Difference between revisions of "Part:BBa K4195130"

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====ClyA====
 
====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''). <br/>
 
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''). <br/>
====r''LvAPN1''====
+
====r''Lv''APN1====
''LvAPN1'', a protein from the aminopeptidase N family, was identified in ''Litopenaeus vannamei'' hemocytes as a receptor for VPAHPND toxin PirA and PirB, which can help the toxins pass through the cell membrane of hemocytes (''2''). <br/>
+
''Lv''APN1, a protein from the aminopeptidase N family, was identified in ''Litopenaeus vannamei'' hemocytes as a receptor for VPAHPND toxin PirA and PirB, which can help the toxins pass through the cell membrane of hemocytes (''2''). <br/>
r''LvAPN1'' is a truncated form of ''LvAPN1'' (residues 205-591) that composes of a crystal insecticidal (Cry) toxin binding region and the active site of peptidase-M1 domain, which was reported to directly bind to both PirA and PirB toxins (''2''). What’s more, there is no glycosylation site in r''LvAPN1'', which makes it easier to obtain the purified protein by using prokaryotic expression system (such as ''E. coli'').<br/>
+
r''Lv''APN1 is a truncated form of ''Lv''APN1 (residues 205-591) that composes of a crystal insecticidal (Cry) toxin binding region and the active site of peptidase-M1 domain, which was reported to directly bind to both PirA and PirB toxins (''2''). What’s more, there is no glycosylation site in r''Lv''APN1, which makes it easier to obtain the purified protein by using prokaryotic expression system (such as ''E. coli'').<br/>
 
===Usage and design===
 
===Usage and design===
 
Engineering outer membrane vesicles (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 was 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.<br/>
 
Engineering outer membrane vesicles (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 was 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.<br/>
Line 17: Line 17:
 
'''Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195130_pSB1C3.'''<br/>
 
'''Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195130_pSB1C3.'''<br/>
 
====2. Ability of binding PirA-his and his-PirB on the surface of engineered bacteria====
 
====2. Ability of binding PirA-his and his-PirB on the surface of engineered bacteria====
The arabinose-induced overnight culture was firstly incubated with purified PirA-his or his-PirB, then FITC-labeled anti-His-tag antibody in turn to characterize whether the displayed r''LvAPN1'' could bind PirA-his and his-PirB or not.<br/>
+
The arabinose-induced overnight culture was firstly incubated with purified PirA-his or his-PirB, then FITC-labeled anti-His-tag antibody in turn to characterize whether the displayed r''Lv''APN1 could bind PirA-his and his-PirB or not.<br/>
 
[[File:T--XMU-China-BBa K4195130 Fig3.png|400px]]<br/>
 
[[File:T--XMU-China-BBa K4195130 Fig3.png|400px]]<br/>
 
'''Fig. 3 The results of immunofluorescence to probe the binding event on the surface of engineered bacteria.'''Purified PirA-his ('''a''') or his-PirB ('''b''') was tested to interact with the displayed receptors (''p'' = 0.0082 for PirA-his, ''p'' = 0.0213 for his-PirB).<br/>
 
'''Fig. 3 The results of immunofluorescence to probe the binding event on the surface of engineered bacteria.'''Purified PirA-his ('''a''') or his-PirB ('''b''') was tested to interact with the displayed receptors (''p'' = 0.0082 for PirA-his, ''p'' = 0.0213 for his-PirB).<br/>
The ratio of fluorescence intensity (λEx = 492 nm, λEm = 528 nm) to OD600 of positive control (culture was incubated with PirA-his or his-PirB) is higher than that of negative control (culture was incubated with 1×TBST) (Fig. 3), which indicates that our surface display system works well and the binding ability of r''LvAPN1'' to PirA-his and his-PirB is retained on the surface of bacteria.<br/>
+
The ratio of fluorescence intensity (λEx = 492 nm, λEm = 528 nm) to OD600 of positive control (culture was incubated with PirA-his or his-PirB) is higher than that of negative control (culture was incubated with 1×TBST) (Fig. 3), which indicates that our surface display system works well and the binding ability of r''Lv''APN1 to PirA-his and his-PirB is retained on the surface of bacteria.<br/>
 
====3. Ability of binding toxins on the surface of OMVs====
 
====3. Ability of binding toxins on the surface of OMVs====
For the test on OMVs, the OMVs were firstly extracted from the culture of engineered bacteria harboring BBa_K4195130 after induction. Subsequently, the OMVs-containing samples were directly spotted onto the nitrocellulose (NC) membrane. Then the NC membrane was incubated with purified his-PirB and anti-His-tag antibody in turn, and finally probed by the HRP-conjugated secondary antibody (3,4). By comparing the chemiluminescence imaging results of OMVs-containing samples of different origins, we could characterize whether the displayed r''LvAPN1'' on OMVs is functional or not.<br/>
+
For the test on OMVs, the OMVs were firstly extracted from the culture of engineered bacteria harboring BBa_K4195130 after induction. Subsequently, the OMVs-containing samples were directly spotted onto the nitrocellulose (NC) membrane. Then the NC membrane was incubated with purified his-PirB and anti-His-tag antibody in turn, and finally probed by the HRP-conjugated secondary antibody (3,4). By comparing the chemiluminescence imaging results of OMVs-containing samples of different origins, we could characterize whether the displayed r''Lv''APN1 on OMVs is functional or not.<br/>
 
[[File:T--XMU-China--BBa K4195130 029 Fig.4.png|400px]]<br/>
 
[[File:T--XMU-China--BBa K4195130 029 Fig.4.png|400px]]<br/>
 
'''Fig. 4 The imaging results of chemiluminescence (dot blot analysis) to probe the binding event on the surface of OMVs.'''<br/>
 
'''Fig. 4 The imaging results of chemiluminescence (dot blot analysis) to probe the binding event on the surface of OMVs.'''<br/>
As with his-PirB added, we observed a strong signal from the OMVs fraction derived from the engineered bacteria harboring BBa_K4195130. In contrast, faint detectable signal was observed from the OMVs fraction derived from the bacteria without r''LvAPN1'' displayed (INPNC-TTPA in this case) (Fig. 4), suggesting that r''LvAPN1'' displayed on OMVs can still bind to toxins PirB specifically. <br/>
+
As with his-PirB added, we observed a strong signal from the OMVs fraction derived from the engineered bacteria harboring BBa_K4195130. In contrast, faint detectable signal was observed from the OMVs fraction derived from the bacteria without r''Lv''APN1 displayed (INPNC-TTPA in this case) (Fig. 4), suggesting that r''Lv''APN1 displayed on OMVs can still bind to toxins PirB specifically. <br/>
For the faint detectable signal observed from the OMVs fraction derived from the bacteria without r''LvAPN1'' displayed, we attributed this to the potential cross-reactivity of our FITC-labeled anti-His-tag antibody to INPNC-TTPA. Learn more information about this from our Proof of Concept page.<br/>
+
For the faint detectable signal observed from the OMVs fraction derived from the bacteria without r''Lv''APN1 displayed, we attributed this to the potential cross-reactivity of our FITC-labeled anti-His-tag antibody to INPNC-TTPA. Learn more information about this from our Proof of Concept page.<br/>
 
===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). 14'', 78 (2022).<br/>
 
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).<br/>

Revision as of 19:22, 10 October 2022

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).

rLvAPN1

LvAPN1, a protein from the aminopeptidase N family, was identified in Litopenaeus vannamei hemocytes as a receptor for VPAHPND toxin PirA and PirB, which can help the toxins pass through the cell membrane of hemocytes (2).
rLvAPN1 is a truncated form of LvAPN1 (residues 205-591) that composes of a crystal insecticidal (Cry) toxin binding region and the active site of peptidase-M1 domain, which was reported to directly bind to both PirA and PirB toxins (2). What’s more, there is no glycosylation site in rLvAPN1, which makes it easier to obtain the purified protein by using prokaryotic expression system (such as E. coli).

Usage and design

Engineering outer membrane vesicles (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 was 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.
T--XMU-China--inpnc-rfet-OMEGA.png
Fig. 1 Graphic description of the expression gene circuits for display cassette designed in OMEGA project.
This composite part was constructed at pSB1C3 to express ClyA-rLvAPN1 (BBa_K4195029), which is induced by L-arabinose. We transformed the 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 PirA and PirB toxins.

Characterization

1.Identification

When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformatants were correct. Target bands (3816 bp) can be observed at the position between 3000 and 5000 bp (Fig. 2).
T--XMU-China-BBa K4195029.png
Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195130_pSB1C3.

2. Ability of binding PirA-his and his-PirB on the surface of engineered bacteria

The arabinose-induced overnight culture was firstly incubated with purified PirA-his or his-PirB, then FITC-labeled anti-His-tag antibody in turn to characterize whether the displayed rLvAPN1 could bind PirA-his and his-PirB or not.
T--XMU-China-BBa K4195130 Fig3.png
Fig. 3 The results of immunofluorescence to probe the binding event on the surface of engineered bacteria.Purified PirA-his (a) or his-PirB (b) was tested to interact with the displayed receptors (p = 0.0082 for PirA-his, p = 0.0213 for his-PirB).
The ratio of fluorescence intensity (λEx = 492 nm, λEm = 528 nm) to OD600 of positive control (culture was incubated with PirA-his or his-PirB) is higher than that of negative control (culture was incubated with 1×TBST) (Fig. 3), which indicates that our surface display system works well and the binding ability of rLvAPN1 to PirA-his and his-PirB is retained on the surface of bacteria.

3. Ability of binding toxins on the surface of OMVs

For the test on OMVs, the OMVs were firstly extracted from the culture of engineered bacteria harboring BBa_K4195130 after induction. Subsequently, the OMVs-containing samples were directly spotted onto the nitrocellulose (NC) membrane. Then the NC membrane was incubated with purified his-PirB and anti-His-tag antibody in turn, and finally probed by the HRP-conjugated secondary antibody (3,4). By comparing the chemiluminescence imaging results of OMVs-containing samples of different origins, we could characterize whether the displayed rLvAPN1 on OMVs is functional or not.
T--XMU-China--BBa K4195130 029 Fig.4.png
Fig. 4 The imaging results of chemiluminescence (dot blot analysis) to probe the binding event on the surface of OMVs.
As with his-PirB added, we observed a strong signal from the OMVs fraction derived from the engineered bacteria harboring BBa_K4195130. In contrast, faint detectable signal was observed from the OMVs fraction derived from the bacteria without rLvAPN1 displayed (INPNC-TTPA in this case) (Fig. 4), suggesting that rLvAPN1 displayed on OMVs can still bind to toxins PirB specifically.
For the faint detectable signal observed from the OMVs fraction derived from the bacteria without rLvAPN1 displayed, we attributed this to the potential cross-reactivity of our FITC-labeled anti-His-tag antibody to INPNC-TTPA. Learn more information about this from our Proof of Concept page.

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. W. Luangtrakul et al., Cytotoxicity of Vibrio parahaemolyticus AHPND toxin on shrimp hemocytes, a newly identified target tissue, involves binding of toxin to aminopeptidase N1 receptor. PLoS Pathog. 17, e1009463 (2021).
3. J. L. Valentine et al., Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies. Cell Chem. Biol. 23, 655-665 (2016).
4.T. C. Stevenson et al., Immunization with outer membrane vesicles displaying conserved surface polysaccharide antigen elicits broadly antimicrobial antibodies. Proc. Natl. Acad. Sci. U. S. A. 115, E3106-E3115 (2018).