Engineering Success

Engineering is essential in iGEM and synthetic biology because it provides a structured, interdisciplinary, and innovative approach to designing, building, and applying biological systems. It enables teams to work towards solutions that are not only scientifically sound but also safe, scalable, and relevant to real-world challenges.

Initial Challenges and Design Flaws

Research

We first did a vast literature review in finding potential protein targets in Plasmodiophora brassicae. The two major problems the Club² team faced was that that genome is not sequenced at all making it even harder to find potential protein targets. Although, recent literature has found certain proteins, PbEL04, which is of interest. After, discovering this protein, our second problem was that since P. brassicae has resting spores which could make it difficult to target certain proteins. PbEL04 being a secreted protein that aids in infection solved this problem (1-7).

Design

After identifying our protein targets, we then began to rationally design chimeric peptides that would bind to and allow the detection of our selected targets. This design process was done rationally based on homology modelling and molecular dynamics simulations of our target proteins.

Build

Following our design of our chimeric protein sequences, we then built a fully-fledged detection system using these sequences by adding either green fluorescent protein or a poly-lysine tag for chemical conjugation with a fluorophore.

• CAPE-GFP (BBa_K4139013)
• FL-PbEL04 (BBa_K4139012)

Test

While we were initially confused by this outcome, we checked the design of our final constructs for mistakes and noticed that the restriction enzyme sites we had chosen for cloning resulted in the excision of the ribosome binding site from the plasmid as the protein coding gene was inserted.

Learn

While we were initially confused by this outcome, we checked the design of our final constructs for mistakes and noticed that the restriction enzyme sites we had chosen for cloning resulted in the excision of the ribosome binding site from the plasmid as the protein coding gene was inserted.

Re-Design, Re-Cloning, and Discoveries

Research

Once we learned of our mistake, we immediately began to look for solutions and noticed that the restriction enzyme we used that resulted in the excision of the ribosome binding site (XbaI) was an isocaudomer to the restriction enzyme cut site we should have used to insert our gene (NheI).

Design

From this information, we realized that we could clone our inserts from our non-functional constructs into the correct position in a new pET28a vector. This could be done by excising our insert with XbaI and XhoI restriction endonucleases and opening a site for insertion on the new expression vectors with NheI and XhoI restriction enzymes.

Build

After designing, we executed our plan and cloned our inserts into a new pET28a vector. This cloning ensured that the ribosome binding site was present, enabling the expression of our protein.

Test

To test that our cloning had been successful, we utilized colony PCR reactions to confirm that our genes had been inserted in the right location. Then, we attempted to expression the protein to confirm that the ribosome binding site was present in these constructs and working properly.

Learn

While we were able to confirm that our cloning was successful with these experiments. We also discovered that the expression levels for our antigen (PbEL04) were low, limiting the experiments we could do with protein expressed from this construct. We also found that while our GFP conjugated chimeric protein (CAPE-GFP) was expressed exclusively into inclusion bodies.

Optimization, Re-Expression, and Path to Prototyping

Research/Design

After determining certain parts did not express or did not purify well, the team went back to the drawing board to optimize the constructs to see better protein expression. It was determined that adding a thioredoxin solubility tag would help in the folding of PbEL04 and lead to easier purification; moreover, the protein was also truncated to improve protein purification, Truncated PbEL04 (BBa_K4139011) (3).

Additionally, we improved CAPE-GFP (BBa_K4139013) by adding a OmpA signaling peptide to transfer the translated protein from the cytoplasm to the periplasm of the cell. This improvement on the part lead to its purification and further work with a Direct ELISA (4).

Build

The team then used the old parts that we had and modified the tags as mentioned in the research/design section with the additions of OmpA and Thioredoxin.

• COAPE-GFP (BBa_K4139015)
• Truncated PbEL04 (BBa_K4139011)

Test

The proteins were then expressed and purified which allowed to carry forward with our direct ELISA which showed interaction between the two proteins.

Learn

• The team learnt that the thioredoxin works as a solubility tag and greatly helps in the stability of proteins needing disulfide bridges. We also learnt that OmpA works as a signaling peptide with this certain construct.

• Furthermore, we learnt the detection of limit of COAPE-GFP (BBa_K4139015) is 291 ng.

• As a proof of principle, we learnt that our designing of chimeric fluorescent probes works since we saw robust interaction in our direct ELISA and that we can continue this method towards a test diagnostic test strip.

• Since the team has confirmed that COAPE-GFP has direct interaction with Truncated PbEL04 (BBa_K4139011) we can now start making prototypes of Club² test strips.

• We have learnt that the test strips would need a fluorescent probe that would be conjugated with colloidal gold that would be activated by visible light upon binding to our antigen of interest (8).

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).

Oliver, C. (2010). Conjugation of colloidal gold to proteins. Methods in Molecular Biology (Clifton, N.J.), 588. https://doi.org/10.1007/978-1-59745-324-0_39