iGEM 2022 — Team unsw-australia

July

!

**Week 1: 25th - 31st**

---

25th July | Eloise & Ezekiel

* Plasmid PCR of the pet19b backbone to prepare for Gibson Assembly.

26th July | Div, Eloise & Ezekiel

* Visualised PCR from day prior on an agarose gel using gel electrophoresis.

27th July | Div, Eloise & Ezekiel

* Repeated PCR with different conditions; ran another gel.

29th July | Div, Eloise & Ezekiel

* Troubleshooted primers for pet19b amplification.

!

August

!

**Week 2: 1st - 7th**

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2nd August | Div, Eloise & Ezekiel

* Plasmid PCR and Gel Visualisation.
* DNA for 4 fungal proteins arrived and were prepared.

3rd August | Div, Eloise & Ezekiel

* Gibson assembly and transformation of PstSCR1, PstHXT1, Pgt\_IaaM and Pst\_12806 into NEB TURBO E.coli.

4th August | Div & Ezekiel

* Checking plates and streaking.

5th August | Div & Ezekiel

* cPCR for SCR1 and IaaM and ran DNA gel electrophoresis.

**Week 3: 8th - 14th**

---

8th August | Div & Ezekiel

* Plate Streaking.
* Prepared overnight culture for the colonies used in the cPCR on the 5th.

9th August | Div, Eloise & Ezekiel

* Miniprep of 4 promising SCR1 colonies from overnight cultures.

12th August | Eloise & Ezekiel

* Repeated cPCR for SCR1 and IaaM.

**Week 4: 15th - 21st**

---

15th August | Eloise & Ezekiel

* Pet19b digest that was forgotten in the incubator (El & Ez).

16th August | Eloise & Ezekiel

* Miniprep of colonies from cPCR on 12th August.
* 3 Colonies of SCR1 in NEB TURBO E.coli were prepared and sent to for Ramaciotti Centre of Genomics for Sequencing.
* Miniprep of pet19b backbone.
* Gibson Assembly for HXT1 and Pst\_12806.

19th August | Div, Eloise & Ezekiel

* Transformed SCR1 into NEB T7 Express E.coli.
* Transformed HXT1 and Pst\_12806 into NEB TUBRO E.coli.

**Week 5: 22nd - 28th**

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22nd August | Div, Eloise & Ezekiel

* Performed cPCR of Pst\_12806 (6 colonies) and PstHXT1 (6 colonies) in NEB TUBRO E.coli.

23rd August | Eloise & Ezekiel

* Repeated cPCR of Pst\_12806 (6 colonies) and PsTHXT1 (6 colonies) in NEB TUBRO E.coli with different colonies.

25th August | Eloise & Ezekiel

* Set up overnight cultures for protein expression of PstSCR1.

26th August | Div, Eloise & Ezekiel

* Day culture and harvest for protein expression of PstSCR1.
* Pet19b gel extraction.

!

September

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**Week 6: 29th August - 4th September**

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29th August | Eloise & Ezekiel

* Gibson assembly & transofrmation of Pst\_12806 and PstHXT1 into NEB TURBO E.coli.
* Prepared for PstSCR1 protein expression optimization on the following day.
* Performed protein expression check (Bugbuster) for PstSCR1 day culture from the 26th of August.

30th August | Eloise, Ezekiel & Roger

* Lysed cells by sonication and purified PstSCR1 protein with gravity columns.
* Set up overnight culture of PstSCR1 in NEB T7 Express E.coli for protein expression optimisation.

31st August | Div, Ezekiel & Ryan

* Performed cPCR on transformed sampled from 29th August; results showed unsuccessful Gibson Assembly.
* Performed day cultures on PstSCR1 NEB T7 Express E.coli for protein expression optimization at different IPTG concentrations and temperatures.
* Harvested the PstSCR1 NEB T7 Express E.coli cultures that were incubated at 30℃ and 37℃ during the day culture; 16℃ culture was incubated overnight.

1st September | Div, Roger & Ryan

* Harvested the 16℃ culture PstSCR1 NEB T7 Express E.coli for protein expression optimization at different IPTG concentrations and temperatures.
* Performed cPCR on PstHXT1 and Pst\_12806 transformations from the29th August.

2nd September | Div, Eloise & Ezekiel

* Performed protein expression check (Bugbuster) for PstSCR1 day culture from the 26th - 27th of August.

**Week 7: 5th - 11th**

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5th September | Div & Ezekiel

* Restriction digest of Pet19b.
* Gibson assembly and transformation of Pst\_12806 into NEB TURBO E.coli.

8th September | Div & Ezekiel

* Set up 2 overnight cultures of Pst\_12806 in NEB TURBO E.coli.

9th September | Div, Eloise & Ryan

* 2 samples of Pst\_12806 in NEB TURBO E.coli were prepared and sent to Ramaciotti Centre of Genomics for sequencing.

**Week 8: 12th - 18th**

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12th September | Eloise & Ezekiel

* Set up overnight culture of PstSCR1 in NEB T7 Express E.coli in optimal conditions (inferred from previous protein optimisation).

13th September | Eloise, Roger & Ryan

* Performed day culture and harvest on PstSCR1 NEB T7 Express E.coli for protein expression.

14th September | Div & Ezekiel

* Performed protein expression check (Bugbuster) for PstSCR1 day culture from the 13th of September.
* Arrival of TaISP and SERK3B gBlocks were prepared and concentration was measured on NanoDrop.

15th September | Div, Eloise, Ezekiel & Ryan

* Gibson assembly and transformation of TaISP and SERK3B into NEB TURBO E.coli.

16th September | Eloise & Ezekiel

* cPCR of TaISP and SERK3B transformation from the 15th of Septmeber and Gel visualisation.

**Week 9: 19th - 25th**

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19th September | Div, Eloise & Ezekiel

* Training on AKTA Start for purification of mCerulean and mVenus.

20th September | Eloise, Ezekiel, Roger & Ryan

* Training on AKTA Start for purification of mCerulean and mVenus.
* Ran SDS-gel for prufication on 19th September.
* Repeated cPCR of TaISP and SERK3B transformation from 15th September and Gel visualisation.

21st September | Div, Roger, & Ryan

* Ran SDS-gel for purification on 20th September.
* Repeated cPCR of TaIS and SERK3B transformation from 15th September and Gel visualisation.

22nd September | Div & Ezekiel

* Repeated Gibson Assembly and transformation of TaISP and SERK3B into NEB TURBO E.coli.
* Transformed Pst\_12806 into NEB T7 Express E.coli.

23rd September | Div, Eloise, Ezekiel & Roger

* cPCR of TaISP and SERK3B transformation from 22nd September and Gel visualisation.

**Week 10: 26th September - 2nd October**

---

26th September | Eloise & Ezekiel

* Repeated cPCR of TaISP and SERK3B transformation from 22nd September and Gel visualisation; observed band of correct bp.
* Set up overnight culture of Pst\_12806 in NEB T7 Express E.coli for protein expression.

27th September | Div, Eloise, Ezekiel & Roger

* Ran DNA gel electrophoresis for cPCR on the 26th of September.
* Preformed day culture and harvest on Pst\_12806 NEB T7 Express E.coli for protein expression.

28th September | Div, Roger & Ryan

* Restreaked colonies for SERK3B in NEB TURBO E.coli.
* Reran DNA gel electrophoresis for cPCR on 26th September.

29th September | Div, Ezekiel & Ryan

* Set up overnight culture of TaISP in NEB TURBO E.coli for miniprep.

30th September | Eloise

* Miniprep of TaISP in NEB TURBO E.coli; low concentration yielded.

!

October

!

**Week 11: 3rd October - 9th October**

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3rd October | Eloise & Ezekiel

* Set up overnight culture of TaISP in NEB TURBO E.coli for minirpep.
* Transofmred TaISP into NEB T7 Express E.coli.
* Set up overnight culture of Pst\_12806 in NEB T7 Express E.coli for protein expression.

4th October | Div, Eloise & Ezekiel

* Miniprep of TaISP in NEB TURBO E.coli.
* Performed day cultures on Pst)12806 NEB T7 Express E.coli for protein expression optimization at different IPTG concentrations and temperatures.
* Harvested the Pst\_12806 NEB T7 Express E.coli cultures that were incubated at 30℃ and 37℃ during the day culture; 16℃ culture was incubared overnight.

5th Ocotber | Div, Roger & Ryan

* Harvested the 16℃ culture of Pst\_12806 NEB T7 Express E.coli culture from day prior.
* Preformed protein expression check (Bugbuster) for PstSCR1 day culture from the 4th - 5th of October.
* 4 colonies of TaISP in NEB TURBO E.coli were prepared and sent to Ramaciotti Centre of Genomics for sequencing.

6th October | Eloise & Ryan

* Set up overnight culture of TaISP in NEB T7 Express E.coli for protein expression.

7th October | Eloise

* Performed day culture and harvest on TaISP NEB T7 Express E.coli for protein expression.

8th October | Div & Ezekiel

* Performed FRET assay between TaISP and PSt\_12806 proteins and MCerulean & mVenus proteins (controls).

!

Integration of the engineering cycle into our project design

.

Implementation of the synthetic biology engineering cycle was at the forefront of our minds when structuring our project for this year. The aim of our project was to use a modelling approach in designing inhibitory peptides against fungal effector proteins in stem rust. Following the de novo design of these peptides, our wet lab team intended to measure their binding with the fungal target proteins in the laboratory using a FRET assay. We had planned for the information gained from this test to feed back into the dry lab and allow further improvement in the peptide design.

Additionally, this cycle could be endlessly repeated to yield several strongly-binding peptides for implementation in future applications. Due to constraints surrounding the time required to design the inhibitory peptides, we were unable to synthesise them in the lab before the deadline. This could possibly be the task of a future iGEM team, allowing completion of our initially intended engineering cycle.

Despite this limitation, our team discovered that there were other aspects of our project that aligned with the criteria for engineering success. These included:

1. Applying various modelling techniques in designing the peptides
2. Wet lab investigation of the native interaction between effector proteins and their plant protein counterparts

With the aid of advice from experts, our wet lab, dry lab and human practices teams worked collaboratively to weave various stages of the synthetic biology engineering cycle into the structure of our project this year.

##

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In silico design of inhibitory peptides against fungal rust effector proteins

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With such a large portion of our project attributed to the modelling efforts of our dry lab team, we came to realise that the process of designing the inhibitory peptides themselves resembled an iteration of the engineering cycle. These short peptides were designed to prevent effector proteins in stem rust from binding to their corresponding protein binding partners in the plants’ immune system. In preventing this binding, the function of the plant immune system should remain intact and promote a more well-rounded immune response to the pathogen.

# Cycle 1 - Identify active sites

---

## Design

Unique fungal protein targets in stem rust were identified through research and a subsequent literature review of effector proteins in *Puccinia* rust species
([Experiments](https://2022.igem.wiki/unsw-australia/experiments)).
Further research of literature allowed us to find a corresponding protein in the plant immune system with which the target protein naturally binds. We then used these known proteins as known templates for the design of our own inhibitory peptides.

## Build

We found the sequence of each fungal protein through literature and used these as templates to produce a sequence with the GGSG linker and mVenus fluorescent protein attached.
Two versions of each fungal protein sequence were created, one with the fluorescent protein attached at the N-terminus and another at the C-terminus (Part: [BBa\_K4467007](http://parts.igem.org/Part%3ABBa_K4467007),
[BBa\_K4467008](http://parts.igem.org/Part%3ABBa_K4467008),
[BBa\_K4467009](http://parts.igem.org/Part%3ABBa_K4467009),
[BBa\_K4467010](http://parts.igem.org/Part%3ABBa_K4467007),
[BBa\_K4467011](http://parts.igem.org/Part%3ABBa_K4467007),
[BBa\_K4467012](http://parts.igem.org/Part%3ABBa_K4467007)).
We then produced 3D structure of both types of sequences using AlphaFold (for more information regarding our AlphaFold modelling, see
[Modelling](https://2022.igem.wiki/unsw-australia/model))

![](https://static.igem.wiki/teams/4467/wiki/engineering-diagrams/engalpha.svg)

## Test

The AlphaFold results were qualitatively assessed by inspecting the structure of each protein, ensuring the fluorescent protein, mVenus, wasn’t obstructing or interacting with the active site of the fungal protein. The confidence/ pLDDT scores of each AlphaFold structure were then assessed to ensure the structure was of adequate accuracy for further modelling simulations. The AlphaFold outputs of best structure and pLDDT scores were selected for subsequent analysis.

![](https://static.igem.wiki/teams/4467/wiki/engineering-diagrams/engnotinteract.svg)

![](https://static.igem.wiki/teams/4467/wiki/engineering-diagrams/enginteracting.svg)

## Learn

These pairs of structures were inputted in ClusPro (Porter et al., 2017) where the protein target was the receptor and the peptide was the ligand for docking simulations. We then took the top 10 balanced docking structures to record which regions of the peptide were interacting with the protein target. These regions were identified using Pymol to view peptide residues that were within 5 Angstroms of the target protein and polar contacts between the peptide and protein target. After determining the main interface areas of the peptide sequence we used this to design peptides.

![](https://static.igem.wiki/teams/4467/wiki/engineering-diagrams/engfcluspro-1.svg)

![](https://static.igem.wiki/teams/4467/wiki/engineering-diagrams/engcollectinterface.svg)

# Cycle 2 - In silico design of peptides

---

## Design

To achieve the goal of producing a peptide to bind to the protein target, we must better understand how the template protein interacts with the protein targets. This was done by using the predicted active regions of the template peptide to design peptides that only possess different combinations of these regions. Furthermore, to better understand how the structure of the active site region behaves, we built peptides that possessed the same combinations but replaced the non active site regions with similar sequences that have known 3D structures.

![](https://static.igem.wiki/teams/4467/wiki/engineering-diagrams/engpepactiveonly.svg)

![](https://static.igem.wiki/teams/4467/wiki/modelling/subpep2.svg)

## Build

These peptide sequences are then used to build 3D structures using AlphaFold. These structures were filtered by pLDDT score and by how well the active site regions are exposed for docking.

## Test

These filtered structures were then run as ligands with the target protein in the docking software, acting as a receptor to generate docking simulations.

## Learn

After generating docking structure data, we learnt that we had multiple issues that needed to be addressed in order to engineer a peptide to interact with the target protein. These issues consist of:

1. Manually annotating the docking structure data to identify a more accurate active site is not feasible due to the size of the data.
2. Relying on polar contacts and an arbitrary distance value between ligand-receptor residues is not sufficient to identify the active site of the peptide.
3. Different protein-protein docking software should have been used to benchmark against known docking structures to identify the most accurate and reliable software.
4. A variety of protein-protein docking software should have been used to generate more data to accurately predict the active site regions.

Knowing this information, we need to produce scripts for active site annotation to handle the data analysis workload, use different docking software such as GalxyPepDock to generate more docking structure data to verify our findings and to assess which docking software is better suited for our protein. Furthermore, we should have also analysed the active sites of the peptide without the GGSG linker and fluorescent protein attached, so we can compare the differences in active sites.

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Understanding the binding of fungal effector proteins and their native plant protein counterparts

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As an initiative to contribute towards future iGEM teams, our wet lab aimed to study the interaction between the selected fungal and plant proteins. The purpose of studying this interaction was to uncover the nature of the native binding between fungal effector proteins and the plant immune system proteins. We hope future iterations of the engineering cycle will result in improved design of the fluorescent tags for FRET analysis and the application of quantitative experimental techniques, for example mass spectrometry.

Throughout our literature search for fungal effector proteins, we found four viable targets:
Pgt\_IaaM (Part: [BBa\_K4467004](http://parts.igem.org/Part%3ABBa_K4467004)),
PstHXT1 (Part: [BBa\_K4467003](http://parts.igem.org/Part%3ABBa_K4467003)),
PstSCR1 (Part: [BBa\_K4467001](http://parts.igem.org/Part%3ABBa_K4467001)) and
Pst\_12806 (Part: [BBa\_K4467002](http://parts.igem.org/Part%3ABBa_K4467002)).
However, due to time constraints and the integration of advice from fungal experts (see Megan Lenardon in
[Integrated Human Practices](https://2022.igem.wiki/unsw-australia/human-practices)
), progress on some of the fungal proteins was discontinued throughout the year. Consequently, the engineering cycle was only fully completed for the fungal protein
Pst\_12806 (Part: [BBa\_K4467002](http://parts.igem.org/Part%3ABBa_K4467002)) and plant protein
TaISP (Part: [BBa\_K4467005](http://parts.igem.org/Part%3ABBa_K4467005)).

## Design

With the aim to investigate the interaction between our selected fungal effector proteins and their binding partners in plants, we began to plan possible methods for our experiments, eventually settling on a FRET assay. As FRET is highly dependent on the presence of fluorescent proteins, a variety of Puccinia effector proteins were modelled using AlphaFold to predict whether the addition of the fluorescent protein mCerulean and Histidine tag would disrupt the proteins’ structure when added to the N or C terminal. Likewise, AlphaFold modelling and inspection of the protein structure was performed on the corresponding plant proteins when mVenus and a Histidine tag was added to either terminal. The highest confidence proteins with little changes in structure following addition of the fluorescent tag were selected for synthesis in the lab
(see [Modelling](https://2022.igem.wiki/unsw-australia/model)). Each of the chosen proteins (Pgt\_IaaM, PstHXT1, PstSCR1, Pst\_12806, TaISP and SERK3B) were designed to allow for Gibson assembly into a pet19b plasmids (Figure 1 & 2). In the design of these inserts, a TEV site was added between the fluorescent protein and the protein coding sequence to ensure mCerulean/ mVenus was able to be cleaved if we decided to perform an experiment not requiring the fluorescent tags.

![](https://static.igem.wiki/teams/4467/wiki/engineering-diagrams/pst-12806.png)

Figure 1: Pst\_12806 + mCerulean in pET-19B (6879bp)

![](https://static.igem.wiki/teams/4467/wiki/engineering-diagrams/taispwheel.png)

Figure 2: TaISP + mVenus in pET-19B

## Build

The DNA sequence we designed for the fluorescent fungal and plant fusion proteins were ordered as gBlocks, inserted into a pet19b plasmid through Gibson assembly and transformed into NEB TURBO *E. coli* . Single colonies were selected for colony PCR screening to ensure the correct length of insert was introduced into the plasmids. The plasmid DNA of successful colonies were isolated by Miniprep, sequenced at the Ramaciotti Centre for Genomics and transformed into NEB T7 Express *E. coli*  cells for subsequent protein expression. The fluorescent fusion proteins for Pst\_12806 and TaISP were expressed in *E. coli*  and the lysate was utilised for FRET experimentation. Additionally, this method was also performed to express the fluorescent proteins, mCerulean and mVenus, by themselves. These protein samples were purified using gravity columns and the AKTA Start purification system, and were used as a negative control for the FRET experiment.

## Test

We tested the binding between proteins through a FRET assay, taking advantage of the overlapping excitation and emission spectra of mCerulean and mVenus to detect interaction between TaISP and Pst\_12806 (Demir et. al., 2017). The samples from prior protein expression were lysed through sonication, centrifuged and filtered. The Nanodrop measured the concentration of protein in each sample, which was then utilised to calculate dilutions for standardising protein concentrations. Serial dilutions the proteins were prepared
(see [Modelling](https://2022.igem.wiki/unsw-australia/model)) in a 96-well FRET plate and analysed inside the plate reader. Light corresponding to the excitation spectra of mCerulean was used to excite the fluorescent proteins, while light with a wavelength corresponding to the emission spectra of mVenus was detected.

## Learn

Our project was not without fault. In undergoing our design-build-test methodology, we have continued to evolve and learn from our mistakes, and plan for the future. Here, one of the first roadblocks we encountered involved the insertion of the gene sequences into the pET-19b plasmid. We used a plasmid backbone that already contained another insert, which had to be cut out. We initially used XbaI and BamHI to cut the insert, though XbaI cut further away from the insert, leaving a small overhang that would disrupt the Gibson assembly process when trying to insert our fungal protein sequence. As such, we decided to use a different restriction enzyme, NcoI, which cut closer to the insert site to avoid such issues.

Furthermore, the expression and purification of the fungal proteins and plant binding partners also presented a challenge. The expression of the TaISP plant protein, tagged with mVenus, realised some fluorescence, though this was after rounds of optimisation of the temperature and IPTG inducer concentration conditions to use. To overcome such low expression, naturally we upscaled the expression system to a larger culture volume to produce larger amounts. On the other hand, the Pst\_12806 fungal protein proved even more difficult. Though there was nothing in the literature accounting for the expression of Pst\_12806 in an E. coli host, we aimed to try, nonetheless. For this protein, there was no fluorescence seen in the sample, perhaps due to low levels of expression. In this case, we would need to upscale into larger culture volumes like we did for TaISP. More worrisomely, perhaps when modelling the proteins on AlphaFold and designing the mCerulean3 tag was there a significant structural change that impacted its ability to fluorescence, something that was unbeknownst to us. If this was the case, we would need to revisit our models and reiterate our design of the conjugate protein, one that has a longer linker sequence connecting the fluorescent tag and the fungal protein sequences. This may be true seeing how when we expressed mVenus and mCerulean3 by themselves as negative controls, we saw large amounts of fluorescence produced, indicative of a high protein yield.

Following on from this, perhaps it is best to heed the advice given by our academic consultants (see [Human Practices](https://2022.igem.wiki/unsw-australia/human-practices)), stating that it is best to use a model eukaryotic fungus like Saccharomyces cerevisiae. In doing so, we may account for post-translational modifications or specific folding that occurs in the presence of chaperone proteins. This may be a focus in the future cycle to generate usable amounts of the Pst\_12806 protein to assay its binding with our peptide candidates.

Lastly, one of the focal points of our experimentation revolved around the use of FRET. When we performed our FRET assay, the presence of errors in our design ensured our results were inaccurate. Here, our lack of sufficient controls proved this true, mostly due to our inexperience with such a technique. In future, by ensuring we have all the necessary parameters to complete a reliable and accurate assay, only then can we measure the binding affinity of our peptide and the fungal protein target to explore its potential as a treatment of in controlling cereal rust infections

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References

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* Demir, HV, Govorov, A & Hernández Martínez, PL 2017, Understanding and Modeling Förster-type Resonance Energy Transfer (FRET) : FRET-Applications, Vol. 3, Springer Singapore, Singapore.
* ROMERA-PAREDES, B., NIKOLOV, S., JAIN, R., ADLER, J., BACK, T., PETERSEN, S., REIMAN, D., CLANCY, E., ZIELINSKI, M., STEINEGGER, M., PACHOLSKA, M., BERGHAMMER, T., BODENSTEIN, S., SILVER, D., VINYALS, O., SENIOR, A. W., KAVUKCUOGLU, K., KOHLI, P. & HASSABIS, D. 2021. Highly accurate protein structure prediction with AlphaFold. Nature, 596, 583-589.
* PORTER, K. A., XIA, B., BEGLOV, D., BOHNUUD, T., ALAM, N., SCHUELER-FURMAN, O. & KOZAKOV, D. 2017. ClusPro PeptiDock: efficient global docking of peptide recognition motifs using FFT. Bioinformatics, 33, 3299-3301.

.

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Introduction

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*Puccinia* are pathogenic fungi known to cause infections in plants known as rusts which include over 120 genera and 6000 species (Sadys et.al, 2017) Their life stages include the production of sexual spores as well as up to four different asexual spores. Due to their large quantities and ultra-light nature, they are aerodynamically efficient and can travel long distances over short periods. Additionally, they possess thick walls and high pigmentation which protects them against UV radiation. This ensures that they remain viable for long distances. On advancement of infection, it manifests as a reddish/brown powder on the leaf surface due to formation of urediniospore masses. This can lead to water and nutrient loss through the restriction of photosynthetic areas, causing large yield losses in crops around the world.

In their obligately parasitic relationship, the fungi develop a specialised organ for the transfer of nutrients, from the plant host cells to the fungal thallus, called the haustorium. Through our initial literature review we found an haustorium-specific effector protein, Pst-12806, which affects chloroplast function in plants upon translocation. Effector proteins are expressed by plant pathogens and are involved in the infection of plant species (Mysore et.al, 2022) Expression of this protein plays a vital role in the pathogenesis of the fungus and in suppressing the basal immunity of the plant. It has been shown to reduce electron transport rate and photosynthesis in plants upon interaction with the wheat TaISP protein - a component of the cytochrome complex (Wang et.al, 2019)

Consequently, Puccinia spp. pose significant threat to wheat crop yields, with potential for disastrous economic and social consequences. Our solution proposes the design of a novel peptide-based inhibitor to prevent the growth of Puccinia spp. We began with a focus on determining appropriate targets to design our peptide-based inhibitor around. In this process, we screened existing literature and analysed protein databases using various bioinformatic tools such as GeneMania and NCBI’s BlastP (Modelling Page). This yielded an initial short list of proteins important to the pathogenesis and function of Puccinia spp. Additionally, we decided early on in our project to use a fluorescence based approach to analysing the interaction/binding affinity between our peptide and target protein. Consequently, we ran AlphaFold models to verify the feasibility of conjugating mVenus fluorophores to our proteins. This helped to narrow down our shortlist of protein targets to: PstSCR1, Pst\_12806, PstHXT1 and Pgt-Iaam – all conjugated to a mVenus fluorophore.

The next stage of our project focussed on designing novel peptides to specifically bind and inhibit our shortlisted proteins. This involved modelling peptide-protein interactions on RossettaDock and PyMol. The feasibility of each peptide-fluorophore combination was not of significant importance – due to the minute differences in sequence/size between peptides. Additionally, we decided to include known protein binding partners for out short-listed proteins. These included TaISP and SERK3B. This resulted in a short list consisting of our novel peptides, TaISP and SERK3B – all conjugated to a mCerulean fluorophore.

Subsequently, we were able to recombinantly express our fluorescently tagged proteins in the laboratory. This involved using NEB TURBO and T7 Express *E.coli* strains transformed with plasmid pET-19b. Following this, the binding affinity between our protein targets and peptides, was tested using Fluorescence Resonance Energy Transfer (FRET). This allowed us to assess the feasibility of using our peptide as a potential Puccinia spp inhibitor.

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Methods

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### Gibson Assembly

---

15 µL of the assembly mixture aliquot consisting of: 320µL 5X Isothermal Master Mix, 0.64 µL 10 U/µL Phusion DNA Polymerase, 160 µL 40 U/µL Taq DNA Ligase and 699.36 µL water, was allowed to thaw in 4 microcentrifuge
tubes and kept on ice. 5 µL DNA fragments of PstSCR1, PstHXTI, Pgt\_IaaM and Pst\_12806 were added to the assembly mixture. The mixture was incubated at 50ºC for 60 minutes.

### Transformation

---

To prepare competent cells, 5 mL of LB media was inoculated in a 50 mL falcon tube with a single colony from each NEB TURBO and T7 Express E. coli strains. The inoculated media was incubated in a shaking incubator for
18 hours, after which they were centrifuged at 3000rcf for 5 minutes. The supernatant was removed and the pellets were resuspended in 5 mL of PBS. 0.5 mL of this culture was used to inoculate a 50 mL flask of LB media
and incubated in a shaking incubator until an OD600 between 0.4 - 0.6 was achieved. Following this, the cells were poured into a sterile 50 mL falcon tube and placed in ice for 10 minutes. They were then centrifuged at
3000rcf for 5 minues and the supernatant was discarded. The resulting pellet was resuspended in 20 mL of 0.1 M CaCl2 and left on ice for 1 hour. The resuspension was centrifuged at 3000rcf for 5 minutes to obtain a pellet
and the supernatant was discarded. The cells were resuspended in 10 mL of 0.1 M CaCl2, 15% glycerol.

2 µl of ligation mixture was chilled in a 1.5 mL microcentrifuge tube. 50 µL of competent cells, prepared earlier, were added to the DNA by gentle mixing. The mixture was placed on ice for 30 minutes and then heat
shocked at 42ºC for 30 seconds. 950 µL of room temperature media was added to the tube which was placed at 37ºC for 60 minutes. The mixture was shaken at 250 rpm and 50 µL was spread onto selection plates. Control
plates were set up according to Table 1. The plates were incubated overnight at 37ºC and inspected for growth the next day. Single colonies were streaked from each of the three transformed cells.

| LB + Ampicillin | LB only (No Ampicillin) |
| --- | --- |
| NEB TURBO cells without plasmid | NEB TURBO cells without plasmid |
| T7 cells without plasmid | T7 cells without plasmid |
| T7 cells with SCR1 plasmid | Dots of the 3 cell cultures with plasmid (SCR1, HXT1, Pst\_12806) |
| TURBO cells with HXT1 plasmid |  |
| TURBO cells with Pst\_12806 plasmid |  |

Table 1: Descriptions of the 8 plates set up for incubation following transformation.

### Colony PCR

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3 plates were prepared with LB and ampicillin for PstHXT1, PstSCR1, and Pst\_12806. A grid was drawn on each plate with 8 cells. 15 µL of cell lysis buffer was added to each of 24 microcentrifuge tubes. A pipette tip
was used to pick a single colony from the relevant cell culture plate and dotted onto cell on the grid plate. The tip was then placed in the cell lysis solution and the resulting samples were boiled at 100 ºC for 15
minutes. Following this, the samples were centrifuged at 13, 000g. PCR was conducted on the supernatant and visualised on agarose gels.

### Protein Purification

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Two protein purification techniques were employed; gravity column purification and the AKTA start chromatography system. SCR1 protein was purified using gravity column purification and then AKTA start chromatography.

### Gravity Column Purification

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For gravity column purification, a chromatography column was loaded with 1 mL of Slurry, composed of equal parts (by volume)of resin and 20% ethanol. 20% ethanol was then allowed to flow through the column. Following
this, the column was equilibriated by adding 5 CV (column volume) of binding buffer. This was done twice. Previously prepared clarified lysate was then loaded into the column, using a syringe. It was prepared via
sonication, then centrifugation and filtration of cell culture. Subsequent, flowthrough was collected for later analysis. Next, 5 CV of binding buffer was loaded and after it flowed through, 5 CV of wash buffer was
added. 5 CV of elution buffer was finally added, with the flow through being collected to catch any protein that was eluted out.

### AKTA Start Purification

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For the AKTA start chromatography system, we first set up the system by running proprietary unicorn software to allow for remote control of the AKTA start from a computer. The tubing was inserted into the peristaltic
pump, the sample inlet was placed into 30 mL of equilibration buffer, Buffer A inlet was placed into equilibration buffer and Buffer B inlet was placed into elution buffer. To connect the column to the system,
a manual run of of 0.5 mL per minute was started. The flowpath of the wash valve was changed from ‘waste’ to ‘column’ and the column was connected, starting with the top first after checking to see if buffer was
dripping from the connector. The bottom was then connected and the column was checked for leaks. To begin purification of the protein, the sample inlet was moved from the equilbriation buffer and placed into the sample
tube. Below the fraction detector, enough tubes were loaded on the a rack. Using the computer, we ran the recommended method of ⅕ mL column 20 CV gradient manual flow through collection. This process is automatic and
our intervention was only needed at the end of the sample loading to avoid drying the column and during the wash phase to hold the wash longer, allowing UV to reach baseline detection.

### Cell Lysis via Sonication

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Due to time constraints, the team decided to use cell lysates in preparation for FRET analysis. NEB T7 E.coli cultures for Pst12806 and TaiSP were sonicated according to the following protocol:

Our SCR1 pellet was suspended in lysis buffer and put on ice. For 1 g of our pellet, we used 10 mL of lysis buffer. Using a serological pipette attached to a pipette gun, the pellet was resuspended.
The resuspended pellet was transferred into a 100 mL glass beaker. The sonicator was then set up by screwing on the sonicator tip on to the bottom of the sonicator probe. We adjusted the table to ensure
the tip was submerged in sample but not contacting the beaker. The beaker on ice was then placed on the table inside the sonicator with parafilm on top. A hole was made to allow tip entry. The sonicator
was then set to pulses of 2 seconds on and 2 seconds off. This is done until the total time a pulse is being applies (‘on’ time) is 8 minutes. Next, we transferred the sample into centrifuge tube and
centrifuged it at 10 000 rcf for 10 minutes.

### FRET Analysis

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Samples of cell lysates containing: Pst12806-mCerulean and TaISP-mVenus were prepared, in duplicate, on a 96 well plate. They were loaded according to an 8 sample serial dilution, at a dilution factor of 4:5, and loaded across two rows of the 96 well plate. Additionally a control containing purified mVenus and mCerulean protein was identically serially diluted in a third row.

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Results

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### Expression

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**cPCR**
Overall, we were able to demonstrate successful transformation of our Pst12806/TaISP ligated pET19b plasmids into NEB T7 E.coli cells.
Figure 1 represents the results of our cPCR screening for cells undergoing transformation with Pst12806 recombinant plasmid. It confirms the successful transformation of Colony 3.
This is indicated by the presence of a DNA band at 1300bp, which corresponds to the predicted length of our Pst12806 DNA (1239bp).

Similarly, Figure 2 represents the results of our cPCR screening for cells undergoing transformation with TaISP recombinant plasmid. It confirms the successful transformation of colony 2 and 6.
This is indicated by the presence of a DNA band at approximately 1595bp which corresponds to the predicted length of our TaISP DNA (1467bp)

![](https://static.igem.wiki/teams/4467/wiki/experiments/gel2.png)

Figure 1: Pst12806-mCerulean Colony PCR Gel. T7 E.coli colonies 1-6 were screened screened for transformation with our pET19b plasmid.

Discussion

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Through our experimental analysis we showed successful recombinant expression and purification of the two fluorescently tagged proteins: Pst12806-mVenus and TaISP-mCerulean. This is supported by colony PCR results, as seen in Figures 1 and 2. In Figure 1, the protein of interest was detected within one colony- indicated by a strong band at1300bp in lane 4 of the gel. Similarly, in Figure 2, bands corresponding to the protein of interest were detected for two colonies.

However, due to constraints within the project, the specific expression levels for Pst12806 and TaISP were not measured. Conventional purification and visualisation steps, were foregone in place of sonication. This allowed us to prepare whole cell lysates for FRET analysis but prevented validation of specific Pst12806 and TaISP expression. This represents a flaw in experimental design. Future iterations of the experiment should implement protein purification followed by quality control via SDS-PAGE. This would allow for verified and enriched expression of our proteins.

Following this, FRET analysis was conducted. Although we had success transforming our colonies, FRET analysis revealed Pst12806 concentrations were not adequately high enough to produce meaningful and statistically significant results. Additionally flaws setting up adequate control samples, voided our FRET results.

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Future Directions and Considerations

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To improve this experiment, we would need to see a significant increase in Pst12806 expression levels. Future rounds of this experiment could focus on the optimisation of Pst12806 expression and implementation
of affinity chromatography. Additionally, optimisation of the Pst12806 fusion protein and pET19b plasmid could play a significant role in enhancing protein expression.

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References

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* Xu, Q., Tang, C., Wang, X., Sun, S., Zhao, J., Kang, Z. and Wang, X., 2019. An effector protein of the wheat stripe rust fungus targets chloroplasts and suppresses chloroplast function. Nature Communications, 10(1).
* Nature.com. 2022. Effectors in plant pathology - Latest research and news | Nature. [online] Available at:https://www.nature.com/subjects/effectors-in-plant-pathology#:~:text=Effectors%20are%20proteins%20expressed%20by,between%20plants%20and%20their%20pathogens. [Accessed 12 October 2022].
* Camacho, I., Leça, R., Sardinha, D., Camacho, R. and Sadyś, M., 2017. Occurrence of Puccinia spp. spores in Madeira Island and their phytopathological importance. European Journal of Plant Pathology, 150(4), pp.955-969.

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