temporary image of the engineering cycle: design, build, test, learn

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.

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.

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).

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).

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).

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).

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.

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.

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

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.

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.

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).

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).

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.