Overview
Three main components were used to construct our product: Surface displayed proteins, which facilitate the entry of our Membrane Vesicles into target fungus cells and their retention on the surface of the plant. The second is the SpyCatcher-SpyTag system and its associated Lpp-OmpA and INPNC. The third is shRNA to induce RNA interference.
Also, The sequences and primers used for the Golden Gate Assembly of surface displayed proteins have been adjusted to achieve various combinations of Surface displayed protein sequences(BBa_K5269003-BBa_K5269008).
Main Parts List
| Part Registry ID | Part Name | Type | Brief Description |
|---|---|---|---|
| BBa_K5269013 | Mgfp-5 | Basic | Mgfp-5 is a freshwater shellfish adhesion protein.[1] |
| BBa_K5269014 | ChitinaseC | Basic | ChitinaseC is an enzyme that hydrolyzes the glycosidic bonds in chitin. |
| BBa_K5269015 | GH19Chitinase | Basic | GH19Chitinase is an endo-type enzyme that hydrolyzes the glycosidic bonds in chitin.[2] |
| BBa_K5269016 | Endo-1, 3(4)-b-glucanase | Basic | This glucanase is an endo-type enzyme that hydrolyzes the 1,3 or 1,4 bond of beta-D-glucan.[3] |
| BBa_K5269021 | SpyTag-ChitinaseC-His-Tag and BsmB I cut site(for GGA) | Composite | SD-sequence, SpyTag, ChitinaseC, His-Tag and BsmB I cleavage sites at both ends of the sequence |
| BBa_K5269022 | SpyTag-GH19Chitinase-His-Tag and BsmB I cut site(for GGA) | Composite | SD-sequence, SpyTag, GH19Chitinase, His-Tag and BsmB I cleavage sites at both ends of the sequence |
| BBa_K5269023 | SpyTag-Mgfp-5-His-Tag and BsmB I cut site(for GGA) | Composite | SD-sequence, SpyTag, Mgfp-5, His-Tag and BsmB I cleavage sites at both ends of the sequence |
| BBa_K5269024 | SpyTag-Glucanase-His-Tag and BsmB I cut site(for GGA) | Composite | SD-sequence, SpyTag, Endo-1, 3(4)-b-glucanase, His-Tag and BsmB I cleavage sites at both ends of the sequence |
| BBa_K5269026 | Lpp_OmpA-SpyCatcher | Composite | Lpp-OmpA and SpyCatcher |
| BBa_K5269027 | INPNC-SpyCatcher | Composite | INPNC and SpyCatcher |
| BBa_K5269019 | GFP targetted shRNA | Basic | This is an shRNA that targets GFP for RNAi |
| BBa_K5269025 | T7 promoter-shRNA-T7 terminator-Golden Gate Assembly Site | Composite | T7 promoter, GFP targeted shRNA, T7 terminator, tac promoter, BsmB I insert, L3S2P21 terminator |
| BBa_K5269020 | Golden Gate Assembly Site | Composite | tac promoter, BsmB I insert, L3S2P21[4] |
Primer or Other Basic Parts
| Part Registry ID | Part Name |
|---|---|
| BBa_K5269003 | Surface displayed protein forward primer 1 |
| BBa_K5269006 | Surface displayed protein reverse primer 2 |
| BBa_K5269004 | Surface displayed protein forward primer 2 |
| BBa_K5269007 | Surface displayed protein reverse primer 3 |
| BBa_K5269005 | Surface displayed protein forward primer 3 |
| BBa_K5269008 | Surface displayed protein reverse primer 4 |
| BBa_K5269010 | pBluescript II SK(-) In-Fusion forward primer 1 |
| BBa_K5269009 | pBluescript II SK(-) In-Fusion reverse primer 1 |
| BBa_K5269011 | Golden Gate Assembly Site In-Fusion forward primer 1 |
| BBa_K5269012 | Golden Gate Assembly Site In-Fusion reverse primer 1 |
| BBa_K5269029 | pBluescript II SK(-) In-Fusion forward primer 2 |
| BBa_K5269028 | pBluescript II SK(-) In-Fusion reverse primer 2 |
| BBa_K5269031 | shRNA-GGA_Site In-Fusion forward primer 2 |
| BBa_K5269030 | shRNA-GGA_Site In-Fusion reverse primer 2 |
| BBa_K5269032 | BsmB I insert |
| BBa_K5269034 | SD-sequence |
References
[1] Dong Soo Hwang, Youngsoo Gim, Dong Gyun Kang, Yeon Kyu Kim, Hyung Joo Cha. Recombinant mussel adhesive protein Mgfp-5 as cell adhesion biomaterial. Journal of Biotechnology. Volume127, Issue4, 727-735 (2007).
[2] Marco Orlando, Patrick C. F. Buchholz, Marina Lotti, Ju ¨rgen PleissI. The GH19 Engineering Database: Sequence diversity, substrate scope, and evolution in glycoside hydrolase family 19. PLoS ONE 16(10), (October 26, 2021).
[3] Dietmar Schomburg, Margit Salzmann,“Endo-1,3(4)-beta-glucanase” in Enzyme Handbook 4(Springer,1991),pp.41-46.
[4] Wenyuan Zhang, Hongrui Wu, Bryan Wei. Golden Gate Assembly of DNA Nanostructures. ACS Materials Letters, 3316-3320(2023 5(12)).