Difference between revisions of "Part:BBa K4195113"

 
 
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<partinfo>BBa_K4195113 short</partinfo>
 
<partinfo>BBa_K4195113 short</partinfo>
  
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===Biology===
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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 <i>Vibrio</i> receptor Vp0980 to mediate phage adsorption. It binds with Vp0980 of ''Vibrio parahaemolyticus'' and then mediates phage adsorption and subsequent bacterial lysis (''1'').
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===Usage and design===
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This circuit was used as the control group to demonstrate surface display of INPNC/ClyA-TTPB and to certify TTPB can interact with the Vp0980 displayed on the surface of ''E. coli''. Both <partinfo>BBa_I0500</partinfo> were used to construct the expression system and obtained the composite <partinfo>BBa_K4195113</partinfo>, which are assembled on the expression vector pSB1C3 by standard assembly(Fig. 1). The constructed plasmids were transformed into ''E. coli'' BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.
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[[File:T--XMU-China--TTPB-his circuit.png|300px]]
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'''Fig. 1 Graphic description of the expression gene circuits for display cassette designed in OMEGA project.'''
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===Characterization===
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====Agarose gel electrophoresis (AGE)====
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When we were constructing this circuit, regular PCR was used to certify the plasmid was correct. We got the target (4048 bp).
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[[File:T--XMU-China-- 113fig.2.png]]
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<b>Fig. 2 The result of regular PCR. Plasmid pSB1C3.</b>
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====SDS-PAGE====
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The plasmid verified by sequencing was successfully transformed into <i>E. coli</i> SHuffle T7. After being cultivated and induced by <i>L</i>-arabinose, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant.
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After the first time of purification, we found that the protein was poorly expressed, so we did not obtain the target protein. Therefore, we constructed this part on the expression vector pET-28a(+) by Gibson assembly, then transformed the plasmid into <i>E. coli</i> BL21(DE3). Learn more specific and detailed information in the parts page of this basic part (<partinfo>BBa_K4195008</partinfo>).
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===Reference===
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1. 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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===Usage and Biology===
 
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4195113 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4195113 SequenceAndFeatures</partinfo>

Latest revision as of 12:09, 12 October 2022


I0500-B0034-ttpB-his-B0015

Biology

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 (1).

Usage and design

This circuit was used as the control group to demonstrate surface display of INPNC/ClyA-TTPB and to certify TTPB can interact with the Vp0980 displayed on the surface of E. coli. Both BBa_I0500 were used to construct the expression system and obtained the composite BBa_K4195113, which are assembled on the expression vector pSB1C3 by standard assembly(Fig. 1). The constructed plasmids were transformed into E. coli BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.

T--XMU-China--TTPB-his circuit.png

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


Characterization

Agarose gel electrophoresis (AGE)

When we were constructing this circuit, regular PCR was used to certify the plasmid was correct. We got the target (4048 bp).

T--XMU-China-- 113fig.2.png

Fig. 2 The result of regular PCR. Plasmid pSB1C3.

SDS-PAGE

The plasmid verified by sequencing was successfully transformed into E. coli SHuffle T7. After being cultivated and induced by L-arabinose, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. After the first time of purification, we found that the protein was poorly expressed, so we did not obtain the target protein. Therefore, we constructed this part on the expression vector pET-28a(+) by Gibson assembly, then transformed the plasmid into E. coli BL21(DE3). Learn more specific and detailed information in the parts page of this basic part (BBa_K4195008).

Reference

1. 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 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
    Illegal BamHI site found at 3348
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2001
    Illegal AgeI site found at 979
    Illegal AgeI site found at 2326
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961
    Illegal SapI site found at 2901