Difference between revisions of "Part:BBa K4195029"

(Ability of binding toxins on the surface of OMVs)
 
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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'').
 
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'').
 
===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 SALAGE]), 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.
+
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 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.
  
 
[[File:T--XMU-China--surface display circuit.png|300px]]
 
[[File:T--XMU-China--surface display circuit.png|300px]]
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For this part (ClyA-r''Lv''APN1), r''Lv''APN1 was fused to the C-terminal of ClyA to surface display for binding PirA and PirB toxins. Arabinose-inducible system was used in the expression circuit of this part in pSB1C3 then composite part <partinfo>BBa_K4195130</partinfo> was obtained. 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 PirA and PirB toxins.
 
For this part (ClyA-r''Lv''APN1), r''Lv''APN1 was fused to the C-terminal of ClyA to surface display for binding PirA and PirB toxins. Arabinose-inducible system was used in the expression circuit of this part in pSB1C3 then composite part <partinfo>BBa_K4195130</partinfo> was obtained. 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 PirA and PirB toxins.
 +
 
===Characterization===
 
===Characterization===
 
====Identification====
 
====Identification====
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'''Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195130_pSB1C3.'''
 
'''Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195130_pSB1C3.'''
  
We used <partinfo>BBa_I0500</partinfo> promoter and RBS (<partinfo>BBa_B0034</partinfo>) to express ClyA- r''Lv''APN1 protein in ''E. coli'' BL21(DE3). 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 verify whether the displayed r''Lv''APN1 could bind PirA-his and his-PirB or not.  
+
====Ability of binding PirA-his and his-PirB on the surface of engineered bacteria====
 +
We used <partinfo>BBa_I0500</partinfo> promoter and RBS (<partinfo>BBa_B0034</partinfo>) to express ClyA-r''Lv''APN1 protein in ''E. coli'' BL21(DE3). 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 verify whether the displayed r''Lv''APN1 could bind PirA-his and his-PirB or not.  
  
[[File:T--XMU-China-BBa K4195130 Fig3.png|200px]]
+
[[File:T--XMU-China-BBa K4195130 Fig3.png|350px]]
  
 
'''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).
 
'''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 (''λ''<sub>Ex</sub> = 492 nm, ''λ''<sub>Em</sub> = 528 nm) to OD<sub>600</sub> 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.
 
The ratio of fluorescence intensity (''λ''<sub>Ex</sub> = 492 nm, ''λ''<sub>Em</sub> = 528 nm) to OD<sub>600</sub> 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.
 +
 +
====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 <partinfo>BBa_K4195130</partinfo> 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.
 +
 +
[[File:T--XMU-China--BBa K4195130 029 Fig.4.png|300px]]
 +
 +
'''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 <partinfo>BBa_K4195130</partinfo>. 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.
 +
 +
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 anti-His-tag antibody to INPNC-TTPA. Learn more information about this from our [https://2022.igem.wiki/xmu-china/proof-of-concept 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).
  
 
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Latest revision as of 02:30, 14 October 2022


clyA-rLvAPN1

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 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--surface display circuit.png

Fig. 1 Graphic description of the expression gene circuits for display cassette designed in OMEGA project.

For this part (ClyA-rLvAPN1), rLvAPN1 was fused to the C-terminal of ClyA to surface display for binding PirA and PirB toxins. Arabinose-inducible system was used in the expression circuit of this part in pSB1C3 then composite part BBa_K4195130 was obtained. 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 PirA and PirB toxins.

Characterization

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.

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

We used BBa_I0500 promoter and RBS (BBa_B0034) to express ClyA-rLvAPN1 protein in E. coli BL21(DE3). 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 verify 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.

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 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).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1773
    Illegal AgeI site found at 1714
    Illegal AgeI site found at 1821
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 645