Results

Club² used a rationally designed, model-based to develop a rapid detection system to overcome the shortcomings of current detection and mitigation strategies against ​Plasmodiophora brassicae​ infected crops including canola, mustard, cabbage, and cauliflower ​(1)​. As seen in the Project Description, current detection methods of Clubroot comes at a higher cost and have a long turnaround time of the results. We have rationally designed and engineered chimeric protein(s) that are linked with fluorescent probes against PbEL04, a Clubroot pathogen. The goal is to create a qualitative rapid detection kit which will eventually let farmers to test their soil samples for Clubroot in field! The Club² team is working towards engineering test strips that will allow farmers to do on-site testing with our rationally designed chimeric fluorescent probes specific to PbEL04 and other protein targets in Clubroot.

Engineering and Designing Chimeric Protein(s)

Abstract

Our long-term goal for this project is the development of a lateral flow test against Plasmodiophora brassicae, while our short-term goal for the 2023 iGEM season was to develop and validate the biochemical parts we would need to create such a test. To accomplish both goals, our first step was to identify target proteins that are highly and specifically expressed by pathogenic P. brassicae. After this, we gathered as much biochemical and structural biology information as we could on our target proteins and used this information to rationally design chimeric fluorophore proteins to bind specific epitopes on our of antigens of interest.

Rationale behind Engineering of Chimeric Proteins against Clubroot protein pathogens

After settling on a long-term goal in the development of a faster and cheaper detection system against Plasmodiophora brassicae in canola, our first step was identifying potential protein targets, as seen in Figure. 1, unique to P. brassicae. After identifying two proteins as potential targets, we began to gather as much information as possible to assist our rational design of potential binders to these targets. The first step in this process was to model both PbEL04 and PRO1 to estimate which residues would be surface exposed. In addition to this, we also used BepiPred 3.0 software to predict potential B-cell epitopes within the sequences of the target proteins ​(2)​. We compared the regions predicted to form epitopes with the regions that appeared to be surface exposed in our models and decided on the most promising epitope regions within our target proteins. In PbEL04, a conserved epitope was found as the protein is made up of four conserved domains that are epidermal growth factor (EGF) like (cysteine rich domains); therefore, this epitope was of great interest for the Club² team ​(3)​.

Figure 1. An overview of the main steps taken in the design of our identification of potential target proteins for a detection system against Plasmodiophora brassicae.

We analyzed the model structure of our target antigen and visualized the orientation of surface exposed residues to determine our epitope., We began to rationally design model-based protein targets. This was done based on the biochemical and structural properties of the exposed residues in the identified epitope region(s). Complementary chimeric peptides were designed manually, with priority being placed on maximizing charge-charge, hydrophobic, and hydrogen bonding interactions. The binding between our chimeric protein paratopes and their epitopes was verified using docking simulations and electrostatic interactions done by our dry lab team (More info here). Following initial dry lab data, we designed and engineered our chimeric proteins with multiple fluorescent reporters for easy detection. Constructs with a reporter of green fluorescent protein (GFP) and a poly-lysine tag for fluorophore conjugation were designed to allow for options if expression and purification for parts that did not work. A list of each of the engineered proteins as well as current progress with each protein is included in Figure 3. We believe that the poly-lysine tagged chimeric protein (CAPE-AFP) represents a better option for our final goal of developing a portable and easy to use lateral flow detection system as seen in Figure 2, though more work is ultimately needed to optimize the purification of this construct.

Figure 2. An overview of the Club² wet lab results from 2023 where the team successfully designed, engineered, and purified chimeric fluorescent probes with its antigen of interest (PbEL04 which is present in Plasmodiophora brassicae). In 2024, the team is planning on utilizing the engineered recombinant tools developed in 2023 by creating a lateral flow test where an enhanced chimeric fluorescent probe will be designed which will be conjugated with colloid gold to create test strips to determine if PbEL04 is present in Brassicacaceae crop fields.

Our GFP-conjugated chimeric protein against PbEL04 (CAPE-GFP) proved easier to express, and as a result we chose to use it to test our chimeric protein sequences in a proof-of-concept direct ELISA assays. In Figure 3, we have all the Club² parts with our two antigens of interest and the chimeric fluorophore probes that were designed for each one, including CAPE-GFP (BBa_4139026) and CAPE-AFP (BBa_4139025). We hope to use the properties of both these parts in the eventual make up the Club² test strips.

Figure 3. The composite parts worked on by the Club² team where the team identified two antigens of interest PbEL04 and PRO1. FL-PbEL04 (BBa_K4139022) was optimized to Truncated PbEL04 (BBa_K4139021) for better expression and purification of the protein. The team also designed six chimeric fluorescence probes to interact with our antigens of interest. The fluorescent probes that are specific to PbEL04 are Chimeric Anti-PbEL04Green Fluorescent Protein (CAPE-GFP) (BBa_K4139026), Chimeric Anti-PbEL04-AlexFlour Protein (BBa_K4139025), Recombinant Chimeric Protein – Mouse Antibody-Framework (BBa_K4139028), and Chimeric Optimized Anti-PbEL04 – Green Fluorescent Protein (BBa_K4139027). The fluorescent probe the team created for Pro1 is called Chimeric Anti-Pro1-Fluorescent Protein (BBa_K4139024) which is bi-specific and binds to both PbEL04 and PRO1.

Protein Expression Test(s) of Engineered Chimeric Fluorescent Probes

Abstract

To test if our chimeric protein constructs were expressing well, we ran an expression screening experiment where we determined what media worked best, the concentration of ​isopropyl ß-D-1-thiogalactopyranoside (​IPTG) needed, and time of incubation. By taking samples of the media containing each construct, from the time of induction to twenty-four hours, determination of best expression was determined by western blot analysis. Based off the expression results as seen in Figure 7, it was determined what constructs would be used for further experimentation, and which ones needed modifications for better expression.

In Depth

Testing Chimeric Protein Expression

Following successful design of our PbEL04 binding chimeric proteins, as confirmed with successful docking simulations, we aimed to optimize the expression of our proteins. To accomplish this, we chose pET-28a and the BL21 LEMO Escherichia coli cell line as our expression vector and cell line due to their ​​versatility and availability to our team and their wide use in protein overexpression ​(4)​.

The first step in this process was to clone the inserts from the pUC19 vectors following synthesis into our chosen expression vector. The first step in this process was to clone the inserts from the pUC19 vectors following synthesis into our chosen expression vector. A general overview of this process is shown in the schematic below​ (Figure 4)​.

Figure 4. Schematic overview of cloning process. To transfer the inserts into a suitable expression vector, the inserts were excised with restriction enzymes, extracted from an agarose gel, and ligated into pET-28a. The pET-28a scaffold provided a T7 promoter and ribosome binding site (RBS) and a lac operon for inducible protein expression.

We first performed maxiprep experiments to obtain maximal amounts of cloning vector. The plasmids were then transformed and isolated following successful transformation. We then excised our plasmids using XbaI and XhoI restriction enzymes, while digesting pET-28a plasmid with NheI and XhoI restriction sites. We then performed an agarose DNA gel extraction to separate inserts from the plasmid backbone and ligated the insert into pET-28a as seen in Fig. ​4.​ Finally, we utilized colony ​polymerase chain reaction (​PCR​)​ experiments with T7 primers to screen transformed colonies from our ligations for successfully ligated plasmid​ (Figure 6)​. After successful ligation was confirmed, we were able to transform our expression vector into BL21 LEMO cells ​as seen in Fig. 5 ​and begin optimizing expression.

Figure 5. (A) DNA gel extractions on an 1.5% agarose gel where protein encoding inserts have been cut out of plasmids pUC19 and Pet28a through restriction enzymes Neh1 and Xho. (B) Plasmid Pet28a has been double digested and linearized with restriction enzymes Neh1 and Xbal1.

Figure 6. Colony PCR results from ten bacterial colonies (only successful PCR reactions are shown) to determine if ligation had taken place. Plasmids had been isolated from seven ligated reactions and consequently had their plasmid used for PCR to determine if ligation was successful. PCR products were run on a 2% agarose gel for 30min at 100V.

​​As ​previous work suggested that PbEL04 may be toxic to some types of cells, we decided to screen the expression of each of our constructs at different time points post-induction ​(5)​. TTo accomplish this, we expressed each construct in 50​ ​mL of LB media by growing cells until an O.D. of 0.6 and inducing with 1mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG). Following this, 2 mL samples were collected at predetermined intervals. These samples were then lysed chemically with lysozyme and sodium deoxycholate and mechanically by sonication. Following this, recombinant his-tagged proteins were detected through western blotting with anti-his antibodies obtained from R&D systems.

Dot-blotting experiments were completed, and results were quantified using ImageJ software following visualization with hydrogen peroxide and luminol. This information was used to inform our decisions on which expression times would produce the largest yields, and also supported our hypothesis that PbEL04 may lead to cell death, as yields were low to begin with and decreased dramatically after 2hrs post induction as seen in ​​Fig. 7. CAPE-AFP showed a similar trend as the full length PbEL04 construct, however in most other cases, it appeared that we got the highest levels of protein expression 20hrs post-induction.

Figure 7. Intensity of the signal detected in cell lysate treated with anti-his antibody during an expression screening experiment. Samples were taken at different intervals following induction and used to optimize expression times for each construct.

Optimization of the Expression and Purification of Club² Parts Leading to Successful Purification

Abstract

Upon successful optimization of PbEL04 expression, purification of the recombinant protein was performed. Since our engineered proteins have a 6x histidine tag on them, we utilized nickel affinity chromatography as our purification technique. This was performed using a 5 mL HisTrap HP column, and more details about this experiment can be found in our protocol Notebook. The resulting chromatogram shows protein eluting with increasing percentages of Buffer B; the elution buffer with a high imidazole concentration, as seen in Figure 8. To confirm that the elution fractions contained PbEL04, anti-his antibody was used for Western blotting.

In Depth

After optimizing expression conditions for both constructs, we attempted to purify ​​CAPE-FP with immobilized metal ​affinity​​ ​chromatography​ (IMAC)​. We quickly observed that no protein appeared to be eluting from the column at an imidazole concentration less than 300mM​​​ (Results not shown)​. We hypothesised that this could be due to the formation of inclusion bodies in our protein and tested this by solubilizing the pellet collected during the clarification step of our purification in 8M urea and running a portion of this in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (​​SDS-PAGE) experiment. ​​We observed a band at the expected molecular weight of our protein, supporting our hypothesis.

When additional expression conditions and purification strategies proved to be ineffective in avoiding the congregation of CAPE-FP in inclusion bodies, we realized we would need optimize our already made composite part to successfully purify functional CAPE-FP protein. As we would already need to order a modified CAPE-FP construct, we decided to also attempt to modify our PbEL04 construct to attempt to increase the yields.

We chose to modify CAPE-FP with an ​​OmpA signalling peptide to influence CAPE-FP to be expressed in the periplasm of the cells to avoid the formation of inclusion bodies. We named the new construct generated form this modification COAPE-FP (BBa_K4139015). To improve the expression of PbEL04 we did extensive literature review and found that thioredoxin is often used as a solubility tag in the expression of epidermal growth factor (EGF) proteins ​(6)​. As our modelling predicted that PbEL04 would fold in a similar manner to these EGF proteins, we decided to employ a similar approach to express PbEL04. We designed a truncated version of PbEL04 containing only the epitope our chimeric proteins would target and the surrounding residues, and then attached a thioredoxin solubility tag to the n-terminal region of this construct (BBa_K4139021).

We ordered these two constructs already cloned into pET-28a at an increased cost in the interest of time (From Biobasic). Upon receiving the constructs, we once again transformed into BL21 LEMO cells and expressed as described previously. Following this, we once again purified with IMAC (Ni^2+^ Affinity Chromatography) and this time observed protein eluting off the resin at a concentration of ~100mM imidazole based on ultraviolet absorbance​ (Figure 8A)​. We confirmed the presence of protein with an SDS-PAGE gel, as seen in Fig. 8​B​, with the first species to elute from the column running to approximately ~21kDa, which was the theoretical molecular weight of our optimized PbEL04 construct.

Figure 8. (A) Chromatogram of the purification of PbEL04 on a HisTrap HP column. Eluted volumes correspond to absorbance readings at 280 nm representing the potential protein being eluted. (B) A 12% SDS-PAGE gel showing fractions run from the chromatogram seen in (A).

As a final quality check measure, we utilized western blotting to further confirm that the proteins seen on the Coomassie stained gels were our proteins of interest. As seen in Fig. 9, all species were observed on our SDS-PAGE gels were also identified on our western blots, suggesting that our PbEL04 construct exists in multiple oligomeric states, and that we may be observing cleavage between our chimeric protein and GFP reporter.

Overall, we were able to express and purify both our modified PbEL04 construct as well as the COAPE-GFP construct, and we were able to confirm the identities of the purified proteins.

Figure 9. Western blots following the purification of truncated PbEL04 (A) and COAPE-GFP (B) to determine purity of the proteins of interest.

Proof-of-Concept:​ ​Direct Fluorescence ELISA

Abstract

We were able to finalize our proof-of-concept experiments by testing binding between our chimeric protein paratopes and their target epitopes with a direct enzyme-linked immunosorbent assay (ELISA).

Following the success of this experiment, we have begun planning our future directions for the following season. Further optimization of the affinity and specificity of our chimeric proteins are among the most important of our goals for this season in terms of accomplishing our goal of developing a functional lateral flow test.

In Depth

Following successful purification of COAPE-GFP and optimized PbEL04 we completed a​ direct ​ELISA as a proof of concept, showing that COAPE-GFP recognized recombinant PbEL04​ (Figure 10)​ ​(7)​. ​ ​Furthermore, we tested our COAPE-GFP against ​bovine serum albumin (​BSA) as a negative control which did see some binding, but when compared to PbEL04, the interaction is much greater. As seen in Figure 10, we see robust significance when compared to blank which was determined by running an unpaired t-test. This represents greater and direct interaction between COAPE-GFP (BBa_K4139015) and Truncated PbEL04 (BBa_K4139011). The calculated limit of detection of 291 ng represents a good initial value, though further computational optimization of our chimeric protein sequence may make it possible to decrease this value even further.

Figure 10. (A) Initial binding data of PbEL04 interacting with COAPE-GFP in a direct ELISA where COAPE-FP was coated to the plate and varying amounts of PbEL04 was added to see specificity of binding. (B) The standard curve used to determine the limit of detection for this construct.

Future Directions: Club² Test Strips

Overall, we have made progress optimizing the biochemistry for the expression and purification of many of the construct designs seen in Fig. 2. While each protein is optimized to a different degree, as seen in Table 1, we have generated a toolbox of chimeric proteins to aid in the detection of Plasmodiophora brassicae proteins PbEL04 and PRO1. Not only did we purify a potential protein target, PbEL04 (BBaK4139011) - which has never been done before - we optimized the original part for better expression. Furthermore, we developed and engineered multiple chimeric fluorescence probes which also needed to be optimized into new composite parts to be purified. Optimizing COAPE-GFP and truncated PbEL04 to a purity at which they represent a viable model to qualitatively test the affinity between our complementary chimeric protein sequence and its target epitope with a direct ELISA assay allowed us to help validate our computational data. This direct correlation between dry lab and wet lab data left us optimistic that our chimeric protein sequences could serve as a building block for a lateral flow test for the detection of _P. brassicae which will need more optimization as we are still in the early stages of creating better fluorescent probes.

Part Number	Type	Name	Cloned	Expressed	Purified	Direct ELISA Assay
BBa_K4139022	Composite	FL-PbEL04	YES	NO	N/D	N/D
BBa_K4139021	Composite	Truncated PbEL04	N/A	YES	YES	YES
BBa_K4139026	Composite	CAPE-GFP	YES	NO	N/D	N/D
BBa_K4139027	Composite	COAPE-GFP	N/A	YES	YES	YES
BBa_K4139024	Composite	CAP-FP	YES	NO	N/D	N/D
BBa_K4139025	Composite	CAPE-AFP	YES	YES	NO	N/D
BBa_K4139029	Composite	PRO-1	YES	YES	NO	N/D
BBa_K4139028	Composite	Rec. CP-MAF	YES	YES	NO	N/D

Table 1. The registry number for each of the composite parts worked on by Club² and if each part was successfully cloned, expressed, and purified. Once antigens and chimeric fluorescent probes were purified, they were carried forward to a preliminary direct ELISA to see binding between Club² designed and engineered chimeric fluorescent probes and our antigen of interest, PbEL04.

In order to accomplish our ultimate goal of developing a lateral flow test, we need to continue to improve the parts we have designed. In the future we hope to improve the affinity between our chimeric proteins and their targets. We hope that this would also reduce our limit of detection, making our testing system more impactful for farmers. By further optimizing the biochemistry for the expression and purification of our constructs that can be conjugated with a fluorophore (BBa_K4139018), we hope to increase our limit of detection by chemically linking more powerful reporter molecules such as colloidal gold that will be easier to detect at lower concentrations which can be activated by visible light ​(8, 9)(6, 7)​.

We also hope to build on our current docking simulations using machine learning to optimize our chimeric protein sequences for higher affinity binding. This would also reduce our limit of detection, making our test strip a more effective tool for farmers. A schematic diagram of the proposed layout and operation of this strip test is shown below in Fig. 11. While some sample preparation would be required in order to isolate PbEL04 from soil samples we believe these steps could be feasible for farmers to complete in the field without access to laboratory equipment. Briefly, we would base our extraction protocol on existing protocols and would use a mild detergent in our extraction buffer to help to solubilize any protein present in a soil sample and a strain to remove the soil once protein had been extracted ​(10, 11)(8, 9)​.

Overall, the development of this lateral flow assay would allow farmers to forgo expensive and time-consuming PCR testing to get a quick answer on the presence of clubroot. One of the biggest advantages our potential kit would have been that it would remove the need for farmers to send out their soil samples for testing. This would mean that farmers would get results back quickly, giving them more time to respond with targeted measures such as crop rotation or lime treatment should some of the tests come back positive. The tests would also allow farmers to have full control of the analysis of the fields, and it is possible that the farmers may have more trust in a test that they do themselves. The Club² team has made significant advances towards making these tests a reality in the near future, and we are excited to pick up where we left off next season.

Figure 11. An overview of the mechanism for the proposed lateral flow assay. In step one, a sample prepared from the soil to be tested is applied to the strip. After this is allowed to dry, buffer containing CAPE-AFP (BBa_K4139025) conjugated with colloidal gold will be applied to the top of the lateral flow assay as seen in step 2. To see the positive a test result - on the Club² test strips - CAPE-AFE will flow down the test strip, where it will reach the processed soil sample and if FL-PbEL04 (BBa_K4139022) is present, it will bind. The conjugated colloidal gold will become be activated by visible light giving a signal. The positive control will have an antibody specific to CAPE-AFP which will ensure that the probe has travelled down the test strip and where it will also provide a signal. A positive test will give off two signals while a negative will give one.

TEM Validation of PbEL04’s Structure

Figure 12.0: On the left, the computationally determined model from AlphaFold showcases the predicted fibrillar structure. On the right, the transmission electron micrograph of PbEL04 with a thyrodexin tag validates this fibrillar morphology. The varying lengths and occasional bundling of the fibril monomers can also be observed against the negatively stained background.

References

J. Engel, EGF-like domains in extracellular matrix proteins: Localized signals for growth and differentiation? FEBS Lett 251 (1989).

S. Gräslund, et al., Protein production and purification. Nat Methods 5 (2008).

A. S. Ferreira, et al., A toolkit for recombinant production of seven human EGF family growth factors in active conformation. Sci Rep 12 (2022).

H. Hayrapetyan, T. Tran, E. Tellez-Corrales, C. Madiraju, Enzyme-Linked Immunosorbent Assay: Types and Applications. Methods Mol Biol 2612 (2023).

A. Moody, Rapid diagnostic tests for malaria parasites. Clin Microbiol Rev 15 (2002).

T. Song, M. Chu, R. Lahlali, F. Yu, G. Peng, Shotgun label-free proteomic analysis of clubroot (Plasmodiophora brassicae) resistance conferred by the gene Rcr1 in Brassica rapa. Front Plant Sci 7 (2016).

K. M. Keiblinger, et al., Soil metaproteomics - Comparative evaluation of protein extraction protocols. Soil Biol Biochem 54 (2012).

J. Hasan, S. Megha, H. Rahman, Clubroot in brassica: Recent advances in genomics, breeding, and disease management. Genome 64 (2021).

J. N. Clifford, et al., BepiPred-3.0: Improved B-cell epitope prediction using protein language models. Protein Science 31 (2022).

J. Engel, EGF-like domains in extracellular matrix proteins: Localized signals for growth and differentiation? FEBS Lett 251 (1989).

S. Gräslund, et al., Protein production and purification. Nat Methods 5 (2008).

X. Jiang, Y. Su, M. Wang, A small cysteine-rich protein identied from the Proteome of clubroot pathogen, Plasmodiophora brassicae, induces cell death in nonhost plants and host plants (2022) https:/doi.org/10.21203/rs.3.rs-1961445/v1.

A. S. Ferreira, et al., A toolkit for recombinant production of seven human EGF family growth factors in active conformation. Sci Rep 12 (2022).

H. Hayrapetyan, T. Tran, E. Tellez-Corrales, C. Madiraju, Enzyme-Linked Immunosorbent Assay: Types and Applications. Methods Mol Biol 2612 (2023).

A. Moody, Rapid diagnostic tests for malaria parasites. Clin Microbiol Rev 15 (2002).

K. M. Koczula, A. Gallotta, Lateral flow assays. Essays Biochem 60 (2016).

​T. Song, M. Chu, R. Lahlali, F. Yu, G. Peng, Shotgun label-free proteomic analysis of clubroot (Plasmodiophora brassicae) resistance conferred by the gene Rcr1 in Brassica rapa. Front Plant Sci 7 (2016).

K. M. Keiblinger, et al., Soil metaproteomics - Comparative evaluation of protein extraction protocols. Soil Biol Biochem 54 (2012).

J. Engel, EGF-like domains in extracellular matrix proteins: Localized signals for growth and differentiation? FEBS Lett 251 (1989).

S. Gräslund, et al., Protein production and purification. Nat Methods 5 (2008).

X. Jiang, Y. Su, M. Wang, A small cysteine-rich protein identied from the Proteome of clubroot pathogen, Plasmodiophora brassicae, induces cell death in nonhost plants and host plants (2022) https:/doi.org/10.21203/rs.3.rs-1961445/v1.

A. S. Ferreira, et al., A toolkit for recombinant production of seven human EGF family growth factors in active conformation. Sci Rep 12 (2022).

H. Hayrapetyan, T. Tran, E. Tellez-Corrales, C. Madiraju, Enzyme-Linked Immunosorbent Assay: Types and Applications. Methods Mol Biol 2612 (2023).

A. Moody, Rapid diagnostic tests for malaria parasites. Clin Microbiol Rev 15 (2002).

K. M. Koczula, A. Gallotta, Lateral flow assays. Essays Biochem 60 (2016).

T. Song, M. Chu, R. Lahlali, F. Yu, G. Peng, Shotgun label-free proteomic analysis of clubroot (Plasmodiophora brassicae) resistance conferred by the gene Rcr1 in Brassica rapa. Front Plant Sci 7 (2016).

K. M. Keiblinger, et al., Soil metaproteomics - Comparative evaluation of protein extraction protocols. Soil Biol Biochem 54 (2012).

Supplementary