Difference between revisions of "Part:BBa K4195010"
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===Usage and design=== | ===Usage and design=== | ||
− | Engineering OMVs for treating and preventing AHPND caused by the pathogen ''V. parahaemolyticus'' are a significant part of '''OMEGA''' project (<u>O</u>perable <u>M</u>agic to <u>E</u>fficiently <u>G</u>etting over <u>A</u>HPND). Based on the efforts of our previous projects in 2020 ([https://2020.igem.org/Team:XMU-China AnTea-Glyphosate]) and 2021 ([https://2021.igem.org/Team:XMU-China SALVAGE]), we further developed the surface display system on the OMVs released by the engineered bacteria. The usage of cargo proteins were no more limited to enzymes that are usually utilized to catalyze series bio-chemical reactions, since some receptors or ligands involved in complex protein-protein interaction (PPI) were selected as the cargo candidates. This year, we chose two classic anchor proteins, ClyA and INPNC, to construct the display cassette with various cargo proteins including rFET (receptor), r''Lv''APN1 (receptor), | + | Engineering OMVs for treating and preventing AHPND caused by the pathogen ''V. parahaemolyticus'' are a significant part of '''OMEGA''' project (<u>O</u>perable <u>M</u>agic to <u>E</u>fficiently <u>G</u>etting over <u>A</u>HPND). Based on the efforts of our previous projects in 2020 ([https://2020.igem.org/Team:XMU-China AnTea-Glyphosate]) and 2021 ([https://2021.igem.org/Team:XMU-China SALVAGE]), we further developed the surface display system on the OMVs released by the engineered bacteria. The usage of cargo proteins were no more limited to enzymes that are usually utilized to catalyze series bio-chemical reactions, since some receptors or ligands involved in complex protein-protein interaction (PPI) were selected as the cargo candidates. This year, we chose two classic anchor proteins, ClyA and INPNC, to construct the display cassette with various cargo proteins including rFET (receptor), r''Lv''APN1 (receptor), TTPA (ligand) and TTPB (ligand) (Fig. 1). On one hand, with the receptors displayed, OMVs will gain the function of neutralizing toxins secreted by ''V. parahaemolyticus''. On the other hand, with the assistance of ligands displayed on the surface, OMVs will become a special vector to deliver antimicrobials for the specific pathogen. In summary, we have taken a step closer to the collections of '''extracellular functional elements (EFE), combining the OMVs, secretion systems and surface display systems''' which we have been dedicated to since 2020. Learn more information from our [https://2022.igem.wiki/xmu-china/design Design] page. |
[[File:T--XMU-China--surface display circuit.png|300px]] | [[File:T--XMU-China--surface display circuit.png|300px]] | ||
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[[File:T--XMU-China--BBa K4195103(ClyA-ttpB,colony PCR,BL21(DE3)).png|200px]] | [[File:T--XMU-China--BBa K4195103(ClyA-ttpB,colony PCR,BL21(DE3)).png|200px]] | ||
− | '''Fig.2 DNA gel electrophoresis of the colony PCR products of BBa_K4195103_pSB1C3.''' | + | '''Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195103_pSB1C3.''' |
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===Reference=== | ===Reference=== | ||
− | 1. K. Murase, Cytolysin A (ClyA): A Bacterial Virulence Factor with Potential Applications in Nanopore Technology, Vaccine Development, and Tumor Therapy. ''Toxins (Basel) ''' | + | 1. K. Murase, Cytolysin A (ClyA): A Bacterial Virulence Factor with Potential Applications in Nanopore Technology, Vaccine Development, and Tumor Therapy. ''Toxins (Basel).'' '''14''', 78 (2022). |
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+ | 2. M. Hu, H. Zhang, D. Gu, Y. Ma, X. Zhou, Identification of a novel bacterial receptor that binds tail tubular proteins and mediates phage infection of ''Vibrio parahaemolyticus''. ''Emerg. Microbes. Infect.'' '''9''', 855-867 (2020). | ||
Latest revision as of 10:30, 13 October 2022
clyA-ttpB
Biology
ClyA
Cytolysin A (ClyA) is a pore-forming toxin that is produced by some bacteria from the Enterobacteriaceae family. When fused to the C-terminal of ClyA, heterologous proteins can be displayed on the surface of the engineered bacteria and OMVs (outer membrane vesicles) released by them (1).
TTPB
TTPB is tail tubular protein B of podophage 7. It has been found that TTPB serves as ligands that recognizes the conserved Vibrio receptor Vp0980 to mediate phage adsorption. It binds with Vp0980 of Vibrio parahaemolyticus and then mediates phage adsorption and subsequent bacterial lysis (2).
Usage and design
Engineering OMVs for treating and preventing AHPND caused by the pathogen V. parahaemolyticus are a significant part of OMEGA project (Operable Magic to Efficiently Getting over AHPND). Based on the efforts of our previous projects in 2020 (AnTea-Glyphosate) and 2021 (SALVAGE), we further developed the surface display system on the OMVs released by the engineered bacteria. The usage of cargo proteins were no more limited to enzymes that are usually utilized to catalyze series bio-chemical reactions, since some receptors or ligands involved in complex protein-protein interaction (PPI) were selected as the cargo candidates. This year, we chose two classic anchor proteins, ClyA and INPNC, to construct the display cassette with various cargo proteins including rFET (receptor), rLvAPN1 (receptor), TTPA (ligand) and TTPB (ligand) (Fig. 1). On one hand, with the receptors displayed, OMVs will gain the function of neutralizing toxins secreted by V. parahaemolyticus. On the other hand, with the assistance of ligands displayed on the surface, OMVs will become a special vector to deliver antimicrobials for the specific pathogen. In summary, we have taken a step closer to the collections of extracellular functional elements (EFE), combining the OMVs, secretion systems and surface display systems which we have been dedicated to since 2020. Learn more information from our Design page.
Fig. 1 Graphic description of the expression gene circuits for display cassette designed in OMEGA project.
For this part (ClyA-TTPB), TTPB was fused to the C-terminal of ClyA to surface display for targeting V. parahaemolyticus. Arabinose-inducible system was used in the expression circuit of this part in pSB1C3 then constructed composite part BBa_K4195103. We transformed the constructed plasmid into E. coli BL21(DE3) for further verification of its expression and function on the surface of E. coli and OMVs, including the interaction between TTPB and Vp0980.
Characterization
1. Identification
When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformatants were correct. Target bands (4670 bp) can be observed at the position around 5000 bp (Fig. 2).
Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195103_pSB1C3.
Reference
1. K. Murase, Cytolysin A (ClyA): A Bacterial Virulence Factor with Potential Applications in Nanopore Technology, Vaccine Development, and Tumor Therapy. Toxins (Basel). 14, 78 (2022).
2. M. Hu, H. Zhang, D. Gu, Y. Ma, X. Zhou, Identification of a novel bacterial receptor that binds tail tubular proteins and mediates phage infection of Vibrio parahaemolyticus. Emerg. Microbes. Infect. 9, 855-867 (2020).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 325
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2166
Illegal BamHI site found at 2088 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1210
Illegal AgeI site found at 2638 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 772