Difference between revisions of "Part:BBa K4195131"

(rLvAPN1)
 
(3 intermediate revisions by one other user not shown)
Line 5: Line 5:
 
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''Lv''APN1====
 
====r''Lv''APN1====
''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/>
+
''Lv''APN1, a protein from the aminopeptidase N family, was identified in ''Litopenaeus'' ''vannamei'' hemocytes as a receptor for VP<sub>AHPND</sub> toxin PirA and PirB, which can help the toxins pass through the cell membrane of hemocytes (''2''). <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/>
+
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 19: Line 20:
 
We used <partinfo>BBa_I0500</partinfo> promoter and RBS (<partinfo>BBa_B0034</partinfo>) to express ClyA-r''Lv''APN1-his protein in ''E. coli'' BL21(DE3). We used <partinfo>BBa_K4195134</partinfo> which has no surface display system (like INPNC or ClyA) as negative control and <partinfo>BBa_K4195131</partinfo> as positive control. The arabinose-induced overnight culture was then incubated with FITC-labeled anti-His-tag antibody to verify whether the display system is functional or not. <br/>
 
We used <partinfo>BBa_I0500</partinfo> promoter and RBS (<partinfo>BBa_B0034</partinfo>) to express ClyA-r''Lv''APN1-his protein in ''E. coli'' BL21(DE3). We used <partinfo>BBa_K4195134</partinfo> which has no surface display system (like INPNC or ClyA) as negative control and <partinfo>BBa_K4195131</partinfo> as positive control. The arabinose-induced overnight culture was then incubated with FITC-labeled anti-His-tag antibody to verify whether the display system is functional or not. <br/>
 
[[File:T--XMU-China--BBa K4195131 030 Fig.3.png|400px]]<br/>
 
[[File:T--XMU-China--BBa K4195131 030 Fig.3.png|400px]]<br/>
'''Fig. 3 The results of immunofluorescence to characterize the function of the display system (p = 0.0092).'''<br/>
+
'''Fig. 3 The results of immunofluorescence to characterize the function of the display system (''p'' = 0.0092).'''<br/>
 
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 (ClyA-r''Lv''APN1-his) is higher than that of negative control (r''Lv''APN1-his) (Fig. 3), which indicates that our surface display system works well that the receptor r''Lv''APN1 is successfully located on the surface of bacteria.<br/>
 
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 (ClyA-r''Lv''APN1-his) is higher than that of negative control (r''Lv''APN1-his) (Fig. 3), which indicates that our surface display system works well that the receptor r''Lv''APN1 is successfully located on the surface of bacteria.<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/>
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).
+
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).
  
  

Latest revision as of 15:44, 12 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 in pSB1C3 to express ClyA-rLvAPN1-his (BBa_K4195030), which is induced by L-arabinose. We transformed the plasmid into E. coli BL21(DE3) for further verification of its location on the surface of E. coli.

Characterization

1.Identification

When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformatants were correct. Target bands (3879 bp) can be observed at the position between 3000 and 5000 bp (Fig. 2).
T--XMU-China--BBa K4195131 030 Fig.2.png
Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195131_pSB1C3.
We used BBa_I0500 promoter and RBS (BBa_B0034) to express ClyA-rLvAPN1-his protein in E. coli BL21(DE3). We used BBa_K4195134 which has no surface display system (like INPNC or ClyA) as negative control and BBa_K4195131 as positive control. The arabinose-induced overnight culture was then incubated with FITC-labeled anti-His-tag antibody to verify whether the display system is functional or not.
T--XMU-China--BBa K4195131 030 Fig.3.png
Fig. 3 The results of immunofluorescence to characterize the function of the display system (p = 0.0092).
The ratio of fluorescence intensity (λEx= 492 nm, λEm = 528 nm) to OD600 of positive control (ClyA-rLvAPN1-his) is higher than that of negative control (rLvAPN1-his) (Fig. 3), which indicates that our surface display system works well that the receptor rLvAPN1 is successfully located on the surface of bacteria.

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).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 3009
    Illegal AgeI site found at 979
    Illegal AgeI site found at 2950
    Illegal AgeI site found at 3057
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961
    Illegal SapI.rc site found at 1881