose the loop in our shRNA, we looked at a study where upon testing in human embryonic kidney 293T cells using a luciferase assay (Schopman et al. 2010), reported that shRNAs based on the mir-17 and mir-25 loops exhibited the strongest target repression, approximately 10-fold stronger than the 9-nt loop.

##### Objective:

We aimed to clone a plasmid that produces shRNA in order to silence specific developmental genes in the Asian hornet. While we were selecting which developmental Asian hornet genes we wanted to target, we started assembling the shRNA producing plasmids: for subsequent cloning, the backbone, promoter and terminator would be the same, and only the shRNA sequence would change. To assemble the archetype plasmid, we have used two control shRNA sequences: the first one corresponds to a DNA sequence used for CRISPR interference in the Schaerli lab (Santos-Moreno et al. 2020), while the second one corresponds to the sequence that was reported to have a high efficiency from the 2023 Estonian iGEM (“Project | Estonia-TUIT - iGEM 2023,” n.d.) pMG36E\_shRNA\_scaffold (Figure 8).

![Map of our shRNA producing plasmid with the pMG36E backbone](https://static.igem.wiki/teams/5047/project-description/shrnas.png )

**Figure 8:** Map of our shRNA producing plasmid with the pMG36E backbone, erythromycin resistance gene for selection, the constitutive PTSIIC promoter and rrnb t1-t7te terminator.

##### Steps:

1. Amplify promoter, shRNA, terminator

2. Amplify backbone

3. 4-fragment Gibson assembly

4. Transformation in L.lactis

##### Results:

We assembled the plasmid for both of our control shRNA sequences. However, upon sequencing, we found out that the newly assembled plasmids had some mismatches. The promoter had a dinucleotide deletion, but we speculated that it might not affect its function, as it was rather distant from the -10 and -35 regions. Both shRNA showed various mismatches. The terminator was assembled correctly (Figure 9).

![ Sequencing results of promoter-shRNA-terminator region](https://static.igem.wiki/teams/5047/lacto/clm.png )

**Figure 9:** Sequencing results of promoter-shRNA-terminator region of pMG36E backbone.

We decided to use the assembled promoter - backbone - terminator construct and replace the shRNA region, to get the correct sequence.

Since all shRNA sequences in different colonies had the exact same mismatches, we hypothesised that the shRNA inverted repeats formed a complex secondary structure during the initial PCR amplification, thereby generating incorrect sequences.
To avoid possible polymerase mistakes, we ordered shRNA sequences as long single stranded oligo with their complementary sequences, and annealed them together to form a double stranded fragment. We have ordered both of our test shRNA sequences and four of our target sequences (identified in the following *V. velutina* genes: 2 Chitin Synthase genes, Vacuolar ATPase and VgR genes).

**To assemble our new plasmids we tried in parallel two different approaches:**

![Schematic representation on the steps for the two cloning strategies ](https://static.igem.wiki/teams/5047/lacto/strats.jpeg)

**Figure 10:** Schematic representation on the steps for the two cloning strategies to assemble our shRNA producing plasmid.

###### **Gibson Assembly** (Fig 10A)

We designed a new primer pair on the [pMG36E\_promoter\_shRNA\*\_terminator] plasmid, with primers binding just outside the mismatched shRNA and facing outwards. With this, we could amplify the backbone with promoter and terminator. We ordered the shRNA sequences with flanking regions matching the new primer pair. In this way, the new shRNA can replace the old one between promoter and terminator.

###### **Restriction-ligation** (Fig 10B)

Since we sought to create a library of different shRNAs, and that potentially we might need to do it for both *L. lactis* and *S. cerevisiae*, we thought about a strategy that could be versatile and efficient. Having two restriction sites flanking the shRNA sequence would allow us to digest the backbone just once and to clone any sequence with the corresponding sticky ends. Thereby, we designed a new primer pair on the [pMG36E\_promoter\_shRNA\*\_terminator] plasmid, with primers binding just outside the mismatched shRNA and facing outwards, and carrying extensions. These extensions contained a SacI and a XhoI restriction site, respectively: the resulting PCR product could thereby be digested with these two restriction enzymes, that did not cut anywhere else on the [pMG36E\_promoter\_shRNA\*\_terminator] plasmid. We ordered the shRNA forward and reverse sequences as ssDNA oligo so that, upon annealing, they would have the appropriate sticky ends. In this way, the new shRNA can replace the old one in between promoter and terminator.

##### Results:

We performed ssDNA oligo annealing, PCR amplifications and Gibson Assembly, and restriction-ligation. Upon transformation of our new constructs, we got some colonies on the plates, suggesting that the cells contained either the correct plasmid, or the template, or a byproduct of the cloning.
Unfortunately, we did not manage to perform colony PCR on the candidates, and we did not manage to get a plasmid concentration high enough for sequencing (neither Sanger sequencing nor whole plasmid sequencing with nanopore). These observations seemed to point towards the fact that most of the cells did not contain the plasmid.

##### Troubleshooting :

We realised that most of our plates and liquid cultures contained an antibiotic concentration 5-10 times lower than the one recommended in the literature. We hypothesised that, if the antibiotic selection was low upon transformation, some cells might have grown even without the plasmid.

Thereby, we did another restreak on plates with higher erythromycin concentration in order to maximise the chances to get a colony with the right plasmid. We also tested other DNA-Clean up and Miniprep kits, to rule out the hypothesis that the issues lied in the sample preparation . We managed to increase DNA concentration by 10 folds, but it remained still very low (4-8ng/ul, measured through QuBit). We tried anyway to send the plasmids for Sanger sequencing, but unfortunately they had too low quality to be analysed.

At this point, we speculated that the shRNA secondary structure might interfere with polymerase amplification or prevent the correct assembly of fragments. Hoping that such an effect might be shRNA-specific, we wanted to try with our real shRNA, as their sequence is different from the controls. We were aware that the issue might lie in the hairpin structure and not within the specific DNA sequence.

We used the Gibson assembly method and assembled four shRNAs with pMG36E backbone and then performed the transformation into *E. coli*.

##### Results:

We obtained colonies on most of the plates. We performed colony PCR, but unfortunately no colony yielded the right band size on the gel, apart from one. We purified the PCR product and sent it for sequencing, however, the sequence had many mismatches.

##### Outlooks:

Our hypothesis is that the complex secondary structure that the shRNA sequence forms makes it hard to amplify and to clone. We would like to continue brainstorming ideas on how to make this cloning possible, and perhaps develop the restriction-ligation strategy further, as it does not require a PCR amplification step.

## Testing in yeast

#### Promoter characterisation

##### Objective:

To test the efficiency of our RNA interference and to avoid testing our shRNA on live animals, we decided to test the efficiency of the target degradation in a yeast model based on the work of the 2023 Estonian iGEM team (TUIT). We aimed to create our own yeast strain that expresses the RNA interference machinery, namely Argonaute proteins (AGO) and Dicer proteins (DICR) that are part of the RISC complex. More concretely, we created plasmids containing the different parts necessary for their subsequent insertion into the yeast genome. Furthermore, to quantify the efficiency of our shRNA, the objective was to introduce the shRNA producing plasmid as well as the plasmid containing the shRNA target downstream of GFP into the yeast model.

##### Design:

To express AGO and DICR in our yeast model, we used constitutive promoters that would ensure a stable expression level of the RISC complex. In our design, AGO is expressed by the promoter pPGK1, which was already characterised and registered in the iGEM registry of parts. However the pTEF1 promoter of the DICR plasmid was not.

To characterise pTEF1, we used an integrative construct that expresses the mCherry reporter, similar to how we wanted to express AGO and DICR proteins in *S.cerevisiae*. We also included the pADH1 promoter and the pCYC1 promoter in similar configurations as the standard for strong and weak promoter in *S. cerevisiae* (Figure 11):

1. Plasmid pDA95 (pTEF1 promoter)
2. Plasmid pDA120 (pCYC1 promoter)
3. Plasmid pDA122 (pADH1 promoter)

We linearized and transformed these constructs into *S.cerevisiae* and measured their expression levels of mCherry using plate reader and flow cytometry.

![Design of the plasmids containing distinct promoters for characterization of fluorescence levels](https://static.igem.wiki/teams/5047/yeast/three-plasmid.png)

**Figure 11:** Design of the plasmids containing distinct promoters for characterization of fluorescence levels.

To transform them in *S.cerevisiae* we learned a new transformation protocol for yeast, in which integrative plasmids have to be linearised before transformation. We did that with the BstBI restriction enzyme. Because these three plasmids can be integrated in the yeast genome multiple times, we first screened 12 colonies and chose the ones that showed the expected fluorescence level of one integration. To choose the right colonies, we were helped by Serge Pelet, an expert on *S.cerevisiae* at the University of Lausanne.

We measured the level of expression of mCherry reporter with both a plate-reader and a flow cytometer, to ensure a more reliable measurement. Plate-reader measures OD and fluorescence from a 96 well plate, by taking a measurement in each well every 10 minutes, allowing for a high throughput analysis of cell concentration and global fluorescence.
Flow cytometer measures fluorescence expression by running a laser through each cell individually, allowing for a more precise quantification of fluorescence. For instance, flow cytometry allowed us to discard colonies that presented two subgroups of cells, suggesting that not all of them had the same number of plasmid integration.

##### Results:

After choosing colonies presenting the expected expression level of mCherry, we prepared four biological replicates for each construct and we did final measurements on plate-reader (Figure 11) and flow cytometer (Figure 12). For plate-reader measurements, we grew cells in Synthetic defined (SD) yeast liquid medium, and used SD medium that is not inoculated by any cells as a control measure of the blank. For flow cytometry, our control is an empty vector of *S.cerevisiae* wild-type that does not contain any fluorescent plasmid.

![Flow cytometry results of mCherry red fluorescence measurements.](https://static.igem.wiki/teams/5047/yeast/four-histograms.png)

**Figure 12:** Flow cytometry results of mCherry red fluorescence measurements. The highest expression level is induced by the promoter pTEF1 (blue), as compared to pADH1 (yellow) and pCYC1 (green). The wild-type control (purple) did not contain any construct.

![Flow cytometry results of mCherry red fluorescence measurements.](https://static.igem.wiki/teams/5047/yeast/three-histograms.png)

**Figure 13:** Plate-reader results of mCherry red fluorescence measurements. The highest expression level is induced by the promoter pTEF1 (blue), as compared to pADH1 (yellow) and pCYC1 (green).

Both measurements (Figure 12 and 13) suggest that pTEF1 induces a higher mCherry expression than pCYC1 and pADH1, with the control in flow cytometer measurements having the lowest fluorescence. Therefore we uploaded pTEF1 as a new basic part (BBa\_K5047036) of the iGEM registry along with the results of the characterisation we performed for it.

#### Assembling AGO and DICR plasmids

##### Design:

Both AGO and DICR genes are native from *S. castelii* (Drinnenberg et al., RNAi in Budding Yeast, Science, 2009) and in the design of our plasmid we made sure to put them under the regulation of constitutive promoters for the insertion into the yeast genome. (Figure 14) To introduce the AGO and DICR plasmids into the genome of *S. cerevisiae* we first assembled two separate plasmids before combining them into one big plasmid including both parts.

![Design of the AGO and DICR plasmids](https://static.igem.wiki/teams/5047/yeast/aplasmid-bplasmid.png)

**Figure 14:** Design of the AGO and DICR plasmids. A. The DICR assembly includes the constitutive promoter pTEF1, the DICR gene and the tSTE2 terminator. B. The AGO assembly includes the constitutive promoter pPGK1, the AGO gene and the tSIF2 promoter.

##### Results:

The first step in the creation of these two plasmids was the amplification through PCR of the backbone, the promoters and the terminators. Both plasmids required pN1-011 as the backbone, additionally the DICR plasmid needed pTEF1, the 2 g-blocks and tSTE2, and for the AGO plasmid we needed pPGK1, the 3 g-blocks and tSIF2.

![gels pN1-011](https://static.igem.wiki/teams/5047/yeast/gelgel.png)

**Figure 15:**
A. Gel of amplified fragments pN1-011 (2603bp), pPGK1 (1060bp) and tSIF2 (552bp).
B. Gel of amplified pN1-011(2603bp).

Some fragments required several rounds of amplifications with changes in certain parameters like elongation time and annealing temperature. However, most fragments were amplified and ended up being at the expected size on the gel. (Figure 15) The only fragment that never looked clean on the gel was tSIF2 (Figure 16), but we simply proceeded with the assembly of the plasmid since the lower band seemed to be the right size.

![Gel of amplified tSIF2 ](https://static.igem.wiki/teams/5047/yeast/gel.png)

**Figure 16:** Gel of amplified tSIF2 (expected band at 552bp lower band, but we see higher band as well).

The DICR plasmid required only 2 rounds of Gibson assemblies, whereas the AGO plasmid required a little more troubleshooting (for details check the “engineering success” page). Ultimately we were able to create both the AGO and the DICR plasmid separately (Figure 17 and 18).

![Sequencing results of DICR plasmid](https://static.igem.wiki/teams/5047/yeast/green-seq.png)

**Figure 17:** Sequencing results of DICR plasmid using primers PR\_VV\_053 and PR\_VV\_054.

![Whole plasmid sequencing results of the final assembled AGO plasmid](https://static.igem.wiki/teams/5047/yeast/purple-seq.png)

**Figure 18:** Whole plasmid sequencing results of the final assembled AGO plasmid.

The next step required the amplification of both the AGO and the DICR transcriptional units (i.e. promoter, gene and terminator) from their respective plasmids, and the subsequent assembly of these fragments with the pSP599 backbone (Figure 19).

![Design of the assembly of AGO and DICR transcriptional units](https://static.igem.wiki/teams/5047/yeast/2baby-onebig.png)

**Figure 19:** Design of the assembly of AGO and DICR transcriptional units into one plasmid using the backbone (pSP599) containing ampicillin resistance.

However, we were unsuccessful in the assembly of the final combined AGO and DICR plasmid, which would have resulted in a plasmid of almost 13kb in size. We suspect this final assembly was unsuccessful due to its large size.

#### shRNA and target production

##### Design:

The next steps to determine how efficiently our shRNA could degrade its target were the design of the target and shRNA plasmids. To do so we created two separate plasmids, one which produces the shRNA, and one that contains the shRNA target downstream of the gene coding for GFP. In our design, we made sure to put the target sequence between the coding sequence of the GFP and the terminator. This ensures that while testing the efficiency of our shRNA if we observe a decrease in GFP production it is purely due to RNA interference and not due to transcriptional repression of the GFP. Indeed, if the target were positioned at the start of the gene, there’s a chance the shRNA could block transcription or translation by binding to the gene or the transcript. However, by positioning it at the end, transcription and translation proceed normally, and the only way the shRNA can function is by triggering the degradation of the transcript (Figure 20).

![Design of the shRNA and target plasmids](https://static.igem.wiki/teams/5047/yeast/two-plasmids.png)

**Figure 20:** Design of the shRNA and target plasmids. The shRNA plasmid includes the pGAL promoter, the shRNA and the tSIF2 terminator. The target gene plasmid included the pGAL promoter, the GFP followed by the target and the tCYC terminator.

To insert all three plasmids and validate their successful insertion and expression in yeast we made sure that each contained an auxotrophic marker. In order to test if all of these different parts were properly expressed within the yeast, we needed a triple auxotrophic yeast strain so we could grow it on minimal media and identify the triple supplemented yeast (Figure 21).

![Schematic of the experimental design for the insertion of three plasmids into yeast](https://static.igem.wiki/teams/5047/yeast/last-one.png)

**Figure 21:** Schematic of the experimental design for the insertion of three plasmids into yeast.

The shRNA plasmid contains a uracil auxotrophic marker, the target plasmid contains a tryptophan auxotrophic marker, the AGO + DICR plasmid contains a leucine auxotrophic marker. *Saccharomyces cerevisiae* is lacking the production of all three amino acids.

We did not have enough time to finalise the assembly of the plasmid containing the target downstream of GFP and the plasmid containing the shRNA. Therefore, the final characterisation of the efficiency of our target gene degradation in yeast could also not be performed.

##### Future perspective:

For the insertion of AGO and DICR into the genome of *S. cerevisiae* we could try to insert both plasmids separately. This would however require a quadruple auxotrophic yeast strain, since the shRNA and target plasmids would still have to be inserted as well. We believe that with sufficient time, this part of the project could be carried out successfully.

## Image recognition software

We successfully wrote a script that would allow us to upload images of hornets, either found online or received from the experts we interviewed. In this process, we learnt that the software worked best on images capturing a top view of the insect. Additionally, the insect size affect the inference of the model (see Figure 1). While we were able to adjust the size of the area containing the Asian hornet by modifying a parameter in the code, this adjustment had to be performed manually for each image. Current image recognition methods do not yet support automated image scaling. For simplicity, instead of performing complex image post-processing, we decided to feed our software with pictures taken at approximately the same distance from the hornet. Thereby, we adjusted the design of our box accordingly (link to relevant section of box).

![Influence of Hornet Orientation and Recognition Area Size on the Accuracy of the VespAI Image Recognition Software](https://static.igem.wiki/teams/5047/results/fig22.png)

**Figure 22:** Influence of Hornet Orientation and Recognition Area Size on the Accuracy of the VespAI Image Recognition Software.
This figure illustrates the impact of hornet orientation and the size of the recognition area on the performance of VespAI, a deep learning-based image recognition model for hornet detection.
**(A)** *Top view* of a hornet with an excessively large recognition area, resulting in decreased detection accuracy.
**(B)** *Top view* with an optimally sized recognition area, yielding the most accurate predictions.
**(C)** *Top view* with a recognition area that is too small, negatively affecting detection performance.
**(D)** *Side view* of a hornet with an optimally sized recognition area; no detection was made as the model struggles to identify side views accurately

According to VESPAI developers, the model demonstrates high accuracy (around 90%) in identifying Vespa velutina (“Andrw3000/Vespai,” n.d.). To further ensure specificity and minimize off-target identifications, we set the threshold for opening the detection box at 90% probability to prevent the software from mistakenly recognizing other species as the Asian hornet.

![Vespa crabro](https://static.igem.wiki/teams/5047/bioinf/vespai/vespa-crabro-01.jpg)

Vespa crabro

![Vespa mandarinia](https://static.igem.wiki/teams/5047/bioinf/vespai/vespa-mandarinia-02.jpeg)

Vespa mandarinia

![](https://static.igem.wiki/teams/5047/bioinf/vespai/vespa-mandarinia-02.jpg)

Vespa mandarinia

![](https://static.igem.wiki/teams/5047/bioinf/vespai/vespa-vulgaris-01.jpg)

Vespa vulgaris

![](https://static.igem.wiki/teams/5047/bioinf/vespai/vespula-germanica-01.jpeg)

Vespula germanica

![](https://static.igem.wiki/teams/5047/bioinf/vespai/vespula-germanica-02.jpeg)

Vespula germanica

![](https://static.igem.wiki/teams/5047/bioinf/vespai/vespula-pensylvanica-01.jpeg)

Vespula pensylvanica

![](https://static.igem.wiki/teams/5047/bioinf/vespai/vespula-pensylvanica-02.jpeg)

Vespula pensylvanica

![](https://static.igem.wiki/teams/5047/bioinf/vespai/anoplius-nigerrimus-01.jpeg)

Anoplius nigerrimus

![](https://static.igem.wiki/teams/5047/bioinf/vespai/anoplius-nigerrimus-02.jpeg)

Anoplius nigerrimus

![](https://static.igem.wiki/teams/5047/bioinf/vespai/cerceris-rybensis.jpg)

Cerceris rybyensis

![](https://static.igem.wiki/teams/5047/bioinf/vespai/chalcis-sispes.jpg)

Chalcis sispes

![](https://static.igem.wiki/teams/5047/bioinf/vespai/drycosmus-kuriphilus-1.jpg)

Dryocosmus kuriphilus

![](https://static.igem.wiki/teams/5047/bioinf/vespai/ganaspis-brasiliensis-male.jpg)

Ganaspis brasiliensis

![](https://static.igem.wiki/teams/5047/bioinf/vespai/netelia-fuscicornis.jpeg)

Netelia fuscicornis

**Figure 6: Image recognition software tested on the off-targets identified in the target gene identification analysis. The box surrouding the insect shows the prediction of the software. In the pictures lacking boxes, the software was not able to make a prediction.**

## Model

Through the design of a bee and hornet population model, we aimed to test the efficiency of the two initial aspects of our solution: First, we wanted to verify our hypothesis that targeting larvae instead of adult workers would be more efficient at reducing hornet pressure on beehives. Then, because we were concerned about our idea of releasing GMOs in the environment as a biocontrol against an invasive species, we wanted to prove that it would be more efficient to deliver shRNA with a gut-commensal bacterium able to spread among an Asian hornet population, instead of directly feeding hornets with shRNA.

To design our model, since we started with a limited knowledge of mathematical models and their use in computational biology, one of our assistants, Gábor Holló, taught us how to solve ordinary differential equations (ODEs).

In parallel, we searched for literature on bee and hornet population dynamics, which led us to find a paper, where they modelled a “simple” bee population with immature and mature bees (Romero-Leiton et al. 2022). This was very useful to us, since we wanted to build a model to compare the effect of our engineered bacteria between immature and mature Asian hornets. Since currently there is little research on Asian hornets, we decided to adapt the “bee model” as a “hornet model”, assuming that both species are evolutionary close enough to have similar population dynamics.

We added a simple infection dynamic to the hornet population to simulate the effect of our engineered bacterium.

To analyse our results, we examined the behaviour of our model in function of the infection rate (k) of adult hornets by our bacterium, and we tested two scenarios, one in which larvae are not infected (kh = 0), the other one in which adult workers transmit the infection to the larvae at a cross-infection rate (kh = 100 (population time)-1).

We also calculated the threshold of hornet concentration at which the bee population collapses. We found that our infection and cross-infection rates must maintain the hornet population under 36.92% of its maximal capacity to save beehives.

![Effect of infection by our engineered bacteria (infection rate (k)) and transmission to the larvae (cross-infection (k<sub>h</sub>))](https://static.igem.wiki/teams/5047/model/plot-abc-final-green-bee-survival.png)

**Figure 24:** Effect of infection by our engineered bacteria (infection rate (k)) and transmission to the larvae (cross-infection kh). In green, we highlight the direction in which we aim to push the population curves to ensure survival of honeybees, by increasing the cross-infection (kh) rate through our gut-commensal bacterium.

(24A, 24B, 24C) - Hornet larvae are not infected (not targeted by our engineered bacteria). In this scenario, transmission rate from infected adult workers to larvae is set to 0 (kh = 0 (population time)-1).

(24D, 24E, 24F) - Hornet larvae are infected. In this scenario, transmission rate from infected adult workers to larvae is set to 100 (kh = 100 (population time)-1).

(24A, 24D) - Mature bee population in function of infection rate (k). (24B and 24E) - Mature hornet population in function of infection rate (k). The solid blue line represents the stable fixed point, the dashed red lines represent the unstable fixed point. The dotted black line represents the maximal hornet concentration (0.3692) under which we want to limit the hornet population, since bee population collapses at this concentration.

(24C, 24F) - Example trajectories of each sub-population plotted over time, starting without mature hornets and with 0.1% of immature hornets, simulating the beginning of an Asian hornet invasion. These trajectories were calculated with a 0.1 (week)-1 infection rate, under which the bee population collapses if there is no cross-infection in the hornet population.

In Figures 24C and 4F, example trajectories of each sub-population are plotted over time, starting without mature hornets and with 0.1% of immature hornets, simulating the beginning of an Asian hornet invasion. These trajectories were calculated with a 0.1 (week)-1 infection rate, under which the bee population collapses if there is no cross-infection in the hornet population.

From Figure 24B and 24E, we see that the hornet population drops lower than the threshold value of critical hornet concentration (H’), as the infection rate (k) of adult workers increases.
When there is cross-infection (kh) between adult workers and larvae, the bee population stays at a much higher value (Figure 24D), even though the infection rate of adults is lower than in the first row.

This suggests that **we would not need a high number of hornets to be infected initially by our engineered bacteria if they spread the infection to the larvae at a high rate**, meaning that our engineered bacteria efficiently colonises the guts of the Asian hornet.
This fits the implementation of our solution in beehives under pressure from the Asian hornet: We are indeed not aiming at infecting as many Asian hornets as possible, since it would make it more difficult to protect other species, but we are rather aiming at allowing **only** Asian hornets to access our engineered bacteria, and thus we need to maximise the transmission of infection in the colony if only a few adult workers get infected.

This supports our idea that **both targeting larvae and delivering shRNA through our engineered bacteria would be a more efficient approach to limiting the spread of the Asian hornet.**

We chose to stick to a “simple” model, as we believe it is already complex enough to answer our questions. However, if we had more time, we would still improve it by confirming the behaviour and dynamics of Asian hornet populations with an expert, since there is currently limited knowledge on this invasive species.

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