Difference between revisions of "Part:BBa K4195011"

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TTPB
 
TTPB
  
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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(''3'').
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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 (''3'').
  
  
 
===Usage and design===
 
===Usage and design===
  
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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 SAVAGE]), 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), TTPB (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.
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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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'''Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195102_pSB1C3.'''
 
'''Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195102_pSB1C3.'''
  
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====2. Ability of binding Vp0980 on the surface of engineered bacteria====
 
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We used <partinfo>BBa_I0500</partinfo> promoter and RBS (<partinfo>BBa_B0034</partinfo>) to express INPNC-TTPB protein in ''E. coli'' BL21(DE3). The arabinose-induced overnight culture was then incubated with purified Vp0980-his and FITC-labeled anti-His-tag antibody in turn to verify whether the displayed TTPB could bind Vp0980 or not.
 
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====3. Ability of binding Vp0980 on the surface of OMVs====
 
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For the test on OMVs, the OMVs were firstly extracted from the culture of engineered bacteria harboring <partinfo>BBa_K4195102</partinfo> after induction. Subsequently, the OMVs-containing samples were directly spotted onto the nitrocellulose (NC) membrane. Then the NC membrane was incubated with purified Vp0980-his and anti-His-tag antibody in turn and finally probed by the HRP-conjugated secondary antibody(''4, 5''). By comparing the chemiluminescence imaging results of OMVs-containing samples of different origins, we could characterize whether the displayed TTPB on OMVs is functional or not.
 
  
  
 
===Reference===
 
===Reference===
  
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1. E. van Bloois, R. T. Winter, H. Kolmar, M. W. Fraaije, Decorating microbes: surface display of proteins on Escherichia coli. ''Trends Biotechnol '''29''''', 79-86 (2011).
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1. E. van Bloois, R. T. Winter, H. Kolmar, M. W. Fraaije, Decorating microbes: surface display of proteins on ''Escherichia coli''. ''Trends. Biotechnol.'' '''29''', 79-86 (2011).
  
 
2. http://2016.igem.org/Team:TJUSLS_China.
 
2. http://2016.igem.org/Team:TJUSLS_China.
  
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3. 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).
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3. 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).
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4. 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).
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5. 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).
 
  
  

Latest revision as of 10:30, 13 October 2022


INPNC-ttpB


Biology

INPNC

INPNC is a truncated form of ice nucleation protein (INP) consisting of N- and C- terminal domains (1). It is a membrane protein commonly used to displayed protein on the cell surface (2).

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 (3).


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 Designpage.

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 (INPNC-TTPB), TTPB was fused to the C-terminal of INPNC 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_K4195102. 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 (4688 bp) can be observed at the position around 5000 bp (Fig. 2).

T--XMU-China--BBa K4195102（INPNC-ttpB,colony PCR,BL21(DE3)).png

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4195102_pSB1C3.


Reference

1. E. van Bloois, R. T. Winter, H. Kolmar, M. W. Fraaije, Decorating microbes: surface display of proteins on Escherichia coli. Trends. Biotechnol. 29, 79-86 (2011).

2. http://2016.igem.org/Team:TJUSLS_China.

3. 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 301
    Illegal NheI site found at 337
    Illegal NheI site found at 2473
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 330
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 72
    Illegal NgoMIV site found at 405
    Illegal NgoMIV site found at 1737
    Illegal AgeI site found at 823
    Illegal AgeI site found at 1093
    Illegal AgeI site found at 1228
    Illegal AgeI site found at 1648
    Illegal AgeI site found at 1996
    Illegal AgeI site found at 2827
    Illegal AgeI site found at 3018
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1726
    Illegal SapI site found at 944