Parts Summary

Introduction

The various parts used in the Club² project can be found in these tabs. Here, a summary can be found of all the parts that we have attempted to express, purify, and use for our proof-of-concept Direct ELISA assay; furthermore, the Basic and Composite Parts are included in the registry and discussed in greater detail.

Basic Parts Overview

Table 1: Each of the Basic Parts worked on by Club² a brief description of the parts

Part Number	Type	Name	Description
BBa_K4139012	Basic	FL-PbEL04	Antigen of interest. Present in canola during infection and gall formation. Fibrillar protein.
BBa_K4139011	Basic	Truncated PbEL04	Truncated antigen of interest with a thioredoxin solubility tag. Present in canola during infection and gall formation. Fibrillar protein.
BBa_K4139013	Basic	CAPE-GFP	Chimeric protein designed to interact with PbEL04 tagged with GFP as fluorescent signal
BBa_K4139015	Basic	COAPE-GFP	Chimeric protein designed with a Ompa solubility made to interact with PbEL04 tagged with GFP as fluorescent signal
BBa_K4139017	Basic	CAP-FP	A bi-specific chimeric protein designed to interact with PbEL04 and PRO1 tagged with GFP as fluorescent signal
BBa_K4139018	Basic	CAPE-AFP	A chimeric protein designed to interact with PbEL04 with a lysine rich region for binding of a Alexa-fluor
BBa_K4139020	Basic	Pro-1	Another antigen of interest present in canola during infection of clubroot, but also present in other related species.
BBa_K4139019	Basic	Rec. CP-MAF	A chimeric protein designed to interact with PbEL04 which was put in the backbone of a mouse antibody

Composite Parts Overview

Table 2. Each of the composite parts worked on by Club² and if each part was successfully cloned, expressed, purified, and carried foreward to the preliminary ELISA test.

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

Part Collection

Figure 1. The composite parts worked on by the Club² team where the team identified two antigens of interest where found. 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.

Summary

To develop a proof of concept for our detection kit, Club², several Basic Parts have been designed and registered separately; furthermore, including parts that were already in the Part Registry, we designed, tested, and registered multiple Composite parts that were all codon-optimized for expression in Escherichia coli cells. All parts were synthesized using IDT, as well as expression vectors offered by BioBasic from collaborators with University of Alberta. Additionally, we created a Part Collection that gives a detailed overview of our ELISA proof of principle, containing our antigen of interest PbEL04, and our probe - Chimeric Optimized Anti-PbEL04 Fluorescent Protein (COAPE-FP). Our favourite parts were these two composite parts.

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