Parts
Introduction
This page provides an overview of the various parts we developed for the C.R.O.P.S project during this iGEM season. Our project focuses on both the detection and mitigation of Plasmodiophora brassicae, the pathogen responsible for Clubroot. On the detection side, we developed parts essential for creating a lateral flow assay, while on the mitigation side, we designed constructs that enhance the production of bioactive compounds like fengycin and salicylic acid to explore sustainable biocontrol options. Below, you will find links to each part, detailing their purpose and contribution to our project’s goals.
Basic Parts
| Part Number | Type | Name | Description |
|---|---|---|---|
| BBa_K5091000 (opens in a new tab) | Basic | Nanobody Scaffold | This nanobody scaffold by Mauricio Aguilar Rangel enables binding to a target antigen with an added sequence, using a small, single-domain antibody. |
| BBa_K5091004 (opens in a new tab) | Basic | Variable Region Binding to PbEL04 | The propose of this region is to bind to our target antigen (BBa_K4139021) or better known as PbEL04. |
| BBa_K5091002 (opens in a new tab) | Basic | Fab Heavy Chain | This part is the heavy chain portion of our FAB antibody. The heavy chain is half of the FAB protein which stands for "fragment antigen binding" |
| BBa_K5091003 (opens in a new tab) | Basic | Fab Light Chain | This part is the light chain portion of our FAB antibody. The light chain is half of the FAB protein which stands for "fragment antigen binding" |
| BBa_K5091005 (opens in a new tab) | Basic | degQ | The degQ gene from Bacillus subtilis XF-1 boosts fengycin production, a powerful antifungal useful for sustainable agricultural biocontro |
| BBa_K5091008 (opens in a new tab) | Basic | pmsC | The pmsC gene from Pseudomonas fluorescens A506 encodes an enzyme essential for salicylic acid production, critical for plant defense and immunity projects. |
| BBa_K5091009 (opens in a new tab) | Basic | pmsB | The pmsB gene from Pseudomonas fluorescens A506 encodes an enzyme that, with pmsC, converts isochorismate to salicylic acid, essential for plant defense. |
Composite Parts
| Part Number | Type | Name | Description |
|---|---|---|---|
| BBa_K5091006 (opens in a new tab) | Composite | Nanobody Scaffold with Chimeric Peptide | A nanobody scaffold was designed with a chimeric peptide in the CDR3 region to bind to truncated PbEl04 (BBa_K4139011), featuring histidine, OmpA, and poly-lysine tags for purification, expression, and chemical binding. |
| BBa_K5091007 (opens in a new tab) | Composite | FAB with Addition of Chimeric Peptide | A FAB was engineered with a chimeric peptide in the CDR3 regions to bind to truncated PbEl04 (BBa_K4139011), incorporating histidine, OmpA, and poly-lysine tags in each domain for purification, expression, and chemical binding. |
| BBa_K5091010 (opens in a new tab) | Composite | degQ Expression Construct | This part is designed to express the degQ gene from Bacillus subtilis XF-1, using a T7 promoter, RBS, and T1 terminator to boost fengycin production, making it ideal for agricultural biocontrol and sustainable agriculture projects. |
| BBa_K5091011 (opens in a new tab) | Composite | Native degQ Expression Construct | This composite part expresses the degQ gene using its native promoter, RBS, and terminator, ensuring natural regulation and efficient production of fengycin for biocontrol and agricultural applications. |
| BBa_K5091012 (opens in a new tab) | Composite | pmsC-pmsB Expression Construct | This composite part co-expresses the pmsC and pmsB genes from Pseudomonas fluorescens to biosynthesize salicylic acid, using an arabinose-inducible promoter for controlled expression, and is ideal for plant defense and biocontrol applications. |
Conclusion
Throughout this iGEM season, we made significant strides in advancing both the detection and mitigation sides of the C.R.O.P.S project. Using the parts developed this year and last, we successfully progressed on the lateral flow assay for pathogen detection, thanks to several newly developed basic and composite parts. These include components specifically designed for binding to PbEL04, which improved the functionality of the assay.
Additionally, we laid the groundwork for the mitigation aspect of our project by developing expression constructs for the degQ and pmsC-pmsB genes, which are critical for boosting the production of bioactive compounds like fengycin and salicylic acid. These compounds are essential for sustainable agricultural biocontrol, aimed at mitigating the impact of Clubroot. While we focused primarily on the detection side, these mitigation parts provide a solid foundation for future research into biocontrol solutions.
By combining existing parts from the registry with our newly developed parts, we optimized codon usage to enhance the success of overexpression and purification, making substantial progress in both the detection and mitigation elements of the C.R.O.P.S project.
References
Rangel, M. A., Bedwell, A., Costanzi, E., Taylor, R., Russo, R., Bernardes, G. J., Ricagno, S., Frydman, J., Vendruscolo, M., & Sormanni, P. (2021). Fragment-Based Computational Design of Antibodies Targeting Structured Epitopes. https://doi.org/10.1101/2021.03.02.433360 (opens in a new tab)
Pelludat, C., Brem, D., & Heesemann, J. (2003). Irp9, encoded by the high-pathogenicity island of Yersinia enterocolitica, is able to convert chorismate into salicylate, the precursor of the siderophore yersiniabactin. Journal of bacteriology, 185(18), 5648–5653. https://doi.org/10.1128/JB.185.18.5648-5653.2003 (opens in a new tab)
Shengye Guo, Xingyu Li, Pengfei He, Honhing Ho, Yixin Wu, Yueqiu He, Whole-genome sequencing of Bacillus subtilis XF-1 reveals mechanisms for biological control and multiple beneficial properties in plants, Journal of Industrial Microbiology and Biotechnology, Volume 42, Issue 6, 1 June 2015, Pages 925–937, https://doi.org/10.1007/s10295-015-1612-y (opens in a new tab)
Banfield, M. J., King, D. J., Mountain, A., & Brady, R. L. (1997). VL:VH domain rotations in engineered antibodies: Crystal Structures of the Fab fragments from two murine antitumor antibodies and their engineered human constructs. Proteins: Structure, Function, and Genetics, 29(2), 161–171. https://doi.org/10.1002/(sici)1097-0134(199710)29:2 (opens in a new tab)