Basic Parts

Basic parts in iGEM are foundational genetic elements that serve as building blocks for synthetic biology projects. They are typically well-characterized and documented, allowing teams to work with confidence in their behavior. These parts are collected in registries and databases like the iGEM Registry of Standard Biological Parts, which makes it easier for teams to access and use them in their projects. Here are the basic parts the Club² team worked on this past year!

CAPE-GFP (BBa_K4139013) (1-2,4)

Figure 1. For this construct it starts with enhanced green fluorescent protein (eGFP) followed by our chimeric anti-PbEL04 (CAPE) which is a complementary to PbEL04. We hypothesized that CAPE was complementary to PbEL04 through simulations between the two proteins. Finally, we added a His-tag to help with the purification of the protein. The reason for using eGFP is to show if proper binding has occurred. If proper binding has occurred; then the protein will glow green when visible blue light excites it in the 470 nm to 498 nm range.

COAPE-GFP (BBa_K4139015) (1-4)

Figure 2. This construct starts with an OmpA tag and is a signaling peptide that will bring the protein from cytoplasm to the periplasm. This is followed by enhanced green fluorescent protein (eGFP) then our Chimeric Anti-PbEL04 (CAPE). The CAPE proportion is the same as our CAPE protein. Finally we added a His-tag to further help with purification. The function of this protein is to bind to PbEL04 and glow once binded correctly. The reason for using eGFP is to show if proper binding has occurred. If proper binding has occurred then the protein will glow green when blue light in the 470 nm to 498 nm range. The difference between CAPE-GFP (BBa_K4139013) and COAPE-GFP (BBa_K4139015) is the OmpA tag which was used as a solubility tag. This construct was successfully purified by our team.

FL-PbEL04 (BBa_K4139012) (4)

Figure 3. This construct was made to have a full version of PbEL04. After identifying two protein targets in Plasmodiophora brassicae, the team needed parts that allowed us to test interactions with the antigens of interest. Here we see full length PbEL04 (our antigen) with the addition of a His-tag to the end to help with purification. Our goal with this protein was to purify the version seen in nature to test the full interaction between it and our COAPE-GFP Protein. PbEL04 is a fibrous protein found in the cell membrane that is secreted from Plasmodiophora Brassicae and aids in the infection of canola.

Truncated PbEL04 (BBa_K4139011) (2-5)

Figure 4. This construct starts with a His-tag, this assists with the purification of the protein. After the His-tag we added thioredoxin to further assist with the purification. Thioredoxin helps with the folding of the protein and acts as a solubility tag as well. This is followed by a T.E.V cut site, which allows us to remove the thioredoxin and His-tag once purified. TEV cut site is cleaved off by TEV protease which is a member of the PA clan of chymotrypsin-like proteases. After the TEV cut site is our truncated version of PbEL04. This is just the target epitope of COAP-FP (BBa_K4139015). The use of this construct is to make a version of PbEL04 that is purified easily. We were able to purify this construct making it a favourite.

CAPE-AFP (BBa_K4139018) (2-4)

Figure 5. This construct begins with a His-tag, which helps with the purification of the protein. After the His-tag is our chimeric Anti-PbEL04 (CAPE) repeated three times, this was done to increase the change of binding with PbEL04 and overall structure to the chimeric fluorophore probe. Finally, we added a lysine rich region. The lysine rich region is present to bind to colloidal gold particles, which when excited, will fluoresce in visible light giving a signal which will be explored with the Club² test strips.

Pro1 (BBa_K4139020) (6)

Figure 6. This construct begins with Pro1. Pro1 is a protein found in fungal and fungal-like pathogens including Plasmodiophora brassicae. After the Pro1 region we added a His-tag to make purification easier. The use of this part was to have other options other than PbEL04 in Plasmodiophora brassicae.

CAP-GFP (BBa_K4139017) (1-2,4,6)

Figure 7. This construct starts with enhanced green fluorescent protein (eGFP) followed by our chimeric anti-PbEL04 (CAPE) which is a complementary to PbEL04. After our CAPE region is our chimeric anti Pro1 (CAP) region which is complementary to the Pro1 protein. Finally, after the CAP region is a His-tag to help with purification. This constructs’ function is to be a bi-spicific protein that can bind to both PbEL04 and Pro1.

CP-MAF (BBa_K4139019) (2,4,6)

Figure 8. This construct begins with a lysine rich region, with this region being included to enhance conjugation with fluorophore reporter molecules. After this, we have utilized the framework three region of a mouse IGG antibody as a scaffold for our chimeric protein against PbEL04. We have inserted our chimeric protein into the CDR3 region of the framework 3 regions of the mouse antibody, and have repeated this framework 3 - chimeric protein motif once with a the only difference being that the chimeric protein utilized was complementary to PRO1 in the second interaction. After this, we included a 6x His-tag to aid in purification.

References

1. Valdivia, R. H., Hromockyj, A. E., Monack, D., Ramakrishnan, L., & Falkow, S. (1996). Applications for green fluorescent protein (GFP) in the study of host-pathogen interactions *. In Gene (Vol. 173).(Vol. 85).

2. Lowe, D. G., Ricketts, M., Levinson, A. D., & Goeddelt, D. V. (1988). Chimeric proteins define variable and essential regions of Ha-ras-encoded protein (guanine nucleotide-binding protein/Ha-ras p21/R-ras p23/mammalian transformation). In Proc. Nail. Acad. Sci. USA

3. Pechsrichuang, P., Songsiriritthigul, C., Haltrich, D., Roytrakul, S., Namvijtr, P., Bonaparte, N., & Yamabhai, M. (2016). OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system. SpringerPlus, 5(1). https://doi.org/10.1186/s40064-016-2893-y

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

5. Ferreira, A. S., Lopacinski, A., Batista, M., Hiraiwa, P. M., Guimarães, B. G., & Zanchin, N. I. T. (2022). A toolkit for recombinant production of seven human EGF family growth factors in active conformation. Scientific Reports, 12(1). https://doi.org/10.1038/s41598-022-09060-9

6. Feng, J., Hwang, R. U., Hwang, S. F., Strelkov, S. E., Gossen, B. D., Zhou, Q. X., & Peng, G. (2010). Molecular characterization of a serine protease Pro1 from Plasmodiophora brassicae that stimulates resting spore germination. Molecular Plant Pathology, 11(4), 503–512. https://doi.org/10.1111/j.1364-3703.2010.00623.x